the role of the transcriptional regulatory protein bcl11b
TRANSCRIPT
AN ABSTRACT OF THE DISSERTATION OF
Kateryna Kyrylkova for the degree of Doctor of Philosophy in Pharmacy
presented on January 16, 2014
Title: The Role of the Transcriptional Regulatory Protein BCL11B in Dental and
Craniofacial Development
Abstract approved:
Mark E. Leid
BCL11B is a transcriptional regulatory protein that plays essential roles during
mouse embryonic development. BCL11B is expressed and functions in the
immune and nervous systems as well as within ectodermal organs. Multiple
studies have characterized the roles of BCL11B in T cells, brain, and skin.
However, very little is known about the mechanistic role of BCL11B during tooth
development, and data are not available on the function of BCL11B in the
craniofacial skeleton.
BCL11B is expressed widely within the or al cavity during development, and mice
lacking BCL11B exhibit a spectrum of to oth developmental defects. The most
striking feature of the Bcl11b-/- dental ph enotype is a defect in development of
enamel-secreting cells, known as ameloblasts, in the mouse incisor. Ameloblasts
are localized exclusively on the labial aspect of the mouse incisor in wild-type
mice. In contrast, Bcl11b-/- mice exhibit defective ameloblasts on the labial and
develop ectopic, ameloblast-like cells on the lingual aspect of the tooth.
BCL11B regulates asymmetric ameloblast formation by regulating the
development of epithelial stem cell niches in the posterior part of the incisor.
Specifically, BCL11B induces proliferation and differentiation of epithelial stem
cells into ameloblasts in the labial cervical loop, whereas BCL11B suppresses
these processes within the lingual epithelium. Such bidirectional actions of
BCL11B are mediated by spatio-specific regulation of a large gene network
comprised of genes that encode members of fibroblast growth factor (FGF) and
transforming growth factor β (TGFβ) superfamilies, Sprouty proteins, and sonic
hedgehog (SHH). In addition, my data integrate BCL11B into FGF and SHH
signaling pathways revealing the molecular mechanisms that suppress
development of ectopic ameloblast-like cells in the lingual epithelium.
In the second half of this dissertation, I show that BCL11B is expressed in the
osteogenic mesenchyme of developing craniofacial skeleton, and loss of
BCL11B in these tissues has striking effects on craniofacial development.
-/-Bcl11b mice exhibit accelerated mineralization of the skull during embryonic
development and synostosis of facial and coronal sutures. My results
demonstrate that BCL11B normally functions to suppress proliferation and
premature differentiation of osteoblasts in the craniofacial complex. I suggest that
the principal mechanistic basis of these actions of BCL11B is the repression of
Fgfr2c expression within the osteogenic mesenchyme.
Taken together, my data demonstrate that BCL11B plays an important role in
proliferation and differentiation of ameloblast and osteoblast lineages. In addition,
my work implicates BCL11B in regulation of FGF and TGFβ signaling pathways.
Therefore, these studies contribute to a better understanding of the molecular
and cellular functions of BCL11B in vivo.
©Copyright by Kateryna Kyrylkova January 16, 2014
All Rights Reserved
The Role of the Transcriptional Regulatory Protein BCL11B in Dental and Craniofacial Development
by Kateryna Kyrylkova
A DISSERTATION
submitted to
Oregon State University
in partial fulfillment of the requirements for the
degree of
Doctor of Philosophy
Presented January 16, 2014 Commencement June 2014
Doctor of Philosophy dissertation of Kateryna Kyrylkova presented on
January 16, 2014.
APPROVED:
Major Professor, representing Pharmacy
Dean of the College of Pharmacy
Dean of the Graduate School
I understand that my dissertation will become part of the permanent collection of
Oregon State University libraries. My signature below authorizes the release of
my dissertation to any reader upon request.
Kateryna Kyrylkova, Author
ACKNOWLEDGEMENTS
Dr. Mark Leid, who was not only my graduate advisor, but a real mentor and a
good friend
Dr. Chrissa Kioussi, who supervised my first steps in developmental biology, and
provided an example of a confident and successful woman
Dr. Michael Freitag, Dr. John Fowler, and Dr. Siva Kolluri, my committee
members, who challenged me throughout the entire PhD program
Dr. Mark Zabriskie, who always believed in me and shared the joy of my
achievements
Dr. Taifo Mahmud, who was always patient and supportive with all my
organizational endeavors and multiple applications
Dr. Urszula Iwaniec, our precious collaborator, without whom our craniofacial
project would not be possible
Dr. Ophir Klein and Dr. Brian Biehs (from UCSF), our collaborators for the tooth
project
Dr. Oleh Taratula, who inspired me to continuously aim for better and achieve
every goal I set
Dr. Andriy Morgun, who was a constant source of encouragement in my
professional development, as well as the postdoctoral position search
Dr. Aleksandra Sikora, who tremendously supported me when Mark was away,
and made me feel as a part of her lab-family
Dr. Jane Ishmael, Dr. Theresa Filtz, Dr. Olena Taratula, Dr. Natalia Shulzhenko,
Drs. Gitali and Arup Indra, Dr. Michael Gross, Dr. Adam Alani, who were always
there for me when I needed any help or support
All students, postdocs, and technicians (especially Dr. Walter Vogel, Dr. Kenneth
Philbrick, Sam Bradford, Jeff Serrill, Katya Distanova, Lina Thomas and
numerous others) for being supportive and creating a great environment at the
department
Former lab members, including Dr. Olga Golonzhka, Dr. Ling-Juan Zhang,
Dr. Anand Venkataraman, for setting an example of what I should aim for
College of Pharmacy and Graduate School for supporting me financially
My family, Volodymyr Kyrylkov, Oksana Kyrylkova, Sergiy Kyrylkov, Hanna
Vayvala, who always emphasized the importance of education as the first priority
of my early life
And finally to a person, who has been with me and supported me in every
possible way throughout my entire long PhD journey, my husband Sergiy
Kyryachenko
CONTRIBUTION OF AUTHORS
Chapter 1: Kateryna Kyrylkova wrote the chapter. Kateryna Kyrylkova and Mark
Leid edited the chapter.
Chapter 2: Kateryna Kyrylkova, Sergiy Kyryachenko, Brian Biehs, Ophir Klein,
Chrissa Kioussi, and Mark Leid conceived and designed experiments. Kateryna
Kyrylkova and Sergiy Kyryachenko performed the experiments and data
analysis. Brian Biehs, Ophir Klein, Chrissa Kioussi, and Mark Leid contributed
reagents and materials. Kateryna Kyrylkova wrote the chapter. Kateryna
Kyrylkova, Ophir Klein, Chrissa Kioussi, and Mark Leid edited the chapter.
Chapter 3: Kateryna Kyrylkova, Urszula Iwaniec, and Mark Leid conceived and
designed experiments. Kateryna Kyrylkova and Urszula Iwaniec performed the
experiments and data analysis. Urszula Iwaniec and Mark Leid contributed
reagents and materials. Kateryna Kyrylkova wrote the chapter. Kateryna
Kyrylkova and Mark Leid edited the chapter.
Chapter 4: Kateryna Kyrylkova wrote the chapter. Kateryna Kyrylkova and Mark
Leid edited the chapter.
Page
Chapter 1. Transcriptional Regulation and in vivo functions of BCL11B ........... 1
BCL11B as a Transcriptional Regulatory Protein ........................................ 3
BCL11B Function in vivo ............................................................................. 6
BCL11B in T Cell Development ............................................................. 6
BCL11B in Neural Development ............................................................ 8
BCL11B in Development of Ectodermal Organs .................................. 11
References ................................................................................................ 13
Chapter 2. BCL11B Regulates Epithelial Proliferation and Asymmetric
Development of the Mouse Mandibular Incisor............................................... 20
Abstract ..................................................................................................... 21
Introduction ............................................................................................... 21
Materials and Methods .............................................................................. 23
Mouse Lines ........................................................................................ 23
Histological Analysis, RNA in situ Hybridization, and
Immunohistochemistry ......................................................................... 24
Cell Proliferation Assay ........................................................................ 24
Apoptosis Assay .................................................................................. 24
TABLE OF CONTENTS
Page
Results ...................................................................................................... 24
BCL11B is Expressed at all Stages of Incisor Development ................ 24
Reduced Epithelial Proliferation between Initiation and Bud Stage in
Bcl11b−/− Incisors ................................................................................. 25
Altered Development of Bcl11b−/− Incisors at Cap Stage ..................... 26
Reduced Size and Disruption of Labial-lingual Asymmetry at Bell
Stage in Bcl11b−/− Incisors ................................................................... 27
Delay in Ameloblast Development and Ectopic Formation of Lingual Ameloblast-like Cells in Bcl11b−/− Incisors ........................................... 28
Alteration of the FGF Signaling at Bell Stage in Bcl11b−/− Incisors ...... 29
−/− Disruption of TGFβ Signaling at Bell Stage in Bcl11b Mice .............. 31
Cell Autonomous Effects of BCL11B in Lingual Epithelium ................. 32
FGF Signaling Negatively Regulates BCL11B Expression in the
Lingual IEE and SR ............................................................................. 34
Discussion ................................................................................................. 36
BCL11B Regulates Proliferation of the Dental Epithelium ................... 36
BCL11B Controls the Expression of FGF and TGFβ Family
Members ............................................................................................. 37
BCL11B Controls Asymmetric Development of the Ameloblasts ......... 39
TABLE OF CONTENTS (Continued)
Page
Integration of BCL11B into FGF and SHH Signaling Pathways ........... 41
Acknowledgements ................................................................................... 42
References ................................................................................................ 67
Chapter 3. BCL11B Regulates Craniofacial Suture Patency through
Repression o f Fgfr2c Expression .................................................................... 71
Abstract ..................................................................................................... 72
Introduction ............................................................................................... 72
Materials and Methods .............................................................................. 74
Mouse Lines ........................................................................................ 74
Micro-CT Analysis ................................................................................ 74
Histological Analysis, RNA in situ Hybridization, BrdU Labeling and
Immunohistochemistry ......................................................................... 74
Results ...................................................................................................... 75
Neural Crest-Specific Ablation of Bcl11b Leads to Growth
Impairment and Craniofacial Synostoses ............................................. 75
Newborn Bcl11b-/- Mice Exhibit Facial and Coronal Synostoses .......... 76
Embryonic Skulls of Bcl11b-/- Exhibit Increased Osteoblast
Proliferation and Maturation, Premature Mineralization, and
Craniofacial Synostosis ....................................................................... 76
TABLE OF CONTENTS (Continued)
TABLE OF CONTENTS (Continued)
Page
BCL11B Is Expressed in Osteogenic Mesenchyme ............................. 77
Runx2 Expression I s Up-Regulated in Osteogenic Mesenchyme of
Bcl11b-/- Skulls ..................................................................................... 78
Fgfr2c is ectopically expressed within facial and coronal sutures of
Bcl11b-/- mice ....................................................................................... 78
Discussion ................................................................................................. 79
References ................................................................................................ 96
Chapter 4. Conclusion .................................................................................. 103
Regenerative Dentistry ............................................................................ 104
Targeted Treatment of Craniofacial Synostosis ...................................... 107
References .............................................................................................. 112
Bibliography .................................................................................................. 117
LIST OF FIGURES
Figure Page
2.1. BCL11B expression during incisor development .................................... 43
2.2. Epithelial invagination defect in Bcl11b−/− developing incisors between
initiation and early bud stage .......................................................................... 44
2.3. Alterations in Bcl11b−/− incisor development at cap stage ....................... 45
2.4. Morphological defects in Bcl11b−/− incisor development at bell stage ...... 46
2.5. Altered ameloblast development in Bcl11b−/− incisors ............................. 48
2.6. Labial to lingual reversal of expression of FGF and Sprouty genes in Bcl11b−/− incisor .............................................................................................. 49
2.7. Altered expression of TGFβ genes and Fst in Bcl11b−/− incisor ............... 50
2.8. Ectopic lingual expression of ameloblast markers and signaling
molecules in Bcl11bep−/− incisor ...................................................................... 51
2.9. Inhibition of Bcl11b expression in the lingual IEE of Spry4−/−; Spry2+/−
mice at E16.5 .................................................................................................. 52
2.10. Model for a reciprocal, inhibitory circuit on the lingual side of the
mouse incisor ................................................................................................. 53
S2.1. Expression of Bcl11b at early bell stage ............................................... 54
S2.2. Expression patterns of selected genes in Bcl11b−/− incisor at early
bud stage ........................................................................................................ 55
LIST OF FIGURES (Continued)
Figure Page
S2.3. Expression patterns of selected genes in Bcl11b−/− incisor at cap
stage ............................................................................................................... 56
S2.4. Delay in the initiation of apoptosis in Bcl11b−/− enamel knot at cap
stage ............................................................................................................... 57
S2.5. Size difference between wild-type and Bcl11b−/− incisors of newborn
mice ................................................................................................................ 58
S2.6. Labial to lingual reversal of expression of Gli1 in Bcl11b−/− incisors ...... 59
S2.7. Expression pattern of Lfrn and Notch1 in Bcl11b−/− incisors .................. 60
S2.8. Expression of ameloblast markers in Bcl11bep−/− and Bcl11bmes/−
incisors at E18.5 ............................................................................................. 61
S2.9. Morphology and mineralization of Bcl11bep−/− incisors at P21 ............... 62
S2.10. Expression patterns of ameloblast markers and signaling molecules in Bcl11bmes−/− incisors at E16.5 ..................................................... 63
S2.11. Labial to lingual reversal of expression of Tbx1 in Bcl11b−/− incisors .. 64
S2.12. BCL11B expression in Fgf3−/−; Fgf10+/− incisors ................................. 65
S2.13. Summary of direct or indirect BCL11B target genes at E16.5 ............. 66
3.1. Impaired postnatal growth, misshapen heads, and craniofacial
synostoses in Bcl11bncc-/- mice ....................................................................... 84
3.2. Craniofacial defects of Bcl11bncc-/- and Bcl11b-/- newborn mice ............... 85
LIST OF FIGURES (Continued)
Figure Page
3.3. Increased osteoblast maturation, mineralization, and craniofacial
synostoses in Bcl11b-/- embryonic skulls ........................................................ 86
3.4. BCL11B expression in osteogenic and sutural mesenchyme .................. 88
3.5. Increased Runx2 expression in the Bcl11b-/- embryonic faces and
coronal sutures ............................................................................................... 89
3.6. Ectopic Fgfr2c expression in facial and coronal sutures of Bcl11b-/-
embryos .......................................................................................................... 90
S3.1. Some Bcl11bncc-/- mice exhibit increased bone porosity in the skull at
P21 ................................................................................................................. 91
S3.2. Craniofacial phenotype development in Bcl11bncc-/- mice at postnatal
stages ............................................................................................................. 92
S3.3. Synostosis of facial sutures in newborn Bcl11b-/- mice .......................... 93
S3.4. Delayed elevation and fusion of palatal shelves in Bcl11b-/- mice ......... 94
S3.5. Neural crest-specific ablation of BCL11B expression in the facial
mesenchyme .................................................................................................. 95
S3.6. Expression patterns of FGF and FGFR genes in the faces of
Bcl11b-/- mice at E14.5 ................................................................................... 96
The Role of the Transcriptional Regulatory Protein BCL11B in Dental and Craniofacial Development
Chapter 1
Transcriptional Regulation and in vivo Functions of BCL11B
2
Cell fates, development of tissues and organs, as well as complex body plans
are governed by large transcriptional and signaling networks that define precise
patterns of gene expression during mammalian embryogenesis (Peter and
Davidson, 2011; Spitz and Furlong, 2012). Loss of a single, key player from such
a system may disrupt the normal developmental program and lead to
morphogenetic and functional defects, as well as early death. Understanding the
precise function of each player and its interaction with the rest of the network
may provide us with invaluable information regarding the genetic, developmental,
and physiological processes. Furthermore, the knowledge obtained can be used
to manipulate genetic and environmental factors and ultimately aid in the
development of targeted therapies and regenerative medicine.
Regulation of transcriptional activity during embryonic development plays an
essential role in cell type specification and organogenesis. Transcriptional
regulatory proteins serve as sequence-specific, DNA-binding factors or adaptor
molecules that determine spatial and temporal expression of ~20,000 protein-
coding genes in humans. Mutations in components of trans-acting transcriptional
machinery or cis-acting regulatory DNA elements, including promoters and distal
elements, are associated with numerous human diseases and developmental
defects (Maston et al., 2006).
In order to study the effects of different genetic mutations that may underlie
human disorders, model organisms are used extensively in many areas of
biological research. Similarities in anatomy, physiology, and genetics between
mice and humans place the mouse at the foremost position as a model to study
biological processes, development, and pathology. Discovery of how
homologous recombination can be utilized to introduce specific, genetic
modifications in mice using embryonic stem cells allowed studying the functions
of individual genes in vivo (Capecchi, 2005). Recently, a new approach of
“genome editing” has emerged that utilizes engineered nucleases (i.e. zinc-finger
nucleases, transcription activator-like effector nucleases, and clustered
3
regulatory interspaced short palindromic repeat/Cas-based RNA-guided DNA
endonucleases) to quickly and efficiently produce mice with mutations in multiple
genes (Gaj et al., 2013; Urnov et al., 2010). This technology will dramatically
accelerate the development of animal models for diseases with simple Mendelian
or even polygenic inheritance. The hope is that linking phenotypic and genotypic
information will allow us to create a functional map of mammalian genome
(Nguyen and Xu, 2008).
BCL11B as a Transcriptional Regulatory Protein
BCL11B (B-cell leukemia/lymphoma 11B) is a Krüppel-like Cys2His2 (C2H2) zinc
finger transcriptional regulatory protein. It is also known as CTIP2 (chicken
ovalbumin upstream promoter transcription factor (COUP-TF)-interacting protein
2), as it, along with its homolog CTIP1, was initially cloned in Dr. Leid’s laboratory
based on its ability to interact with all members of COUP-TF family of orphan
nuclear receptors in yeast two-hybrid screening (Avram et al., 2000). Later,
Bcl11a/Ctip1 was demonstrated to serve as a site of retroviral integration that
was associated with murine myeloid leukemia (Nakamura et al., 2000).
Moreover, human BCL11A was shown to be involved in B-cell lymphoid
malignancies through chromosomal translocation (Satterwhite et al., 2001).
Subsequently, Ctip2 was re-named to Bcl11b based on its sequence homology
to Bcl11a, although it has never been implicated in B-cell leukemia and is not
expressed in B cells (Avram et al., 2000; Satterwhite et al., 2001).
The Bcl11b gene is located on mouse chromosome 12 (52.0 cM) and human
chromosome 14 (q32.1). Both mouse and human genes contain four exons, and
three splice variants, composed of exons 1-2-4, 1-2-3-4 (long isoform), and 1-4
(short isoform), have been described (Wakabayashi et al., 2003).
BCL11B harbors seven C2H2 zinc finger motifs that represent the most common
DNA-binding domains found in eukaryotic transcription factors (Avram et al.,
2000). A single C2H2 zinc finger, composed of a ȕ-hairpin and an α-helix held
4
together by a tetrahedrally coordinated zinc ion, spans a DNA sequence of three
or four consecutive base pairs in the major groove. DNA recognition usually
requires two to four tandemly arranged zinc fingers. When only one or two finger
motifs are present, additional secondary structure elements are generally used to
augment DNA recognition (Wolfe et al., 2000). Extensive studies of zinc finger
binding to DNA made possible to generate “designer” zinc finger proteins that
can target specific genomic sites. Moreover, zinc finger motifs are now known to
have additional activities, such as recognition of RNA and proteins (Brown, 2005;
Gamsjaeger et al., 2007; Hall, 2005).
Initially, BCL11B was shown to act as a transcriptional repressor when recruited
to the template either by interaction with COUP-TF family members or by direct
sequence-specific DNA-binding to a GC-rich response element (Avram et al.,
2000; Avram et al., 2002). BCL11B-mediated transcriptional repression is
dependent on the trichostatin A (TSA)-insensitive class III histone deacetylase
(HDAC) sirtuin 1 as well as TSA-sensitive class I and/or II HDACs (Cismasiu et
al., 2005; Senawong et al., 2003; Topark-Ngarm et al., 2006). HDACs promote
the repressed transcriptional state by facilitating formation of a compact form of
chromatin, thereby making genes less accessible to the general transcriptional
machinery and/or activators (Dai and Faller, 2008). In addition, BCL11B was
shown to interact with the histone methyltransferase SUV39H1, which, along with
HDAC1 and HDAC2, promotes HIV-1 transcriptional silencing in the microglia
cells (Marban et al., 2007). Genes encoding the cyclin-dependent kinase
inhibitors p21 and p57, transcription factor FOXP3, anti-inflammatory cytokine IL
10, as well as ubiquitin ligase for p53 degradation HDM2 are transcriptionally
repressed by BCL11B (Cherrier et al., 2009; Obata et al., 2012; Topark-Ngarm et
al., 2006; Vanvalkenburgh et al., 2011). More recent reports suggest that
BCL11B can act not only as a transcriptional repressor, but also as an activator
of the target genes in a cell type and promoter context-dependent manner. For
example, BCL11B associates with the p300 co-activator and augments
expression from the IL2 promoter in T cells (Cismasiu et al., 2006).
5
BCL11B was found to associate with the nucleosome remodeling and
deacetylation (NuRD) co-repressor complex in T lymphocytes and
neuroblastoma cells (Cismasiu et al., 2005; Topark-Ngarm et al., 2006).
Multisubunit complexes, such as the NuRD complex, function in spatially and
temporarily coordinated manner by acting as “writers”, “erasers”, or “readers” of
the histone code. The “writer” and “eraser” functions reside in subunits containing
catalytic activity that add or remove the mark of histones, whereas the “reader”
function requires signature domains that specifically recognize and bind to these
marks (Ruthenburg et al., 2007; Taverna et al., 2007). For example, the NuRD
complex contains HDAC1 and HDAC2 that act as “erasers” of histone acetylation
marks, leading to transcriptional repression (Zhang et al., 1999). Non-enzymatic
subunits that perform “reader” function in the NuRD complex are methyl-CpG
binding domain 2 protein (MBD2) and MBD3 that convert the information
represented by DNA methylation patterns into the appropriate functional state
(Fatemi and Wade, 2006; Zhang et al., 1999). In addition, the NuRD complex
contains chromodomain-helicase-DNA-binding protein CHD3 or CHD4 that
participate in chromatin remodeling and facilitate transcriptional activation (Zhang
et al., 1998). Consistent with this, recent studies demonstrate that the NuRD
complex, traditionally known to regulate transcriptional repression, is also
required for transcriptional activation in certain contexts (Hung et al., 2012;
Miccio and Blobel, 2010; Miccio et al., 2010). This provides one possible
explanation for how BCL11B, in association with the NuRD complex, may act as
a transcriptional repressor or activator in a promoter context-dependent manner.
Bimodal function of BCL11B as a transcriptional repressor or activator depends
also on its post-translational modifications. ERK1/2-mediated phosphorylation of
BCL11B stimulates its repressive activity on the Id2 promoter, whereas
sumoylation of BCL11B results in recruitment of p300 to the promoter with
subsequent induction of transcription. These post-translational modifications are
mutually exclusive, which suggests the existence of a phospho-deSUMO switch
within the protein (Zhang et al., 2012b).
6
BCL11B Function in vivo
BCL11B is highly conserved at the amino acid level across a wide range of
vertebrate species, suggesting that it plays an important role in these organisms
(Satterwhite et al., 2001). It is expressed during mouse development and
adulthood, and the most notable expression is observed in the central nervous
system, thymus, and ectodermal structures (Enomoto et al., 2011; Golonzhka et
al., 2007; Golonzhka et al., 2009b; Leid et al., 2004). Mice without functional
Bcl11b (Bcl11b-/-) were generated by “floxing” and deletion of exon 4, which
encodes ~75% of the open reading frame and six zinc finger motifs. The mutants
exhibit perinatal death and severe phenotypes in the tissues that express Bcl11b
(Golonzhka et al., 2009a).
BCL11B in T Cell Development
The common lymphoid progenitor gives rise to the lymphocytes: T cells, B cells,
and natural killer cells. Progenitors of T cells are produced in the bone marrow,
but migrate to the thymus, in which they undergo maturation prior to release into
general circulation. The two major subtypes of T cells differ in their T-cell
receptor (TCR) structure and functional properties in the immune system: αȕ and
Ȗβ cells. T cells expressing Ȗβ TCR represent only a small fraction of thymocytes
(no more than 5%), largely mediate mucosal immunity, and are localized
predominantly in the epithelium of intestine, skin, lungs, and reproductive organs.
In contrast, αȕ T cells are abundant in lymphoid organs, play a major role in cell-
mediated, adaptive immunity, and require antigen presentation by major
histocompatibility complex (Mertsching et al., 2002).
Development of αȕ T cells proceeds through multiple, sequential stages that are
distinguished by the expression of cell surface markers and increasingly
restricted differentiation potential (Liu et al., 2010; Rothenberg and Taghon,
2005). Early, committed T cells lack expression of TCR and are termed double-
negative (DN; cluster of differentiation CD4 - CD8-) thymocytes. As they progress
7
through their development they become double-positive T cells (DP; CD4+ CD8+)
and finally mature to single-positive (CD4+ CD8- or CD4 - CD8+) thymocytes that
are then released from thymus to peripheral tissues (Germain, 2002). The DN
population can be further subdivided by the expression of CD44 and CD25:
CD44+ CD25− (DN1) cells differentiate into CD44+ CD25+ (DN2) cells, which give
rise to CD44− CD25+ (DN3) cells, which finally become the most mature
CD44− CD25− (DN4) DN population (Ceredig and Rolink, 2002).
Bcl11b is expressed in the mouse developing thymus as early as embryonic day
(E) 14.5, when most thymocytes are DN T-cell precursors (Leid et al., 2004).
Specifically, steep onset of Bcl11b expression is observed in the early DN2
stage, just preceding commitment of hematopoietic precursors to the T
lymphocyte lineage (Li et al., 2010a). The Bcl11b expression persists in thymus
throughout all stages of mouse development and in mature T lymphocytes (Leid
et al., 2004; Wakabayashi et al., 2003). Germline deletion of Bcl11b results in a
blockade of thymocyte development at the DN stage, enhanced susceptibility to
apoptosis, and a complete absence of αȕ T cells without impairment of the B
and Ȗβ T-cell lineages (Wakabayashi et al., 2003). The role of BCL11B in later
stages of T-cell development was examined by conditionally deleting Bcl11b in
DP thymocytes using CD4-cre transgenic mice. This approach revealed that
BCL11B also plays a critical role in a positive selection of both CD4+ and CD8+
lineages (Albu et al., 2007). Therefore, BCL11B serves as a key regulator of both
survival and differentiation during thymocyte development. Moreover, BCL11B is
necessary for T-cell lineage commitment in mice, as it is specifically required to
repress natural killer and stem cell-associated genes and induce T-cell-specific
gene expression (Li et al., 2010a; Li et al., 2010b).
Conditional deletion of Bcl11b at the DP stage of T-cell development or in
regulatory T cells causes inflammatory bowel disease (Vanvalkenburgh et al.,
2011). This condition is a chronic disorder that includes Crohn’s disease and
ulcerative colitis, both of which are characterized by infiltration of highly reactive
8
CD4+ T cells into the gut. Bcl11b-deficient regulatory T cells exhibit reduced
protein levels of a lineage specification factor FOXP3 and an anti-inflammatory
cytokine IL-10. At the same time, genes encoding pro-inflammatory cytokines are
up-regulated in the mutant CD4+ T cells that infiltrate the colon (Vanvalkenburgh
et al., 2011).
BCL11B was shown to function as a tumor suppressor gene in
lymphomagenesis. The Bcl11b locus was once named Rit1 (radiation-induced
tumor suppressor gene 1), because it was characterized by homozygous
deletions and point mutations in Ȗ-ray-induced mouse thymic lymphomas
(Wakabayashi et al., 2003). The paradigm for the role of tumor suppressors in
cancer is that they are trans-acting and recessive in nature (Cavenee et al.,
1983). Although Bcl11b+/- heterozygous mice rarely develop thymic lymphomas
spontaneously, Bcl11b+/-; p53+/- double heterozygotes are characterized by
significantly increased lymphomagenesis. This suggests cooperativity between
BCL11B and p53 in cancer development (Kamimura et al., 2007). BCL11B has
also been implicated in the pathogenesis of 9-16% of human T-cell acute
lymphoblastic leukemia (T-ALL) (De Keersmaecker et al., 2010; Gutierrez et al.,
2011). BCL11B is involved in recurrent cryptic t(5;14)(q35;q32) translocations
with the TLX3 locus, in which BCL11B gene regulatory elements drive aberrant
over-expression of the TLX3 oncogene (Bernard et al., 2001; Van Vlierberghe et
al., 2008). Moreover, BCL11B acts as a haploinsufficient tumor suppressor that
collaborates with all major T-ALL oncogenic lesions in human thymocyte
transformation (De Keersmaecker et al., 2010; Gutierrez et al., 2011).
BCL11B in Neural Development
High levels of Bcl11b expression are detected in the central nervous system
during the course of fetal development, predominantly through the hippocampal
subregions, olfactory bulb, limbic system, basal ganglia, frontal cortex of the
9
developing brain, and in dorsal cells of the spinal cord. The brain expression
domains of Bcl11b are also maintained into adulthood (Leid et al., 2004).
The mammalian cerebral cortex is organized into six layers. BCL11B is highly
expressed in corticospinal motor neurons that are located primarily in cortical
layer V and extend extremely long axons to precise locations within the spinal
cord. Developmental analysis of Bcl11b-/- mice showed defects in the
organization and fasciculation of subcerebral fiber tracts. Particularly, loss of
Bcl11b results in failure of the corticospinal motor neurons to extend projections
to the spinal cord, with striking pathfinding errors along the corticospinal tract
(Arlotta et al., 2005). Moreover, ectopic expression of Bcl11b in layer II/III cells
causes their axons to project subcortically. Therefore, BCL11B is both required
and sufficient to form subcortical projections (Chen et al., 2008).
The striatum is the largest and major receptive component of the basal ganglia
that control motor and cognitive functions. Within the striatum BCL11B is
uniquely expressed by medium-sized spiny neurons, specifically labeling this
neuronal population from early post-mitotic stages (Arlotta et al., 2008).
GABAergic, medium-spiny neurons account for the vast majority (~90-95%) of
striatal neurons and are critically involved in motor control (Gerfen, 1992).
Degeneration of this neuronal population is a critical component of Huntington’s
and Parkinson’s diseases (Albin et al., 1989). Loss of BCL11B results in a failure
of differentiation of medium-spiny neurons and distinct changes in the expression
of multiple genes. As a result, Bcl11b-/- medium-spiny neurons fail to aggregate
into patches, which results in invasion of heterotopic cellular aggregates (Arlotta
et al., 2008). Abundant striatal expression of BCL11B is also observed in
adulthood, and BCL11B is responsible for transcriptional control of important
genes in the adult striatum (Desplats et al., 2006). This has important
implications in Huntington's disease as sequestration of BCL11B by huntingtin
and down-regulation of Bcl11b expression results in dysregulation of striatal gene
expression in this disease (Desplats et al., 2008). BCL11B regulates the
10
expression of genes in the brain-derived neurotrophic factor signaling pathway,
alterations in which are implicated in several neurodegenerative diseases,
including Huntington’s, Alzheimer’s, and Parkinson’s disease (Tang et al., 2011).
The hippocampus plays an important role in memory and learning as well as in
emotional behavior. The dentate gyrus, the primary gateway for input information
into the hippocampus, is one of only two brain regions with continuous
neurogenesis in adult mammals. BCL11B is expressed in post-mitotic granule
neurons and is involved in the regulation of both progenitor proliferation as well
as granule cell differentiation. BCL11B directly suppresses the expression of
Desmoplakin, a gene that encodes an obligate component of desmosomes; and
re-expression of Desmoplakin in Bcl11b-/- mice rescues impaired neurogenesis
(Simon et al., 2012).
The vomeronasal organ is an auxiliary olfactory sense organ that functions by
detecting pheromones to mediate social and reproductive behavior and is found
in most terrestrial vertebrates (Keverne, 1999). Vomeronasal sensory neurons
are classified into two major types: vomeronasal 1 receptor/Gαi2- and
vomeronasal 2 receptor/Gαo-positive cells (Jia and Halpern, 1996). Bcl11b is
highly expressed in the developing vomeronasal system in mice and is required
for its proper development. Bcl11b-/- mice display various olfactory phenotypes,
such as disorganization of layer formation of the accessory olfactory bulb;
impaired differentiation and axonal projections of vomeronasal sensory neurons;
and impaired balance of the two neuronal types (Enomoto et al., 2011).
The hair cells are sensory neurons acting as receptors for auditory sensation in
the cochlea of the inner ear. Degeneration of these cells is a major cause of
deafness and age-related hearing loss. Outer hair cells are the mechanical
amplifiers for sound sensitivity and frequency selectivity, whereas inner hair cells
serve as passive detectors of the amplified vibratory signal (Fettiplace and
Hackney, 2006). BCL11B is expressed in the outer hair cells, and heterozygous
11
deletion of Bcl11b leads to degeneration of these cells and progressive age-
related hearing loss, which is evident at 3 months of age. Therefore, BCL11B
activity is required for maintenance of the outer hair cells and normal hearing
(Okumura et al., 2011).
BCL11B in Development of Ectodermal Organs
The ectoderm is the outermost of the three primitive germ layers of an animal
embryo. In vertebrates, the ectoderm gives rise to the epidermis, hair, teeth,
olfactory epithelium, and many exocrine glands. Bcl11b is expressed in most of
these tissues including the developing skin, hair follicles, teeth, mammary glands,
and olfactory epithelium (Golonzhka et al., 2007; Golonzhka et al., 2009b; Leid et
al., 2004).
BCL11B expression in the ectoderm is first detected at E10.5, and becomes
increasingly restricted to the basal cells of the developing epidermis at later
stages of fetal development and in adult skin. BCL11B expression is also
detected is suprabasal layer of developing epidermis, dermal cells, as well as
developing and adult hair follicles (Golonzhka et al., 2007). BCL11B plays critical
roles in epidermal proliferation and terminal differentiation, as well as barrier
formation during mouse embryonic development in both cell autonomous and
non-cell autonomous manners (Golonzhka et al., 2009a). BCL11B functions
through interaction with the promoter regions of several genes involved in lipid
metabolism and epidermal development, such as epidermal growth factor
receptor 1 and Notch1 (Wang et al., 2013; Zhang et al., 2012a). Moreover,
BCL11B modulates cell migration, proliferation and differentiation, as well as
maintains expression of hair follicle stem cell markers during cutaneous wound
healing (Liang et al., 2012). Selective deletion of Bcl11b in epidermal
keratinocytes (Bcl11bep-/- mice) reveal cell-autonomous role of BCL11B in barrier
maintenance and epidermal homeostasis in adult mouse skin. In addition,
12
BCL11B suppresses inflammatory responses in skin by repressing expression of
T-helper 2-type cytokines and thymic stromal lymphopoietin (Wang et al., 2012).
BCL11B is highly expressed in the ectodermal components of the developing
tooth, including inner and outer enamel epithelia, stellate reticulum, stratum
intermedium, and the ameloblasts. Bcl11b-/- molars and incisor are poorly
developed and exhibit hypoplastic stellate reticulum. Ameloblasts do not
differentiate properly on the labial side, and ectopic, ameloblast-like cells form on
the lingual side of the Bcl11b-null incisor. Therefore BCL11B is required for
proper tooth development and differentiation of ameloblast lineage (Golonzhka et
al., 2009b).
In the studies described below, I further elucidate the role of BCL11B in mouse
incisor development and describe molecular mechanisms that underlie the dental
phenotype of Bcl11b-/- mice. In addition, I describe a novel role of BCL11B in the
craniofacial development, and show that germline and neural crest-specific
ablation of Bcl11b leads to craniofacial synostoses in mice.
13
References
Albin, R. L., Young, A. B. and Penney, J. B. (1989). The functional anatomy of basal ganglia disorders. Trends Neurosci 12, 366-75.
Albu, D. I., Feng, D., Bhattacharya, D., Jenkins, N. A., Copeland, N. G., Liu, P. and Avram, D. (2007). BCL11B is required for positive selection and survival of double-positive thymocytes. J Exp Med 204, 3003-15.
Arlotta, P., Molyneaux, B. J., Chen, J., Inoue, J., Kominami, R. and Macklis, J. D. (2005). Neuronal subtype-specific genes that control corticospinal motor neuron development in vivo. Neuron 45, 207-21.
Arlotta, P., Molyneaux, B. J., Jabaudon, D., Yoshida, Y. and Macklis, J. D. (2008). Ctip2 controls the differentiation of medium spiny neurons and the establishment of the cellular architecture of the striatum. J Neurosci 28, 622-32.
Avram, D., Fields, A., Pretty On Top, K., Nevrivy, D. J., Ishmael, J. E. and Leid, M. (2000). Isolation of a novel family of C(2)H(2) zinc finger proteins implicated in transcriptional repression mediated by chicken ovalbumin upstream promoter transcription factor (COUP-TF) orphan nuclear receptors. J Biol Chem 275, 10315-22.
Avram, D., Fields, A., Senawong, T., Topark-Ngarm, A. and Leid, M. (2002). COUP-TF (chicken ovalbumin upstream promoter transcription factor)-interacting protein 1 (CTIP1) is a sequence-specific DNA binding protein. Biochem J 368, 555-63.
Bernard, O. A., Busson-LeConiat, M., Ballerini, P., Mauchauffe, M., Della Valle, V., Monni, R., Nguyen Khac, F., Mercher, T., Penard-Lacronique, V., Pasturaud, P. et al. (2001). A new recurrent and specific cryptic translocation, t(5;14)(q35;q32), is associated with expression of the Hox11L2 gene in T acute lymphoblastic leukemia. Leukemia 15, 1495-504.
Brown, R. S. (2005). Zinc finger proteins: getting a grip on RNA. Curr Opin Struct Biol 15, 94-8.
Capecchi, M. R. (2005). Gene targeting in mice: functional analysis of the mammalian genome for the twenty-first century. Nat Rev Genet 6, 507-12.
Cavenee, W. K., Dryja, T. P., Phillips, R. A., Benedict, W. F., Godbout, R., Gallie, B. L., Murphree, A. L., Strong, L. C. and White, R. L. (1983). Expression of recessive alleles by chromosomal mechanisms in retinoblastoma. Nature 305, 779-84.
14
Ceredig, R. and Rolink, T. (2002). A positive look at double-negative thymocytes. Nat Rev Immunol 2, 888-97.
Chen, B., Wang, S. S., Hattox, A. M., Rayburn, H., Nelson, S. B. and McConnell, S. K. (2008). The Fezf2-Ctip2 genetic pathway regulates the fate choice of subcortical projection neurons in the developing cerebral cortex. Proc Natl Acad Sci U S A 105, 11382-7.
Cherrier, T., Suzanne, S., Redel, L., Calao, M., Marban, C., Samah, B., Mukerjee, R., Schwartz, C., Gras, G., Sawaya, B. E. et al. (2009). p21(WAF1) gene promoter is epigenetically silenced by CTIP2 and SUV39H1. Oncogene 28, 3380-9.
Cismasiu, V. B., Adamo, K., Gecewicz, J., Duque, J., Lin, Q. and Avram, D. (2005). BCL11B functionally associates with the NuRD complex in T lymphocytes to repress targeted promoter. Oncogene 24, 6753-64.
Cismasiu, V. B., Ghanta, S., Duque, J., Albu, D. I., Chen, H. M., Kasturi, R. and Avram, D. (2006). BCL11B participates in the activation of IL2 gene expression in CD4+ T lymphocytes. Blood 108, 2695-702.
Dai, Y. and Faller, D. V. (2008). Transcription Regulation by Class III Histone Deacetylases (HDACs)-Sirtuins. Transl Oncogenomics 3, 53-65.
De Keersmaecker, K., Real, P. J., Gatta, G. D., Palomero, T., Sulis, M. L., Tosello, V., Van Vlierberghe, P., Barnes, K., Castillo, M., Sole, X. et al. (2010). The TLX1 oncogene drives aneuploidy in T cell transformation. Nat Med 16, 1321-7.
Desplats, P. A., Kass, K. E., Gilmartin, T., Stanwood, G. D., Woodward, E. L., Head, S. R., Sutcliffe, J. G. and Thomas, E. A. (2006). Selective deficits in the expression of striatal-enriched mRNAs in Huntington's disease. J Neurochem 96, 743-57.
Desplats, P. A., Lambert, J. R. and Thomas, E. A. (2008). Functional roles for the striatal-enriched transcription factor, Bcl11b, in the control of striatal gene expression and transcriptional dysregulation in Huntington's disease. Neurobiol Dis 31, 298-308.
Enomoto, T., Ohmoto, M., Iwata, T., Uno, A., Saitou, M., Yamaguchi, T., Kominami, R., Matsumoto, I. and Hirota, J. (2011). Bcl11b/Ctip2 controls the differentiation of vomeronasal sensory neurons in mice. J Neurosci 31, 10159-73.
Fatemi, M. and Wade, P. A. (2006). MBD family proteins: reading the epigenetic code. J Cell Sci 119, 3033-7.
15
Fettiplace, R. and Hackney, C. M. (2006). The sensory and motor roles of auditory hair cells. Nat Rev Neurosci 7, 19-29.
Gaj, T., Gersbach, C. A. and Barbas, C. F., 3rd. (2013). ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering. Trends Biotechnol 31, 397-405.
Gamsjaeger, R., Liew, C. K., Loughlin, F. E., Crossley, M. and Mackay, J. P. (2007). Sticky fingers: zinc-fingers as protein-recognition motifs. Trends Biochem Sci 32, 63-70.
Gerfen, C. R. (1992). The neostriatal mosaic: multiple levels of compartmental organization. Trends Neurosci 15, 133-9.
Germain, R. N. (2002). T-cell development and the CD4-CD8 lineage decision. Nat Rev Immunol 2, 309-22.
Golonzhka, O., Leid, M., Indra, G. and Indra, A. K. (2007). Expression of COUPTF-interacting protein 2 (CTIP2) in mouse skin during development and in adulthood. Gene Expr Patterns 7, 754-60.
Golonzhka, O., Liang, X., Messaddeq, N., Bornert, J. M., Campbell, A. L., Metzger, D., Chambon, P., Ganguli-Indra, G., Leid, M. and Indra, A. K. (2009a). Dual role of COUP-TF-interacting protein 2 in epidermal homeostasis and permeability barrier formation. J Invest Dermatol 129, 1459-70.
Golonzhka, O., Metzger, D., Bornert, J. M., Bay, B. K., Gross, M. K., Kioussi, C. and Leid, M. (2009b). Ctip2/Bcl11b controls ameloblast formation during mammalian odontogenesis. Proc Natl Acad Sci U S A 106, 4278-83.
Gutierrez, A., Kentsis, A., Sanda, T., Holmfeldt, L., Chen, S. C., Zhang, J., Protopopov, A., Chin, L., Dahlberg, S. E., Neuberg, D. S. et al. (2011). The BCL11B tumor suppressor is mutated across the major molecular subtypes of T-cell acute lymphoblastic leukemia. Blood 118, 4169-73.
Hall, T. M. (2005). Multiple modes of RNA recognition by zinc finger proteins. Curr Opin Struct Biol 15, 367-73.
Hung, H., Kohnken, R. and Svaren, J. (2012). The nucleosome remodeling and deacetylase chromatin remodeling (NuRD) complex is required for peripheral nerve myelination. J Neurosci 32, 1517-27.
Jia, C. and Halpern, M. (1996). Subclasses of vomeronasal receptor neurons: differential expression of G proteins (Gi alpha 2 and G(o alpha)) and segregated projections to the accessory olfactory bulb. Brain Res 719, 117-28.
16
Kamimura, K., Ohi, H., Kubota, T., Okazuka, K., Yoshikai, Y., Wakabayashi, Y., Aoyagi, Y., Mishima, Y. and Kominami, R. (2007). Haploinsufficiency of Bcl11b for suppression of lymphomagenesis and thymocyte development. Biochem Biophys Res Commun 355, 538-42.
Keverne, E. B. (1999). The vomeronasal organ. Science 286, 716-20.
Leid, M., Ishmael, J. E., Avram, D., Shepherd, D., Fraulob, V. and Dolle, P. (2004). CTIP1 and CTIP2 are differentially expressed during mouse embryogenesis. Gene Expr Patterns 4, 733-9.
Li, L., Leid, M. and Rothenberg, E. V. (2010a). An early T cell lineage commitment checkpoint dependent on the transcription factor Bcl11b. Science 329, 89-93.
Li, P., Burke, S., Wang, J., Chen, X., Ortiz, M., Lee, S. C., Lu, D., Campos, L., Goulding, D., Ng, B. L. et al. (2010b). Reprogramming of T cells to natural killer-like cells upon Bcl11b deletion. Science 329, 85-9.
Liang, X., Bhattacharya, S., Bajaj, G., Guha, G., Wang, Z., Jang, H. S., Leid, M., Indra, A. K. and Ganguli-Indra, G. (2012). Delayed cutaneous wound healing and aberrant expression of hair follicle stem cell markers in mice selectively lacking Ctip2 in epidermis. PLoS One 7, e29999.
Liu, P., Li, P. and Burke, S. (2010). Critical roles of Bcl11b in T-cell development and maintenance of T-cell identity. Immunol Rev 238, 138-49.
Marban, C., Suzanne, S., Dequiedt, F., de Walque, S., Redel, L., Van Lint, C., Aunis, D. and Rohr, O. (2007). Recruitment of chromatin-modifying enzymes by CTIP2 promotes HIV-1 transcriptional silencing. EMBO J 26, 412-23.
Maston, G. A., Evans, S. K. and Green, M. R. (2006). Transcriptional regulatory elements in the human genome. Annu Rev Genomics Hum Genet 7, 29-59.
Mertsching, E., Wurster, A. L., Katayama, C., Esko, J., Ramsdell, F., Marth, J. D. and Hedrick, S. M. (2002). A mouse strain defective for alphabeta versus gammadelta T cell lineage commitment. Int Immunol 14, 1039-53.
Miccio, A. and Blobel, G. A. (2010). Role of the GATA-1/FOG-1/NuRD pathway in the expression of human beta-like globin genes. Mol Cell Biol 30, 3460-70.
Miccio, A., Wang, Y., Hong, W., Gregory, G. D., Wang, H., Yu, X., Choi, J. K., Shelat, S., Tong, W., Poncz, M. et al. (2010). NuRD mediates activating and repressive functions of GATA-1 and FOG-1 during blood development. EMBO J 29, 442-56.
17
Nakamura, T., Yamazaki, Y., Saiki, Y., Moriyama, M., Largaespada, D. A., Jenkins, N. A. and Copeland, N. G. (2000). Evi9 encodes a novel zinc finger protein that physically interacts with BCL6, a known human B-cell protooncogene product. Mol Cell Biol 20, 3178-86.
Nguyen, D. and Xu, T. (2008). The expanding role of mouse genetics for understanding human biology and disease. Dis Model Mech 1, 56-66.
Obata, M., Kominami, R. and Mishima, Y. (2012). BCL11B tumor suppressor inhibits HDM2 expression in a p53-dependent manner. Cell Signal 24, 1047-52.
Okumura, H., Miyasaka, Y., Morita, Y., Nomura, T., Mishima, Y., Takahashi, S. and Kominami, R. (2011). Bcl11b heterozygosity leads to age-related hearing loss and degeneration of outer hair cells of the mouse cochlea. Exp Anim 60, 355-61.
Peter, I. S. and Davidson, E. H. (2011). Evolution of gene regulatory networks controlling body plan development. Cell 144, 970-85.
Rothenberg, E. V. and Taghon, T. (2005). Molecular genetics of T cell development. Annu Rev Immunol 23, 601-49.
Ruthenburg, A. J., Li, H., Patel, D. J. and Allis, C. D. (2007). Multivalent engagement of chromatin modifications by linked binding modules. Nat Rev Mol Cell Biol 8, 983-94.
Satterwhite, E., Sonoki, T., Willis, T. G., Harder, L., Nowak, R., Arriola, E. L., Liu, H., Price, H. P., Gesk, S., Steinemann, D. et al. (2001). The BCL11 gene family: involvement of BCL11A in lymphoid malignancies. Blood 98, 3413-20.
Senawong, T., Peterson, V. J., Avram, D., Shepherd, D. M., Frye, R. A., Minucci, S. and Leid, M. (2003). Involvement of the histone deacetylase SIRT1 in chicken ovalbumin upstream promoter transcription factor (COUP-TF)-interacting protein 2-mediated transcriptional repression. J Biol Chem 278, 43041-50.
Simon, R., Brylka, H., Schwegler, H., Venkataramanappa, S., Andratschke, J., Wiegreffe, C., Liu, P., Fuchs, E., Jenkins, N. A., Copeland, N. G. et al. (2012). A dual function of Bcl11b/Ctip2 in hippocampal neurogenesis. EMBO J 31, 292236.
Spitz, F. and Furlong, E. E. (2012). Transcription factors: from enhancer binding to developmental control. Nat Rev Genet 13, 613-26.
18
Tang, B., Di Lena, P., Schaffer, L., Head, S. R., Baldi, P. and Thomas, E. A. (2011). Genome-wide identification of Bcl11b gene targets reveals role in brain-derived neurotrophic factor signaling. PLoS One 6, e23691.
Taverna, S. D., Li, H., Ruthenburg, A. J., Allis, C. D. and Patel, D. J. (2007). How chromatin-binding modules interpret histone modifications: lessons from professional pocket pickers. Nat Struct Mol Biol 14, 1025-40.
Topark-Ngarm, A., Golonzhka, O., Peterson, V. J., Barrett, B., Jr., Martinez, B., Crofoot, K., Filtz, T. M. and Leid, M. (2006). CTIP2 associates with the NuRD complex on the promoter of p57KIP2, a newly identified CTIP2 target gene. J Biol Chem 281, 32272-83.
Urnov, F. D., Rebar, E. J., Holmes, M. C., Zhang, H. S. and Gregory, P. D. (2010). Genome editing with engineered zinc finger nucleases. Nat Rev Genet 11, 636-46.
Van Vlierberghe, P., Homminga, I., Zuurbier, L., Gladdines-Buijs, J., van Wering, E. R., Horstmann, M., Beverloo, H. B., Pieters, R. and Meijerink, J. P. (2008). Cooperative genetic defects in TLX3 rearranged pediatric T-ALL. Leukemia 22, 762-70.
Vanvalkenburgh, J., Albu, D. I., Bapanpally, C., Casanova, S., Califano, D., Jones, D. M., Ignatowicz, L., Kawamoto, S., Fagarasan, S., Jenkins, N. A. et al. (2011). Critical role of Bcl11b in suppressor function of T regulatory cells and prevention of inflammatory bowel disease. J Exp Med 208, 2069-81.
Wakabayashi, Y., Watanabe, H., Inoue, J., Takeda, N., Sakata, J., Mishima, Y., Hitomi, J., Yamamoto, T., Utsuyama, M., Niwa, O. et al. (2003). Bcl11b is required for differentiation and survival of alphabeta T lymphocytes. Nat Immunol 4, 533-9.
Wang, Z., Kirkwood, J. S., Taylor, A. W., Stevens, J. F., Leid, M., Ganguli-Indra, G. and Indra, A. K. (2013). Transcription factor Ctip2 controls epidermal lipid metabolism and regulates expression of genes involved in sphingolipid biosynthesis during skin development. J Invest Dermatol 133, 668-76.
Wang, Z., Zhang, L. J., Guha, G., Li, S., Kyrylkova, K., Kioussi, C., Leid, M., Ganguli-Indra, G. and Indra, A. K. (2012). Selective ablation of Ctip2/Bcl11b in epidermal keratinocytes triggers atopic dermatitis-like skin inflammatory responses in adult mice. PLoS One 7, e51262.
Wolfe, S. A., Nekludova, L. and Pabo, C. O. (2000). DNA recognition by Cys2His2 zinc finger proteins. Annu Rev Biophys Biomol Struct 29, 183-212.
19
Zhang, L. J., Bhattacharya, S., Leid, M., Ganguli-Indra, G. and Indra, A. K. (2012a). Ctip2 is a dynamic regulator of epidermal proliferation and differentiation by integrating EGFR and Notch signaling. J Cell Sci 125, 5733-44.
Zhang, L. J., Vogel, W. K., Liu, X., Topark-Ngarm, A., Arbogast, B. L., Maier, C. S., Filtz, T. M. and Leid, M. (2012b). Coordinated regulation of transcription factor Bcl11b activity in thymocytes by the mitogen-activated protein kinase (MAPK) pathways and protein sumoylation. J Biol Chem 287, 26971-88.
Zhang, Y., LeRoy, G., Seelig, H. P., Lane, W. S. and Reinberg, D. (1998). The dermatomyositis-specific autoantigen Mi2 is a component of a complex containing histone deacetylase and nucleosome remodeling activities. Cell 95, 279-89.
Zhang, Y., Ng, H. H., Erdjument-Bromage, H., Tempst, P., Bird, A. and Reinberg, D. (1999). Analysis of the NuRD subunits reveals a histone deacetylase core complex and a connection with DNA methylation. Genes Dev 13, 1924-35.
The Role of the Transcriptional Regulatory Protein BCL11B in Dental and Craniofacial Development
Chapter 2
BCL11B Regulates Epithelial Proliferation and Asymmetric Development of the Mouse Mandibular Incisor
Kateryna Kyrylkova, Sergiy Kyryachenko, Brian Biehs, Ophir Klein, Chrissa Kioussi, Mark Leid
PLoS One Published May 22, 2012 PLoS One 7(5):e37670 doi: 10.1371/journal.pone.0037670
21
Abstract
Mouse incisors grow continuously throughout life with enamel deposition
uniquely on the outer, or labial, side of the tooth. Asymmetric enamel deposition
is due to the presence of enamel-secreting ameloblasts exclusively within the
labial epithelium of the incisor. We have previously shown that mice lacking the
transcription factor BCL11B/CTIP2 (BCL11B hereafter) exhibit severely disrupted
ameloblast formation in the developing incisor. We now report that BCL11B is a
key factor controlling epithelial proliferation and overall developmental
asymmetry of the mouse incisor: BCL11B is necessary for proliferation of the
labial epithelium and development of the epithelial stem cell niche, which gives
rise to ameloblasts; conversely, BCL11B suppresses epithelial proliferation, and
development of stem cells and ameloblasts on the inner, or lingual, side of the
incisor. This bidirectional action of BCL11B in the incisor epithelia appears
responsible for the asymmetry of ameloblast localization in developing incisor.
Underlying these spatio-specific functions of BCL11B in incisor development is
the regulation of a large gene network comprised of genes encoding several
members of the FGF and TGFβ superfamilies, Sprouty proteins, and Sonic
hedgehog. Our data integrate BCL11B into these pathways during incisor
development and reveal the molecular mechanisms that underlie phenotypes of
both Bcl11b−/− and Sprouty mutant mice.
Introduction
Tooth initiation in the mouse is characterized by a thickening of the oral
epithelium at embryonic day (E) 11.5. The proliferating epithelium invaginates
into the underlying neural crest-derived mesenchyme and forms a bud at E12.5–
E13.5 (bud stage). The epithelium expands and folds around the condensed
mesenchyme to form a cap-like structure at E14.5 (cap stage). The cap stage is
characterized by formation of the enamel knot, a critical signaling center, and
lateral protrusions of the epithelium, known as cervical loops (CLs). CLs extend
22
during bell stage (E16.5–E18.5), at which point cytodifferentiation begins (Kerley,
1975; Lumsden, 1988; Peters and Balling, 1999; Tucker and Sharpe, 2004).
Continuous growth of the rodent incisor requires the presence of epithelial and
mesenchymal stem cells that provide a continuous supply of enamel-producing
ameloblasts and dentin-producing odontoblasts, respectively. Epithelial stem
cells (EpSCs) are slow-cycling cells located in the CLs (Harada et al., 1999; Klein
et al., 2008; Seidel et al., 2010). The labial CL consists of a core stellate
reticulum (SR) and stratum intermedium cells surrounded by basal epithelial
cells, known as the inner and outer enamel epithelium (IEE and OEE,
respectively) (Tummers and Thesleff, 2009). EpSCs reside in the labial CL and
give rise to transit amplifying cells that migrate anteriorly along the IEE while
sequentially differentiating to mitotic pre-ameloblasts, post-mitotic secretory
ameloblasts, and mature ameloblasts (Ryan et al., 1999). The lingual CL
contains a smaller EpSC niche, which does not give rise to ameloblasts, resulting
in a complete lack of enamel deposition on the lingual aspect of the rodent
incisor. Thus, enamel, the hardest substance in the body, is secreted uniquely on
the labial aspect of the incisor. This leads, to preferential abrasion of the lingual
incisor surface during feeding, counteracting the continuous growth of the mouse
incisor to produce an incisor of fixed length (Tummers and Thesleff, 2003).
Tooth development is regulated by sequential and reciprocal signaling between
the epithelium and mesenchyme and is accompanied by patterning and
differentiation of specialized cell types at distinct anatomical locations. A complex
network of fibroblast (FGFs) and transforming (TGFβ) growth factors regulates
proliferation and differentiation of EpSCs during development. The antagonists of
these pathways, Sprouty (Spry) proteins and Follistatin (FST), respectively, also
regulate EpSC niche development, and growth and asymmetry of the mouse
incisor (Klein et al., 2008; Tummers and Thesleff, 2009).
23
CTIP2/BCL11B (BCL11B hereafter) is a transcription factor that plays essential
roles in the development of the immune (Li et al., 2010; Wakabayashi et al.,
2003), central nervous (Arlotta et al., 2005; Arlotta et al., 2008), and cutaneous
(Golonzhka et al., 2009a) systems and is required for perinatal survival
(Wakabayashi et al., 2003). Bcl11b−/− incisors and molars are poorly developed,
and exhibit a hypoplastic SR. Ameloblasts do not differentiate properly on the
labial side, and ectopic ameloblast-like cells form on the lingual side of the
Bcl11b-null incisor (Golonzhka et al., 2009b).
Our analyses of Bcl11b−/− mice revealed that BCL11B plays important roles
throughout incisor development. Mice lacking Bcl11b exhibit epithelial
proliferation defects early in development, which ultimately impact incisor size
and shape. BCL11B also controls formation of both labial and lingual epithelial
stem cell niches and differentiation of ameloblasts. However, BCL11B does so in
a bidirectional manner: promoting development and differentiation of the
epithelium on the labial side while suppressing that on the lingual side, which
strongly enforces asymmetric ameloblast development in the mouse mandibular
incisor.
Materials and Methods
Mouse Lines
/− cl11b− B and Bcl11bL2/L2 mice have been described (Golonzhka et al., 2009a).
Lines carrying mutant alleles of Spry2 (Shim et al., 2005), Spry4 (Klein et al.,
2006), Fgf3 (Alvarez et al., 2003), and Fgf10 (Sekine et al., 1999), as well as
K14-cre (Dassule et al., 2000) and Wnt1-cre (Danielian et al., 1998) transgenes,
were maintained as reported. Animal experiments were approved by the Oregon
State University Institutional Animal Care and Use Committee, protocol 4279.
24
Histological Analysis, RNA in situ Hybridization, and
Immunohistochemistry
Embryonic heads were fixed in 4% paraformaldehyde, cryopreserved in 30%
sucrose, and frozen in O.C.T. Hematoxylin and eosin (H&E) staining and RNA in
situ hybridization (ISH) with digoxigenin-labeled probes were performed
according to standard protocols on 16 µm-thick sagittal sections.
Immunohistochemistry using anti-BCL11B (Abcam, 1:300) was performed as
described (Golonzhka et al., 2007).
Cell Proliferation Assay
Pregnant mice (E11.5–E16.5) were injected intraperitoneally with 100 µl of 5
mg/ml BrdU solution per 100 g of body weight and sacrificed after 2 h.
Cryopreserved heads were serially sectioned (10 µm), and an anti-BrdU antibody
(Accurate Chemical, 1:100) was used to detect BrdU incorporation. The BrdU
index was calculated as the mean relative amount of BrdU-positive cells as a
fraction of total, DAPI-positive cells. An unpaired, two-tailed Student’s t-test was
used to determine statistical significance. At least six sections from a minimum of
three animals per genotype and age were analyzed.
Apoptosis Assay
Apoptosis in sagittal sections (16 µm) was determined with the DeadEnd
Colorimetric TUNEL System (Promega) using Cy3-conjugated streptavidin (SA
Cy3; Sigma 1:250).
Results
BCL11B is Expressed at all Stages of Incisor Development
BCL11B is expressed in the ectoderm of the first branchial arch at E9.5 and
E10.5 and in the molar at all stages of development (Golonzhka et al., 2009b).
25
To determine the function of BCL11B in the developing incisor, we analyzed
BCL11B expression on sagittal sections of the mandibular incisor from E11.5 to
birth. At initiation (E11.5) and early bud (E12.5) stages, BCL11B was expressed
in the thickened epithelium; lower levels of BCL11B were detected in the
underlying mesenchyme (Figs. 2.1A and B). High levels of BCL11B persisted in
the dental epithelium at cap stage (E14.5), whereas mesenchymal cells
surrounding CLs and the follicle continued to express lower levels (Fig. 2.1C). At
early (E16.5) and late (E18.5) bell stages, BCL11B was detected in the lingual
epithelium and in the labial OEE, and at lower levels in the papillary
mesenchyme surrounding both CLs, dental follicle, SR, and ameloblasts at all
stages of differentiation (Figs. 2.1D, E and S2.1). BCL11B was expressed in the
tissue surrounding the tip of the incisor and the vestibular lamina, an invagination
of the oral epithelium that gives rise to the oral vestibule (Figs. 2.1C and D).
Reduced Epithelial Proliferation between Initiation and Bud Stage in
Bcl11b−/− Incisors
The first morphological sign of tooth development is the thickening of the oral
epithelium at E11.5. At this stage, wild-type and Bcl11b−/− incisors were
morphologically indistinguishable (Figs. 2.2A, B, E, F, and I; wild-type BrdU index
= 33.2±5.4%; Bcl11b−/− BrdU index = 33.2±4.0%). By E12.5 the mutant
epithelium appeared approximately one-half the thickness of the wild-type, and
cells at the leading edge of the mutant epithelium were less elongated and poorly
polarized (Fig. 2.2C, D). A 2.7-fold decrease in epithelial proliferation of the
Bcl11b−/− incisor was detected at E12.5 (Figs. 2.2G, H, and J; wild-type BrdU
index = 61.9±3.8%; Bcl11b−/− BrdU index = 23.2±1.4%).
Several signaling molecules and transcription factors orchestrate invagination of
the epithelium between initiation and early bud stages. For example, BMP4, a
critical signaling molecule that regulates tooth initiation and morphogenesis
(Zhang et al., 2005), is expressed in the dental epithelium and underlying
26
mesenchyme during the initiation of tooth development at E11.5 (Fig. 2.2K).
Bmp4expression largely shifts to the dental mesenchyme by early bud stage in
wild-type mice (Fig. 2.2M) (Peters and Balling, 1999; Zhang et al., 2000).
Alterations in Bmp4 expression were not detected in Bcl11b−/− incisors at E11.5
(Fig. 2.2L). However, the Bcl11b−/− epithelium failed to down-regulate expression
of Bmp4 at E12.5 (Fig. 2.2N). The expression patterns of other critical signaling
molecules and transcription factors, including Activin, Shh, Pax9, and Msx1, were
not altered in Bcl11b−/− incisors at the bud stage (Fig. S2.2).
− Altered Development of Bcl11b /− Incisors at Cap Stage
The wild-type, mandibular incisor is characterized by a cap-like shape of the
dental epithelium at E14.5, with an enamel knot in the center and protruding CLs
(Fig. 2.3A). The enamel knot is a transitory signaling center that is characterized
by minimal proliferation and clearly defined apoptosis (Vaahtokari et al., 1996a;
Vaahtokari et al., 1996b). In contrast, the CLs are highly proliferative with a low
−/− apoptotic index (Wang et al., 2007). The Bcl11b incisor exhibited a delay in
epithelial invagination and protrusion of both CLs at E14.5 (Fig. 2.3B). BrdU
labeling studies revealed that the incisor epithelium of Bcl11b−/− mice was
hypoproliferative compared to that of wild-type mice (27.6±4.1% and 51.8±2.7%
BrdU-positive cells, respectively), whereas mesenchymal proliferation appeared
unchanged from controls (Fig. 2.3D and E).
Proliferation of the dental epithelium at cap stage is controlled in part by FGF10,
which is derived from mesenchymal cells of the dental papilla (Kettunen et al.,
2000). The Bcl11b−/− papillary mesenchyme was essentially devoid of Fgf10
transcripts at early E14 and ectopic Fgf10 expression was noted between the
dental epithelium and vestibular lamina, the latter of which exhibited impaired
invagination (Fig. 2.3F, G). The delay of initiation of mesenchymal Fgf10
expression may contribute to decreased dental epithelial proliferation, delayed
27
invagination of the dental epithelium, and subsequently decreased size of the
mutant incisor.
The expression patterns of Shh, Gli1, Fgf3, Fgf9, Spry2, Spry4, Bmp4, activin,
Fst, and Tbx1, were unaltered in Bcl11b−/− incisors at cap stage (Fig. S2.3).
Apoptotic cells were predominantly localized in the enamel knot of wild-type
incisors at E14.5 (Fig. S2.4A). However, very few apoptotic cells were detected
in the Bcl11b−/− enamel knot (Fig. S2.4B), consistent with delayed incisor
development in Bcl11b−/− mice.
Reduced Size and Disruption of Labial-lingual Asymmetry at Bell Stage in
Bcl11b−/− Incisors
Wild-type incisors at E16.5 are characterized by a large labial CL, which contains
stem cells that give rise to ameloblasts (Figs. 2.4A and B). In contrast, the lingual
CL of wild-type incisors is relatively smaller, consistent with reduced
developmental potential on the lingual side of the incisor (Fig. 2.4C). Bcl11b−/−
incisors were reduced in size by approximately half at this stage (compare Figs.
2.4A and E) and were characterized by a hypocellular labial CL (compare Figs.
2.4B and F), an enlarged lingual CL (compare Figs. 2.4C and G), and elongated
cells resembling ameloblasts along the length of the lingual epithelium (compare
Figs. 2.4D and H).
The posterior basal epithelium of the labial CL of Bcl11b−/− mice was
hypoproliferative relative to that of wild-type mice at E16.5 (23.2±4.9% and
42.0±5.0% BrdU-positive cells, respectively; Figs. 2.4I-K). However, neither the
apoptotic index (Figs. S2.4C and D) nor proliferation in the lingual CL (Fig. 2.4K)
of mutant incisors was significantly different from wild-type.
By E18.5, wild-type incisors developed a large labial (region I, Figs. 2.4L and P)
and a small lingual (Figs. 2.4L and M) CL. Ameloblast differentiation occurs
Bcl11 −/− b incisors were reduced in size at E18.5 (compare Figs. 2.4L and N).
However, the mutant lingual CL was enlarged (compare Figs. 2.4M and O), and
the labial CL was markedly hypoplastic (compare Figs. 2.4P and U) to the point
of resembling the lingual CL of wild-type mice in both size and morphology
(compare Figs. 2.4M and U). Mutant ameloblasts were smaller and disorganized
at all stages of differentiation along the labial epithelium (compare Figs. 2.4Q-S
and V-X), and an abnormal layer of polarized cells resembling ameloblasts was
28
sequentially in the labial IEE with mitotic pre-ameloblasts (Figs. 2.4L and Q),
post-mitotic secretory ameloblasts (Figs. 2.4L and R), and mature ameloblasts
(Figs. 2.4L and S) in regions II, III, and IV, respectively (Ryan et al., 1999).
Ameloblasts are not present on the lingual side of the wild-type incisor, but rather
a thin layer of non-polarized epithelial cells is found on this aspect of the
developing tooth (Figs. 2.4L and T).
observed in the anterior region of the lingual epithelium (compare Figs. 2.4T and
Y). Finally, Bcl11b−/− incisors were approximately one-half the length of wild-type
incisors at birth and correspondingly narrower across the entire tooth (Fig. S2.5).
These results demonstrate that BCL11B plays an important role in development
of the labial CL and differentiation of ameloblasts, while simultaneously
suppressing these processes on the lingual side of the incisor.
Delay in Ameloblast Development and Ectopic Formation of Lingual
Ameloblast-like Cells in Bcl11b−/− Incisors
To determine if labial ameloblasts and lingual ameloblast-like cells underwent
differentiation in Bcl11b−/− incisors, we examined expression of sonic hedgehog
(Shh) and amelogenin (Amelx), markers of pre-ameloblasts (Bitgood and
McMahon, 1995; Klein et al., 2008) and mature ameloblasts (Zeichner-David et
al., 1995), respectively. Shh expression was observed in a gradient along the
length of the labial IEE of wild-type incisors at E16.5 and E18.5, with the most
intense staining in the posterior region (Fig. 2.5A, C). Shh expression was greatly
29
reduced in the labial epithelium of Bcl11b−/− mice, and ectopic Shh transcripts
were detected in the lingual epithelium at E16.5 and E18.5 (Figs. 2.5B and D).
The expression pattern of Gli1, a mediator of SHH signaling (Ruiz i Altaba,
1999), reflected changes in Shh expression in the mutant incisor (Fig. S2.6).
Amelx expression was greatly reduced in the labial epithelium of Bcl11b−/− mice
at E16.5 (compare Figs. 2.5E and F) but recovered to a level similar to that of
wild-type mice by E18.5 (Fig. 2.5G). Ectopic Amelx expression was observed in
the anterior lingual IEE of Bcl11b−/− mice at E18.5 (compare Figs. 2.5G and H),
consistent with the presence of terminally differentiated ameloblasts in the lingual
epithelium.
These results demonstrate that BCL11B plays a key role in the establishment
and/or enforcement of developmental incisor asymmetry and cellular
differentiation within the ameloblast lineage.
− Alteration of the FGF Signaling at Bell Stage in Bcl11b /− Incisors
Asymmetric development of the CLs is controlled by several signaling pathways.
FGFs and their intracellular antagonists, the Sprouty proteins, are crucial for
proper development of the labial and lingual CLs (Klein et al., 2008). FGF3 and
FGF10 are key mesenchymal instructive signals that cooperatively stimulate
proliferation of the incisor epithelium at bell stage (Harada et al., 1999; Harada et
al., 2002; Yokohama-Tamaki et al., 2006). Fgf3 is expressed exclusively within
the posterior labial mesenchyme in wild-type incisors (Figs. 2.6A and C),
whereas Fgf10 transcripts are more widely distributed around the labial CL and
to a lesser extent in the lingual mesenchyme (Figs. 2.6E and G).
The Fgf3 expression domain, which is located in the mesenchyme just anterior to
the labial CL in wild-type mice, was absent in Bcl11b−/− mutants at E16.5 and
E18.5. However, Fgf3 was ectopically expressed in the mesenchyme adjacent to
the lingual CL in Bcl11b−/− mice (Figs. 2.6B and D). The expression pattern of
30
Fgf10 was altered in Bcl11b−/− incisors in a manner that was qualitatively similar
to that of Fgf3 (Figs. 2.6F and H).
Epithelial FGF9 forms a positive-feedback signaling loop with mesenchymal
FGF3 and FGF10 on the labial side of the wild-type incisor (Klein et al., 2008).
Fgf9 RNA was detected anterior to the labial CL of the wild-type incisor (Figs.
2.6I and K). This Fgf9-positive domain was reduced in Bcl11b−/−incisors, and
ectopic expression of Fgf9 was detected in the lingual epithelium at E16.5 and
E18.5 (Figs. 2.6J and L).
Sprouty proteins are responsible, in part, for inhibition of ameloblast
differentiation in the lingual epithelium (Klein et al., 2006). Spry4 RNA was
detected in the mesenchyme adjacent to the labial CL, and at lower levels in the
posterior lingual and labial epithelium in wild-type mice at E16.5 and E18.5 (Figs.
2.6M and O). Spry2 expression was detected predominantly in the posterior
lingual and labial epithelium of the wild-type incisor (Figs. 2.6Q and S).
Spry4 expression was up-regulated in the lingual basal epithelium and underlying
mesenchyme of Bcl11b−/− incisors at E16.5 and E18.5, and down-regulated on
the labial side of the developing Bcl11b−/− incisor at both developmental stages
(Figs. 2.6N and P). Spry2 expression was up-regulated in the lingual CL and
slightly down-regulated in the labial epithelium of Bcl11b−/−mice at E16.5 and
E18.5 (Figs. 2.6R and T).
Mesenchymal FGF10 stimulates expression of Lunatic Fringe (Lfrn), which
encodes a secretory molecule that modulates the Notch pathway (Harada et al.,
1999). To determine if the Notch pathway was altered inBcl11b−/− incisors at bell
stage, we examined expression patterns of Lfrn and Notch1. Lfrn RNA was
detected predominantly along the length of IEE and in the posterior OEE of wild-
type incisors at E16.5 and E18.5 (Figs. S2.7A and C). Lfrn expression was down-
regulated at the posterior end of the labial CL of Bcl11b−/− incisors at both E16.5
and E18.5 (Figs. S2.7B and D). Ectopic Lfrn expression was detected in the
31
posterior part of the mutant lingual epithelium at E18.5 (Fig. S2.7D). Loss of
BCL11B did not affect the level of expression or localization of Notch1
transcripts. However, Notch1 expression reflected the morphological expansion
and contraction of lingual and labial SR, respectively, in Bcl11b−/− incisors (Figs.
S2.7E-H).
These findings highlight dysregulation of the FGF signaling pathways as being
central to the incisor phenotype of Bcl11b−/− mice. As asymmetric expression of
Fgf3 and Fgf10 contribute to asymmetric development of labial and lingual EpSC
niches (Wang et al., 2007). Thus, the complete reversal of asymmetric Fgf3 and
Fgf10 expression, together with that of Fgf9, likely underlies the enhanced and
repressed development of the lingual and labial CLs, respectively, in Bcl11b−/−
mice.
Dis ruption of TGFβ Signaling at Bell Stage in Bcl11b−/− Mice
The TGFβ family members, BMP4 and activin βA, and the antagonist FST play
key roles in the generation and maintenance of asymmetric ameloblast
localization during incisor development. For example, FST inhibits ameloblast
differentiation on the lingual side of the incisor, whereas BMP4 promotes it on the
labial side. In contrast, activin enhances development of the labial CL, whereas
BMP4 limits CL growth (Wang et al., 2007; Wang et al., 2004; Zhang et al.,
2005).
Bmp4 expression was detected predominantly in the labial mesenchyme, anterior
to the labial CL, in wild-type mice at E16.5. Lower levels of Bmp4 transcripts
were present in the mesenchyme underlying the lingual epithelium and in an
anterior region of the ameloblast layer (Fig. 2.7A). The boundaries of
mesenchymal Bmp4 expression were disrupted in Bcl11b−/−incisors at E16.5,
with ectopic expression noted in the mesenchyme posterior to the lingual CL.
Expression of Bmp4 in the ameloblast layer appeared reduced in mutants at this
stage (Fig. 2.7B). At E18.5, Bmp4 transcripts were detected predominantly in the
32
labial epithelium, in a wide region of labial mesenchyme, and at lower levels on
the lingual side of the wild-type incisor (Fig. 2.7C). Bmp4 expression increased
uniformly in all of these domains in Bcl11b−/− mutants at E18.5 (Fig. 2.7D).
Activin expression was restricted to the labial mesenchyme directly underlying
the posterior epithelium, within the tip of the labial CL, and in the posterior part of
the dental follicle in wild-type mice at E16.5 and E18.5 (Figs. 2.7E and G). Activin
expression was lost within the labial mesenchyme and epithelium in Bcl11b−/−
mice at both developmental stages. However, ectopic mesenchymal expression
of activin was observed around the lingual CL and follicular expression of activin
appeared to be delocalized in the Bcl11b−/− incisors at E16.5 and E18.5
(asterisks in Figs. 2.7F and H).
Fst transcripts were observed in the OEE on the labial and lingual sides at E16.5
(Fig. 2.7I; see also (Wang et al., 2004)). Fst expression in the OEE persisted at
E18.5, and Fst transcripts were also detected in highly-defined domains at the
anterior epithelial tip of the incisor on both labial and lingual sides (Fig. 2.7K; data
not shown). In contrast, Fst transcripts were diffusely distributed throughout the
labial and lingual epithelium of Bcl11b−/− incisors, particularly at the anterior
(incisal) tip of the epithelium, and in the papillary mesenchyme at E16.5 (Fig.
2.7J). Fst expression within the posterior region of the wild-type incisor at E18.5
was indistinguishable from that of Bcl11b−/−mice (data not shown). However, we
noted a dramatic expansion of the Fst expression domain within the anterior
labial epithelium at E18.5. Additionally, Bcl11b−/− incisors failed to extinguish Fst
expression along the length of the labial OEE at E18.5 (Fig. 2.7L).
Cell Autonomous Effects of BCL11B in Lingual Epithelium
BCL11B is expressed in both ectodermal-derived epithelium and neural crest-
derived mesenchyme (Fig. 2.1). We created lines conditionally null for Bcl11b
expression in both germinal layers to determine the expression domain
responsible for BCL11B-mediated suppression of ameloblast differentiation in the
33
lingual epithelium. Mice harboring an epithelial-specific deletion of Bcl11b
(Bcl11bep−/−), which were created by crossing floxed Bcl11bL2/L2 mice with the
K14-cre deleter strain (Dassule et al., 2000), clearly lacked BCL11B in the entire
dental epithelium (Figs. 2.8A and B). However, specific BCL11B expression
persisted in the dental mesenchyme and other non-epithelium-derived tissues.
Bcl11bep−/− mice expressed the pre-ameloblast marker Shh with an ectopic
gradient along the length of the lingual epithelium at E16.5 (Figs. 2.8C and D),
and this persisted at a lower level at E18.5 (Fig. S2.8B) However, Bcl11bep−/−
mice did not express Amelxin the lingual epithelium at either E16.5 (Fig. 2.8F) or
E18.5 (Fig. S2.8E). Considered together, these results suggest that Bcl11bep−/−
mice initiate but do not complete ameloblast differentiation within the lingual
dental epithelium.
Next, we examined the expression of several genes encoding signaling
molecules to determine the effect of epithelium-specific inactivation of Bcl11b on
generation of labial-lingual asymmetry at E16.5. A low level of ectopic expression
of Fgf3, Fgf9, and activin was detected on the lingual side of the Bcl11bep−/−
incisor, and this was qualitatively similar to Bcl11b−/− incisors (compare Figs.
2.8G-L, 2.6B and F, and 2.7F). However, we did not observe alterations in the
expression patterns of these signaling molecules on the labial side of the
Bcl11bep−/− incisor (Figs. 2.8G-L), as described previously for Bcl11b−/− mice (see
Figs. 2.6 and 2.7).
The size and shape of the Bcl11bep−/− incisors were similar to control incisors
(see Fig. 2.8), and the slight variations in lingual gene expression in Bcl11bep−/−
incisors did not result in altered amelogenesis as determined by X-ray micro-CT
radiography performed on P21 mandibles (Fig. S2.9).
Excision of the Bcl11b locus in neural crest-derived mesenchyme using the
Wnt1-cre deleter line (Bcl11bmes−/−; see Figs. S2.10A and B) did not result in
34
altered morphology or disrupted gene expression patterns (Shh, Amelx, Fgf3,
and activin; Figs. S2.10C-J; see also S2.8C and F).
These data indicate that epithelial, but not mesenchymal Bcl11b expression is
required for suppression of ectopic pre-ameloblast formation in the lingual
epithelium. However, loss of Bcl11b in the epithelium is not sufficient for the
lingual pre-ameloblasts to persist or to undergo further differentiation into mature,
Amelx-positive ameloblasts.
FGF Signaling Negatively Regulates BCL11B Expression in the Lingual IEE
and SR
−/− The ectopic development of lingual pre-ameloblasts expressing Shh in Bcl11b
mice is similar to that reported in Spry4−/−; Spry2+/− mice. Loss of Sprouty gene
expression results in abnormal FGF gene expression and establishment of a
FGF positive-feedback signaling loop on the lingual side of the incisor. In
addition, Spry4−/−; Spry2+/− mice were characterized by up-regulated expression
of Etv4 and Etv5 (previously known as Pea3 and Erm), which are considered to
be transcriptional targets of FGF signaling, and indicative of activation of the FGF
signaling pathway(s) in mutant incisors (Klein et al., 2008; O'Hagan and Hassell,
1998; Roehl and Nusslein-Volhard, 2001). We assessed expression of BCL11B
in incisors from Spry4−/−; Spry2+/− embryos in order to determine if the FGF
signaling pathway(s) regulates BCL11B expression.
BCL11B was highly expressed in the entirety of the wild-type lingual epithelium at
E16.5, including the CL, anterior OEE, and IEE (Fig. 2.9A; see also 2.1D). In
contrast, BCL11B protein levels were dramatically decreased in the Spry4−/−;
Spry2+/− incisor, particularly within the lingual IEE and SR, while BCL11B levels
in the lingual OEE and labial epithelium were largely unaffected (Fig. 2.9B).
Expression of Tbx1, which is also important for incisor developmental
asymmetry, was increased in the lingual epithelium of Spry4−/−; Spry2+/− incisors
35
(Caton et al., 2009). These findings prompted us to examine Tbx1 expression in
Bcl11b−/− mice. Tbx1 was predominantly expressed in the posterior basal
epithelium on the labial side of wild-type incisors at both E16.5 and E18.5, and
diffusely at a much lower level in the lingual epithelium (Figs. S2.11A and C). We
observed striking up-regulation of Tbx1 expression in the lingual IEE of Bcl11b−/−
mice at E16.5 and E18.5 (Figs. S2.11B and D), suggesting that BCL11B directly
or indirectly represses the Tbx1 expression in the lingual epithelium, and that up-
regulation of Tbx1 expression in Spry4−/−; Spry2+/− mice (Caton et al., 2009) may
occur through down-regulation of BCL11B protein levels. These findings place
BCL11B downstream of FGF signaling and upstream of Tbx1 expression in the
lingual epithelium of the developing incisor. Tbx1 expression was severely
decreased in the labial epithelium of Bcl11b−/− mice at E16.5 (Fig. S2.11B).
Labial expression of Tbx1 in the Bcl11b−/− incisor recovered by E18.5 (Fig.
S2.11D), suggesting that another factor(s) may compensate for loss of BCL11B
expression in the control of expression of Tbx1 in the labial epithelium.
These above findings suggest that the FGF signaling pathways regulate BCL11B
expression in the lingual epithelium, and we hypothesized that inactivation of
FGF signaling may lead to up-regulation of BCL11B expression within the labial
IEE. In order to test this hypothesis, we assessed BCL11B expression in Fgf3−/−;
Fgf10+/− incisors; however, BCL11B immunostaining was indistinguishable from
wild-type incisors (Fig. S2.12). It is conceivable that another FGF family
member(s) may compensate for loss of Fgf3 expression and partial loss of Fgf10
expression by enforcing the repression of BCL11B expression within the labial
IEE (Porntaveetus et al., 2011). Indeed, Fgf3−/−; Fgf10+/− and wild-type incisors
are nearly identical in size (Fig. S2.12), suggesting that loss or partial loss of
these two signaling molecules did not compromise proliferation during incisor
development. Finally, it is possible that regulation of BCL11B expression within
the labial epithelium may not involve the FGF signaling pathways, as was clearly
evident on the lingual side (Fig. 2.9B).
36
Discussion
The studies reported here demonstrate that the transcription factor BCL11B
participates in several essential aspects of mouse incisor development. First,
BCL11B controls epithelial proliferation, which ultimately impacts the size and
shape of the incisor. Second, BCL11B plays a key role in the establishment and
maintenance of labial-lingual asymmetry by regulating the expression of several
key signaling molecules and transcription factors. Third, BCL11B is essential for
the proper formation, differentiation, and localization of ameloblasts.
To our knowledge, this is the first report of a transcription factor that integrates
developmental control of both labial and lingual EpSC niches and ameloblasts.
Such regulation appears to be bidirectional: BCL11B stimulates the development
of the labial CL by enhancing the expression of key signaling molecules on the
labial side while limiting development of the lingual CL by repressing the
expression of the same signaling molecules on the lingual aspect. Subsequently,
BCL11B promotes differentiation of the labial, EpSC-derived IEE cells into
mature ameloblasts and blocks ectopic formation and differentiation of the lingual
IEE into cells of the ameloblast lineage.
BCL11B Regulates Proliferation of the Dental Epithelium
BCL11B is initially required for proper transition from initiation to early bud stage
of tooth development. Specifically, BCL11B is necessary for the proper timing of
epithelial proliferation, invagination, and down-regulation of epithelial Bmp4
expression. During tooth initiation, BMP4 is secreted by the epithelium and
induces the mesenchymal expression of genes (Msx1, Msx2, and Bmp4) that
further direct incisor formation (Tucker and Sharpe, 2004). While the significance
of down-regulation ofBmp4 expression in the dental epithelium at bud stage is
unknown, overexpression of Bmp4 in the distal respiratory epithelium results in
decreased epithelial proliferation (Bellusci et al., 1996). Thus, a delay in down
37
regulation of epithelial Bmp4 expression may contribute to reduced proliferation
of the dental epithelium between initiation and bud stages.
The size and shape of the incisor is tightly regulated by the opposing forces of
cellular proliferation and apoptosis, both of which are controlled by signaling
pathways. FGF10 induces a mitogenic response in dental epithelial cells
(Kettunen et al., 2000), and a delay in induction of Fgf10 in Bcl11b−/−incisors may
further contribute to the proliferation defect in Bcl11b−/− dental epithelium.
Therefore, a combination of at least two molecular dysregulations, a delay in
down-regulation of epithelial Bmp4 expression at E12.5 and in the induction of
mesenchymal Fgf10 expression at E14, may contribute to decreased proliferation
of Bcl11b−/− dental epithelium. We further propose that altered epithelial
proliferation in the absence of BCL11B may account for the slowed invagination
of this tissue, resulting in an incisor that is approximately one-half of the size of a
wild-type incisor at birth.
Apoptosis of epithelial cells comprising the enamel knot at cap stage also
regulates the overall shape of the incisor (Vaahtokari et al., 1996a). Thus,
delayed initiation of apoptosis in Bcl11b−/− incisors may also contribute to altered
morphology in the mutant.
BCL11B Controls the Expression of FGF and TGFβ Family Members
Transition from the cap to bell stage of incisor development is accompanied by
establishment of an asymmetric shape. The size and asymmetry of the mouse
incisor is dictated by transcription factor networks, which control the expression
of genes encoding components of various signaling pathways that play
deterministic roles in cellular specification and organogenesis. The FGF and
TGFβ signaling pathways, and their respective antagonists–the Sprouty proteins
and FST–are particularly important in incisor development.
38
FGF3 and FGF10, both of which are expressed predominantly in the labial
mesenchyme, maintain proliferation of EpSCs and, thus, directly contribute to the
asymmetric shape of the incisor (Harada et al., 1999; Harada et al., 2002; Wang
et al., 2007). Furthermore, both FGF3 and FGF10, together with epithelial FGF9,
form a positive-feedback loop on the labial aspect of the tooth. This FGF
feedback loop is inhibited by Sprouty proteins on the lingual side, resulting in the
limited development of the lingual EpSC niche (Klein et al., 2008). In turn, the
expression of the Sprouty genes can be induced by FGF signaling (Hacohen et
al., 1998).
In the absence of BCL11B, a remarkable inversion of the expression patterns of
FGF genes relative to the labial-lingual axis occurred, such that these genes
were expressed predominantly on the lingual side, with no or little expression
was observed on the labial side. Therefore, BCL11B may function as a spatial
switch governing expression of FGF signaling pathway members (Fig. S2.13).
Expression of Sprouty genes was altered in a similar manner in Bcl11b−/−incisors,
possibly in a compensatory or feedback manner. Consistent with this, expression
of Tbx1, which is positively regulated by FGF signaling in the developing incisor
(Caton et al., 2009), was similarly altered. Our findings strongly suggest that
complete loss of expression of FGF family members on the labial side coupled
with ectopic expression of these signaling proteins on the lingual side of the
Bcl11b−/− incisor underlies the abnormal morphology of the Bcl11b−/− tooth.
The FGF and TGFβ signaling pathways are closely interweaved during incisor
development. For example, BMP4 represses Fgf3 expression in the
mesenchyme; however, activin abrogates this repression on the labial side of the
incisor, which allows FGF3 expression within this domain. Low activin expression
in the lingual mesenchyme allows BMP4 to inhibit Fgf3 expression on the lingual
side in an unopposed fashion (Kettunen et al., 2000). In addition, BMP4
promotes ameloblast differentiation within the labial epithelium, possibly by
inducing expression of p21 and ameloblastin. Ameloblast-inducing activity of
39
BMP4 is inhibited on the lingual side by FST, but the relative lack of
Fstexpression in the labial epithelium facilitates terminal differentiation of
ameloblasts (Wang et al., 2004). Alteration of Bmp4 expression in Bcl11b−/−
incisor generally paralleled those observed with FGF family members. Thus,
BCL11B appears to be required for Bmp4 expression on the labial side of the
developing incisor and for suppression of Bmp4 expression in the lingual
mesenchyme.
Expression of activin also exhibited complete reversal in the mutants at the bell
stage. Activin expression was completely lost on the labial side of the mutant
incisor, perhaps allowing BMP4 to inhibit Fgf3 expression within this domain. In
contrast, activin transcripts were detected in the lingual mesenchyme of Bcl11b−/−
incisors, suggesting that this ectopic expression domain allows activin to block
the repressive action of BMP4 on Fgf3 expression in this tissue. Because FGF3
functions in a positive-feedback loop with FGF10 and FGF9 (Klein et al., 2008),
the expression patterns of the latter were also altered. FGF3, and possibly FGF
family members, could then induce the development of the lingual EpSC niche in
Bcl11b−/− mice. Thus, we propose that activin contributes to the establishment of
new borders of expression of FGF family members in the Bcl11b−/− incisor.
BCL11B Controls Asymmetric Development of the Ameloblasts
The asymmetric pattern of expression of FGF and TGFβ signaling molecules is
thought to lead to the asymmetric development of ameloblasts in wild-type
incisors. Therefore, dysregulated expression of these signaling pathways in
Bcl11b−/− incisors likely contributes to the ectopic development of lingual,
ameloblast-like cells and delayed development of the labial ameloblasts. The
expanded lingual EpSC niche in Bcl11b−/− incisors gave rise to ectopic Shh-
expressing pre-ameloblasts, which further differentiated into mature, Amelx
positive ameloblasts. The mutant labial EpSC niche also gave rise to some pre
ameloblasts, which were characterized by down-regulated Shh expression.
40
These pre-ameloblasts failed to differentiate into mature ameloblasts at the early
bell stage. Although the pool of Shh-positive pre-ameloblasts was greatly
reduced in Bcl11b−/− incisors at late bell stage, it was remarkable that
differentiation to Amelx-positive ameloblasts occurred in the labial epithelium by
E18.5. This observation suggests that another transcription factor(s) may
compensate for loss of Bcl11b expression in the ameloblast lineage, allowing
ameloblast development to occur, albeit in a delayed manner.
Ectopic Shh-positive pre-ameloblasts were abundant on the lingual aspect of the
Bcl11bep−/−incisor at early bell stage. Low levels of ectopic lingual expression of
Fgf3, Fgf9, and activin might contribute to such differentiation of IEE. However,
the ectopic, lingual Shh-positive domain was dramatically reduced in size and
was present only in the posterior epithelium by late bell stage, and mature
ameloblast-like cells were not observed on the lingual aspect of the Bcl11bep−/−
incisor, suggesting that other factors and/or mesenchymal BCL11B may be
sufficient to suppress terminal differentiation in the ameloblast lineage within the
lingual epithelium. Deletion of Bcl11b in either the epithelium or mesenchyme did
not affect labial expression of ameloblast markers and key signaling molecules,
the morphology of the labial CL, ameloblast development or amelogenesis,
suggesting that both epithelial and mesenchymal BCL11B may contribute to
developmental processes on the labial side of the incisor. BCL11B regulates
expression of signaling molecules and transcription factors that are essential for
establishment and maintenance of asymmetric incisor development. The majority
of these changes in expression of key genes in Bcl11b−/− mice were qualitatively
similar and characterized by down-regulation on the labial side and up-regulation
on the lingual aspect of the developing incisor. The single exception to this
observation was Fst, the expression domain of which appeared to be maintained
by BCL11B. Collectively, these data suggest that BCL11B regulates the
expression of downstream signaling molecules and transcription factors
bidirectionally, activating expression on the labial side while repressing
expression of the same genes on the lingual side (Fig. S2.13).
41
Integration of BCL11B into FGF and SHH Signaling Pathways
Combined deletion of Spry4 and one allele of Spry2 (Klein et al., 2008) also
results in ectopic development of Shh-positive pre-ameloblasts along the lingual
epithelium of the developing incisor, suggesting a possible convergence in the
FGF signaling pathways and BCL11B-dependent transcriptional regulation. This
interpretation was supported by the strongly decreased expression of BCL11B
within the lingual IEE of Spry4−/−; Spry2+/− incisors, indicating that unrestrained
activity of the FGF signaling pathways results in pronounced down-regulation of
BCL11B expression in the lingual IEE and subsequent de-repression of Shh
expression. Based on these findings, we propose a model (Fig. 2.10) to explain
the role of BCL11B in the FGF signaling pathways within the lingual epithelium at
E16.5 (Fig. 2.10A) and in the Spry4−/−; Spry2+/− incisor (Fig. 2.10B). This model
posits that:
1. SPRY4 and SPRY2 inhibit establishment of a FGF positive-feedback
signaling loop (Klein et al., 2008) and the repressive effect of FGFs on
BCL11B expression in the wild-type lingual IEE.
2. As a result, BCL11B is highly expressed in the entire lingual epithelium in
wild-type mice and directly or indirectly inhibits Fgf9 and Shh expression in
the lingual epithelium, as was demonstrated in both Bcl11b−/− and Bcl11bep−/−
incisors. FGF9 acts in the mesenchyme to induce expression of Fgf3 and
Fgf10 (Klein et al., 2008; Wang et al., 2007). Therefore, the repression of Fgf9
expression by BCL11B in the lingual epithelium prevents activation of Fgf3
and Fgf10 expression in the dental mesenchyme. (Fig. 2.10A).
3. In contrast, Sprouty gene inactivation leads to up-regulation of FGF gene
expression on the lingual side (Klein et al., 2008) and subsequent repression
of BCL11B expression in the lingual IEE. This down-regulation of BCL11B
expression leads to up-regulation of FGF family members, as well as that of
Shh, which induces development of ectopic pre-ameloblasts in the lingual
epithelium (Seidel et al., 2010; Takahashi et al., 2007) (Fig. 2.10B).
42
These data, which suggest that FGFs and BCL11B form a reciprocal, inhibitory
circuit that is upstream of SHH, integrate FGFs, BCL11B, and SHH in a single
pathway and provide insight into the molecular mechanisms underlying both the
Sprouty and Bcl11b−/− phenotypes.
Acknowledgments
We thank I. Thesleff, A. P. McMahon, M.-J. Tsai, S. M. K. Lee, P.-T. Chuang,
R. G. Kelly, S. Bellusci, and J. Smith for activin, Shh, Notch1, Bmp4, Gli1, Tbx1,
Fgf10, and Fst probes, respectively. We are also grateful to O. Golonzhka for
valuable contributions and support.
43
Figure 2.1. BCL11B expression during incisor development. BCL11B immunostaining in sections of wild-type mice at indicated developmental stages. The epithelium is outlined by white dots. Scale bars: (A-B) 100 µm; (C) 200 µm; (D-E) 500 µm. a, ameloblasts; an, anterior; cl, cervical loop; de, dental epithelium; df, dental follicle; dm, dental mesenchyme; iee, inner enamel epithelium; lab, labial; lin, lingual; oee, outer enamel epithelium; pm, papillary mesenchyme; po, posterior; sr, stellate reticulum; vl, vestibular lamina.
44
Figure 2.2. Epithelial invagination defect in Bcl11b−/− developing incisors between initiation and early bud stage.
(A-D) H&E staining in sections of wild-type and Bcl11b−/− mice at indicated developmental stages. The epithelium is outlined in black. (E-H) BrdU immunostaining (green) in sections of wild-type and Bcl11b−/− mice. All sections were counterstained with DAPI (blue). The epithelium is outlined in white. (I-J) BrdU index of wild-type and Bcl11b−/− dental epithelium; *** denotes statistical significance at p ≤ 0.001, n = 3. (K-N) RNA ISH using a Bmp4 probe in sections of wild-type and Bcl11b−/− mice at indicated developmental stages. The epithelium is outlined by red dots. Red arrows denote epithelial staining. Scale bar, 100 µm.
Figure 2.3. Alterations in Bcl11b−/− incisor development at cap stage.
(A-B) H&E staining in sections of wild-type and Bcl11b−/− mice at E14.5. The
epithelium is outlined in black. (C-D) BrdU immunostaining (green) in sections of wild-type andBcl11b−/− mice at E14.5. All sections were counterstained with DAPI
(blue). The epithelium is outlined in white. (E) BrdU index of wild-type and Bcl11b−/− CLs; *** denotes statistical significance at p ≤ 0.001, n = 3. (F-G)
RNA ISH using an Fgf10 probe in sections of wild-type and Bcl11b−/− mice at E14. Black arrows indicate mesenchymal staining; black asterisks denote the staining between dental epithelium and vestibular lamina. Scale bar, 200 µm. cl, cervical loop; ek, enamel knot; vl, vestibular lamina.
45
46
47
Figure 2.4. Morphological defects in Bcl11b−/− incisor development at bell stage.
(A-H, L-Y) H&E staining in sections of wild-type and Bcl11b−/− mice at indicated stages. The epithelium is outlined in black. Brackets indicate the posterior end of the incisors in A, E, L, N. Panels D, H, T, Y are higher magnification images of boxes in A, E, L, N, respectively. White arrows indicate lingual epithelium. I denotes the labial CL; II-IV denote stages of progression of ameloblast differentiation in L, N, P-S, U-X. The Bcl11b−/− labial CL was smaller than that of wild-type mice by 60% at E16.5 and by 69% at E18.5 (p ≤ 0.001, n = 11), whereas the mutant lingual CL was enlarged by 35% at E16.5 and by 64% at E18.5 relative to wild-type tissue (p ≤ 0.001, n = 11). (I-J) BrdU immunostaining (green) in sections of wild-type and Bcl11b−/− mice at E16.5. All sections were counterstained with DAPI (blue). The epithelium is outlined in white. (K) BrdU index of wild-type and Bcl11b−/− basal epithelium of labial and lingual CLs; ns, not significant; *** denotes statistical significance at p ≤ 0.001, n = 3. Scale bars: (AS, U-X) 200 µm; other panels, 100 µm. a, ameloblasts; cl, cervical loop; lab, labial; lin, lingual.
48
Figure 2.5. Altered ameloblast development in Bcl11b−/− incisors.
RNA ISH using the indicated probes in sections of wild-type and Bcl11b−/− mice at indicated developmental stages. The epithelium is outlined by red dots. Red arrows and arrowheads denote labial and lingual epithelial staining, respectively. Scale bars: (A-B, E-F) 500 µm; other panels, 200 µm.
49
Figure 2.6. Labial to lingual reversal of expression of FGF and Sprouty genes in Bcl11b−/− incisor.
RNA ISH using the indicated probes in sections of wild-type and Bcl11b−/−
incisors at indicated developmental stages. The epithelium is outlined by red dots. Black and red arrows denote labial mesenchymal and epithelial staining, respectively, and black and red arrowheads indicate lingual mesenchymal and epithelial staining, respectively. Scale bars: (A-B, E-F, I-J, M-N, Q-R) 500 µm; other panels, 200 µm.
Figure 2.7. Altered expression of TGFβ genes and Fst in Bcl11b−/− incisor.
RNA ISH using the indicated probes in sections of wild-type
and Bcl11b−/− incisors at indicated developmental stages. The epithelium is outlined by red dots. Black and red arrows denote labial mesenchymal and epithelial staining, respectively, and black and red arrowheads indicate lingual mesenchymal and epithelial staining, respectively. Black and red asterisks denote staining in the posterior part of the dental follicle and epithelial tip of the incisor, respectively. Scale bars: (A-B, E-F, I-J) 500 µm; other panels, 200 µm.
50
Figure 2.8. Ectopic lingual expression of ameloblast markers and signaling molecules in Bcl11bep−/− incisor.
(A-B) BCL11B immunostaining in sections of Bcl11bL2/L2 and Bcl11bep−/− mice at E16.5. The epithelium is outlined by white dots. (C-L) RNA ISH using the
indicated probes in sections of Bcl11bL2/L2 and Bcl11bep−/− incisors at E16.5. The epithelium is outlined by red dots. Black and red arrowheads denote lingual mesenchymal and epithelial staining, respectively. Scale bar, 500 µm.
51
52
Figure 2.9. Inhibition of Bcl11b expression in the lingual IEE of Spry4−/−; Spry2+/− mice at E16.5.
BCL11B immunostaining in sections of wild-type and Spry4−/−; Spry2+/− mice at E16.5. The epithelium is outlined by white dots. White arrowheads denote lingual epithelial staining. Scale bar, 500 µm.
53
Figure 2.10. Model for a reciprocal, inhibitory circuit on the lingual side of the mouse incisor. (A) Proposed model for integration of BCL11B into FGF signaling pathways in the lingual epithelium of wild-type incisors. This model suggests that BCL11B expression in the lingual epithelium is facilitated by SPRY2- and SPRY4mediated inhibition of the FGF signaling pathways. The resulting, high levels of BCL11B directly or indirectly suppress expression of Shh and subsequently lead to disruption of pre-ameloblast differentiation and ameloblast formation. It is also proposed that BCL11B inhibits expression of Fgf9 in the lingual epithelium, and this serves to enforce disruption of the FGF signaling loop that is mediated by the Sprouty proteins. The repressive effect of BCL11B on Fgf3 and Fgf10expression is most likely a secondary effect and is depicted by a dashed line. Therefore, BCL11B participates in the suppression of both FGF-mediated stimulation of lingual EpSC proliferation and differentiation of EpSC into Shh-positive preameloblasts within the lingual IEE. (B) In the absence of Spry4 and one allele of Spry2, FGF signaling is enhanced, which extinguishes expression of BCL11B in the lingual IEE. Loss of BCL11B results in derepression of Fgf9 and Shh expression in the epithelium. FGF9 drives Fgf3 and Fgf10 expression in the lingual mesenchyme, whereas Shh expression is required for development of ectopic pre-ameloblasts within the lingual IEE.
54
Figure S2.1. Expression of Bcl11b at early bell stage. (A) RNA ISH using Bcl11b probe in sections of wild-type mice at E16.5. (B-D) Sections of wild-type mice stained with DAPI and immunostained for BCL11B. Scale bar, 500 µm.
55
Figure S2.2. Expression patterns of selected genes in Bcl11b−/− incisor at early bud stage.
RNA ISH using the indicated probes in sections of wild-type and Bcl11b−/− mice at E12.5. The epithelium is outlined by red dots. Scale bar, 100 µm.
56
Figure S2.3. Expression patterns of selected genes in Bcl11b−/− incisor at cap stage.
RNA ISH using the indicated probes in sections of wild-type and Bcl11b−/− mice at E14.5. The epithelium is outlined by red dots. Scale bar, 200 µm.
57
Figure S2.4. Delay in the initiation of apoptosis in Bcl11b−/− enamel knot at cap stage.
TUNEL immunostaining in sections of wild-type and Bcl11b−/− mice at indicated stages. The epithelium is outlined by white dots. White arrows denote apoptosis in the enamel knot. Scale bars: (A-B) 200 µm; other panels, 500 µm.
58
Figure S2.5. Size difference between wild-type and Bcl11b−/− incisors of newborn mice.
Alizarin red staining of wild-type and Bcl11b−/− mandibular incisors at P0. Blue brackets indicate the posterior end of the incisor. Scale bar, 500 µm.
59
Figure S2.6. Labial to lingual reversal of expression of Gli1 in Bcl11b−/−
incisors.
RNA ISH using aGli1 probe in sections of wild-type and Bcl11b−/− mice at indicated stages. The epithelium is outlined by red dots. Black and red arrows denote labial mesenchymal and epithelial staining, respectively, and red arrowheads indicate lingual epithelial staining. Scale bars: (A-B) 500 µm; other panels, 200 µm.
60
Figure S2.7. Expression pattern of Lfrn and Notch1 in Bcl11b−/− incisors.
RNA ISH using the indicated probes in sections of wild-type and Bcl11b−/− mice at indicated stages. The epithelium is outlined by red dots. Red arrows and arrowheads denote labial and lingual epithelial staining, respectively. Scale bars: (A-B, E-F) 500 µm; other panels, 200 µm.
61
Figure S2.8. Expression of ameloblast markers in Bcl11bep−/− and Bcl11bmes/− incisors at E18.5.
RNA ISH using the indicated probes in sections of Bcl11bL2/L2 , Bcl11bep−/−,
and Bcl11bmes−/−mice at E18.5. The epithelium is outlined by red dots. Red arrowheads denote lingual epithelial staining. Scale bar, 200 µm.
62
Figure S2.9. Morphology and mineralization of Bcl11b incisors at P21. Micro-CT analysis of Bcl11bL2/L2 and Bcl11bep−/− jaws at P21.
ep−/−
63
Figure S2.10. Expression patterns of ameloblast markers and signaling molecules in Bcl11bmes−/− incisors at E16.5.
(A-B) BCL11B immunostaining (red) in sections of Bcl11bL2/L2 and Bcl11bmes−/−
mice at E16.5. The epithelium is outlined by white dots. White asterisks denote BCL11B staining in the posterior mesenchyme. (C-J) RNA ISH using the indicated probes in sections of Bcl11bL2/L2 and Bcl11bmes−/− mice at E16.5. The epithelium is outlined by red dots. Scale bar, 500 µm.
64
Figure S2.11. Labial to lingual reversal of expression of Tbx1 in Bcl11b−/−
incisors.
RNA ISH using aTbx1 probe in sections of wild-type and Bcl11b−/− mice at indicated stages. The epithelium is outlined by red dots. Red arrows and arrowheads denote labial and lingual epithelial staining, respectively. Scale bars: (A-B) 500 µm; other panels, 200 µm.
65
Figure S2.12. BCL11B expression in Fgf3−/−; Fgf10+/− incisors.
BCL11B immunostaining in sections of wild-type and Fgf3−/−; Fgf10+/− mice at E16.5. The epithelium is outlined by white dots. Scale bar, 500 µm.
66
Figure S2.13. Summary of direct or indirect BCL11B target genes at E16.5.
This model is based on RNA ISH studies presented in Figs. 5, 6, 7 and Suppl. Figs. S5, S6, and S10. The red staining of the incisor is a pseudo-color representation of BCL11B immunohistochemical staining experiment. The epithelium is outlined by black dots. Green and red arrows indicate induction and inhibition of gene expression, respectively; blue dots denote the enforcement of gene expression domains by BCL11B.
67
References
Alvarez, Y., Alonso, M. T., Vendrell, V., Zelarayan, L. C., Chamero, P., Theil, T., Bosl, M. R., Kato, S., Maconochie, M., Riethmacher, D. et al. (2003). Requirements for FGF3 and FGF10 during inner ear formation. Development 130, 6329-38.
Arlotta, P., Molyneaux, B. J., Chen, J., Inoue, J., Kominami, R. and Macklis, J. D. (2005). Neuronal subtype-specific genes that control corticospinal motor neuron development in vivo. Neuron 45, 207-21.
Arlotta, P., Molyneaux, B. J., Jabaudon, D., Yoshida, Y. and Macklis, J. D. (2008). Ctip2 controls the differentiation of medium spiny neurons and the establishment of the cellular architecture of the striatum. J Neurosci 28, 622-32.
Bellusci, S., Henderson, R., Winnier, G., Oikawa, T. and Hogan, B. L. (1996). Evidence from normal expression and targeted misexpression that bone morphogenetic protein (Bmp-4) plays a role in mouse embryonic lung morphogenesis. Development 122, 1693-702.
Bitgood, M. J. and McMahon, A. P. (1995). Hedgehog and Bmp genes are coexpressed at many diverse sites of cell-cell interaction in the mouse embryo. Dev Biol 172, 126-38.
Caton, J., Luder, H. U., Zoupa, M., Bradman, M., Bluteau, G., Tucker, A. S., Klein, O. and Mitsiadis, T. A. (2009). Enamel-free teeth: Tbx1 deletion affects amelogenesis in rodent incisors. Dev Biol 328, 493-505.
Danielian, P. S., Muccino, D., Rowitch, D. H., Michael, S. K. and McMahon, A. P. (1998). Modification of gene activity in mouse embryos in utero by a tamoxifeninducible form of Cre recombinase. Curr Biol 8, 1323-6.
Dassule, H. R., Lewis, P., Bei, M., Maas, R. and McMahon, A. P. (2000). Sonic hedgehog regulates growth and morphogenesis of the tooth. Development 127, 4775-85.
Golonzhka, O., Leid, M., Indra, G. and Indra, A. K. (2007). Expression of COUPTF-interacting protein 2 (CTIP2) in mouse skin during development and in adulthood. Gene Expr Patterns 7, 754-60.
Golonzhka, O., Liang, X., Messaddeq, N., Bornert, J. M., Campbell, A. L., Metzger, D., Chambon, P., Ganguli-Indra, G., Leid, M. and Indra, A. K. (2009a). Dual role of COUP-TF-interacting protein 2 in epidermal homeostasis and permeability barrier formation. J Invest Dermatol 129, 1459-70.
68
Golonzhka, O., Metzger, D., Bornert, J. M., Bay, B. K., Gross, M. K., Kioussi, C. and Leid, M. (2009b). Ctip2/Bcl11b controls ameloblast formation during mammalian odontogenesis. Proc Natl Acad Sci U S A 106, 4278-83.
Hacohen, N., Kramer, S., Sutherland, D., Hiromi, Y. and Krasnow, M. A. (1998). sprouty encodes a novel antagonist of FGF signaling that patterns apical branching of the Drosophila airways. Cell 92, 253-63.
Harada, H., Kettunen, P., Jung, H. S., Mustonen, T., Wang, Y. A. and Thesleff, I. (1999). Localization of putative stem cells in dental epithelium and their association with Notch and FGF signaling. J Cell Biol 147, 105-20.
Harada, H., Toyono, T., Toyoshima, K., Yamasaki, M., Itoh, N., Kato, S., Sekine, K. and Ohuchi, H. (2002). FGF10 maintains stem cell compartment in developing mouse incisors. Development 129, 1533-41.
Kerley, M. A. (1975). Pre-natal development of the mouse incisor. Proc Natl Acad Sci USA 55, 6–10.
Kettunen, P., Laurikkala, J., Itaranta, P., Vainio, S., Itoh, N. and Thesleff, I. (2000). Associations of FGF-3 and FGF-10 with signaling networks regulating tooth morphogenesis. Dev Dyn 219, 322-32.
Klein, O. D., Lyons, D. B., Balooch, G., Marshall, G. W., Basson, M. A., Peterka, M., Boran, T., Peterkova, R. and Martin, G. R. (2008). An FGF signaling loop sustains the generation of differentiated progeny from stem cells in mouse incisors. Development 135, 377-85.
Klein, O. D., Minowada, G., Peterkova, R., Kangas, A., Yu, B. D., Lesot, H., Peterka, M., Jernvall, J. and Martin, G. R. (2006). Sprouty genes control diastema tooth development via bidirectional antagonism of epithelialmesenchymal FGF signaling. Dev Cell 11, 181-90.
Li, L., Leid, M. and Rothenberg, E. V. (2010). An early T cell lineage commitment checkpoint dependent on the transcription factor Bcl11b. Science 329, 89-93.
Lumsden, A. G. (1988). Spatial organization of the epithelium and the role of neural crest cells in the initiation of the mammalian tooth germ. Development 103 Suppl, 155-69.
O'Hagan, R. C. and Hassell, J. A. (1998). The PEA3 Ets transcription factor is a downstream target of the HER2/Neu receptor tyrosine kinase. Oncogene 16, 301-10.
Peters, H. and Balling, R. (1999). Teeth. Where and how to make them. Trends
69
Genet 15, 59-65.
Porntaveetus, T., Otsuka-Tanaka, Y., Basson, M. A., Moon, A. M., Sharpe, P. T. and Ohazama, A. (2011). Expression of fibroblast growth factors (Fgfs) in murine tooth development. J Anat 218, 534-43.
Roehl, H. and Nusslein-Volhard, C. (2001). Zebrafish pea3 and erm are general targets of FGF8 signaling. Curr Biol 11, 503-7.
Ruiz i Altaba, A. (1999). Gli proteins encode context-dependent positive and negative functions: implications for development and disease. Development 126, 3205-16.
Ryan, M. C., Lee, K., Miyashita, Y. and Carter, W. G. (1999). Targeted disruption of the LAMA3 gene in mice reveals abnormalities in survival and late stage differentiation of epithelial cells. J Cell Biol 145, 1309-23.
Seidel, K., Ahn, C. P., Lyons, D., Nee, A., Ting, K., Brownell, I., Cao, T., Carano, R. A., Curran, T., Schober, M. et al. (2010). Hedgehog signaling regulates the generation of ameloblast progenitors in the continuously growing mouse incisor. Development 137, 3753-61.
Sekine, K., Ohuchi, H., Fujiwara, M., Yamasaki, M., Yoshizawa, T., Sato, T., Yagishita, N., Matsui, D., Koga, Y., Itoh, N. et al. (1999). Fgf10 is essential for limb and lung formation. Nat Genet 21, 138-41.
Shim, K., Minowada, G., Coling, D. E. and Martin, G. R. (2005). Sprouty2, a mouse deafness gene, regulates cell fate decisions in the auditory sensory epithelium by antagonizing FGF signaling. Dev Cell 8, 553-64.
Takahashi, S., Kawashima, N., Sakamoto, K., Nakata, A., Kameda, T., Sugiyama, T., Katsube, K. and Suda, H. (2007). Differentiation of an ameloblast-lineage cell line (ALC) is induced by Sonic hedgehog signaling. Biochem Biophys Res Commun 353, 405-11.
Tucker, A. and Sharpe, P. (2004). The cutting-edge of mammalian development; how the embryo makes teeth. Nat Rev Genet 5, 499-508.
Tummers, M. and Thesleff, I. (2003). Root or crown: a developmental choice orchestrated by the differential regulation of the epithelial stem cell niche in the tooth of two rodent species. Development 130, 1049-57.
Tummers, M. and Thesleff, I. (2009). The importance of signal pathway modulation in all aspects of tooth development. J Exp Zool B Mol Dev Evol 312B, 309-19.
70
Vaahtokari, A., Aberg, T., Jernvall, J., Keranen, S. and Thesleff, I. (1996a). The enamel knot as a signaling center in the developing mouse tooth. Mech Dev 54, 39-43.
Vaahtokari, A., Aberg, T. and Thesleff, I. (1996b). Apoptosis in the developing tooth: association with an embryonic signaling center and suppression by EGF and FGF-4. Development 122, 121-9.
Wakabayashi, Y., Watanabe, H., Inoue, J., Takeda, N., Sakata, J., Mishima, Y., Hitomi, J., Yamamoto, T., Utsuyama, M., Niwa, O. et al. (2003). Bcl11b is required for differentiation and survival of alphabeta T lymphocytes. Nat Immunol 4, 533-9.
Wang, X. P., Suomalainen, M., Felszeghy, S., Zelarayan, L. C., Alonso, M. T., Plikus, M. V., Maas, R. L., Chuong, C. M., Schimmang, T. and Thesleff, I. (2007). An integrated gene regulatory network controls stem cell proliferation in teeth. PLoS Biol 5, e159.
Wang, X. P., Suomalainen, M., Jorgez, C. J., Matzuk, M. M., Werner, S. and Thesleff, I. (2004). Follistatin regulates enamel patterning in mouse incisors by asymmetrically inhibiting BMP signaling and ameloblast differentiation. Dev Cell 7, 719-30.
Yokohama-Tamaki, T., Ohshima, H., Fujiwara, N., Takada, Y., Ichimori, Y., Wakisaka, S., Ohuchi, H. and Harada, H. (2006). Cessation of Fgf10 signaling, resulting in a defective dental epithelial stem cell compartment, leads to the transition from crown to root formation. Development 133, 1359-66.
Zeichner-David, M., Diekwisch, T., Fincham, A., Lau, E., MacDougall, M., Moradian-Oldak, J., Simmer, J., Snead, M. and Slavkin, H. C. (1995). Control of ameloblast differentiation. Int J Dev Biol 39, 69-92.
Zhang, Y., Zhang, Z., Zhao, X., Yu, X., Hu, Y., Geronimo, B., Fromm, S. H. and Chen, Y. P. (2000). A new function of BMP4: dual role for BMP4 in regulation of Sonic hedgehog expression in the mouse tooth germ. Development 127, 143143.
Zhang, Y. D., Chen, Z., Song, Y. Q., Liu, C. and Chen, Y. P. (2005). Making a tooth: growth factors, transcription factors, and stem cells. Cell Res 15, 301-16.
The Role of the Transcriptional Regulatory Protein BCL11B in Dental and Craniofacial Development
Chapter 3
BCL11B Regulates Craniofacial Suture Patency through Repression of Fgfr2c Expression
Kateryna Kyrylkova, Urszula Iwaniec, Mark Leid
To be submitted to: Development
72
Abstract
BCL11B is a transcription factor that plays essential roles during development of
the immune, nervous and cutaneous systems. Here we show that BCL11B is
expressed in the osteogenic and sutural mesenchyme of the developing
craniofacial complex. Bcl11b-/- mice exhibit increased proliferation of
osteoprogenitors, premature osteoblast differentiation, and skull mineralization.
These processes lead to synostosis of facial and coronal sutures in the Bcl11b-/-
skulls. Expression of Fgfr2c, a gene implicated in craniosynostosis in mice and
humans, was up-regulated and Fgfr2c transcripts were detected ectopically
within these sutures in Bcl11b-/- mice. These data suggest that overexpression of
Fgfr2c in the sutural mesenchyme, without concomitant changes in the
expression of FGF ligands, leads to craniosynostosis in Bcl11b-/- mice.
Introduction
Flat bones of the skull and face develop by intramembranous ossification of
mesenchyme derived from the mesoderm and neural crest (Jiang et al., 2002). In
this process, mesenchymal cells condense, differentiate, and form ossification
centers (Opperman, 2000; Rice, 2008). Craniofacial sutures are formed when the
margins of the developing bones approach each other. While human sutures
remain patent, they harbor proliferating progenitors that give rise to osteoblasts
at the osteogenic fronts of the skull bones. Therefore, craniofacial sutures serve
as important sites of facial and calvarial bone growth during fetal development
and into young adulthood, when sutures undergo ossification (Rice, 2008).
Suture development is precisely controlled and synchronized with brain growth.
Premature osteoblast differentiation and suture ossification underlie the condition
known as craniosynostosis, which occurs in 1 out of 2,200 live births.
Craniosynostosis is associated with restricted skull expansion, increased
intracranial pressure, and craniofacial dysmorphologies, all of which may
negatively impact respiration, vision, hearing, and cognition. Midfacial hypoplasia
73
(MFH) often accompanies craniosynostosis and until recently was thought to
result from premature ossification of sutures at the cranial base (Nie, 2005;
Rosenberg et al., 1997). However, it was shown that MFH, in the context of
syndromic craniosynostosis, likely arises from premature fusion of facial sutures
(Purushothaman et al., 2011).
Mutations in fibroblast growth factor receptors (FGFRs) are the most common
cause of syndromic cranyosynostosis, of which the most prevalent forms are
Crouzon and Apert syndromes (Purushothaman et al., 2011). Both conditions are
the result of activating mutations within FGFR2 isoform IIIc (FGFR2c). The
FGFR2c-C342Y mutation, which affects immunoglobulin-like (Ig) domain III,
results in constitutive activation of the receptor and leads to development of
Crouzon syndrome. In contrast, Apert syndrome is caused by FGFR2c-S252W
mutation in Ig II-III linker region and is characterized by increased ligand affinity
and altered specificity of FGFR2 (Cunningham et al., 2007). FGFRs act through
the mitogen-activated protein kinase (MAPK) and protein kinase C (PKC)
pathways to stimulate transcription factor RUNX2, a master-regulator of
osteogenesis (Kim et al., 2003a; Park et al., 2010). As a result, constitutive
activation of FGFR2c leads to enhanced osteoblast differentiation and premature
ossification of the sutures. However, little is known about molecular control of
FGFR2c expression and function during craniofacial development.
We showed previously that the transcription factor BCL11B controls mouse
incisor development by regulating the expression of some of the individual
components of the FGF signaling network in the labial and lingual cervical loops
(Kyrylkova et al., 2012a). Our analyses of neural crest-specific and germline
Bcl11b knockouts (Bcl11bncc-/- and Bcl11b-/-, respectively) reveal that BCL11B
plays an essential role in craniofacial development and maintenance of suture
patency. Mice lacking Bcl11b exhibit craniosynostosis of facial and coronal
sutures, severe midfacial hypoplasia, and malocclusion. We show that BCL11B
regulates timely proliferation and differentiation of osteoprogenitors by repressing
74
Fgfr2c expression directly or indirectly in the craniofacial sutures.
Materials and Methods
Mouse Lines
− cl11 − B b / , Bcl11bL2/L2 , and Bcl11bncc-/- (same as Bcl11bmes-/-) mice have been
described (Golonzhka et al., 2009; Kyrylkova et al., 2012a). Lines carrying Wnt1
cre transgene were maintained as reported (Danielian et al., 1998). Animal
experiments were approved by the Oregon State University Institutional Animal
Care and Use Committee, protocol 4279.
Micro-CT Analysis
Mouse skulls were scanned using a Scanco μCT40 scanner (Scanco Medical
AG, Basserdorf, Switzerland) at a voxel size of 12 to 20 μm. The threshold for
analysis was determined empirically and set at 245 (scale 0 – 1000). At least
three mouse skulls were scanned for each genotype and age group.
Histological Analysis, RNA in situ Hybridization, BrdU labeling, and
Immunohistochemistry
Embryonic heads were fixed in 4% paraformaldehyde, cryopreserved in 30%
sucrose, and frozen in O.C.T. Von Kossa as well as hematoxylin and eosin
(H&E) stainings were performed according to standard protocols. RNA in situ
hybridization using digoxigenin-labeled probes and immunohistochemistry using
anti-BCL11B (Abcam, 1:300) were performed on 16 µm-thick sagittal (for coronal
suture) and horizontal (for facial suture) sections as described previously
(Kyryachenko et al., 2012; Kyrylkova et al., 2012b). At least three mice were
used for each genotype and experiment. At least ten serial sections across the
facial and coronal sutures were analyzed for each experiment.
75
Results
Neural Crest-Specific Ablation of Bcl11b Leads to Growth Impairment and
Craniofacial Synostoses
In our previous study on the role of BCL11B in tooth development, we generated
a mouse line conditionally null for Bcl11b expression in neural crest-derived
dental mesenchyme (Bcl11bmes-/-; referred to as Bcl11bncc-/- in a current
manuscript) by crossing floxed Bcl11bL2/L2 mice with the Wnt1-cre deleter strain
(Danielian et al., 1998; Kyrylkova et al., 2012a). Wnt1-expressing neural crest
cells contribute not only to the tooth, but also to a plethora of different tissues and
organs, including components of enteric and peripheral nervous system,
endocrine and cardiac derivatives, melanocytes, as well as cephalic
Bcl11bncc-/-mesenchyme (Pietri et al., 2003). mice did not exhibit dental
abnormalities, as the incisor morphology and gene expression patterns were
indistinguishable from those of control mice (Kyrylkova et al., 2012a). However,
-/- ncc-/-unlike Bcl11b mice, Bcl11b mice survived after birth for around three weeks
(P21). At this age, Bcl11bncc-/- mice were characterized by abnormally small and
misshapen heads, severe MFH, and malocclusion (compare Figs. 3.1A, C with B,
D). We observed fusion of multiple craniofacial sutures, including the interfrontal,
internasal, temporal, premaxillary-maxillary, and premaxillary-nasal sutures. In
addition, the Bcl11bncc-/- nasofrontal suture exhibited abnormal morphology and
lacked its canonical, fractal interdigitation (compare Figs. 3.1E, G with F, H).
Bcl11bncc-/-Some skulls were characterized by a high degree of porosity, which
was accompanied by up-regulated expression of pro-inflammatory cytokines in
frontal and parietal bones (Fig. S3.1 and data not shown).
We traced back the developmental dynamics of the conditional knockout
Bcl11bncc-/-phenotype by conducting micro-CT analyses of multiple skulls at
different postnatal stages. At P5, synostosis of temporal and frontomaxillary
Bcl11bncc-/-sutures was observed in 50% of mice. In addition, the skulls of
76
Bcl11bncc-/- mice were characterized by expansion of sagittal, lambdoid, and
coronal sutures, and these expansions appeared to be of a compensatory
nature. The onset of bone porosity was visible at P5 in the anterior region of
frontal bones and at the base of the Bcl11bncc-/- skulls. A similar but more severe
craniofacial phenotype was observed in the conditional mutants at P10. The
progression of synostosis was further evident in the anterior part of interfrontal
and posterior part of internasal sutures in Bcl11bncc-/- mice at P14 (Fig. S3.2).
Newborn Bcl11b-/- Mice Exhibit Facial and Coronal Synostosis
Premaxillary-maxillary and premaxillary-nasal sutures in Bcl11bncc-/- mice were
already fused at birth, at which time mild MFH and a reduced gap between
frontal and maxillary bones were also evident (compare Figs. 3.2A, D with B, E).
Bcl11bncc-/-The early onset of the craniofacial phenotype of mice led us to
examine Bcl11b-/- mice, which harbor a germline deletion of Bcl11b and die
perinatally (Golonzhka et al., 2009). Bcl11b-/- skulls exhibited facial synostoses at
ncc-/-P0 that were indistinguishable from those of Bcl11b mice (compare Figs.
3.2B, E with C, F). However, premature fusion of the temporal and coronal
sutures was also evident in Bcl11b-/- mice at P0, a phenotype that we have never
Bcl11bncc-/-observed in mice (compare Figs. 3.2A, D with C, F). Moreover,
enhanced ossification within the anterior fontanelle was noted in Bcl11b-/- mice
Bcl11bncc-/-relative to either control or mice (compare Figs. 3.2A, B with C).
Enhanced mineralization of the facial and cranial skeleton in Bcl11b-/- mice was
further confirmed by von Kossa staining at P0 (Fig. S3.3). Collectively, the above
findings indicate that facial and cranial synostoses in mice lacking Bcl11b initiate
during embryonic development.
Embryonic Skulls of Bcl11b-/- Exhibit Increased Osteoblast Proliferation and
Maturation, Premature Mineralization, and Craniofacial Synostosis
Mineralization within control and Bcl11b-/- heads was observed at E14.5 as
detected by von Kossa staining. However, Bcl11b-/- mice exhibited increased
77
mineralization within the facial skeleton (compare Figs. 3.3A, C with B, D).
Moreover, expression of bone sialoprotein (Bsp), a marker of osteoblast
maturation, was dramatically up-regulated within Bcl11b-/- faces (compare Figs.
3.3E, G with F, H). Osteoblasts of Bcl11b-/- facial mesenchyme exhibited ~30%
increase in the proliferation rate compared to the control cells (data not shown).
Micro-CT analyses of Bcl11b-/- skulls at E16.5 and E18.5 also revealed increased
mineralization within the facial skeleton and calvaria of the mutant skulls (Figs.
3.3I-P). The premaxillary-maxillary suture was fused in the mutants at this
developmental stage (compare Figs. 3.3I, K with J, L). However, initiation of
coronal and temporal synostoses was not observed until E18.5 in the Bcl11b-/-
skulls (compare Figs. 3.3M, O with N, P).
Palatogenesis is a key component of midface development (Weinzweig et al.,
2006). Bcl11bncc-/- embryos did not exhibit defects in palatal development (data
not shown). However, we detected a delay in palatal shelf elevation and fusion in
-/-Bcl11b mice at E14.5 but this defect was completely resolved by E15.5.
BCL11B expression was localized predominantly in the palatal epithelium prior to
and after shelf elevation and fusion (Fig. S3.4).
BCL11B Is Expressed in Osteogenic and Sutural Mesenchyme
BCL11B expression within osteogenic progenitors of the face was first detected
at E13.5 (Fig. S3.5C). High levels of BCL11B expression persisted at E14.5 and
E16.5 within facial and calvarial osteogenic and sutural mesenchyme, including
osteogenic fronts of frontal and parietal bonas as well as the coronal suture
(Figs. 3.4A-F). However, by E18.5 BCL11B expression was down-regulated and
barely detectable within either facial or cranial bones and sutures (Figs. 3.4G-I),
suggesting that BCL11B functions predominantly during embryonic stages of
skull development.
78
BCL11B expression was ablated specifically in neural crest-derived facial
mesenchyme, including dermal and osteogenic mesenchyme, in Bcl11bncc-/- mice
at E12.5. The expression of BCL11B within the osteogenic mesenchyme of the
face was also absent in Bcl11bncc-/- at E13.5. At the same time, levels of BCL11B
protein were unaltered in all epithelial structures in Bcl11bncc-/- mice (Fig. S3.5).
Runx2 Expression Is Up-Regulated in Osteogenic Mesenchyme of Bcl11b-/-
Skulls
RUNX2 is the transcripition factor required for determination of the osteoblast
lineage and is a master regulator of osteogenesis (Komori, 2010; Komori et al.,
1997; Otto et al., 1997). Runx2 expression was strongly detected in
differentiating osteoblasts of the facial and calvarial osteogenic mesenchyme at
E14.5 and E16.5, respectively, in control mice. At the same time, low levels of
Runx2 transcripts were observed in the premaxillary-maxillary and coronal
sutures of control skulls (Figs. 3.5A, C, E). An expansion of Runx2-positive cells
-/-was observed in the osteogenic mesenchyme of the Bcl11b face, along with
ectopic Runx2 transcripts within coronal suture of these mutants at E16.5 (Figs.
3.5B, D, F). This observation suggests that BCL11B limits osteoblast expansion
and restricts Runx2 expression within sutural mesenchyme.
Fgfr2c Is Ectopically Expressed Within Facial and Coronal Sutures of
Bcl11b-/- Mice
The FGF signaling pathways play important roles in regulation of craniofacial
suture patency, and activating mutations within FGFR receptors are associated
with syndromic and non-syndromic craniosynostosis in mice and humans (Nie et
al., 2006). We showed previously that BCL11B regulates FGF signaling during
tooth development (Kyrylkova et al., 2012a). Therefore, we analyzed Bcl11b-/-
mice for expression of multiple FGF ligands and receptors that play roles during
craniofacial development. Surprisingly, we did not detect alterations in
expression of any genes encoding FGF or FGFR family members in Bcl11b-/-
79
mice (Fig. S3.6), with the exception of Fgfr2c (Fig. 3.6) Fgfr2c expression, was
detected widely within differentiating osteoblasts of the face and head of control
mice but was generally excluded from sutural mesenchyme (Fig. 3.6). Fgfr2c
transcripts were ectopically expressed within facial and coronal sutures of
Bcl11b-/- mice at E14.5 and E16.5, respectively (compare Figs. 3.6A, C, E with B,
D, F). However, Fgfr2c transcripts were excluded from the more differentiated
mesenchyme within central osteoids of Bcl11b-/- facial bones. These results
suggest that BCL11B normally represses Fgfr2c expression within craniofacial
sutures, and its absence is associated with ectopic expression of this key Fgfr2
splice variant.
Discussion
Data presented in this paper demonstrate that BCL11B plays an important role in
maintaining sutural patency from the earliest stages of craniofacial development.
BCL11B is expressed in osteogenic and sutural mesenchyme beginning at E13.5
and is down-regulated by E18.5, suggesting that it functions primarily during a
finite window of embryogenesis. BCL11B controls timely proliferation and
differentiation of osteoprogenitors, limits premature bone mineralization, and
suppresses ossification of facial and coronal sutures. We speculate that the
principal mechanistic basis of these actions of BCL11B is the repression of
Fgfr2c expression within osteogenic and sutural mesenchyme.
The craniofacial skeleton is derived from two distinct embryonic sources: neural
crest cells and the paraxial mesoderm. Neural crest cells give rise to the facial
skeleton, frontal and squamosal bones, as well as central part of the interparietal
bone, as determined by lineage tracing analyses (Jiang et al., 2002). In contrast,
the parietal bone and most of the interparietal bone are mesodermal in origin
(Yoshida et al., 2008). Therefore, the coronal suture, a major growth center of the
skull vault, is formed at the interface of the neural crest-derived frontal bone and
mesoderm-derived parietal bone. However, the mesenchyme of the coronal
80
suture is derived from the mesoderm (Yoshida et al., 2008), and this likely
explains our observation of coronal synostosis in Bcl11b-/-, but not in Bcl11bncc-/-,
mice.
Bcl11bncc-/- mice survive after birth and exhibit severe MFH, a condition that often
accompanies syndromic craniosynostosis in humans. Until recently, the most
common theory on etiology of MFH was premature ossification of one or more
sutures in the cranial base (Rosenberg et al., 1997; Stewart et al., 1977).
However, analyses of mice harboring constitutively active mutations in Fgfr1 and
Fgfr2 genes, which underlie Apert and Pfeiffer syndromes, revealed that MFH is
a consequence of synostoses of facial sutures (Purushothaman et al., 2011).
Consistent with this, Bcl11bncc-/- mice did not exhibit premature ossification of the
cranial base. Instead, Bcl11bncc-/- mice were characterized by synostoses of facial
sutures, including premaxillary-maxillary and premaxillary-nasal, further
supporting the role of facial skeletal pathology in etiology of MFH. In comparison
to the biology of cranial sutures, development of facial sutures is understudied
and poorly understood. While growth at the osteogenic fronts of the facial bones
is similar to that of the cranial vault bones, the facial bones differ in that they
overlie the cartilaginous elements of the facial complex rather than the dura
mater. Moreover, development of some facial sutures, such as frontonasal, is
characterized by complex interdigitation patterns that are not observed within any
sutures of the cranial vault (Nelson and Williams, 2004). Therefore, Bcl11bncc-/-
mice may serve as a valuable model to study pathogenesis of facial synostoses
and development of MFH.
The FGF signaling serves as a positive regulator of osteogenesis and plays a
major role in craniofacial development. The FGF family is comprised of 22 genes
encoding structurally related proteins (Ornitz and Itoh, 2001). Fgf2, Fgf9, Fgf10
and Fgf18 are expressed in developing bones and/or sutural mesenchyme and
have been directly or indirectly associated with enhanced ossification and
associated skeletal defects including craniosynostosis (Behr et al., 2010;
81
Hajihosseini et al., 2009; Harada et al., 2009; Kim et al., 1998; Liu et al., 2002;
Montero et al., 2000; Ohbayashi et al., 2002; Rice et al., 2000). In addition,
although Fgf4 is not expressed in the developing facial or cranial skeleton, direct
application of FGF4 in the vicinity of sutures promotes osteoblast proliferation
and/or differentiation and subsequent suture closure (Greenwald et al., 2001;
Kim et al., 2003b; Kim et al., 1998; Nagayama et al., 2013). It is noteworthy that
neither the expression level nor localization of Fgf transcripts that are relevant for
craniofacial development was altered in the osteogenic mesenchyme of Bcl11b-/-
mice.
FGFs bind four high-affinity, receptor tyrosine kinases (FGFR1 to FGFR4). The
ligand-binding specificity of FGFRs is mediated by specific extracellular regions
of these receptors known as immune globulin-like (Ig) domains. Alternative
splicing of transcripts derived from Fgfr1, Fgfr2 and Fgfr3 results in two different
versions of Ig-domain III within each receptor (referred to as domain IIIb and IIIc
or b and c isoforms, respectively). These alternative splicing events have
functional consequences as the b and c isoforms of FGFRs are characterized by
unique ligand-binding properties (Ornitz et al., 1996). Expression of Fgfr2c, as
well as low levels of Fgfr1c, Fgfr2b, and Fgfr3c, has been reported in developing
calvarial bones and particularly at the osteogenic fronts of these bones (Rice et
al., 2003). Mutations in genes encoding FGFR1-3, but not FGFR4, are
associated with craniosynostosis in mice and humans (Chim et al., 2011). In
general, all of these mutations result in receptors with enhanced and/or
constitutive activity (Cunningham et al., 2007). With the exception of Fgfr2c, we
did not observe alterations of expression of Fgfr family members in Bcl11b-/-
mice.
Fgfr2c is highly expressed in proliferating osteoprogenitors at the osteogenic
fronts (Iseki et al., 1997; Rice et al., 2003) and is of particular relevance in the
etiology of craniosynostosis. Expression of Fgfr2 is mutually exclusive with that of
Secreted phosphoprotein 1 (Spp1), a marker of differentiating osteoblasts (Iseki
82
et al., 1997), suggesting that FGFR2 isoforms are expressed early in the
osteoblast lineage and are down-regulated as osteoprogenitors mature toward
terminal differentiation. Loss-of-function mutation in the IIIc exon of Fgfr2 gene
results in defective ossification and reduced expression of Runx2 and Spp1,
indicating altered osteoblast differentiation (Eswarakumar et al., 2002). In
contrast, mice harboring gain-of-function mutation of Fgfr2c (Fgfr2cc342y/+) exhibit
craniosynostosis, cleft lip and palate, MFH, and other skeletal defects. The mice
are characterized by increased proliferation of osteoprogenitor cells and up-
regulation of Runx2 and Spp1 expression (Eswarakumar et al., 2004). RUNX2 is
an indispensible transcription factor for osteogenesis and skeletal development
(Kim et al., 2003a; Park et al., 2010). FGF signaling induces Runx2 expression
as well as enhances RUNX2 stability, DNA-binding, and transcriptional activity
through PKC and extracellular signal-regulated kinase MAPK pathways (Kim et
al., 2003a; Park et al., 2010; Twigg et al., 2013; Xiao et al., 2002). Some effects
of FGF activity on RUNX2 activity are mediated by a complicated cascade of
downstream factors, including the transcription factors Twist1, TCF12, and ERF
(Connerney et al., 2006; Fitzpatrick, 2013; Sharma et al., 2013; Twigg et al.,
2013).
Fgfr2c expression was up-regulated and Fgfr2c transcripts were ectopically
localized in the facial and coronal sutures of Bcl11b-/- mice. However, the central
osteoid of both facial and cranial bones was largely devoid of Fgfr2c transcripts,
consistent with enhanced osteoblast differentiation in these areas of the Bcl11b-/-
craniofacial skeleton. In addition, we observed enhanced osteoprogenitor
proliferation and increased Runx2 expression, both of which phenocopy defects
Fgfr2cc342y/+ of mice. However, we assume that Bcl11b-/- mice over- and
ectopically-express a wild-type form of Fgfr2c, yet exhibit a craniosynostosis
phenotype that is much more severe than that of mice heterozygously expressing
Fgfr2cc342y/+ a constitutive active form of the receptor, e.g., . This may be
explained by the fact that Fgfr2 over-expression results in ligand-independent
activation of the receptor in other cell systems (Turner et al., 2010). In addition,
83
overexpression of Fgfr1, driven by transient transfection, results in FGF2
mediated proliferation in neonatal cardiac myocyte cultures (Sheikh et al., 1999).
In the latter case, the over-expressed FGFR1 would appear to be stimulated by
an FGF ligand(s) that is in the vicinity. Therefore, we suggest that overexpression
of Fgfr2c in the sutural mesenchyme, without concomitant changes in the
expression of FGF ligands, leads to craniosynostosis in Bcl11b-/- mice.
Craniosynostosis affects 1 in 2,200 individuals and may be isolated
(nonsyndromic) or associated with other clinical signs as part of a syndrome.
Plausible causative mutations were identified only in ~30% of the cohort that
comprised over 300 cases of craniosynostosis. Genes that are predominantly
affected in craniosynostosis are FGFR2, FGFR3, TWIST1, and EFNB1
(Fitzpatrick, 2013; Twigg et al., 2004; Wilkie et al., 2010). Recently, heterozygous
loss-of-function mutations in TCF12 and ERF, both of which encode transcription
factors, were also found in the cohort (Sharma et al., 2013; Twigg et al., 2013).
However, ~70% of craniosynostosis cases still have unknown genetic etiology
(Fitzpatrick, 2013). We do not presently know if mutations at the BCL11B locus
contributes to craniosynostosis in humans as less than optimal exome coverage
of this locus hinders identification of possible mutations in humans. Given the
severity of the craniosynostosis phenotype of the Bcl11b-/- mice, it seems
plausible to hypothesize that dysregulated expression of the transcription factor
BCL11B may be implicated in craniosynostosis in humans.
84
Figure 3.1. Impaired postnatal growth, misshapen heads, and craniofacial synostoses in Bcl11bncc-/- mice. (A-D) Ventral (A, B) and lateral (C, D) views of mouse heads show abnormal shape, short snouts, and severe malocclusion in Bcl11bncc-/- mice at P21. (E-H) Dorsal (E, F) and lateral (G, H) views of the micro-CT images of control and Bcl11bncc-/-mouse skulls at P21. Asterisks denote sutures affected by synostosis in Bcl11bncc-/- skulls and corresponding patent sutures in the control mice. Arrow points to the lack of fractal interdigitation in Bcl11bncc-/- fronto-premaxillary suture. Red lines indicate dental occlusion. Scale bar: (E-H) 1 mm.
85
Figure 3.2. Craniofacial defects of Bcl11bncc-/- and Bcl11b-/- newborn mice. Dorsal (A-C) and lateral (D-F) views of the micro-CT images of control, Bcl11bncc-/-, and Bcl11b-/- mouse skulls at birth. Asterisks denote sutures affected by synostosis in Bcl11bncc-/- and Bcl11b-/- skulls and corresponding patent sutures in the control mice. Arrowhead points to increased mineralization and reduced anterior fontanelle within Bcl11b-/- calvaria. Red lines indicate dental occlusion. Scale bars: 1 mm.
86
87
Figure 3.3. Increased osteoblast maturation, mineralization, and craniofacial synostoses in Bcl11b-/- embryonic skulls.
(A-D) Von Kossa staining for mineralization with Nuclear Fast Red counterstain in horizontal sections of control and Bcl11b-/- embryos at E14.5. Arrowhead points to increased mineralization in Bcl11b-/- facial mesenchyme. (E-H) RNA in situ hybridization using a Bsp probe in sections of control and Bcl11bncc-/- heads at E14.5. Arrowheads point to enhanced osteoblast maturation in Bcl11b-/- facial mesenchyme. (I-P) Dorsal (third row) and lateral (fourth row) views of the micro-CT images of control and Bcl11b-/- mouse skulls at E16.5 (A-D) and E18.5 (E-H). Arrowheads point to increased mineralization in cranial bones in Bcl11b-/- skulls. Asterisks denote sutures affected by synostosis in Bcl11b-/- skulls and corresponding patent sutures in the control mice. Scale bars: (A-H) 500 um; (IL), (M-P) 1 mm.
88
Figure 3.4. BCL11B expression in osteogenic and sutural mesenchyme. BCL11B immunostaining in sections of embryonic faces (two upper rows represent sections from two different planes) and coronal sutures (lower row) at indicated stages. Osteogenic mesenchyme is denoted by asterisk in the facial sections and outlined by dashed line in the section of coronal suture. Scale bars: (two upper rows) 500 um; (C), (F, I) 200 um.
89
Figure 3.5. Increased Runx2 expression in the Bcl11b-/- embryonic faces and coronal sutures. RNA in situ hybridization using a Runx2 probe in sections of embryonic faces at E14.5 (two upper rows represent sections from two different planes) and coronal sutures at E16.5 (lower row). Arrowheads denote up-regulation and expansion of Runx2 expression in Bcl11b-/- face. Asterisks indicate ectopic expression of Runx2 in Bcl11b-/- coronal suture. Scale bars: (A-D) 500 um; (E-F) 200 um.
90
Figure 3.6. Ectopic Fgfr2c expression in facial and coronal sutures of Bcl11b-/- embryos. RNA in situ hybridization using an Fgfr2c probe in sections of embryonic faces at E14.5 (two upper rows represent sections from two different planes) and coronal sutures at E16.5 (lower row). Arrowheads denote up-regulation and expansion of Fgfr2c expression in Bcl11b-/- face. Asterisks indicate ectopic expression of Fgfr2c in Bcl11b-/- facial and coronal sutures. Scale bars: (A-D) 500 um; (E-F) 200 um.
91
92
Figure S3.1. Some Bcl11bncc-/- mice exhibit increased bone porosity in the skull at P21. (A-F) Dorsal (A-B), lateral (C-D), and ventral (E-F) views of the micro-CT images of control and Bcl11bncc-/-mouse skulls at P21. Asterisks denote sutures affected by synostosis in Bcl11bncc-/- skulls and corresponding patent sutures in the control mice. Arrows point to increased porosity in multiple Bcl11bncc-/- skull bones. Red lines indicate dental occlusion. Scale bar: (A-F) 1 mm.
Figure S3.2. Craniofacial phenotype development in Bcl11bncc-/- mice at postnatal stages. Dorsal (upper row), lateral (middle row), and ventral (bottom row) views of the micro-CT images of control and Bcl11bncc-/- at indicated stages. Asterisks denote sutures affected by synostosis in Bcl11bncc-/- skulls and corresponding patent sutures in the control mice. Arrows point to increased porosity in multiple Bcl11bncc-/- skull bones. Red lines indicate dental occlusion. Red brackets indicate compensatory expansion in the sagittal suture of Bcl11bncc-/- heads. Scale bars: (A-F), (G-L), (M-R) 1 mm.
93
94
Figure S3.3. Synostosis of facial sutures in newborn Bcl11b-/- mice.
Von Kossa staining for mineralization with Nuclear Fast Red counterstain in horizontal sections of control and Bcl11b-/- newborn mice. Arrows and arrowheads point to premaxillary-nasal and premaxillary-maxillary sutures, respectively, in control mice and synostosis of these sutures in Bcl11b-/- skulls. Scale bar: 500 um.
95
Figure S3.4. Delayed elevation and fusion of palatal shelves in Bcl11b-/-
mice.
(A-D) Hematoxylin and eosin staining in sections of wild-type and Bcl11b-/- mice at indicated stages. Arrows point to the palatal shelves that are fused in control embryos at E14.5, and exhibit delay in elevation and fusion in Bcl11b-/- mice. (EF) Immunostaining for BCL11B (red) in the sections of palatal shelf at E13.5 and in the palate at E15.5. The sections were counterstained with DAPI (blue). BCL11B is expressed predominantly in the palatal epithelium. Scale bars: (A-D) 500 um; (E), (F) 100 um.
96
Figure S3.5. Neural crest-specific ablation of BCL11B expression in the facial mesenchyme.
Bcl11bncc-/-BCL11B immunostaining in horizontal sections of control and embryonic faces at indicated stages. Asterisk denotes BCL11B expression in osteogenic mesenchyme at E13.5. Notice the lack of BCL11B staining in neural
Bcl11bncc-/-crest-derived facial mesenchyme in ; whereas BCL11B expression persists in all epithelial structures. Scale bar: 500 um.
97
Figure S3.6. Expression patterns of FGF and FGFR genes in the faces of Bcl11b-/- mice at E14.5.
RNA in situ hybridization using indicated probes in sections of control and Bcl11b-/- embryonic faces at E14.5. Scale bar: 500 um.
98
References
Behr, B., Leucht, P., Longaker, M. T. and Quarto, N. (2010). Fgf-9 is required for angiogenesis and osteogenesis in long bone repair. Proc Natl Acad Sci U S A 107, 11853-8.
Chim, H., Manjila, S., Cohen, A. R. and Gosain, A. K. (2011). Molecular signaling in pathogenesis of craniosynostosis: the role of fibroblast growth factor and transforming growth factor-beta. Neurosurg Focus 31, E7.
Connerney, J., Andreeva, V., Leshem, Y., Muentener, C., Mercado, M. A. and Spicer, D. B. (2006). Twist1 dimer selection regulates cranial suture patterning and fusion. Dev Dyn 235, 1345-57.
Cunningham, M. L., Seto, M. L., Ratisoontorn, C., Heike, C. L. and Hing, A. V. (2007). Syndromic craniosynostosis: from history to hydrogen bonds. Orthod Craniofac Res 10, 67-81.
Danielian, P. S., Muccino, D., Rowitch, D. H., Michael, S. K. and McMahon, A. P. (1998). Modification of gene activity in mouse embryos in utero by a tamoxifeninducible form of Cre recombinase. Curr Biol 8, 1323-6.
Eswarakumar, V. P., Horowitz, M. C., Locklin, R., Morriss-Kay, G. M. and Lonai, P. (2004). A gain-of-function mutation of Fgfr2c demonstrates the roles of this receptor variant in osteogenesis. Proc Natl Acad Sci U S A 101, 12555-60.
Eswarakumar, V. P., Monsonego-Ornan, E., Pines, M., Antonopoulou, I., Morriss-Kay, G. M. and Lonai, P. (2002). The IIIc alternative of Fgfr2 is a positive regulator of bone formation. Development 129, 3783-93.
Fitzpatrick, D. R. (2013). Filling in the gaps in cranial suture biology. Nat Genet 45, 231-2.
Golonzhka, O., Liang, X., Messaddeq, N., Bornert, J. M., Campbell, A. L., Metzger, D., Chambon, P., Ganguli-Indra, G., Leid, M. and Indra, A. K. (2009). Dual role of COUP-TF-interacting protein 2 in epidermal homeostasis and permeability barrier formation. J Invest Dermatol 129, 1459-70.
Greenwald, J. A., Mehrara, B. J., Spector, J. A., Warren, S. M., Fagenholz, P. J., Smith, L. E., Bouletreau, P. J., Crisera, F. E., Ueno, H. and Longaker, M. T. (2001). In vivo modulation of FGF biological activity alters cranial suture fate. Am J Pathol 158, 441-52.
Hajihosseini, M. K., Duarte, R., Pegrum, J., Donjacour, A., Lana-Elola, E., Rice, D. P., Sharpe, J. and Dickson, C. (2009). Evidence that Fgf10 contributes to the
99
skeletal and visceral defects of an Apert syndrome mouse model. Dev Dyn 238, 376-85.
Harada, M., Murakami, H., Okawa, A., Okimoto, N., Hiraoka, S., Nakahara, T., Akasaka, R., Shiraishi, Y., Futatsugi, N., Mizutani-Koseki, Y. et al. (2009). FGF9 monomer-dimer equilibrium regulates extracellular matrix affinity and tissue diffusion. Nat Genet 41, 289-98.
Iseki, S., Wilkie, A. O., Heath, J. K., Ishimaru, T., Eto, K. and Morriss-Kay, G. M. (1997). Fgfr2 and osteopontin domains in the developing skull vault are mutually exclusive and can be altered by locally applied FGF2. Development 124, 337584.
Jiang, X., Iseki, S., Maxson, R. E., Sucov, H. M. and Morriss-Kay, G. M. (2002). Tissue origins and interactions in the mammalian skull vault. Dev Biol 241, 10616.
Kim, H. J., Kim, J. H., Bae, S. C., Choi, J. Y. and Ryoo, H. M. (2003a). The protein kinase C pathway plays a central role in the fibroblast growth factor-stimulated expression and transactivation activity of Runx2. J Biol Chem 278, 319-26.
Kim, H. J., Lee, M. H., Park, H. S., Park, M. H., Lee, S. W., Kim, S. Y., Choi, J. Y., Shin, H. I. and Ryoo, H. M. (2003b). Erk pathway and activator protein 1 play crucial roles in FGF2-stimulated premature cranial suture closure. Dev Dyn 227, 335-46.
Kim, H. J., Rice, D. P., Kettunen, P. J. and Thesleff, I. (1998). FGF-, BMP- and Shh-mediated signalling pathways in the regulation of cranial suture morphogenesis and calvarial bone development. Development 125, 1241-51.
Komori, T. (2010). Regulation of osteoblast differentiation by Runx2. Adv Exp Med Biol 658, 43-9.
Komori, T., Yagi, H., Nomura, S., Yamaguchi, A., Sasaki, K., Deguchi, K., Shimizu, Y., Bronson, R. T., Gao, Y. H., Inada, M. et al. (1997). Targeted disruption of Cbfa1 results in a complete lack of bone formation owing to maturational arrest of osteoblasts. Cell 89, 755-64.
Kyryachenko, S., Kyrylkova, K., Leid, M. and Kioussi, C. (2012). Immunohistochemistry and detection of proliferating cells by BrdU. Methods Mol Biol 887, 33-9.
Kyrylkova, K., Kyryachenko, S., Biehs, B., Klein, O., Kioussi, C. and Leid, M. (2012a). BCL11B regulates epithelial proliferation and asymmetric development
100
of the mouse mandibular incisor. PLoS One 7, e37670.
Kyrylkova, K., Kyryachenko, S., Kioussi, C. and Leid, M. (2012b). Determination of gene expression patterns by in situ hybridization in sections. Methods Mol Biol 887, 23-31.
Liu, Z., Xu, J., Colvin, J. S. and Ornitz, D. M. (2002). Coordination of chondrogenesis and osteogenesis by fibroblast growth factor 18. Genes Dev 16, 859-69.
Montero, A., Okada, Y., Tomita, M., Ito, M., Tsurukami, H., Nakamura, T., Doetschman, T., Coffin, J. D. and Hurley, M. M. (2000). Disruption of the fibroblast growth factor-2 gene results in decreased bone mass and bone formation. J Clin Invest 105, 1085-93.
Nagayama, T., Okuhara, S., Ota, M. S., Tachikawa, N., Kasugai, S. and Iseki, S. (2013). FGF18 accelerates osteoblast differentiation by upregulating Bmp2 expression. Congenit Anom (Kyoto) 53, 83-8.
Nelson, D. K. and Williams, T. (2004). Frontonasal process-specific disruption of AP-2alpha results in postnatal midfacial hypoplasia, vascular anomalies, and nasal cavity defects. Dev Biol 267, 72-92.
Nie, X. (2005). Cranial base in craniofacial development: developmental features, influence on facial growth, anomaly, and molecular basis. Acta Odontol Scand 63, 127-35.
Nie, X., Luukko, K. and Kettunen, P. (2006). FGF signalling in craniofacial development and developmental disorders. Oral Dis 12, 102-11.
Ohbayashi, N., Shibayama, M., Kurotaki, Y., Imanishi, M., Fujimori, T., Itoh, N. and Takada, S. (2002). FGF18 is required for normal cell proliferation and differentiation during osteogenesis and chondrogenesis. Genes Dev 16, 870-9.
Opperman, L. A. (2000). Cranial sutures as intramembranous bone growth sites. Dev Dyn 219, 472-85.
Ornitz, D. M. and Itoh, N. (2001). Fibroblast growth factors. Genome Biol 2, REVIEWS3005.
Ornitz, D. M., Xu, J., Colvin, J. S., McEwen, D. G., MacArthur, C. A., Coulier, F., Gao, G. and Goldfarb, M. (1996). Receptor specificity of the fibroblast growth factor family. J Biol Chem 271, 15292-7.
Otto, F., Thornell, A. P., Crompton, T., Denzel, A., Gilmour, K. C., Rosewell, I. R.,
101
Stamp, G. W., Beddington, R. S., Mundlos, S., Olsen, B. R. et al. (1997). Cbfa1, a candidate gene for cleidocranial dysplasia syndrome, is essential for osteoblast differentiation and bone development. Cell 89, 765-71.
Park, O. J., Kim, H. J., Woo, K. M., Baek, J. H. and Ryoo, H. M. (2010). FGF2activated ERK mitogen-activated protein kinase enhances Runx2 acetylation and stabilization. J Biol Chem 285, 3568-74.
Pietri, T., Eder, O., Blanche, M., Thiery, J. P. and Dufour, S. (2003). The human tissue plasminogen activator-Cre mouse: a new tool for targeting specifically neural crest cells and their derivatives in vivo. Dev Biol 259, 176-87.
Purushothaman, R., Cox, T. C., Maga, A. M. and Cunningham, M. L. (2011). Facial suture synostosis of newborn Fgfr1(P250R/+) and Fgfr2(S252W/+) mouse models of Pfeiffer and Apert syndromes. Birth Defects Res A Clin Mol Teratol 91, 603-9.
Rice, D. P. (2008). Developmental anatomy of craniofacial sutures. Front Oral Biol 12, 1-21.
Rice, D. P., Aberg, T., Chan, Y., Tang, Z., Kettunen, P. J., Pakarinen, L., Maxson, R. E. and Thesleff, I. (2000). Integration of FGF and TWIST in calvarial bone and suture development. Development 127, 1845-55.
Rice, D. P., Rice, R. and Thesleff, I. (2003). Fgfr mRNA isoforms in craniofacial bone development. Bone 33, 14-27.
Rosenberg, P., Arlis, H. R., Haworth, R. D., Heier, L., Hoffman, L. and LaTrenta, G. (1997). The role of the cranial base in facial growth: experimental craniofacial synostosis in the rabbit. Plast Reconstr Surg 99, 1396-407.
Sharma, V. P., Fenwick, A. L., Brockop, M. S., McGowan, S. J., Goos, J. A., Hoogeboom, A. J., Brady, A. F., Jeelani, N. O., Lynch, S. A., Mulliken, J. B. et al. (2013). Mutations in TCF12, encoding a basic helix-loop-helix partner of TWIST1, are a frequent cause of coronal craniosynostosis. Nat Genet 45, 304-7.
Sheikh, F., Fandrich, R. R., Kardami, E. and Cattini, P. A. (1999). Overexpression of long or short FGFR-1 results in FGF-2-mediated proliferation in neonatal cardiac myocyte cultures. Cardiovasc Res 42, 696-705.
Stewart, R. E., Dixon, G. and Cohen, A. (1977). The pathogenesis of premature craniosynostosis in acrocephalosyndactyly (Apert's syndrome). A reconsideration. Plast Reconstr Surg 59, 699-707.
Turner, N., Lambros, M. B., Horlings, H. M., Pearson, A., Sharpe, R., Natrajan,
102
R., Geyer, F. C., van Kouwenhove, M., Kreike, B., Mackay, A. et al. (2010). Integrative molecular profiling of triple negative breast cancers identifies amplicon drivers and potential therapeutic targets. Oncogene 29, 2013-23.
Twigg, S. R., Kan, R., Babbs, C., Bochukova, E. G., Robertson, S. P., Wall, S. A., Morriss-Kay, G. M. and Wilkie, A. O. (2004). Mutations of ephrin-B1 (EFNB1), a marker of tissue boundary formation, cause craniofrontonasal syndrome. Proc Natl Acad Sci U S A 101, 8652-7.
Twigg, S. R., Vorgia, E., McGowan, S. J., Peraki, I., Fenwick, A. L., Sharma, V. P., Allegra, M., Zaragkoulias, A., Sadighi Akha, E., Knight, S. J. et al. (2013). Reduced dosage of ERF causes complex craniosynostosis in humans and mice and links ERK1/2 signaling to regulation of osteogenesis. Nat Genet 45, 308-13.
Weinzweig, J., Panter, K. E., Seki, J., Pantaloni, M., Spangenberger, A. and Harper, J. S. (2006). The fetal cleft palate: IV. Midfacial growth and bony palatal development following in utero and neonatal repair of the congenital caprine model. Plast Reconstr Surg 118, 81-93.
Wilkie, A. O., Byren, J. C., Hurst, J. A., Jayamohan, J., Johnson, D., Knight, S. J., Lester, T., Richards, P. G., Twigg, S. R. and Wall, S. A. (2010). Prevalence and complications of single-gene and chromosomal disorders in craniosynostosis. Pediatrics 126, e391-400.
Xiao, G., Jiang, D., Gopalakrishnan, R. and Franceschi, R. T. (2002). Fibroblast growth factor 2 induction of the osteocalcin gene requires MAPK activity and phosphorylation of the osteoblast transcription factor, Cbfa1/Runx2. J Biol Chem 277, 36181-7.
Yoshida, T., Vivatbutsiri, P., Morriss-Kay, G., Saga, Y. and Iseki, S. (2008). Cell lineage in mammalian craniofacial mesenchyme. Mech Dev 125, 797-808.
The Role of the Transcriptional Regulatory Protein BCL11B in Dental and Craniofacial Development
Chapter 4
Conclusion
104
A deeper understanding of developmental biology can foster greater progress in
regenerative medicine and treatment of congenital disorders. I elucidated the role
of the transcriptional regulatory protein BCL11B during tooth development and
showed that BCL11B functions in craniofacial development. Lack of Bcl11b
results in abnormal incisor morphology and craniofacial synostoses in mice.
BCL11B represses cell proliferation and premature differentiation of epithelial
cells in the lingual cervical loop of the incisor and osteogenic mesenchymal cells
of the craniofacial complex. BCL11B is simultaneously necessary to promote
these processes in the cells of the labial cervical loop. This observation
demonstrates context-dependent function of BCL11B. In all instances, ablation of
BCL11B function results in dysregulation of FGF signaling, and in the case of
tooth development TGF-β signaling is also affected. Moreover, ectopic FGF
signaling results in loss of BCL11B localization in the lingual. This is the first
report that integrates BCL11B in the FGF signaling network and illuminates the
molecular mechanisms that underlie dental and craniofacial phenotypes of
Bcl11b-/- mice. The studies described here are of significance because they will
contribute to a broader understanding of how the activities of transcription factors
and growth factors are regulated and how these pathways can be modulated as
an approach to therapy for developmental disorders that are not well treated by
current drugs.
Regenerative Dentistry
Substantial effort has been devoted to the understanding of the mechanisms of
tooth development, pathology, and therapy over the last several decades. The
immense interest in this subject is quite justified, as congenital disturbances in
tooth formation, acquired dental diseases, and odontogenic tumors affect millions
of people. Tooth agenesis is one of the most commonly inherited disorders,
which affect up to 20% of the population, whereas oral pathology is ranked as the
second most frequent clinical problem. In addition, tooth loss affects almost 10%
of the population and adversely influences mastication, articulation, facial
105
esthetics, and psychological health (Kapadia et al., 2007; Koussoulakou et al.,
2009; Tucker and Sharpe, 2004).
Historically, surgeons have used several procedures to replace lost and repair
damaged teeth, including tooth allotransplantation, autotransplantation, and
artificial dentures (Ferreira et al., 2007; Koussoulakou et al., 2009). The current
state-of-the-art approach to replace a missing tooth is by means of a dental
implant. During the last half of the 20th century, dental implantation procedures
have improved significantly due to innovations and discoveries from basic and
translational research, material sciences, and clinical techniques (Chai and
Slavkin, 2003). Traditionally, the implants have been made from titanium alloys,
and the implant surface has undergone numerous modifications over the years
aiming to promote successful osseointegration within the host bone. However, in
comparison to the natural dentition, the metal implant is more prone to
mechanical and biological failure because it lacks periodontal ligament and
crevicular sulcus, which provide mechanical stress absorption and local anti-
bacterial defense, respectively. In addition, dental implants require a minimum
level of bone, and in cases of severe bone loss the use of the implants is very
limited and must be preceded by bone grafts (Ferreira et al., 2007; Volponi et al.,
2010). Technological advances in dental implantology have slowed substantially
since inception of this procedure, and relatively more effort is currently devoted to
development of biological methods of tooth replacement that could overcome
difficulties associated with traditional dental implants.
The future practice of dentistry is likely to be revolutionized by regenerative
therapies, which will rely on the bioengineering of dental tissues and even a
whole tooth. Bioengineered teeth will serve as a genuine replica of the damaged
tooth. In addition, because teeth are nonessential for life, bioengineered teeth will
be an excellent model to optimize new, cell-based treatments that can also be
used for replacement of major, internal organs (Sartaj and Sharpe, 2006; Volponi
et al., 2010).
106
The development of new approaches in regenerative dentistry relies significantly
on our understanding of embryonic development, stem cell biology, and tissue
engineering technology. The mouse has proven to be an invaluable research
model for study of the molecular mechanisms that control tooth organogenesis.
Tooth development in mice is similar to that of humans, with the same set of
genes being expressed during tooth organogenesis; and mutations in the
counterpart genes often cause similar defective phenotypes (Fleischmannova et
al., 2008; Koussoulakou et al., 2009). However, unlike human teeth, mouse
incisors grow continuously throughout the life of the animal and are characterized
by the presence of epithelial and mesenchymal stem cell niches in the posterior
part of the tooth (Harada et al., 1999). This makes the mouse incisor an excellent
model to study several aspects of the dental stem cells.
Several methods have been proposed to achieve biological tooth replacement in
mice and other model organisms. These include stimulation of the formation of a
third dentition, construction of a tooth by bioengineering different component
parts separately, seeding of tooth-shaped biodegradable scaffolds with stem
cells, and producing embryonic-like tooth primordia from cultured cell populations
(Duailibi et al., 2004; Ferreira et al., 2007; Ohazama et al., 2004; Young et al.,
2005a; Young et al., 2005b). The latter approach has been used successfully by
several groups, which demonstrated that embryonic tooth primordia can form
teeth following dissociation into single-cell populations, subsequent
recombination, and transplantation into the adult mouse jaw (Angelova Volponi et
al., 2013; Oshima et al., 2011). However, embryonic cell sources have little rel-
evance for the development of a clinical therapy. Therefore, it is important to
identify the adult sources of human epithelial and mesenchymal cells that can be
obtained in sufficient numbers to render bioengineered teeth a viable alternative
to dental implants. To date, the only two non-dental sources of adult cells that
have been successfully used in tooth bioengineering are mesenchymal cells
derived from bone marrow stroma and human gingival epithelial cells that were
recombined with embryonic dental epithelium or mesenchyme, respectively
107
(Angelova Volponi et al., 2013; Ohazama et al., 2004). In each case, embryonic
tissue produced instructive signals to the non-embryonic counterpart. Therefore,
it is important to understand the complex networks of signaling molecules and
transcription factors that regulate embryonic tooth morphogenesis in order to
induce odontogenic potential in adult non-dental tissue in vitro.
Due to the rapid progress of research in molecular and developmental biology,
we are starting to understand how the development of dental tissues is regulated
at the level of proteins and signaling molecules. The transcriptional mechanisms
underlying tooth development are only beginning to be understood. We
demonstrate that BCL11B is a key component of the complex genetic network,
which regulates the development of the dental epithelial stem cells and their
differentiation into enamel-secreting ameloblasts in the mouse incisor. Therefore,
our findings on the role of BCL11B in tooth development is equally applicable to
research in emerging areas of regenerative dental medicine as it is in
developmental biology.
Targeted Treatment of Craniofacial Synostosis
With recent strides in medical genetics and developmental biology, it is evident
that advances in the treatment of craniosynostosis are dependent upon
elucidating the molecular events leading to this condition (Wan et al., 2008).
Craniosynostosis, defined as the premature fusion of one or more sutures in the
skull, is a common congenital craniofacial abnormality, with an overall incidence
of 1 in 2,200 live births (Fitzpatrick, 2013).
Craniofacial sutures serve as the major sites of bone growth along the leading
margins of the bones in the skull (Opperman, 2000). During childbirth, the
sutures allow the calvarial bones to overlap and mold, allowing for the passage of
the head through the birth canal (Warren and Longaker, 2001). Normal growth of
the skull within the sutures matches the spatial requirements of the developing
brain during fetal development and after birth. In the first year of life, head
108
circumference grows at an average of 2.4 mm per week and then drops down to
0.5 and 0.24 mm per week, respectively, in the second and third years of life
(Fitzpatrick, 2013). Therefore, one of the greatest concerns of the
craniosynostosis is elevated intracranial pressure that can result in deafness,
blindness, seizures, and cognitive deficits (Warren and Longaker, 2001).
Midfacial hypoplasia often accompanies craniosynostosis and can result from
premature ossification of facial sutures (Purushothaman et al., 2011). The most
common and debilitating concerns associated with midfacial hypoplasia are
malocclusion, respiratory difficulty related to nasopharyngeal stenosis, and ocular
conditions, such as ocular keratitis, corneal ulcers, globe herniation, and
blindness (Panchal and Uttchin, 2003; Raulo and Tessier, 1981).
Current treatment of craniofacial synostosis consists almost exclusively of
surgical modalities. Surgical approaches involve excision of the prematurely
fused suture(s) and/or parts of cranial bones, as well as advancement
procedures to remodel the skull. In addition, external and internal devices can be
used to separate the components of the skull after surgery (Cohen et al., 2008).
Ultimately, these invasive procedures are designed to increase intracranial
volume and to prevent possible complications associated with elevated
intracranial pressure. As a result, surgical correction of craniosynostosis should
be performed in infancy, within the first six months of life to maximize therapeutic
outcomes at an early stage of development (Panchal and Uttchin, 2003).
However, surgical treatment of craniofacial synostosis remains an invasive
procedure with commensurate risks, including infection, optic nerve ischemia,
seizures, and bleeding (Whitaker et al., 1979). Some 80-100% of patients who
undergo surgical correction of craniosynostosis need blood transfusions (Meara
et al., 2005). In addition, more than 6% of infants require a second operation to
separate the bones again, and 25% of those require third surgery (Panchal and
Uttchin, 2003). For these reasons, the development of minimally invasive,
biologically-based and targeted therapies offer an attractive option to be pursued
for the treatment of craniofacial synostoses (Wan et al., 2008).
109
The study of human sutural material obtained after surgeries is not sufficient for
experimentation to understand the etiology of craniofacial synostoses, but use of
mouse models of craniofacial synostoses obviates this concern. In mice, all
sutures, with the exception of the posterior frontal suture, remain patent
throughout life (Holmes, 2012). Mouse and human genetics revealed that
interactions between growth factors, their receptors, and transcription factors all
play integral roles in craniofacial suture biology. At least 150 syndromes have
been identified with specific mutations resulting in craniosynostosis, and most of
the common ones exhibit dominant inheritance (Cohen, 2005; Wan et al., 2008;
Wilkie, 1997). Many of the syndromes, such as Apert, Pfeiffer, and Crouzon
syndromes, can be traced to gain-of-function mutations in FGFRs (Wilkie, 1997).
TGFβ signaling pathway also has a well-established role in bone biology and
regulation of suture patency (Rawlins and Opperman, 2008). However, unlike the
FGF signaling, TGFβ family members have not been closely associated with
craniosynostosis in humans (Senarath-Yapa et al., 2012). Other less frequent
mutations linked to this condition are found in TWIST, MSX2, EFNB1, RAB23,
GLI3, TCF12, ERF and other genes (el Ghouzzi et al., 1997; Howard et al., 1997;
Jabs et al., 1993; Jenkins et al., 2007; Sharma et al., 2013; Twigg et al., 2004;
Twigg et al., 2013; Vortkamp et al., 1992; Wieland et al., 2005).
The goal of the biologically-based targeted treatment of craniofacial synostosis
would be to suppress the expression of genes promoting sutural fusion or to
increase the expression of those which support sutural patency. Gene therapy
holds great promise for development of such treatment for craniofacial synostosis
(Wan et al., 2008). Several attempts have been made to utilize gene therapy in
mice. Gene transfer of bone morphogenetic proteins-2 and -9 using adenoviral
vectors demonstrated promising effect in the promotion of osteogenesis,
specifically for craniofacial bone repair in rats (Alden et al., 2000; Lindsey, 2001).
Local adenoviral delivery of noggin, a TGF antagonist, to the posterior frontal
suture in mice prevented normal sutural fusion and resulted in broad snouts and
widely-spaced eyes (Warren et al., 2003). Similar experiments were performed
110
with adenoviral delivery of the dominant-negative forms of the TGF-β receptor II
and Fgfr1 to prevent fusion of the posterior frontal suture in organ culture
(Greenwald et al., 2001; Mehrara et al., 2002; Song et al., 2004). Conversely,
adenoviral infection of the normally patent coronal suture with an Fgf2 expression
vector resulted in pathologic suture fusion (Greenwald et al., 2001). It is
important to note that viral vectors suffer from several drawbacks, such as
toxicity, imunnogenicity, and the lack of cell-specific targeting. Therefore, delivery
of gene products using non-viral techniques would be desirable in humans. Such
approaches may involve direct injection of DNA, liposomes, electroporation,
particle bombardment, and nanoparticles (Schmidt-Wolf and Schmidt-Wolf,
2003).
Prevention of craniosynostosis in mouse models was also demonstrated using
pharmacologic strategies. Mouse calvaria obtained from the Crouzon-like
Fgfr2C342Y/+ mouse model cultured in the presence of FGFR tyrosine kinase
inhibitors (PD173074 or PLX052) exhibited coronal suture patency, as opposed
to the premature suture fusion seen in untreated mutants (Eswarakumar et al.,
2006; Perlyn et al., 2006). An in vitro calvarial culture model of the Apert mouse
harboring the Fgfr2 P253R mutation showed that treatment with MEK1 inhibitor
PD98059 partially alleviated fusion of the coronal suture (Yin et al., 2008).
Furthermore, treatment of the Apert Fgfr2S252W/+ mice with U0126, an inhibitor of
MEK1/2, during pregnancy and early postnatal development significantly
inhibited development of craniosynostosis in vivo (Shukla et al., 2007). These
studies suggest that small-molecule inhibitors can be used for treatment of
specific gain-of-function mutations observed in syndromic craniofacial synostosis
(Melville et al., 2010).
Several other strategies were used to modulate protein levels involved in
aberrant FGFR or TGFβ signaling. Infection of the posterior frontal suture of fetal
rats by introduction of the dominant-negative Fgfr1 construct in utero abrogated
overactive FGFR signaling and prevented suture closure (Greenwald et al.,
111
2001). shRNA targeting a mutant form of Fgfr2 was shown to prevent an Apert-
like syndrome in mice (Shukla et al., 2007). Delivery of neutralizing antibodies to
TGFβ3 resulted in synostosis of coronal suture, whereas antibodies targeted to
TGFβ2 prevented sutural closure (Opperman et al., 1999). Overall, these
strategies further demonstrate that targeted treatment may be promising for
prevention of the craniofacial synostosis.
As our understanding of the molecular and genetic basis of craniofacial
synostosis improves, the prospect of genetic and pharmacological therapy for the
treatment and prevention of premature suture fusion becomes more tangible.
However, such targeted treatment has yet to overcome significant obstacles,
such as development of targeted methods of delivery (including in utero), as well
as establishment of proper dosage and timing. In addition, more investigations
are still required to determine which protein or group of proteins would serve as
the optimal target in each case. Studies presented here demonstrate that
BCL11B plays an important role in maintaining of sutural patency in mice.
Abrogation of BCL11B function leads to accelerated mineralization of the skull
during development and subsequent facial and coronal synostoses that are
already evident at birth. Although BCL11B has not been previously associated
with the craniosynostosis in humans, the data presented here will be useful in
defining alternative therapeutic approaches to maintain sutural patency in patiens
with this condition.
112
References
Alden, T. D., Beres, E. J., Laurent, J. S., Engh, J. A., Das, S., London, S. D., Jane, J. A., Jr., Hudson, S. B. and Helm, G. A. (2000). The use of bone morphogenetic protein gene therapy in craniofacial bone repair. J Craniofac Surg 11, 24-30.
Angelova Volponi, A., Kawasaki, M. and Sharpe, P. T. (2013). Adult human gingival epithelial cells as a source for whole-tooth bioengineering. J Dent Res 92, 329-34.
Chai, Y. and Slavkin, H. C. (2003). Prospects for tooth regeneration in the 21st century: a perspective. Microsc Res Tech 60, 469-79.
Cohen, M. M., Jr. (2005). Editorial: perspectives on craniosynostosis. Am J Med Genet A 136A, 313-26.
Cohen, S. R., Pryor, L., Mittermiller, P. A., Meltzer, H. S., Levy, M. L., Broder, K. W. and Ozgur, B. M. (2008). Nonsyndromic craniosynostosis: current treatment options. Plast Surg Nurs 28, 79-91.
Duailibi, M. T., Duailibi, S. E., Young, C. S., Bartlett, J. D., Vacanti, J. P. and Yelick, P. C. (2004). Bioengineered teeth from cultured rat tooth bud cells. J Dent Res 83, 523-8.
el Ghouzzi, V., Le Merrer, M., Perrin-Schmitt, F., Lajeunie, E., Benit, P., Renier, D., Bourgeois, P., Bolcato-Bellemin, A. L., Munnich, A. and Bonaventure, J. (1997). Mutations of the TWIST gene in the Saethre-Chotzen syndrome. Nat Genet 15, 42-6.
Eswarakumar, V. P., Ozcan, F., Lew, E. D., Bae, J. H., Tome, F., Booth, C. J., Adams, D. J., Lax, I. and Schlessinger, J. (2006). Attenuation of signaling pathways stimulated by pathologically activated FGF-receptor 2 mutants prevents craniosynostosis. Proc Natl Acad Sci U S A 103, 18603-8.
Ferreira, C. F., Magini, R. S. and Sharpe, P. T. (2007). Biological tooth replacement and repair. J Oral Rehabil 34, 933-9.
Fitzpatrick, D. R. (2013). Filling in the gaps in cranial suture biology. Nat Genet 45, 231-2.
Fleischmannova, J., Matalova, E., Tucker, A. S. and Sharpe, P. T. (2008). Mouse models of tooth abnormalities. Eur J Oral Sci 116, 1-10.
113
Greenwald, J. A., Mehrara, B. J., Spector, J. A., Warren, S. M., Fagenholz, P. J., Smith, L. E., Bouletreau, P. J., Crisera, F. E., Ueno, H. and Longaker, M. T. (2001). In vivo modulation of FGF biological activity alters cranial suture fate. Am J Pathol 158, 441-52.
Harada, H., Kettunen, P., Jung, H. S., Mustonen, T., Wang, Y. A. and Thesleff, I. (1999). Localization of putative stem cells in dental epithelium and their association with Notch and FGF signaling. J Cell Biol 147, 105-20.
Holmes, G. (2012). The role of vertebrate models in understanding craniosynostosis. Childs Nerv Syst 28, 1471-81.
Howard, T. D., Paznekas, W. A., Green, E. D., Chiang, L. C., Ma, N., Ortiz de Luna, R. I., Garcia Delgado, C., Gonzalez-Ramos, M., Kline, A. D. and Jabs, E. W. (1997). Mutations in TWIST, a basic helix-loop-helix transcription factor, in Saethre-Chotzen syndrome. Nat Genet 15, 36-41.
Jabs, E. W., Muller, U., Li, X., Ma, L., Luo, W., Haworth, I. S., Klisak, I., Sparkes, R., Warman, M. L., Mulliken, J. B. et al. (1993). A mutation in the homeodomain of the human MSX2 gene in a family affected with autosomal dominant craniosynostosis. Cell 75, 443-50.
Jenkins, D., Seelow, D., Jehee, F. S., Perlyn, C. A., Alonso, L. G., Bueno, D. F., Donnai, D., Josifova, D., Mathijssen, I. M., Morton, J. E. et al. (2007). RAB23 mutations in Carpenter syndrome imply an unexpected role for hedgehog signaling in cranial-suture development and obesity. Am J Hum Genet 80, 1162-70.
Kapadia, H., Mues, G. and D'Souza, R. (2007). Genes affecting tooth morphogenesis. Orthod Craniofac Res 10, 105-13.
Koussoulakou, D. S., Margaritis, L. H. and Koussoulakos, S. L. (2009). A curriculum vitae of teeth: evolution, generation, regeneration. Int J Biol Sci 5, 226-43.
Lindsey, W. H. (2001). Osseous tissue engineering with gene therapy for facial bone reconstruction. Laryngoscope 111, 1128-36.
Meara, J. G., Smith, E. M., Harshbarger, R. J., Farlo, J. N., Matar, M. M. and Levy, M. L. (2005). Blood-conservation techniques in craniofacial surgery. Ann Plast Surg 54, 525-9.
Mehrara, B. J., Spector, J. A., Greenwald, J. A., Ueno, H. and Longaker, M. T. (2002). Adenovirus-mediated transmission of a dominant negative transforming
114
growth factor-beta receptor inhibits in vitro mouse cranial suture fusion. Plast Reconstr Surg 110, 506-14.
Melville, H., Wang, Y., Taub, P. J. and Jabs, E. W. (2010). Genetic basis of potential therapeutic strategies for craniosynostosis. Am J Med Genet A 152A, 3007-15.
Ohazama, A., Modino, S. A., Miletich, I. and Sharpe, P. T. (2004). Stem-cell-based tissue engineering of murine teeth. J Dent Res 83, 518-22.
Opperman, L. A. (2000). Cranial sutures as intramembranous bone growth sites. Dev Dyn 219, 472-85.
Opperman, L. A., Chhabra, A., Cho, R. W. and Ogle, R. C. (1999). Cranial suture obliteration is induced by removal of transforming growth factor (TGF)-beta 3 activity and prevented by removal of TGF-beta 2 activity from fetal rat calvaria in vitro. J Craniofac Genet Dev Biol 19, 164-73.
Oshima, M., Mizuno, M., Imamura, A., Ogawa, M., Yasukawa, M., Yamazaki, H., Morita, R., Ikeda, E., Nakao, K., Takano-Yamamoto, T. et al. (2011). Functional tooth regeneration using a bioengineered tooth unit as a mature organ replacement regenerative therapy. PLoS One 6, e21531.
Panchal, J. and Uttchin, V. (2003). Management of craniosynostosis. Plast Reconstr Surg 111, 2032-48; quiz 2049.
Perlyn, C. A., Morriss-Kay, G., Darvann, T., Tenenbaum, M. and Ornitz, D. M. (2006). A model for the pharmacological treatment of crouzon syndrome. Neurosurgery 59, 210-5; discussion 210-5.
Purushothaman, R., Cox, T. C., Maga, A. M. and Cunningham, M. L. (2011). Facial suture synostosis of newborn Fgfr1(P250R/+) and Fgfr2(S252W/+) mouse models of Pfeiffer and Apert syndromes. Birth Defects Res A Clin Mol Teratol 91, 603-9.
Raulo, Y. and Tessier, P. (1981). Fronto-facial advancement for Crouzon's and Apert's syndromes. Scand J Plast Reconstr Surg 15, 245-50.
Rawlins, J. T. and Opperman, L. A. (2008). Tgf-beta regulation of suture morphogenesis and growth. Front Oral Biol 12, 178-96.
Sartaj, R. and Sharpe, P. (2006). Biological tooth replacement. J Anat 209, 503-9.
115
Schmidt-Wolf, G. D. and Schmidt-Wolf, I. G. (2003). Non-viral and hybrid vectors in human gene therapy: an update. Trends Mol Med 9, 67-72.
Senarath-Yapa, K., Chung, M. T., McArdle, A., Wong, V. W., Quarto, N., Longaker, M. T. and Wan, D. C. (2012). Craniosynostosis: molecular pathways and future pharmacologic therapy. Organogenesis 8, 103-13.
Sharma, V. P., Fenwick, A. L., Brockop, M. S., McGowan, S. J., Goos, J. A., Hoogeboom, A. J., Brady, A. F., Jeelani, N. O., Lynch, S. A., Mulliken, J. B. et al. (2013). Mutations in TCF12, encoding a basic helix-loop-helix partner of TWIST1, are a frequent cause of coronal craniosynostosis. Nat Genet 45, 304-7.
Shukla, V., Coumoul, X., Wang, R. H., Kim, H. S. and Deng, C. X. (2007). RNA interference and inhibition of MEK-ERK signaling prevent abnormal skeletal phenotypes in a mouse model of craniosynostosis. Nat Genet 39, 1145-50.
Song, H. M., Fong, K. D., Nacamuli, R. P., Warren, S. M., Fang, T. D., Mathy, J. A., Cowan, C. M., Aalami, O. O. and Longaker, M. T. (2004). Mechanisms of murine cranial suture patency mediated by a dominant negative transforming growth factor-beta receptor adenovirus. Plast Reconstr Surg 113, 1685-97.
Tucker, A. and Sharpe, P. (2004). The cutting-edge of mammalian development; how the embryo makes teeth. Nat Rev Genet 5, 499-508.
Twigg, S. R., Kan, R., Babbs, C., Bochukova, E. G., Robertson, S. P., Wall, S. A., Morriss-Kay, G. M. and Wilkie, A. O. (2004). Mutations of ephrin-B1 (EFNB1), a marker of tissue boundary formation, cause craniofrontonasal syndrome. Proc Natl Acad Sci U S A 101, 8652-7.
Twigg, S. R., Vorgia, E., McGowan, S. J., Peraki, I., Fenwick, A. L., Sharma, V. P., Allegra, M., Zaragkoulias, A., Sadighi Akha, E., Knight, S. J. et al. (2013). Reduced dosage of ERF causes complex craniosynostosis in humans and mice and links ERK1/2 signaling to regulation of osteogenesis. Nat Genet 45, 308-13.
Volponi, A. A., Pang, Y. and Sharpe, P. T. (2010). Stem cell-based biological tooth repair and regeneration. Trends Cell Biol 20, 715-22.
Vortkamp, A., Franz, T., Gessler, M. and Grzeschik, K. H. (1992). Deletion of GLI3 supports the homology of the human Greig cephalopolysyndactyly syndrome (GCPS) and the mouse mutant extra toes (Xt). Mamm Genome 3, 461-3.
Wan, D. C., Kwan, M. D., Lorenz, H. P. and Longaker, M. T. (2008). Current treatment of craniosynostosis and future therapeutic directions. Front Oral Biol 12, 209-30.
116
Warren, S. M., Brunet, L. J., Harland, R. M., Economides, A. N. and Longaker, M. T. (2003). The BMP antagonist noggin regulates cranial suture fusion. Nature 422, 625-9.
Warren, S. M. and Longaker, M. T. (2001). The pathogenesis of craniosynostosis in the fetus. Yonsei Med J 42, 646-59.
Whitaker, L. A., Munro, I. R., Salyer, K. E., Jackson, I. T., Ortiz-Monasterio, F. and Marchac, D. (1979). Combined report of problems and complications in 793 craniofacial operations. Plast Reconstr Surg 64, 198-203.
Wieland, I., Reardon, W., Jakubiczka, S., Franco, B., Kress, W., Vincent-Delorme, C., Thierry, P., Edwards, M., Konig, R., Rusu, C. et al. (2005). Twenty-six novel EFNB1 mutations in familial and sporadic craniofrontonasal syndrome (CFNS). Hum Mutat 26, 113-8.
Wilkie, A. O. (1997). Craniosynostosis: genes and mechanisms. Hum Mol Genet 6, 1647-56.
Yin, L., Du, X., Li, C., Xu, X., Chen, Z., Su, N., Zhao, L., Qi, H., Li, F., Xue, J. et al. (2008). A Pro253Arg mutation in fibroblast growth factor receptor 2 (Fgfr2) causes skeleton malformation mimicking human Apert syndrome by affecting both chondrogenesis and osteogenesis. Bone 42, 631-43.
Young, C. S., Abukawa, H., Asrican, R., Ravens, M., Troulis, M. J., Kaban, L. B., Vacanti, J. P. and Yelick, P. C. (2005a). Tissue-engineered hybrid tooth and bone. Tissue Eng 11, 1599-610.
Young, C. S., Kim, S. W., Qin, C., Baba, O., Butler, W. T., Taylor, R. R., Bartlett, J. D., Vacanti, J. P. and Yelick, P. C. (2005b). Developmental analysis and computer modelling of bioengineered teeth. Arch Oral Biol 50, 259-65.
117
Bibliography
Albin, R. L., Young, A. B. and Penney, J. B. (1989). The functional anatomy of basal ganglia disorders. Trends Neurosci 12, 366-75.
Albu, D. I., Feng, D., Bhattacharya, D., Jenkins, N. A., Copeland, N. G., Liu, P. and Avram, D. (2007). BCL11B is required for positive selection and survival of double-positive thymocytes. J Exp Med 204, 3003-15.
Alden, T. D., Beres, E. J., Laurent, J. S., Engh, J. A., Das, S., London, S. D., Jane, J. A., Jr., Hudson, S. B. and Helm, G. A. (2000). The use of bone morphogenetic protein gene therapy in craniofacial bone repair. J Craniofac Surg 11, 24-30.
Alvarez, Y., Alonso, M. T., Vendrell, V., Zelarayan, L. C., Chamero, P., Theil, T., Bosl, M. R., Kato, S., Maconochie, M., Riethmacher, D. et al. (2003). Requirements for FGF3 and FGF10 during inner ear formation. Development 130, 6329-38.
Angelova Volponi, A., Kawasaki, M. and Sharpe, P. T. (2013). Adult human gingival epithelial cells as a source for whole-tooth bioengineering. J Dent Res 92, 329-34.
Arlotta, P., Molyneaux, B. J., Chen, J., Inoue, J., Kominami, R. and Macklis, J. D. (2005). Neuronal subtype-specific genes that control corticospinal motor neuron development in vivo. Neuron 45, 207-21.
Arlotta, P., Molyneaux, B. J., Jabaudon, D., Yoshida, Y. and Macklis, J. D. (2008). Ctip2 controls the differentiation of medium spiny neurons and the establishment of the cellular architecture of the striatum. J Neurosci 28, 622-32.
Avram, D., Fields, A., Pretty On Top, K., Nevrivy, D. J., Ishmael, J. E. and Leid, M. (2000). Isolation of a novel family of C(2)H(2) zinc finger proteins implicated in transcriptional repression mediated by chicken ovalbumin upstream promoter transcription factor (COUP-TF) orphan nuclear receptors. J Biol Chem 275, 10315-22.
Avram, D., Fields, A., Senawong, T., Topark-Ngarm, A. and Leid, M. (2002). COUP-TF (chicken ovalbumin upstream promoter transcription factor)-interacting protein 1 (CTIP1) is a sequence-specific DNA binding protein. Biochem J 368, 555-63.
Behr, B., Leucht, P., Longaker, M. T. and Quarto, N. (2010). Fgf-9 is required for angiogenesis and osteogenesis in long bone repair. Proc Natl Acad Sci U S A 107, 11853-8.
118
Bellusci, S., Henderson, R., Winnier, G., Oikawa, T. and Hogan, B. L. (1996). Evidence from normal expression and targeted misexpression that bone morphogenetic protein (Bmp-4) plays a role in mouse embryonic lung morphogenesis. Development 122, 1693-702.
Bernard, O. A., Busson-LeConiat, M., Ballerini, P., Mauchauffe, M., Della Valle, V., Monni, R., Nguyen Khac, F., Mercher, T., Penard-Lacronique, V., Pasturaud, P. et al. (2001). A new recurrent and specific cryptic translocation, t(5;14)(q35;q32), is associated with expression of the Hox11L2 gene in T acute lymphoblastic leukemia. Leukemia 15, 1495-504.
Bitgood, M. J. and McMahon, A. P. (1995). Hedgehog and Bmp genes are coexpressed at many diverse sites of cell-cell interaction in the mouse embryo. Dev Biol 172, 126-38.
Brown, R. S. (2005). Zinc finger proteins: getting a grip on RNA. Curr Opin Struct Biol 15, 94-8.
Capecchi, M. R. (2005). Gene targeting in mice: functional analysis of the mammalian genome for the twenty-first century. Nat Rev Genet 6, 507-12.
Caton, J., Luder, H. U., Zoupa, M., Bradman, M., Bluteau, G., Tucker, A. S., Klein, O. and Mitsiadis, T. A. (2009). Enamel-free teeth: Tbx1 deletion affects amelogenesis in rodent incisors. Dev Biol 328, 493-505.
Cavenee, W. K., Dryja, T. P., Phillips, R. A., Benedict, W. F., Godbout, R., Gallie, B. L., Murphree, A. L., Strong, L. C. and White, R. L. (1983). Expression of recessive alleles by chromosomal mechanisms in retinoblastoma. Nature 305, 779-84.
Ceredig, R. and Rolink, T. (2002). A positive look at double-negative thymocytes. Nat Rev Immunol 2, 888-97.
Chai, Y. and Slavkin, H. C. (2003). Prospects for tooth regeneration in the 21st century: a perspective. Microsc Res Tech 60, 469-79.
Chen, B., Wang, S. S., Hattox, A. M., Rayburn, H., Nelson, S. B. and McConnell, S. K. (2008). The Fezf2-Ctip2 genetic pathway regulates the fate choice of subcortical projection neurons in the developing cerebral cortex. Proc Natl Acad Sci U S A 105, 11382-7.
Cherrier, T., Suzanne, S., Redel, L., Calao, M., Marban, C., Samah, B., Mukerjee, R., Schwartz, C., Gras, G., Sawaya, B. E. et al. (2009). p21(WAF1) gene promoter is epigenetically silenced by CTIP2 and SUV39H1. Oncogene 28, 3380-9.
119
Chim, H., Manjila, S., Cohen, A. R. and Gosain, A. K. (2011). Molecular signaling in pathogenesis of craniosynostosis: the role of fibroblast growth factor and transforming growth factor-beta. Neurosurg Focus 31, E7.
Cismasiu, V. B., Adamo, K., Gecewicz, J., Duque, J., Lin, Q. and Avram, D. (2005). BCL11B functionally associates with the NuRD complex in T lymphocytes to repress targeted promoter. Oncogene 24, 6753-64.
Cismasiu, V. B., Ghanta, S., Duque, J., Albu, D. I., Chen, H. M., Kasturi, R. and Avram, D. (2006). BCL11B participates in the activation of IL2 gene expression in CD4+ T lymphocytes. Blood 108, 2695-702.
Cohen, M. M., Jr. (2005). Editorial: perspectives on craniosynostosis. Am J Med Genet A 136A, 313-26.
Cohen, S. R., Pryor, L., Mittermiller, P. A., Meltzer, H. S., Levy, M. L., Broder, K. W. and Ozgur, B. M. (2008). Nonsyndromic craniosynostosis: current treatment options. Plast Surg Nurs 28, 79-91.
Connerney, J., Andreeva, V., Leshem, Y., Muentener, C., Mercado, M. A. and Spicer, D. B. (2006). Twist1 dimer selection regulates cranial suture patterning and fusion. Dev Dyn 235, 1345-57.
Cunningham, M. L., Seto, M. L., Ratisoontorn, C., Heike, C. L. and Hing, A. V. (2007). Syndromic craniosynostosis: from history to hydrogen bonds. Orthod Craniofac Res 10, 67-81.
Dai, Y. and Faller, D. V. (2008). Transcription Regulation by Class III Histone Deacetylases (HDACs)-Sirtuins. Transl Oncogenomics 3, 53-65.
Danielian, P. S., Muccino, D., Rowitch, D. H., Michael, S. K. and McMahon, A. P. (1998). Modification of gene activity in mouse embryos in utero by a tamoxifen-inducible form of Cre recombinase. Curr Biol 8, 1323-6.
Dassule, H. R., Lewis, P., Bei, M., Maas, R. and McMahon, A. P. (2000). Sonic hedgehog regulates growth and morphogenesis of the tooth. Development 127, 4775-85.
De Keersmaecker, K., Real, P. J., Gatta, G. D., Palomero, T., Sulis, M. L., Tosello, V., Van Vlierberghe, P., Barnes, K., Castillo, M., Sole, X. et al. (2010). The TLX1 oncogene drives aneuploidy in T cell transformation. Nat Med 16, 1321-7.
Desplats, P. A., Kass, K. E., Gilmartin, T., Stanwood, G. D., Woodward, E. L., Head, S. R., Sutcliffe, J. G. and Thomas, E. A. (2006). Selective deficits in the
120
expression of striatal-enriched mRNAs in Huntington's disease. J Neurochem 96, 743-57.
Desplats, P. A., Lambert, J. R. and Thomas, E. A. (2008). Functional roles for the striatal-enriched transcription factor, Bcl11b, in the control of striatal gene expression and transcriptional dysregulation in Huntington's disease. Neurobiol Dis 31, 298-308.
Duailibi, M. T., Duailibi, S. E., Young, C. S., Bartlett, J. D., Vacanti, J. P. and Yelick, P. C. (2004). Bioengineered teeth from cultured rat tooth bud cells. J Dent Res 83, 523-8.
el Ghouzzi, V., Le Merrer, M., Perrin-Schmitt, F., Lajeunie, E., Benit, P., Renier, D., Bourgeois, P., Bolcato-Bellemin, A. L., Munnich, A. and Bonaventure, J. (1997). Mutations of the TWIST gene in the Saethre-Chotzen syndrome. Nat Genet 15, 42-6.
Enomoto, T., Ohmoto, M., Iwata, T., Uno, A., Saitou, M., Yamaguchi, T., Kominami, R., Matsumoto, I. and Hirota, J. (2011). Bcl11b/Ctip2 controls the differentiation of vomeronasal sensory neurons in mice. J Neurosci 31, 10159-73.
Eswarakumar, V. P., Horowitz, M. C., Locklin, R., Morriss-Kay, G. M. and Lonai, P. (2004). A gain-of-function mutation of Fgfr2c demonstrates the roles of this receptor variant in osteogenesis. Proc Natl Acad Sci U S A 101, 12555-60.
Eswarakumar, V. P., Monsonego-Ornan, E., Pines, M., Antonopoulou, I., Morriss-Kay, G. M. and Lonai, P. (2002). The IIIc alternative of Fgfr2 is a positive regulator of bone formation. Development 129, 3783-93.
Eswarakumar, V. P., Ozcan, F., Lew, E. D., Bae, J. H., Tome, F., Booth, C. J., Adams, D. J., Lax, I. and Schlessinger, J. (2006). Attenuation of signaling pathways stimulated by pathologically activated FGF-receptor 2 mutants prevents craniosynostosis. Proc Natl Acad Sci U S A 103, 18603-8.
Fatemi, M. and Wade, P. A. (2006). MBD family proteins: reading the epigenetic code. J Cell Sci 119, 3033-7.
Ferreira, C. F., Magini, R. S. and Sharpe, P. T. (2007). Biological tooth replacement and repair. J Oral Rehabil 34, 933-9.
Fettiplace, R. and Hackney, C. M. (2006). The sensory and motor roles of auditory hair cells. Nat Rev Neurosci 7, 19-29.
Fitzpatrick, D. R. (2013). Filling in the gaps in cranial suture biology. Nat Genet 45, 231-2.
121
Fleischmannova, J., Matalova, E., Tucker, A. S. and Sharpe, P. T. (2008). Mouse models of tooth abnormalities. Eur J Oral Sci 116, 1-10.
Gaj, T., Gersbach, C. A. and Barbas, C. F., 3rd. (2013). ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering. Trends Biotechnol 31, 397-405.
Gamsjaeger, R., Liew, C. K., Loughlin, F. E., Crossley, M. and Mackay, J. P. (2007). Sticky fingers: zinc-fingers as protein-recognition motifs. Trends Biochem Sci 32, 63-70.
Gerfen, C. R. (1992). The neostriatal mosaic: multiple levels of compartmental organization. Trends Neurosci 15, 133-9.
Germain, R. N. (2002). T-cell development and the CD4-CD8 lineage decision. Nat Rev Immunol 2, 309-22.
Golonzhka, O., Leid, M., Indra, G. and Indra, A. K. (2007). Expression of COUP-TF-interacting protein 2 (CTIP2) in mouse skin during development and in adulthood. Gene Expr Patterns 7, 754-60.
Golonzhka, O., Liang, X., Messaddeq, N., Bornert, J. M., Campbell, A. L., Metzger, D., Chambon, P., Ganguli-Indra, G., Leid, M. and Indra, A. K. (2009a). Dual role of COUP-TF-interacting protein 2 in epidermal homeostasis and permeability barrier formation. J Invest Dermatol 129, 1459-70.
Golonzhka, O., Metzger, D., Bornert, J. M., Bay, B. K., Gross, M. K., Kioussi, C. and Leid, M. (2009b). Ctip2/Bcl11b controls ameloblast formation during mammalian odontogenesis. Proc Natl Acad Sci U S A 106, 4278-83.
Greenwald, J. A., Mehrara, B. J., Spector, J. A., Warren, S. M., Fagenholz, P. J., Smith, L. E., Bouletreau, P. J., Crisera, F. E., Ueno, H. and Longaker, M. T. (2001). In vivo modulation of FGF biological activity alters cranial suture fate. Am J Pathol 158, 441-52.
Gutierrez, A., Kentsis, A., Sanda, T., Holmfeldt, L., Chen, S. C., Zhang, J., Protopopov, A., Chin, L., Dahlberg, S. E., Neuberg, D. S. et al. (2011). The BCL11B tumor suppressor is mutated across the major molecular subtypes of T-cell acute lymphoblastic leukemia. Blood 118, 4169-73.
Hacohen, N., Kramer, S., Sutherland, D., Hiromi, Y. and Krasnow, M. A. (1998). sprouty encodes a novel antagonist of FGF signaling that patterns apical branching of the Drosophila airways. Cell 92, 253-63.
122
Hajihosseini, M. K., Duarte, R., Pegrum, J., Donjacour, A., Lana-Elola, E., Rice, D. P., Sharpe, J. and Dickson, C. (2009). Evidence that Fgf10 contributes to the skeletal and visceral defects of an Apert syndrome mouse model. Dev Dyn 238, 376-85.
Hall, T. M. (2005). Multiple modes of RNA recognition by zinc finger proteins. Curr Opin Struct Biol 15, 367-73.
Harada, H., Kettunen, P., Jung, H. S., Mustonen, T., Wang, Y. A. and Thesleff, I. (1999). Localization of putative stem cells in dental epithelium and their association with Notch and FGF signaling. J Cell Biol 147, 105-20.
Harada, H., Toyono, T., Toyoshima, K., Yamasaki, M., Itoh, N., Kato, S., Sekine, K. and Ohuchi, H. (2002). FGF10 maintains stem cell compartment in developing mouse incisors. Development 129, 1533-41.
Harada, M., Murakami, H., Okawa, A., Okimoto, N., Hiraoka, S., Nakahara, T., Akasaka, R., Shiraishi, Y., Futatsugi, N., Mizutani-Koseki, Y. et al. (2009). FGF9 monomer-dimer equilibrium regulates extracellular matrix affinity and tissue diffusion. Nat Genet 41, 289-98.
Holmes, G. (2012). The role of vertebrate models in understanding craniosynostosis. Childs Nerv Syst 28, 1471-81.
Howard, T. D., Paznekas, W. A., Green, E. D., Chiang, L. C., Ma, N., Ortiz de Luna, R. I., Garcia Delgado, C., Gonzalez-Ramos, M., Kline, A. D. and Jabs, E. W. (1997). Mutations in TWIST, a basic helix-loop-helix transcription factor, in Saethre-Chotzen syndrome. Nat Genet 15, 36-41.
Hung, H., Kohnken, R. and Svaren, J. (2012). The nucleosome remodeling and deacetylase chromatin remodeling (NuRD) complex is required for peripheral nerve myelination. J Neurosci 32, 1517-27.
Iseki, S., Wilkie, A. O., Heath, J. K., Ishimaru, T., Eto, K. and Morriss-Kay, G. M. (1997). Fgfr2 and osteopontin domains in the developing skull vault are mutually exclusive and can be altered by locally applied FGF2. Development 124, 3375-84.
Jabs, E. W., Muller, U., Li, X., Ma, L., Luo, W., Haworth, I. S., Klisak, I., Sparkes, R., Warman, M. L., Mulliken, J. B. et al. (1993). A mutation in the homeodomain of the human MSX2 gene in a family affected with autosomal dominant craniosynostosis. Cell 75, 443-50.
Jenkins, D., Seelow, D., Jehee, F. S., Perlyn, C. A., Alonso, L. G., Bueno, D. F., Donnai, D., Josifova, D., Mathijssen, I. M., Morton, J. E. et al. (2007). RAB23
123
mutations in Carpenter syndrome imply an unexpected role for hedgehog signaling in cranial-suture development and obesity. Am J Hum Genet 80, 1162-70.
Jia, C. and Halpern, M. (1996). Subclasses of vomeronasal receptor neurons: differential expression of G proteins (Gi alpha 2 and G(o alpha)) and segregated projections to the accessory olfactory bulb. Brain Res 719, 117-28.
Jiang, X., Iseki, S., Maxson, R. E., Sucov, H. M. and Morriss-Kay, G. M. (2002). Tissue origins and interactions in the mammalian skull vault. Dev Biol 241, 106-16.
Kamimura, K., Ohi, H., Kubota, T., Okazuka, K., Yoshikai, Y., Wakabayashi, Y., Aoyagi, Y., Mishima, Y. and Kominami, R. (2007). Haploinsufficiency of Bcl11b for suppression of lymphomagenesis and thymocyte development. Biochem Biophys Res Commun 355, 538-42.
Kapadia, H., Mues, G. and D'Souza, R. (2007). Genes affecting tooth morphogenesis. Orthod Craniofac Res 10, 105-13.
Kerley, M. A. (1975). Pre-natal development of the mouse incisor. Proc Natl Acad Sci USA 55, 6–10.
Kettunen, P., Laurikkala, J., Itaranta, P., Vainio, S., Itoh, N. and Thesleff, I. (2000). Associations of FGF-3 and FGF-10 with signaling networks regulating tooth morphogenesis. Dev Dyn 219, 322-32.
Keverne, E. B. (1999). The vomeronasal organ. Science 286, 716-20.
Kim, H. J., Kim, J. H., Bae, S. C., Choi, J. Y. and Ryoo, H. M. (2003a). The protein kinase C pathway plays a central role in the fibroblast growth factor-stimulated expression and transactivation activity of Runx2. J Biol Chem 278, 319-26.
Kim, H. J., Lee, M. H., Park, H. S., Park, M. H., Lee, S. W., Kim, S. Y., Choi, J. Y., Shin, H. I. and Ryoo, H. M. (2003b). Erk pathway and activator protein 1 play crucial roles in FGF2-stimulated premature cranial suture closure. Dev Dyn 227, 335-46.
Kim, H. J., Rice, D. P., Kettunen, P. J. and Thesleff, I. (1998). FGF-, BMP- and Shh-mediated signalling pathways in the regulation of cranial suture morphogenesis and calvarial bone development. Development 125, 1241-51.
Klein, O. D., Lyons, D. B., Balooch, G., Marshall, G. W., Basson, M. A., Peterka, M., Boran, T., Peterkova, R. and Martin, G. R. (2008). An FGF signaling loop
124
sustains the generation of differentiated progeny from stem cells in mouse incisors. Development 135, 377-85.
Klein, O. D., Minowada, G., Peterkova, R., Kangas, A., Yu, B. D., Lesot, H., Peterka, M., Jernvall, J. and Martin, G. R. (2006). Sprouty genes control diastema tooth development via bidirectional antagonism of epithelial-mesenchymal FGF signaling. Dev Cell 11, 181-90.
Komori, T. (2010). Regulation of osteoblast differentiation by Runx2. Adv Exp Med Biol 658, 43-9.
Komori, T., Yagi, H., Nomura, S., Yamaguchi, A., Sasaki, K., Deguchi, K., Shimizu, Y., Bronson, R. T., Gao, Y. H., Inada, M. et al. (1997). Targeted disruption of Cbfa1 results in a complete lack of bone formation owing to maturational arrest of osteoblasts. Cell 89, 755-64.
Koussoulakou, D. S., Margaritis, L. H. and Koussoulakos, S. L. (2009). A curriculum vitae of teeth: evolution, generation, regeneration. Int J Biol Sci 5, 226-43.
Kyryachenko, S., Kyrylkova, K., Leid, M. and Kioussi, C. (2012). Immunohistochemistry and detection of proliferating cells by BrdU. Methods Mol Biol 887, 33-9.
Kyrylkova, K., Kyryachenko, S., Biehs, B., Klein, O., Kioussi, C. and Leid, M. (2012a). BCL11B regulates epithelial proliferation and asymmetric development of the mouse mandibular incisor. PLoS One 7, e37670.
Kyrylkova, K., Kyryachenko, S., Kioussi, C. and Leid, M. (2012b). Determination of gene expression patterns by in situ hybridization in sections. Methods Mol Biol 887, 23-31.
Leid, M., Ishmael, J. E., Avram, D., Shepherd, D., Fraulob, V. and Dolle, P. (2004). CTIP1 and CTIP2 are differentially expressed during mouse embryogenesis. Gene Expr Patterns 4, 733-9.
Li, L., Leid, M. and Rothenberg, E. V. (2010a). An early T cell lineage commitment checkpoint dependent on the transcription factor Bcl11b. Science 329, 89-93.
Li, P., Burke, S., Wang, J., Chen, X., Ortiz, M., Lee, S. C., Lu, D., Campos, L., Goulding, D., Ng, B. L. et al. (2010b). Reprogramming of T cells to natural killer-like cells upon Bcl11b deletion. Science 329, 85-9.
125
Liang, X., Bhattacharya, S., Bajaj, G., Guha, G., Wang, Z., Jang, H. S., Leid, M., Indra, A. K. and Ganguli-Indra, G. (2012). Delayed cutaneous wound healing and aberrant expression of hair follicle stem cell markers in mice selectively lacking Ctip2 in epidermis. PLoS One 7, e29999.
Lindsey, W. H. (2001). Osseous tissue engineering with gene therapy for facial bone reconstruction. Laryngoscope 111, 1128-36.
Liu, P., Li, P. and Burke, S. (2010). Critical roles of Bcl11b in T-cell development and maintenance of T-cell identity. Immunol Rev 238, 138-49.
Liu, Z., Xu, J., Colvin, J. S. and Ornitz, D. M. (2002). Coordination of chondrogenesis and osteogenesis by fibroblast growth factor 18. Genes Dev 16, 859-69.
Lumsden, A. G. (1988). Spatial organization of the epithelium and the role of neural crest cells in the initiation of the mammalian tooth germ. Development 103 Suppl, 155-69.
Marban, C., Suzanne, S., Dequiedt, F., de Walque, S., Redel, L., Van Lint, C., Aunis, D. and Rohr, O. (2007). Recruitment of chromatin-modifying enzymes by CTIP2 promotes HIV-1 transcriptional silencing. EMBO J 26, 412-23.
Maston, G. A., Evans, S. K. and Green, M. R. (2006). Transcriptional regulatory elements in the human genome. Annu Rev Genomics Hum Genet 7, 29-59.
Meara, J. G., Smith, E. M., Harshbarger, R. J., Farlo, J. N., Matar, M. M. and Levy, M. L. (2005). Blood-conservation techniques in craniofacial surgery. Ann Plast Surg 54, 525-9.
Mehrara, B. J., Spector, J. A., Greenwald, J. A., Ueno, H. and Longaker, M. T. (2002). Adenovirus-mediated transmission of a dominant negative transforming growth factor-beta receptor inhibits in vitro mouse cranial suture fusion. Plast Reconstr Surg 110, 506-14.
Melville, H., Wang, Y., Taub, P. J. and Jabs, E. W. (2010). Genetic basis of potential therapeutic strategies for craniosynostosis. Am J Med Genet A 152A, 3007-15.
Mertsching, E., Wurster, A. L., Katayama, C., Esko, J., Ramsdell, F., Marth, J. D. and Hedrick, S. M. (2002). A mouse strain defective for alphabeta versus gammadelta T cell lineage commitment. Int Immunol 14, 1039-53.
Miccio, A. and Blobel, G. A. (2010). Role of the GATA-1/FOG-1/NuRD pathway in the expression of human beta-like globin genes. Mol Cell Biol 30, 3460-70.
126
Miccio, A., Wang, Y., Hong, W., Gregory, G. D., Wang, H., Yu, X., Choi, J. K., Shelat, S., Tong, W., Poncz, M. et al. (2010). NuRD mediates activating and repressive functions of GATA-1 and FOG-1 during blood development. EMBO J 29, 442-56.
Montero, A., Okada, Y., Tomita, M., Ito, M., Tsurukami, H., Nakamura, T., Doetschman, T., Coffin, J. D. and Hurley, M. M. (2000). Disruption of the fibroblast growth factor-2 gene results in decreased bone mass and bone formation. J Clin Invest 105, 1085-93.
Nagayama, T., Okuhara, S., Ota, M. S., Tachikawa, N., Kasugai, S. and Iseki, S. (2013). FGF18 accelerates osteoblast differentiation by upregulating Bmp2 expression. Congenit Anom (Kyoto) 53, 83-8.
Nakamura, T., Yamazaki, Y., Saiki, Y., Moriyama, M., Largaespada, D. A., Jenkins, N. A. and Copeland, N. G. (2000). Evi9 encodes a novel zinc finger protein that physically interacts with BCL6, a known human B-cell proto-oncogene product. Mol Cell Biol 20, 3178-86.
Nelson, D. K. and Williams, T. (2004). Frontonasal process-specific disruption of AP-2alpha results in postnatal midfacial hypoplasia, vascular anomalies, and nasal cavity defects. Dev Biol 267, 72-92.
Nguyen, D. and Xu, T. (2008). The expanding role of mouse genetics for understanding human biology and disease. Dis Model Mech 1, 56-66.
Nie, X. (2005). Cranial base in craniofacial development: developmental features, influence on facial growth, anomaly, and molecular basis. Acta Odontol Scand 63, 127-35.
Nie, X., Luukko, K. and Kettunen, P. (2006). FGF signalling in craniofacial development and developmental disorders. Oral Dis 12, 102-11.
Obata, M., Kominami, R. and Mishima, Y. (2012). BCL11B tumor suppressor inhibits HDM2 expression in a p53-dependent manner. Cell Signal 24, 1047-52.
O'Hagan, R. C. and Hassell, J. A. (1998). The PEA3 Ets transcription factor is a downstream target of the HER2/Neu receptor tyrosine kinase. Oncogene 16, 301-10.
Ohazama, A., Modino, S. A., Miletich, I. and Sharpe, P. T. (2004). Stem-cell-based tissue engineering of murine teeth. J Dent Res 83, 518-22.
127
Ohbayashi, N., Shibayama, M., Kurotaki, Y., Imanishi, M., Fujimori, T., Itoh, N. and Takada, S. (2002). FGF18 is required for normal cell proliferation and differentiation during osteogenesis and chondrogenesis. Genes Dev 16, 870-9.
Okumura, H., Miyasaka, Y., Morita, Y., Nomura, T., Mishima, Y., Takahashi, S. and Kominami, R. (2011). Bcl11b heterozygosity leads to age-related hearing loss and degeneration of outer hair cells of the mouse cochlea. Exp Anim 60, 355-61.
Opperman, L. A. (2000). Cranial sutures as intramembranous bone growth sites. Dev Dyn 219, 472-85.
Opperman, L. A., Chhabra, A., Cho, R. W. and Ogle, R. C. (1999). Cranial suture obliteration is induced by removal of transforming growth factor (TGF)-beta 3 activity and prevented by removal of TGF-beta 2 activity from fetal rat calvaria in vitro. J Craniofac Genet Dev Biol 19, 164-73.
Ornitz, D. M. and Itoh, N. (2001). Fibroblast growth factors. Genome Biol 2, REVIEWS3005.
Ornitz, D. M., Xu, J., Colvin, J. S., McEwen, D. G., MacArthur, C. A., Coulier, F., Gao, G. and Goldfarb, M. (1996). Receptor specificity of the fibroblast growth factor family. J Biol Chem 271, 15292-7.
Oshima, M., Mizuno, M., Imamura, A., Ogawa, M., Yasukawa, M., Yamazaki, H., Morita, R., Ikeda, E., Nakao, K., Takano-Yamamoto, T. et al. (2011). Functional tooth regeneration using a bioengineered tooth unit as a mature organ replacement regenerative therapy. PLoS One 6, e21531.
Otto, F., Thornell, A. P., Crompton, T., Denzel, A., Gilmour, K. C., Rosewell, I. R., Stamp, G. W., Beddington, R. S., Mundlos, S., Olsen, B. R. et al. (1997). Cbfa1, a candidate gene for cleidocranial dysplasia syndrome, is essential for osteoblast differentiation and bone development. Cell 89, 765-71.
Panchal, J. and Uttchin, V. (2003). Management of craniosynostosis. Plast Reconstr Surg 111, 2032-48; quiz 2049.
Park, O. J., Kim, H. J., Woo, K. M., Baek, J. H. and Ryoo, H. M. (2010). FGF2-activated ERK mitogen-activated protein kinase enhances Runx2 acetylation and stabilization. J Biol Chem 285, 3568-74.
Perlyn, C. A., Morriss-Kay, G., Darvann, T., Tenenbaum, M. and Ornitz, D. M. (2006). A model for the pharmacological treatment of crouzon syndrome. Neurosurgery 59, 210-5; discussion 210-5.
128
Peter, I. S. and Davidson, E. H. (2011). Evolution of gene regulatory networks controlling body plan development. Cell 144, 970-85.
Peters, H. and Balling, R. (1999). Teeth. Where and how to make them. Trends Genet 15, 59-65.
Pietri, T., Eder, O., Blanche, M., Thiery, J. P. and Dufour, S. (2003). The human tissue plasminogen activator-Cre mouse: a new tool for targeting specifically neural crest cells and their derivatives in vivo. Dev Biol 259, 176-87.
Porntaveetus, T., Otsuka-Tanaka, Y., Basson, M. A., Moon, A. M., Sharpe, P. T. and Ohazama, A. (2011). Expression of fibroblast growth factors (Fgfs) in murine tooth development. J Anat 218, 534-43.
Purushothaman, R., Cox, T. C., Maga, A. M. and Cunningham, M. L. (2011). Facial suture synostosis of newborn Fgfr1(P250R/+) and Fgfr2(S252W/+) mouse models of Pfeiffer and Apert syndromes. Birth Defects Res A Clin Mol Teratol 91, 603-9.
Raulo, Y. and Tessier, P. (1981). Fronto-facial advancement for Crouzon's and Apert's syndromes. Scand J Plast Reconstr Surg 15, 245-50.
Rawlins, J. T. and Opperman, L. A. (2008). Tgf-beta regulation of suture morphogenesis and growth. Front Oral Biol 12, 178-96.
Rice, D. P. (2008). Developmental anatomy of craniofacial sutures. Front Oral Biol 12, 1-21.
Rice, D. P., Aberg, T., Chan, Y., Tang, Z., Kettunen, P. J., Pakarinen, L., Maxson, R. E. and Thesleff, I. (2000). Integration of FGF and TWIST in calvarial bone and suture development. Development 127, 1845-55.
Rice, D. P., Rice, R. and Thesleff, I. (2003). Fgfr mRNA isoforms in craniofacial bone development. Bone 33, 14-27.
Roehl, H. and Nusslein-Volhard, C. (2001). Zebrafish pea3 and erm are general targets of FGF8 signaling. Curr Biol 11, 503-7.
Rosenberg, P., Arlis, H. R., Haworth, R. D., Heier, L., Hoffman, L. and LaTrenta, G. (1997). The role of the cranial base in facial growth: experimental craniofacial synostosis in the rabbit. Plast Reconstr Surg 99, 1396-407.
Rothenberg, E. V. and Taghon, T. (2005). Molecular genetics of T cell development. Annu Rev Immunol 23, 601-49.
129
Ruiz i Altaba, A. (1999). Gli proteins encode context-dependent positive and negative functions: implications for development and disease. Development 126, 3205-16.
Ruthenburg, A. J., Li, H., Patel, D. J. and Allis, C. D. (2007). Multivalent engagement of chromatin modifications by linked binding modules. Nat Rev Mol Cell Biol 8, 983-94.
Ryan, M. C., Lee, K., Miyashita, Y. and Carter, W. G. (1999). Targeted disruption of the LAMA3 gene in mice reveals abnormalities in survival and late stage differentiation of epithelial cells. J Cell Biol 145, 1309-23.
Sartaj, R. and Sharpe, P. (2006). Biological tooth replacement. J Anat 209, 503-9.
Satterwhite, E., Sonoki, T., Willis, T. G., Harder, L., Nowak, R., Arriola, E. L., Liu, H., Price, H. P., Gesk, S., Steinemann, D. et al. (2001). The BCL11 gene family: involvement of BCL11A in lymphoid malignancies. Blood 98, 3413-20.
Schmidt-Wolf, G. D. and Schmidt-Wolf, I. G. (2003). Non-viral and hybrid vectors in human gene therapy: an update. Trends Mol Med 9, 67-72.
Seidel, K., Ahn, C. P., Lyons, D., Nee, A., Ting, K., Brownell, I., Cao, T., Carano, R. A., Curran, T., Schober, M. et al. (2010). Hedgehog signaling regulates the generation of ameloblast progenitors in the continuously growing mouse incisor. Development 137, 3753-61.
Sekine, K., Ohuchi, H., Fujiwara, M., Yamasaki, M., Yoshizawa, T., Sato, T., Yagishita, N., Matsui, D., Koga, Y., Itoh, N. et al. (1999). Fgf10 is essential for limb and lung formation. Nat Genet 21, 138-41.
Senarath-Yapa, K., Chung, M. T., McArdle, A., Wong, V. W., Quarto, N., Longaker, M. T. and Wan, D. C. (2012). Craniosynostosis: molecular pathways and future pharmacologic therapy. Organogenesis 8, 103-13.
Senawong, T., Peterson, V. J., Avram, D., Shepherd, D. M., Frye, R. A., Minucci, S. and Leid, M. (2003). Involvement of the histone deacetylase SIRT1 in chicken ovalbumin upstream promoter transcription factor (COUP-TF)-interacting protein 2-mediated transcriptional repression. J Biol Chem 278, 43041-50.
Sharma, V. P., Fenwick, A. L., Brockop, M. S., McGowan, S. J., Goos, J. A., Hoogeboom, A. J., Brady, A. F., Jeelani, N. O., Lynch, S. A., Mulliken, J. B. et al. (2013). Mutations in TCF12, encoding a basic helix-loop-helix partner of TWIST1, are a frequent cause of coronal craniosynostosis. Nat Genet 45, 304-7.
130
Sheikh, F., Fandrich, R. R., Kardami, E. and Cattini, P. A. (1999). Overexpression of long or short FGFR-1 results in FGF-2-mediated proliferation in neonatal cardiac myocyte cultures. Cardiovasc Res 42, 696-705.
Shim, K., Minowada, G., Coling, D. E. and Martin, G. R. (2005). Sprouty2, a mouse deafness gene, regulates cell fate decisions in the auditory sensory epithelium by antagonizing FGF signaling. Dev Cell 8, 553-64.
Shukla, V., Coumoul, X., Wang, R. H., Kim, H. S. and Deng, C. X. (2007). RNA interference and inhibition of MEK-ERK signaling prevent abnormal skeletal phenotypes in a mouse model of craniosynostosis. Nat Genet 39, 1145-50.
Simon, R., Brylka, H., Schwegler, H., Venkataramanappa, S., Andratschke, J., Wiegreffe, C., Liu, P., Fuchs, E., Jenkins, N. A., Copeland, N. G. et al. (2012). A dual function of Bcl11b/Ctip2 in hippocampal neurogenesis. EMBO J 31, 2922-36.
Song, H. M., Fong, K. D., Nacamuli, R. P., Warren, S. M., Fang, T. D., Mathy, J. A., Cowan, C. M., Aalami, O. O. and Longaker, M. T. (2004). Mechanisms of murine cranial suture patency mediated by a dominant negative transforming growth factor-beta receptor adenovirus. Plast Reconstr Surg 113, 1685-97.
Spitz, F. and Furlong, E. E. (2012). Transcription factors: from enhancer binding to developmental control. Nat Rev Genet 13, 613-26.
Stewart, R. E., Dixon, G. and Cohen, A. (1977). The pathogenesis of premature craniosynostosis in acrocephalosyndactyly (Apert's syndrome). A reconsideration. Plast Reconstr Surg 59, 699-707.
Takahashi, S., Kawashima, N., Sakamoto, K., Nakata, A., Kameda, T., Sugiyama, T., Katsube, K. and Suda, H. (2007). Differentiation of an ameloblast-lineage cell line (ALC) is induced by Sonic hedgehog signaling. Biochem Biophys Res Commun 353, 405-11.
Tang, B., Di Lena, P., Schaffer, L., Head, S. R., Baldi, P. and Thomas, E. A. (2011). Genome-wide identification of Bcl11b gene targets reveals role in brain-derived neurotrophic factor signaling. PLoS One 6, e23691.
Taverna, S. D., Li, H., Ruthenburg, A. J., Allis, C. D. and Patel, D. J. (2007). How chromatin-binding modules interpret histone modifications: lessons from professional pocket pickers. Nat Struct Mol Biol 14, 1025-40.
Topark-Ngarm, A., Golonzhka, O., Peterson, V. J., Barrett, B., Jr., Martinez, B., Crofoot, K., Filtz, T. M. and Leid, M. (2006). CTIP2 associates with the NuRD
131
complex on the promoter of p57KIP2, a newly identified CTIP2 target gene. J Biol Chem 281, 32272-83.
Tucker, A. and Sharpe, P. (2004). The cutting-edge of mammalian development; how the embryo makes teeth. Nat Rev Genet 5, 499-508.
Tummers, M. and Thesleff, I. (2003). Root or crown: a developmental choice orchestrated by the differential regulation of the epithelial stem cell niche in the tooth of two rodent species. Development 130, 1049-57.
Tummers, M. and Thesleff, I. (2009). The importance of signal pathway modulation in all aspects of tooth development. J Exp Zool B Mol Dev Evol 312B, 309-19.
Turner, N., Lambros, M. B., Horlings, H. M., Pearson, A., Sharpe, R., Natrajan, R., Geyer, F. C., van Kouwenhove, M., Kreike, B., Mackay, A. et al. (2010). Integrative molecular profiling of triple negative breast cancers identifies amplicon drivers and potential therapeutic targets. Oncogene 29, 2013-23.
Twigg, S. R., Kan, R., Babbs, C., Bochukova, E. G., Robertson, S. P., Wall, S. A., Morriss-Kay, G. M. and Wilkie, A. O. (2004). Mutations of ephrin-B1 (EFNB1), a marker of tissue boundary formation, cause craniofrontonasal syndrome. Proc Natl Acad Sci U S A 101, 8652-7.
Twigg, S. R., Vorgia, E., McGowan, S. J., Peraki, I., Fenwick, A. L., Sharma, V. P., Allegra, M., Zaragkoulias, A., Sadighi Akha, E., Knight, S. J. et al. (2013). Reduced dosage of ERF causes complex craniosynostosis in humans and mice and links ERK1/2 signaling to regulation of osteogenesis. Nat Genet 45, 308-13.
Urnov, F. D., Rebar, E. J., Holmes, M. C., Zhang, H. S. and Gregory, P. D. (2010). Genome editing with engineered zinc finger nucleases. Nat Rev Genet 11, 636-46.
Vaahtokari, A., Aberg, T., Jernvall, J., Keranen, S. and Thesleff, I. (1996a). The enamel knot as a signaling center in the developing mouse tooth. Mech Dev 54, 39-43.
Vaahtokari, A., Aberg, T. and Thesleff, I. (1996b). Apoptosis in the developing tooth: association with an embryonic signaling center and suppression by EGF and FGF-4. Development 122, 121-9.
Van Vlierberghe, P., Homminga, I., Zuurbier, L., Gladdines-Buijs, J., van Wering, E. R., Horstmann, M., Beverloo, H. B., Pieters, R. and Meijerink, J. P. (2008). Cooperative genetic defects in TLX3 rearranged pediatric T-ALL. Leukemia 22, 762-70.
132
Vanvalkenburgh, J., Albu, D. I., Bapanpally, C., Casanova, S., Califano, D., Jones, D. M., Ignatowicz, L., Kawamoto, S., Fagarasan, S., Jenkins, N. A. et al. (2011). Critical role of Bcl11b in suppressor function of T regulatory cells and prevention of inflammatory bowel disease. J Exp Med 208, 2069-81.
Volponi, A. A., Pang, Y. and Sharpe, P. T. (2010). Stem cell-based biological tooth repair and regeneration. Trends Cell Biol 20, 715-22.
Vortkamp, A., Franz, T., Gessler, M. and Grzeschik, K. H. (1992). Deletion of GLI3 supports the homology of the human Greig cephalopolysyndactyly syndrome (GCPS) and the mouse mutant extra toes (Xt). Mamm Genome 3, 461-3.
Wakabayashi, Y., Inoue, J., Takahashi, Y., Matsuki, A., Kosugi-Okano, H., Shinbo, T., Mishima, Y., Niwa, O. and Kominami, R. (2003a). Homozygous deletions and point mutations of the Rit1/Bcl11b gene in gamma-ray induced mouse thymic lymphomas. Biochem Biophys Res Commun 301, 598-603.
Wakabayashi, Y., Watanabe, H., Inoue, J., Takeda, N., Sakata, J., Mishima, Y., Hitomi, J., Yamamoto, T., Utsuyama, M., Niwa, O. et al. (2003b). Bcl11b is required for differentiation and survival of alphabeta T lymphocytes. Nat Immunol 4, 533-9.
Wan, D. C., Kwan, M. D., Lorenz, H. P. and Longaker, M. T. (2008). Current treatment of craniosynostosis and future therapeutic directions. Front Oral Biol 12, 209-30.
Wang, X. P., Suomalainen, M., Felszeghy, S., Zelarayan, L. C., Alonso, M. T., Plikus, M. V., Maas, R. L., Chuong, C. M., Schimmang, T. and Thesleff, I. (2007). An integrated gene regulatory network controls stem cell proliferation in teeth. PLoS Biol 5, e159.
Wang, X. P., Suomalainen, M., Jorgez, C. J., Matzuk, M. M., Werner, S. and Thesleff, I. (2004). Follistatin regulates enamel patterning in mouse incisors by asymmetrically inhibiting BMP signaling and ameloblast differentiation. Dev Cell 7, 719-30.
Wang, Z., Kirkwood, J. S., Taylor, A. W., Stevens, J. F., Leid, M., Ganguli-Indra, G. and Indra, A. K. (2013). Transcription factor Ctip2 controls epidermal lipid metabolism and regulates expression of genes involved in sphingolipid biosynthesis during skin development. J Invest Dermatol 133, 668-76.
Wang, Z., Zhang, L. J., Guha, G., Li, S., Kyrylkova, K., Kioussi, C., Leid, M., Ganguli-Indra, G. and Indra, A. K. (2012). Selective ablation of Ctip2/Bcl11b in
133
epidermal keratinocytes triggers atopic dermatitis-like skin inflammatory responses in adult mice. PLoS One 7, e51262.
Warren, S. M. and Longaker, M. T. (2001). The pathogenesis of craniosynostosis in the fetus. Yonsei Med J 42, 646-59.
Warren, S. M., Brunet, L. J., Harland, R. M., Economides, A. N. and Longaker, M. T. (2003). The BMP antagonist noggin regulates cranial suture fusion. Nature 422, 625-9.
Weinzweig, J., Panter, K. E., Seki, J., Pantaloni, M., Spangenberger, A. and Harper, J. S. (2006). The fetal cleft palate: IV. Midfacial growth and bony palatal development following in utero and neonatal repair of the congenital caprine model. Plast Reconstr Surg 118, 81-93.
Whitaker, L. A., Munro, I. R., Salyer, K. E., Jackson, I. T., Ortiz-Monasterio, F. and Marchac, D. (1979). Combined report of problems and complications in 793 craniofacial operations. Plast Reconstr Surg 64, 198-203.
Wieland, I., Reardon, W., Jakubiczka, S., Franco, B., Kress, W., Vincent-Delorme, C., Thierry, P., Edwards, M., Konig, R., Rusu, C. et al. (2005). Twenty-six novel EFNB1 mutations in familial and sporadic craniofrontonasal syndrome (CFNS). Hum Mutat 26, 113-8.
Wilkie, A. O. (1997). Craniosynostosis: genes and mechanisms. Hum Mol Genet 6, 1647-56.
Wilkie, A. O., Byren, J. C., Hurst, J. A., Jayamohan, J., Johnson, D., Knight, S. J., Lester, T., Richards, P. G., Twigg, S. R. and Wall, S. A. (2010). Prevalence and complications of single-gene and chromosomal disorders in craniosynostosis. Pediatrics 126, e391-400.
Wolfe, S. A., Nekludova, L. and Pabo, C. O. (2000). DNA recognition by Cys2His2 zinc finger proteins. Annu Rev Biophys Biomol Struct 29, 183-212.
Xiao, G., Jiang, D., Gopalakrishnan, R. and Franceschi, R. T. (2002). Fibroblast growth factor 2 induction of the osteocalcin gene requires MAPK activity and phosphorylation of the osteoblast transcription factor, Cbfa1/Runx2. J Biol Chem 277, 36181-7.
Yin, L., Du, X., Li, C., Xu, X., Chen, Z., Su, N., Zhao, L., Qi, H., Li, F., Xue, J. et al. (2008). A Pro253Arg mutation in fibroblast growth factor receptor 2 (Fgfr2) causes skeleton malformation mimicking human Apert syndrome by affecting both chondrogenesis and osteogenesis. Bone 42, 631-43.
134
Yokohama-Tamaki, T., Ohshima, H., Fujiwara, N., Takada, Y., Ichimori, Y., Wakisaka, S., Ohuchi, H. and Harada, H. (2006). Cessation of Fgf10 signaling, resulting in a defective dental epithelial stem cell compartment, leads to the transition from crown to root formation. Development 133, 1359-66.
Yoshida, T., Vivatbutsiri, P., Morriss-Kay, G., Saga, Y. and Iseki, S. (2008). Cell lineage in mammalian craniofacial mesenchyme. Mech Dev 125, 797-808.
Young, C. S., Abukawa, H., Asrican, R., Ravens, M., Troulis, M. J., Kaban, L. B., Vacanti, J. P. and Yelick, P. C. (2005a). Tissue-engineered hybrid tooth and bone. Tissue Eng 11, 1599-610.
Young, C. S., Kim, S. W., Qin, C., Baba, O., Butler, W. T., Taylor, R. R., Bartlett, J. D., Vacanti, J. P. and Yelick, P. C. (2005b). Developmental analysis and computer modelling of bioengineered teeth. Arch Oral Biol 50, 259-65.
Zeichner-David, M., Diekwisch, T., Fincham, A., Lau, E., MacDougall, M., Moradian-Oldak, J., Simmer, J., Snead, M. and Slavkin, H. C. (1995). Control of ameloblast differentiation. Int J Dev Biol 39, 69-92.
Zhang, L. J., Bhattacharya, S., Leid, M., Ganguli-Indra, G. and Indra, A. K. (2012a). Ctip2 is a dynamic regulator of epidermal proliferation and differentiation by integrating EGFR and Notch signaling. J Cell Sci 125, 5733-44.
Zhang, L. J., Vogel, W. K., Liu, X., Topark-Ngarm, A., Arbogast, B. L., Maier, C. S., Filtz, T. M. and Leid, M. (2012b). Coordinated regulation of transcription factor Bcl11b activity in thymocytes by the mitogen-activated protein kinase (MAPK) pathways and protein sumoylation. J Biol Chem 287, 26971-88.
Zhang, Y. D., Chen, Z., Song, Y. Q., Liu, C. and Chen, Y. P. (2005). Making a tooth: growth factors, transcription factors, and stem cells. Cell Res 15, 301-16.
Zhang, Y., LeRoy, G., Seelig, H. P., Lane, W. S. and Reinberg, D. (1998). The dermatomyositis-specific autoantigen Mi2 is a component of a complex containing histone deacetylase and nucleosome remodeling activities. Cell 95, 279-89.
Zhang, Y., Ng, H. H., Erdjument-Bromage, H., Tempst, P., Bird, A. and Reinberg, D. (1999). Analysis of the NuRD subunits reveals a histone deacetylase core complex and a connection with DNA methylation. Genes Dev 13, 1924-35.
Zhang, Y., Zhang, Z., Zhao, X., Yu, X., Hu, Y., Geronimo, B., Fromm, S. H. and Chen, Y. P. (2000). A new function of BMP4: dual role for BMP4 in regulation of Sonic hedgehog expression in the mouse tooth germ. Development 127, 1431-43.