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Developmental Biology 2
Differential susceptibility of midbrain and spinal cord patterning to
floor plate defects in the talpid2 mutant
Seema Agarwala a,*, Galina V. Aglyamova a, Amanda K. Marma b,
John F. Fallon c, Clifton W. Ragsdale b
a Section of Neurobiology, University of Texas at Austin, Austin, TX 78712-0248, USAb Department of Neurobiology, Pharmacology and Physiology, University of Chicago, Chicago, IL 60637-1431, USA
c Department of Anatomy, University of Wisconsin at Madison, Madison, WI 53706-1510, USA
Received for publication 12 July 2005, revised 13 September 2005, accepted 14 September 2005
Available online 21 October 2005
Abstract
The chick talpid2 mutant displays polydactylous digits attributed to defects of the Hedgehog (HH) signaling pathway. We examined the talpid2
neural tube and show that patterning defects in the spinal cord and the midbrain are distinct from each other and from the limb. Unlike the Sonic
Hedgehog (SHH) source in the limb, the SHH-rich floor plate (FP) is reduced in the talpid2 midbrain. This is accompanied by a severe depletion
of medial cell populations that encounter high concentrations of SHH, an expansion of lateral cell populations that experience low concentrations
of SHH and a broad deregulation of HH’s principal effectors (PTC1, GLI1, GLI2, GLI3). Together with the failure of SHH misexpression to
rescue the talpid2 phenotype, these results suggest that talpid2 is likely to have a tissue-autonomous, bidirectional (positive and negative) role in
HH signaling that cannot be attributed to the altered expression of several newly cloned HH pathway genes (SUFU, DZIP1, DISP1, BTRC).
Strikingly, FP defects in the spinal cord are accompanied by relatively normal patterning in the talpid2 mutant. We propose that this differential FP
dependence may be due to the prolonged apposition of the notochord to the spinal cord, but not the midbrain during development.
D 2005 Elsevier Inc. All rights reserved.
Keywords: Sonic Hedgehog; Midbrain; Spinal cord; Floor plate; Motor neurons; Dopaminergic neurons; Notochord
Introduction
The role of the Hedgehog (HH) signaling cascade in
directing cellular diversity and patterning in the embryo is
well established. The HH pathway has been implicated in the
specification of a wide array of embryonic systems ranging
from segment patterning in Drosophila, limb development, the
establishment of left–right asymmetry and dorsoventral pat-
terning in the vertebrate neural tube (Ingham and McMahon,
2001). In humans, misregulation of the HH pathway causes
congenital defects (spina bifida, cyclopia, holoprosencephaly,
limb polydactyly) and can lead to various types of tumors of
the skin and brain (basal cell carcinomas, medulloblastomas,
glioblastomas; McMahon et al., 2003; Roessler et al., 1996;
Villavicencio et al., 2000). Given its importance in develop-
0012-1606/$ - see front matter D 2005 Elsevier Inc. All rights reserved.
doi:10.1016/j.ydbio.2005.09.034
* Corresponding author.
E-mail address: agarwala@mail.utexas.edu (S. Agarwala).
ment and disease, HH signal transduction and its modulation
have received intense scrutiny.
The core HH signaling machinery appears to be conserved
across species and embryonic tissues (Aza-Blanc et al., 2000;
Hooper and Scott, 2005; Nybakken and Perrimon, 2002). The
HH ligand is bound by a 12-pass transmembrane receptor
protein, Patched (PTC; Marigo et al., 1996a,b; Stone et al.,
1996). In the absence of the HH ligand, PTC inhibits the
activity of the 7-pass membrane protein Smoothened (SMO),
blocking HH pathway activation (Akiyama et al., 1997; Alcedo
and Noll, 1997). The intracellular signal transduction beyond
these key events is complex and not fully understood, but the
activation of HH target genes is dependent upon a single
effector, cubitus interruptus (Ci) in flies, and upon three Ci
homologs in vertebrates, GLI1, GLI2 and GLI3 (Methot and
Basler, 2001).
In Drosophila, Ci forms a cytoplasmic complex with the
kinase Fused and Costal2 (Cos2) and is processed into a
truncated repressor when levels of HH signaling are low (Aza-
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S. Agarwala et al. / Developmental Biology 288 (2005) 206–220 207
Blanc et al., 1997; Lum et al., 2003). This proteolytic step is
mediated sequentially by protein kinase A (PKA), glycogen
synthase kinase 3h and casein kinase 1, eventually leading to
the degradation of the carboxy-terminal end of Ci/GLI and the
formation of a Ci/GLI repressor (Jiang, 2002). In the presence
of HH, cleavage of Ci/GLI is inhibited, and the full-length
activator form of the Ci/GLI predominates (Ohlmeyer and
Kalderon, 1998). Modulation of the GLI gene family by
nucleo-cytoplasmic shuttling is also provided by Suppressor of
Fused (SUFU) and the newly identified zinc finger transcrip-
tion factor iguana (DZIP1; Kogerman et al., 1999; Sekimizu et
al., 2004; Wolff et al., 2004). Paradoxically, a number of genes
involved in the production of the Ci repressor (e.g., Cos2,
PKA) have additional roles as positive modulators of HH
signaling (Jia et al., 2004; Zhang et al., 2005). Recent mutant
analysis in the fly has revealed several new contributors to the
HH signaling cascade, a number of which (e.g., Slimb, Nedd8,
Cul1 and 3, Roc1) are dedicated to the proteolysis of GLI
proteins and the modulation of GLI function (Jiang, 2002).
Despite their importance in the HH signaling cascade,
vertebrate counterparts of these genes have yet to be described.
In this paper, we have examined the autosomal recessive
chick talpid2 mutant where defects in GLI protein modulation
are seen (Wang et al., 2000). Phenotypic defects in talpid2
include decreased interocular distance with occasional cyclo-
pia, defects in craniofacial development and, most strikingly,
the presence of polydactylous limbs that exhibit aberrant digit
size and identity (Dvorak and Fallon, 1991; Schneider et al.,
1999). Thus, although the molecular basis of the talpid2
mutation is as yet uncharacterized, the overall talpid2
phenotype is consistent with a defect in the Hedgehog (HH)
signaling cascade. Recent studies in the talpid2 limb have
further suggested a broad constitutive activation of the
Hedgehog signaling machinery (Caruccio et al., 1999).
Biochemical studies have confirmed this idea by showing that
a disruption in the ratio of the GLI3 repressor and activator
across the limb may account for the observed supernumerary
preaxial digits seen in the talpid2 (Litingtung et al., 2002;
Wang et al., 2000). In this report, we have studied the effects of
the talpid2 mutation on neural tube development, with a focus
on neuronal patterning defects, and the expression of a battery
of HH pathway components including the HH ligand, GLI
transcription factors and a panel of mediators including PTC1,
SUFU, DZIP1, DISP1, HHIP and BTRC.
We have found that the talpid2 mutation produces a unique
and complex phenotype in the neural tube. Unlike the SHH-
rich zone of polarizing activity of the limb, the SHH-rich floor
plate (FP) is markedly reduced in size. In the midbrain, cell
types found close to the ventral midline are reduced in number,
and those arising at a distance from the FP are expanded and
inappropriately distributed. PCT1, GLI1, GLI2 and GLI3 are
all upregulated in a manner consistent with an increased range
and reduced magnitude of HH signaling. Taken together with
the craniofacial, eye and limb phenotypes, our results suggest
that talpid2 is likely to be a novel component of the vertebrate
HH signaling cascade capable of both positive and negative
regulation (Caruccio et al., 1999; Jia et al., 2004; Litingtung et
al., 2002; Park et al., 2000; Schneider et al., 1999; Wang et al.,
2000; Zhang et al., 2005).
Strikingly, we found profound rostrocaudal differences in
the effects of talpid2 on dorsoventral neuronal patterning: in
the spinal cord, although the FP is also compromised, neuronal
fate specification appears relatively normal. Our results thus
provide evidence that proper cell fate specification requires an
intact FP in the midbrain but not in the spinal cord, where the
proximity of the notochord may compensate for disrupted floor
plate development (Jeong and McMahon, 2005).
Materials and methods
Chick embryos
Fertilized eggs were obtained from talpid2 flocks maintained at the
University of Wisconsin, Madison and the University of California, Davis. All
eggs were incubated at 38-C in a humidified forced draft chamber and were
staged according to Hamburger and Hamilton (1951). Mutant embryos were
seen in Mendelian ratios and identified by the shape of their limb buds at
embryonic day 4 (E4)–E6. As we characterized the mutant midbrain, we
identified diagnostic gene expression defects that then allowed us to recognize
mutant embryos at earlier stages by in situ hybridization. A comparison of the
Wisconsin and Davis flocks with gene expression in the midbrain showed that,
in general, the talpid2 phenotype of the Wisconsin flock was slightly more
severe compared to the Davis flock. However, since the phenotype was
qualitatively similar, observations from both flocks were pooled. Figs. 1E, F
and 2B were drawn from in situ hybridizations done on Wisconsin embryos,
with the remaining data derived from embryos of the Davis flock.
In ovo electroporation
Protein-coated implants in a target tissue result in the delivery of variable
and declining protein concentrations over time, releasing >50% of their protein
within the first 24 h following implantation (Fallon et al., 1994). By contrast,
when delivered by electroporation, ectopic expression continues for at least 4
days (Agarwala and Ragsdale, 2002; Fig. 2). To obtain a prolonged and
continuous source of SHH, micro-electroporation was employed to introduce
the SHH-expressing plasmid pXeX-SHH into the midbrain of talpid2 embryos
and controls (Agarwala et al., 2001; Agarwala and Ragsdale, 2002; Momose et
al., 1999). Fifty to 250 nl of plasmid DNA solution (1 Ag/Al in 10 mM Tris, 1
mM EDTA, 0.02% Fast Green) was injected through a glass capillary needle
into the midbrain vesicle of stages 10–15 chick embryos displayed by
windowing. Electrodes were held at a distant of 1 mm, and 3 electric pulses (50
ms, 7 V) were delivered. The embryos were returned to the incubator for 3–4
days before harvesting. Our conclusions are based on an analyses of 41
successfully electroporated embryos (n = 34 control, n = 7 talpid2).
Cloning of HH pathway genes
Chicken HH pathways gene fragments for BTRC, DISP1, DZIP1, HHIP
and WNT11 were isolated by PCR from E7 chick ventral midbrain cDNA and
subcloned into pCR-TOPO (Invitrogen). Primers were designed with Mac
Vector 7.2 (Accelrys) based on sequences culled from BLAST searches of chick
EST and genomic databases (Boardman et al., 2002; Caldwell et al., 2005;
Hilier et al., 2004). The primer pairs successfully employed for our cDNA
cloning include BTRC-forward, 5V-TCTGAATGGGCACAAGCGTG-3V;BTRC-reverse, 5V-AGACGACCACTGAGAGCAGGAAAG-3V; DISP1-for-
ward, 5 V-GGACTCGTGGATTTCAAGTGAGC-3 V; DISP1-reverse,
5V-AAGGTCTGAGAGAAGGCATTACACC-3V; DZIP1-forward, 5V-CCAAGTCGGCTTCAGGATTGTAG-3V; DZIP1-reverse, 5V-TCAGGGC-TAAAAGACACAGGAGC-3V; HHIP-forward, 5V-AGAACGGTGGGC-
TATTGGACC-3V; HHIP-reverse, 5V-GCAGCATTTTCCTGTGGGAGTG-3V;WNT11-forward, 5V-AGACCTGCCGTAAAACTTTCTCAG-3V; WNT11 re-
verse, 5V-TGCGACCCCACTTTCTCATTC-3V.
Fig. 1. Midbrain arcuate cell types are differentially affected in the talpid2 mutant. Left column: flock mate controls (wild type or heterozygote); right column:
talpid2 mutants. Images are of wholemounts (open book preparation) processed for one- or two-color in situ hybridization. Top is rostral, and the ventricular surface
faces the reader. (A, B) Loss of motor neurons (PHOX2A+, brown) and expansion of EVX1+ cells (blue) in the E5 talpid2 midbrain. (C, D) Reduction of TH+ (blue)
and PAX6+ cells (brown, arrowheads) in E6 midbrains. Note the increased cell dispersal in the talpid2 mutant (arrowheads, D). (E, F) Loss of the SHH+ (brown)
rostral floor plate (rFP) and the PHOX2A+ oculomotor neurons by E3 in the talpid2 mutant. 1: medial or 1st arc; MHB, midbrain–hindbrain boundary; rFP, rostral
floor plate.
S. Agarwala et al. / Developmental Biology 288 (2005) 206–220208
In situ hybridization
Embryos were harvested at E3 to E6 and immersion-fixed in 4%
paraformaldehyde in a phosphate-buffered saline solution. Digoxigenin
(DIG)- and fluorescein-labeled riboprobes were synthesized from chick cDNA
plasmids for BTRC/SLIMB , CLASS III b-TUBULIN, CHOX10, DISP1,
DZIP1, EN1, EVX1, FOXA2/HNF3b, GLI1, GLI2, GLI3, ISL1, LMX1B,
NKX2.2, OTX2, PAX6, PAX7, PHOX2A, PTC1, SHH, SIM1, SUFU, TH and
WNT11 (Agarwala et al., 2001; Agarwala and Ragsdale, 2002; Sanders et al.,
2002). High-stringency one and two-color wholemount and section in situ
hybridizations were carried out as described on brain, spinal cord and limbs
(Agarwala and Ragsdale, 2002).
Cell death assay
Wholemount cell death assays were performed with modifications of the
Yamamoto and Henderson (1999) method (Sanders, 2001). E5–E6 embryos
were harvested and immersion-fixed for 2–3 h in 4% paraformaldehyde
containing 0.1% Tween. Dissected midbrains were dehydrated and rehydrated
through graded ethanols and permeabilized with detergents. TUNEL
(Terminal deoxytransferase (Tdt)-mediated dUTP-digoxigenin nick end
labeling) was accomplished by incubation in a Tdt (Chemicon, CA) reaction
at 37-C for 40 min. Incorporation of DIG-labeled nucleotide allowed
detection as with non-radioactive in situ hybridization histochemistry.
Results
The SHH source and arc patterning are disrupted in the talpid2
mutant midbrain
In the midbrain, arcuate stripes of molecularly distinct cell
types (midbrain arcs) are arrayed bilateral to the midbrain or
rostral floor plate source of SHH (Fig. 1; Agarwala et al., 2001;
Kingsbury, 1922, 1930; Sanders et al., 2002). Previous work
has shown that an ectopic source of SHH can generate a full
complement of midbrain arcs in a size- and position-dependent
manner (Agarwala et al., 2001). In the limb, SHH-mediated
elements of pattern such as the size of the autopod, digit
number and digit identity are disrupted in the talpid2 mutant
(Caruccio et al., 1999). We asked whether the SHH-dependent
midbrain arcs are disrupted in the talpid2 mutant as well.
The anlagen of the oculomotor complex (OMC, PHOX2A+),
the red nucleus (RN, BRN3A+) and midbrain dopaminergic
neurons (MDN, TH+) arise from themost medial (1st) arc which
abuts the SHH source and thus constitutes cell fates specified by
Fig. 2. SHH misexpression does not restore the arcuate pattern of cell fates in the talpid2 midbrain. Orientation for this and subsequent midbrain wholemounts as in
Fig. 1. (A, B) Homeobox gene expression of PHOX2A, PAX6 and EVX1 (together referred to as HX; blue) at E6 confirms the downregulation of PHOX2A+ and
PAX6+ cell types closer to the SHH source (brown) compared to the massive upregulation of the more lateral EVX1+ cell types. Red arrow in panel A marks the
lateral edge of SHH gene expression in control midbrains. Note that the reduction of SHH gene expression in the talpid2 mutant (B) that was obvious at E3 (Fig. 1F)
continues at E6, being retained only along the ventral midline. (C, D) Unilateral (right side) overexpression of SHH at E5 following electroporation at E2. The left
half of the midbrain in each panel serves as the unelectroporated control. Since chick SHH was electroporated, SHH (brown) expression on the right in excess of the
left side represents the misexpressed SHH. (C) Ectopic induction of a SHH+ FP (brown, arrowhead) and an expansion of the arc-specific homeobox (HX), PHOX2A,
PAX6 and EVX1 (blue) genes at E5. (D) SHH overexpression in the talpid2 midbrain (right half) does not elicit an orderly pattern of arcuate territories when assayed
by HX gene expression at E5 nor does it produce a consolidated SHH source (brown, arrow and arrowheads) as seen in the controls (C). Instead, clumps of
ectopically expressed SHH (brown) surrounded by HX gene expression (blue) are seen throughout the electroporated region (arrowheads).
S. Agarwala et al. / Developmental Biology 288 (2005) 206–220 209
high concentrations of SHH (Agarwala and Ragsdale, 2002).
PAX6+ andEVX1+ arcuate stripes occupymore lateral positions
in ventral midbrain and are thought to encounter progressively
lower concentrations of SHH. In the talpid2midbrain, themedial
(OMC, RN and MDN) and intermediate (PAX6+) arcuate
populations are severely reduced, while lateral cell fates
(EVX1+) are expanded and expressed at ectopic locations (Figs.
1A–D, data not shown). In marked contrast with the normal
SHH source seen in the talpid2 and talpid3 limbs (Caruccio et
al., 1999; Francis-West et al., 1995; Lewis et al., 1999;
Schneider et al., 1999), the rostral floor plate (rFP) source of
SHH is severely disrupted in the midbrain by E3 (Figs. 1E, F).
Taken together, these data suggest that a reduced SHH
signaling source compromises medial cell fates normally
induced by high concentrations of SHH (‘‘high’’ HH fates)
but expands lateral cell fates that normally encounter low
concentrations of SHH (‘‘low’’ HH fates).
SHH misexpression cannot rescue the talpid2 midbrain
phenotype: talpid2 is required autonomously in HH-responsive
tissue
The dramatic reduction in size of the midbrain FP, which is
apparent at E3 (Fig. 1F) and maintained through E6 (Figs. 2A,
B), is sufficient to explain a loss of medial cell fates that are
induced by high HH concentrations. To address the mechan-
isms of the impaired talpid2 FP development, we took
advantage of our earlier finding that misexpression of SHH
by electroporation is able to induce an ectopic floor plate along
with a complete ectopic arc pattern (Agarwala et al., 2001). Our
prediction was, if talpid2 FP development is compromised
because of a deficit in the inductive signals from axial
mesoderm, ectopic SHH should rescue the FP development
program (Placzek et al., 1993). By contrast, a failure of ectopic
SHH to elicit an ectopic FP and ectopic arcs would demonstrate
an HH signaling defect intrinsic to the HH-responsive cells of
the midbrain.
We found in control experiments that, as expected, unilateral
overexpression of SHH elicited an expanded, uninterrupted
SHH-rich FP, surrounded by ectopic arcs (Fig. 2C; Agarwala et
al., 2001). In the talpid2 midbrain, however, SHH over-
expression is unable to elicit a coherent SHH-rich source (Fig.
2D). Patches of arc-specific gene expression were seen around
the ectopic SHH+ patches (Fig. 2D), demonstrating that,
although the talpid2 midbrain can respond to HH signaling,
this signaling is defective and results in the inability of ectopic
SHH to produce the FP expansion and the organized arc pattern
elicited in controls (Fig. 2C).
Talpid2 limbs grafted to wild type limbs self-differentiate
and form talpid2 digits (Dvorak and Fallon, 1992; MacCabe
and Abbott, 1974). Furthermore, talpid2, but not wild type
limbs, can regenerate the resected 2/3rd of the posterior limb
Fig. 3. The principal effectors of HH are upregulated in the talpid2 midbrain. (A–H) Gene expression of PTC1 (A, B), GLI1 (C, D), GLI2 (E, F) and GLI3 (G, H) is
greatly upregulated in the E5 talpid2 midbrain (B, D, F, H) in comparison with controls (A, C, E, G). Note that, for all four genes, the talpid2 ventral midline remains
unlabeled and that, particularly for GLI3, the expansion is very patterned (also seen in Figs. 8E, F). (I, J) Talpid2 midbraiin showing a strictly segregated mosaicism
of SHH (brown) and GLI3 (blue) mRNA expression wherever the two genes are co-expressed. (I) Unilateral SHH misexpression on the right side of the embryo with
the left side serving as the unelectroporated control. As with Fig. 2, SHH (brown) expression on the right in excess of the left side represents misexpressed chick
SHH. Clumps of ectopic SHH+ tissue seen beyond the domain of ectopic GLI3 expression (arrowheads, I) indicate that the ectopic upregulation of GLI3 cannot
explain the failure in talpid2 midbrains of ectopic SHH deposits to coalesce. Panel J illustrates at high power the boxed region in panel I.
S. Agarwala et al. / Developmental Biology 288 (2005) 206–220210
S. Agarwala et al. / Developmental Biology 288 (2005) 206–220 211
bud in the absence of detectable SHH mRNA expression
(Caruccio et al., 1999). Taken together with our SHH
misexpression experiments (Figs. 2C, D), these results indicate
that talpid2 function is likely to be cell-autonomous in multiple
embryonic systems. Thus, the FP defect and the consequent arc
patterning defects are likely to result from incorrect HH signal
transduction in the HH-responsive cells of the midbrain such
that FP induction by axial mesoderm and the homeogenetic
induction of FP by itself are compromised (Placzek et al.,
1993).
Deregulation of HH effector genes in the talpid2 mutant
midbrain
To further characterize the nature of the defect in the
talpid2 mutant, we next examined gene expression of the
principal effectors of HH signal transduction. As in the
talpid2 limb, gene expression of PTC1, a receptor and
negative regulator of HH signaling, is massively upregulated
in the mutant midbrain (Figs. 3A, B). Notably, the dynamic
spatial modulation of PTC1 seen along the mediolateral axis
in controls is replaced by uniformly high expression that only
avoids the midline of rFP (Figs. 3A, B). Gene expression of
PTC1 is a reliable readout of the range of HH signaling
(Goodrich et al., 1997). The increased PTC1 gene expression
thus suggests that a broader than normal expanse of the
talpid2 midbrain behaves as if responding to HH signaling
(Caruccio et al., 1999).
GLI transcription factors are required for HH signal
transduction (Bai et al., 2004; Krishnan et al., 1997; Methot
and Basler, 2001). In the talpid2 limb, Caruccio et al. (1999)
found that GLI1 gene expression is increased. We found in the
talpid2 midbrain that expression levels of all three GLI genes
are raised (Figs. 3C–H). The domains of GLI1 and GLI2
occupy most of midbrain with a notable lack of any GLI
expression in the PTC1-poor ventral midline (Figs. 3C–F).
Although GLI3 expression also avoids the midline, its ectopic
upregulation is more restrained, being concentrated along the
midbrain–hindbrain junction (MHB), the dorsoventral (DV)
border of the midbrain, and, most prominently, flanking the
ventral midline where ectopic GLI3 expression forms a non-
overlapping mosaic pattern with SHH (Figs. 3G–J; see also
Figs. 8E, F). Interestingly, we also saw this mosaicism between
SHH and GLI3 expression patterns when we misexpressed
SHH in the talpid2 midbrain by electroporation (Figs. 3I, J).
Misexpression of SHH, however, elicits clumps of ectopic
SHH-expressing cells even outside the regions expressing
ectopic GLI3, indicating that the failure of the talpid2 midbrain
to support normal rFP development is unlikely to be due
simply to interference from pools of GLI3-rich cells (Fig. 3I).
Not all HH pathway genes are disrupted in the talpid2 mutant
Our observations suggest that the talpid2 mutation is likely
to involve the HH transport, reception or signal transduction
machinery. We therefore isolated cDNAs for chicken Dis-
patched (DISP1) and Hedgehog Interacting Protein (HHIP),
which are known to regulate HH transport (Ingham and
McMahon, 2001; Jeong and McMahon, 2005; Tian et al.,
2004). Gene expression for HHIP1, a glycoprotein that binds
HH and attenuates the range and magnitude of HH signaling
(Chuang and McMahon, 1999; Jeong and McMahon, 2005), is
lost from the talpid2 midbrain (Figs. 4A, B). DISP1 is required
for cholesterol-modified HH to be secreted from the HH-
producing cells, and lowering the levels of DISP produces a
dose-dependent reduction in HH signaling (Tian et al., 2004).
Normal levels of DISP1 transcripts are distributed along the
ventral midline in both the control and mutant midbrain,
suggesting that the talpid2 mutation does not interfere with
DISP1 expression (Figs. 4C, D).
Biochemical evidence from the talpid2 limb suggests that
one consequence of the mutation is the misregulation of GLI3
such that excess full-length GLI3 (presumed activator) is
produced and abnormally distributed in the anterior (preaxial)
limb bud, while the amount of GLI3 repressor is reduced
(Litingtung et al., 2002; Wang et al., 2000). To examine GLI
gene modulation in the talpid2 midbrain, we isolated from
chickens the cDNAs for several genes involved in GLI gene
processing. We found that both SUFU, whose protein product
is involved in the cytoplasmic retention of GLI proteins, and
BTRC/SLIMB, which is involved in targeting hyperphosphory-
lated Ci/GLI proteins for degradation (Jiang, 2002; Kogerman
et al., 1999), are expressed normally in the talpid2 mutant
midbrain (Figs. 4E–H).
A recently identified member of the HH family is the
zinc finger transcription factor DZIP1 which may participate
in the nuclear-cytoplasmic shuttling of GLI proteins, thereby
regulating the ratio of GLI activator and repressor (Sekimizu
et al., 2004; Vokes and McMahon, 2004; Wolff et al.,
2004). In the zebrafish dzip1 mutant iguana, cell fates
triggered by high levels of HH signaling are compromised,
while those promoted by low levels of HH are expanded, a
feature shared by the talpid2 mutant (Sekimizu et al., 2004).
In addition, the lateral floor plate is absent in the igu mutant
(Karlstrom et al., 1996; Odenthal et al., 2000). Given the
similarities between the iguana and the talpid2 phenotypes,
we asked if chick DZIP1 gene expression was misregulated
in the talpid2 embryo. We found that DZIP1 expression was
not perturbed in the talpid2 midbrain, spinal cord or limb
bud (Figs. 4I, J, data not shown).
Cell death, tissue size and thickness are compromised in the
talpid2 mutant
The loss of the rFP and medial midbrain cell populations
could be due to increased cell death, possibly triggered by
upregulation of PTC1 expression in the talpid2 mutant
(Thibert et al., 2003). We found increased cell death in the
midbrain of the talpid2 mutant with TUNEL labeling (Figs.
5A, B). Unlike control midbrains, where cell death was
largely confined to the ventricular layer (Fig. 5A, data not
shown), apoptotic cells in the talpid2 midbrain were spread
across the ventricular and mantle layer and concentrated in
the FP along the midline (Fig. 5B, data not shown).
Fig. 4. Expression of key modulators of HH function in the talpid2 midbrain. (A, B) Hedgehog-interacting protein (HHIP) gene expression is lost from the midbrain
of the talpid2 mutant, although it can be seen in the forebrain (arrowhead). (C–J) Expression patterns of DISPATCHED 1 (DISP1), SUPPRESSOR OF FUSED
(SUFU), BTRC/SLIMB and DZIP1/IGUANA are largely unaffected by the talpid2 mutation. E4 (A, B, I, J) and E5 (C–H) midbrains harvested from control (A, C, E,
G, I) and talpid2 (B, D, F, H, J) embryos. Identification of talpid2 mutants in I, J was based on SHH expression since they could not be distinguished by their limb
phenotype at this stage. III: 3rd ventricle. rFP: rostral floor plate, MHB: midbrain–hindbrain junction.
S. Agarwala et al. / Developmental Biology 288 (2005) 206–220212
Fig. 5. Cell death, tissue size and thickness are compromised in the talpid2 midbrain. (A, B) TUNEL staining demonstrates increased numbers of apoptotic cells in
the E5 talpid2 midbrain (B) over those of control (A). (C, D) Increased size of the talpid2 ventral midbrain at E5 (D) photographed at the same magnification as an
E5 control (C). Midbrain dorsoventral and midbrain– forebrain boundaries are identified by gene expression of GLI3 (blue) in tectum (tec) and PAX6 (brown) in
caudal forebrain (FB). (E, F) CLASS III b-TUBULIN labeling demonstrates largely normal neuronal differentiation in the E5 talpid2 midbrain (F) as compared with
controls (E). (G–H) Transverse sections of the wholemounts shown in panels E and F demonstrate the reduced thickness of the talpid2 midbrain. Images of the
controls and the corresponding talpid2 midbrains are taken at the same magnification. III: 3rd ventricle; FB: forebrain; tec: tectum or dorsal midbrain.
S. Agarwala et al. / Developmental Biology 288 (2005) 206–220 213
Intriguingly, this increased cell death was accompanied by
an increase in the width (DV axis) of the midbrain (Figs.
5C, D). An increase in tissue size is also reported to occur
along the anteroposterior (AP) axis of the talpid2 and the
talpid3 limbs (Caruccio et al., 1999; Lewis et al., 1999).
CLASS III b-TUBULIN gene expression, which identifies
differentiated neurons of the mantle layer (Figs. 5E–H),
demonstrated a marked reduction in the thickness of
midbrain tissue in cross-sections (Figs. 5G, H). These
results indicate that, although the talpid2 midbrain is wider
compared to the controls, the mantle layer is thinner.
Anteroposterior patterning is preserved in talpid2 midbrain,
but dorsoventral territory boundaries are no longer sharp
AP and DV patterning
The midbrain phenotype observed in the talpid2 mutant
could be a consequence of defects in other non-HH-mediated
signaling cascades originating along the boundary regions of
the ventral midbrain. The orthodenticle homolog OTX2 is
expressed throughout anterior neural tube (forebrain and
midbrain), and its caudal limit coincides with the midbrain–
hindbrain boundary (Fig. 6A). We found that OTX2 expression
Fig. 6. The midbrain–hindbrain boundary remains sharp, but the tectotegmental (DV) boundary becomes irregular in the talpid2 midbrain. (A, B) OTX2 gene
expression demonstrates that the sharp and smooth contours of the midbrain–hindbrain boundary (A) are retained in the E5 talpid2 mutant (B). (C, D) PAX7
expression distinguishes the dorsal midbrain (tec, tectum) from the ventral midbrain and documents that the talpid2 dorsoventral boundary is broad and disrupted
(arrowheads, D) compared to that of controls (C). MHB: midbrain–hindbrain boundary; tec: tectum.
S. Agarwala et al. / Developmental Biology 288 (2005) 206–220214
is appropriately regionalized along the AP axis of the neural
tube, with its caudal expression terminating sharply at the
MHB (Figs. 6A, B). We also observed the normal localization
of BMP7 (rostral) and FGF8 (caudal) expression at the
midbrain–hindbrain boundary (MHB; data not shown). We
next examined the tectotegmental (DV) boundary of the
midbrain with PAX7 gene expression identifying the dorsal
midbrain or tectum. In controls as well as the mutants, PAX7
gene expression remained restricted to the tectum, suggesting
that the talpid2 midbrain was divided as usual into dorsal and
ventral domains (Figs. 6C, D). The confinement of PAX3/PAX7
to dorsal neural tube is thought to be accomplished by low
levels of HH signaling, confirming yet again that low level HH
signaling can occur in the talpid2 midbrain (Ericson et al.,
1996) and that the principal effects of the talpid2 mutation are
confined to the ventral midbrain. Interestingly, unlike the
caudal midbrain boundary, which remained sharp and regular
in the talpid2 mutant, the DV boundary presented a ‘‘wiggly’’,
irregular border (Figs. 6C, D; data not shown; Lawrence,
1997). Along with the lack of coherent arcs and the inability of
ectopic SHH to assemble into a coalesced morphogen source
(Figs. 1A–D and 2C, D), these results suggest changes in
midbrain cells’ adhesive properties or increased cell death of
HH target cells or both (Figs. 5A, B; Jarov et al., 2003;
Lawrence et al., 1999; Rodriguez and Basler, 1997).
Cell fate specification is normal in the talpid2 spinal cord
In the spinal cord, motor neurons and multiple classes of
ventral interneurons (V3–V0) are specified in response to
graded HH signaling (Ericson et al., 1996; Roelink et al.,
1995). We found that all ventral spinal neurons including motor
neurons (ISL1+) as well as the V3 (SIM1+), V2 (CHOX10+),
V1 (EN1+) and VO (EVX1+) interneurons were specified in
near-normal numbers along the dorsoventral axis of the talpid2
mutant with only a modest disturbance in their spatial
patterning (Figs. 7A–J). Furthermore, an SHH-rich FP and
notochord were readily detected in the talpid2 mutant (Figs.
7K, L) and were accompanied by a normal distribution of
PTC1 and GLI3 transcripts in the spinal cord (Figs. 7M–P).
Thus, unlike the midbrain, neuronal specification proceeds
normally in the talpid2 spinal cord, although, as shown below,
the caudal talpid2 FP is compromised in a manner similar to
that of midbrain FP.
Floor plate defects in the talpid2 midbrain and spinal cord
By gene expression and embryonic origin, the avian floor
plate has been partitioned into medial (node-derived, SHH+,
NKX2.2�) and lateral (neural-plate-derived, SHH+, NKX2.2+)
subdivisions at spinal cord levels (Charrier et al., 2002). We
cloned out chick WNT11, a marker of medial-most FP at spinal
cord levels, and found that, though slightly reduced in extent, it
was retained in the talpid2 ventral midline (Figs. 8A, B; see
also Figs. 7K, L). The NKX2.2+ lateral floor plate region
(LFP), however, was severely reduced in the talpid2 caudal
spinal cord (Figs. 8C, D). This was accompanied by an
increased overlap between SHH and NKX2.2 expression more
medially, suggesting a defect in LFP induction, maintenance or
segregation from the medial FP (MFP; Figs. 8C, D). In
zebrafish and rats, a midline HH signal is required for the
proper homeogenetic induction of lateral regions of FP and for
Fig. 7. Cell fate specification in the talpid2 spinal cord. Transverse sections demonstrating the relatively normal specification of SIM1+ V3 interneurons (A, B),
ISL1+ motor neurons (C, D), CHOX10+ V2 (E, F), EN1+ V1 (G, H) and EVX1+ VO (I, J) interneurons in control (columns 1 and 3) and talpid2 spinal cords
(columns 2 and 4). (K, L) SHH gene expression demonstrates that the floor plate (FP) and notochord (nc) are retained in the talpid2 mutant. (M, N) PTC1 expression
appears normal in the talpid2 spinal cord. (O, P) Normal relationship between GLI3 (blue) and SHH (brown) domains in the talpid2 spinal cord (P), relative to
controls (O). CHX10: CHOX10; DR: dorsal root ganglion; MN, motor neurons. A, B, E–H: E5 spinal cords; C, D, I–N: E4 spinal cords; O, P: E3 spinal cords.
S. Agarwala et al. / Developmental Biology 288 (2005) 206–220 215
the survival of cells within the medial regions of FP (Le
Douarin and Halpern, 2000; Odenthal et al., 2000; Placzek et
al., 1993). Defective HH signaling in the talpid2 may therefore
prevent the proper induction of the spinal cord LFP by MFP or
its segregation from MFP. In addition, it may result in the
observed reduction of MFP due to increased cell death (Figs.
7K, L and 8A, B; see also Figs. 5A, B).
The enormous lateral expansion of midbrain FP along with
its distinctive mode of induction and embryonic origin
precluded the use of medial MFP and LFP definitions derived
from spinal cord studies (Kingsbury, 1922, 1930; Placzek and
Briscoe, 2005). However, based on high and low levels of SHH
expression, clear medial and lateral divisions could be
discerned in the E3 midbrain (Figs. 8E, F). The lateral region,
Fig. 8. Disruption of lateral floor plate (LFP) in the spinal cord and midbrain of the talpid2 mutant. (A, B) Cross-section of E5 spinal cord demonstrating that the
medial-most region of the medial FP (MFP) stained by WNT11 in the control (A) is retained in the talpid2 spinal cord (B). (C, D) Cross-sections through E5 spinal
cord demonstrating a reduced LFP (NKX2.2+, brown) in the talpid2 mutant, while the MFP (SHH+) is retained. (E, F) E3 midbrain wholemounts demonstrate
downregulation of SHH (brown) in LFP in the talpid2 mutant (F) concomitant to the ectopic expression of GLI3 (blue) in this region. (G, H) Expression of FOXA2
(brown), which extends lateral to the SHH domain in E5 control midbrains (G), is repressed in the talpid2 midbrain (H). (I, J) LMX1B staining of E5 midbrain
demonstrates a retained MFP in the talpid2 midbrain (J). LFP: lateral floor plate; MFP: medial floor plate; MHB: midbrain–hindbrain junction.
S. Agarwala et al. / Developmental Biology 288 (2005) 206–220216
marked by the loss of SHH and ectopic GLI3 expression, was
severely disrupted in the talpid2 mutant (Figs. 8E, F, see also
Figs. 4C, D; I, J). By E5, the enormous growth and complex
subarchitecture of the rFP foreclosed any simple analysis of the
midbrain FP into lateral and medial subdivisions. Even so, the
talpid2 E5 rFP resembled in quality the talpid2 spinal cord FP:
a disruption of lateral FP regions with FP tissue along the
ventral midline remaining relatively intact, displaying only a
modest increase in cell death (Figs. 8G, H; Figs. 5A, B).
LMX1B and DISP1 gene expression corroborated the retention
of medial-most rFP at E5 (Figs. 8I, J and 4C, D). In the
zebrafish as well as in the rat, LFP specification and MFP cell
survival require proper HH signaling (Le Douarin and Halpern,
2000; Odenthal et al., 2000; Placzek et al., 1993). The failure of
LFP to be correctly specified in the midbrain and spinal cord
and the increased cell death in the medial regions of the FP
together suggest that talpid2 may function as a component of
the HH signaling cascade without which the lateral regions of
S. Agarwala et al. / Developmental Biology 288 (2005) 206–220 217
FP cannot be correctly specified and the medial regions of FP
suffer increased cell death.
Discussion
In this study, we show that talpid2 is likely to be a novel
component of the avian HH signal transduction pathway, its
misregulation resulting in dramatically different phenotypes in
the limb, spinal cord and the midbrain. In the limb, the readout
is loss of growth control and polydactyly with indeterminate
digit identity (Caruccio et al., 1999). In the spinal cord, the
principal defect is a disruption of floor plate development,
while cell fate specification of motor and interneurons remains
fairly normal. In the midbrain, by contrast, multiple facets of
patterning associated with HH signaling are disrupted. These
disruptions include reduced cell survival, loss of size regula-
tion, deregulation of cellular affinities and, most profoundly, a
disruption in cell fate specification.
Defective HH signaling in the talpid2 mutant
HH signaling is sufficient for normal midbrain patterning
and for the segregation of ventral (PAX7�) from dorsal
(PAX7+) neural tube (Agarwala et al., 2001; Ericson et al.,
1996). In the talpid2 midbrain, the correct dorsal (tectal)
localization of PAX7 and the residual presence of HH target cell
fates suggest that some HH signaling does indeed occur in the
midbrain (Figs. 1, 2, 5, 6; Ericson et al., 1996). However, HH
signaling in the talpid2 is fundamentally defective as demon-
strated by the autonomous defects in HH signal transduction
within the HH-responsive tissue (Fig. 2; Caruccio et al., 1999;
Dvorak and Fallon, 1992; MacCabe and Abbott, 1974), the
ligand-independent regulation of several HH effector genes
(PTC, GLIs), by the upregulation of ‘‘low’’ HH cell fates and
by the reduction of ‘‘high’’ HH cell fates. Several features of
the talpid2 mutant could account for this phenotype.
Upregulation of PTC1 and the loss of HHIP
HH signal transduction requires the function of its receptor
PTC1 (Ingham et al., 1991). A product of this signal
transduction is the transcriptional upregulation of PTC1
expression, making PTC1 expression a reliable readout of the
occurrence of HH signaling (Marigo et al., 1996b; Goodrich et
al., 1997). Paradoxically, PTC1 also sequesters HH and
attenuates HH signaling (Marigo et al., 1996b; Goodrich et
al., 1997). The glycoprotein HHIP, also an HH binding protein,
a transcriptional target of HH and a negative regulator of HH
signaling, serves much the same function in vertebrates as does
PTC1 (Chuang and McMahon, 1999; Jeong and McMahon,
2005). The simultaneous removal of PTC and HHIP in mice
results in a massively ventralized neural tube and in a loss of
size regulation (Jeong and McMahon, 2005). In the mouse
Ptc1�/� mutant, the absence of PTC1 function results in
increased HH signaling and consequently in increased levels of
ptc1 expression (Goodrich et al., 1997). The upregulated PTC1
mRNA levels in the talpid2 midbrain may therefore reflect
lowered PTC function and, together with the loss of HHIP
expression, suggests an increased range and magnitude of HH
signaling in the midbrain. These findings fit well with the
ectopic upregulation of low HH fates (EVX1+; Fig. 1) and the
loss of size regulation seen in the midbrain (Figs. 5C, D). But,
they cannot readily account for the loss of ‘‘high’’ HH fates in
the talpid2 midbrain and are not concordant with the lowered
levels of SHH observed (Fig. 1). These results suggest instead
that the upregulation of PTC1 expression in the talpid2 may
occur in a ligand-independent manner as suggested in the
talpid2 limb (Caruccio et al., 1999). However, as discussed
below, additional explanation is required to explain the full
talpid2 midbrain phenotype.
Defective GLI modulation
The talpid2 midbrain exhibits lowered levels of HH gene
expression along with the upregulation of all three GLI genes
(Fig. 3). This is accompanied by a complex midbrain
phenotype with the widespread upregulation of ‘‘low’’ HH
fates (e.g., EVX1+ cells) and a depletion of ‘‘high’’ HH fates
such as the FP, oculomotor and dopaminergic neurons (Fig.
1). In all embryonic systems examined, GLI1 ubiquitously
exhibits activator function, and GLI3 protein can serve as an
activator or a repressor depending on the presence or absence
of HH signaling respectively (Ohlmeyer and Kalderon, 1998;
Dai et al., 1999). The increased GLI1 and PTC1 expression is
thus in conflict with the low levels of SHH expression
observed in the talpid2 midbrain. Furthermore, the readout of
GLI1 would be in conflict with the increased GLI3 levels,
which would be presumably be predominantly of the
repressor variety due to the lowered levels of HH. What
could the function of talpid2 be to produce a phenotype
consistent with both positive and negative regulation of HH
signaling?
To our knowledge, DZIP1/iguana, which modulates GLI
activity, is the only other HH pathway gene in vertebrates,
whose defect is known to result in the differential regulation of
‘‘high’’ (neural tube) and ‘‘low’’ (somites) HH target cell fates
(Sekimizu et al., 2004). However, the gene expression of
DZIP1 in the midbrain, spinal cord and limb of the talpid2
mutant appears to be normal, although a point mutation or a
mis-sense mutation cannot be ruled out (Figs. 4I, J; data not
shown). Unlike vertebrates, several components of the Dro-
sophila HH pathway required for Ci/GLI processing including
Cos2 and PKA display a similar dual (positive and negative)
regulation of the HH signaling cascade. Mutations of such Ci-
modulating genes result not only in the derepression of the HH
pathway, but also attenuate ‘‘high’’ HH activity (e.g., Jia et al.,
2004). For example, PKA, which is required for the phosphor-
ylation and proteolysis of Ci into its repressor form, also
phosphorylates and targets SMO to the cell surface, an event
essential for the upregulation of ‘‘high’’ HH cell fates (Jia et al.,
2004). Thus, the differential regulation of high vs. low HH
fates, the misregulation of GLI gene expression and the
incorrect processing of GLI3 protein in the talpid2 limb
together lead us to predict that the talpid2 is likely to be a
novel component of the vertebrate HH signaling cascade that
can modulate HH-GLI activity bidirectionally.
S. Agarwala et al. / Developmental Biology 288 (2005) 206–220218
Floor plate specification is defective in the talpid2 neural tube
A major finding of this study is the selective disruption of
the lateral regions of floor plate in the midbrain and spinal cord
(Fig. 8). A subdivision of the FP into medial and lateral regions
is well established in the teleosts and has also been proposed in
the chick and other amniotes (Ang and Rossant, 1994; Charrier
et al., 2002; Placzek and Briscoe, 2005; Odenthal et al., 2000;
Placzek et al., 1991; Roelink et al., 1995; Sasaki and Hogan,
1993; Yamada et al., 1991). Studies involving quail–chick
chimeras suggest that, as with the zebrafish, the avian MFP
(SHH+, NKX2.2�) is derived from the node (embryonic shield
in fish), while LFP (SHH+, NKX2.2+) has its origin within the
neurectoderm and requires a midline HH signal for its
induction (Charrier et al., 2002; Melby et al., 1996; Odenthal
et al., 2000; Placzek et al., 1993; Shih and Fraser, 1996). Our
studies in control animals suggest that SHH misexpression can
indeed induce an ectopic FP in the midbrain that expands
through homeogenetic induction (Fig. 2; Agarwala et al., 2001;
Placzek et al., 1993). However, similar misexpression of SHH
in the talpid2 midbrain cannot sustain such an FP expansion,
consistent with the idea that SHH signaling is defective in the
talpid2 mutant and that the induction of LFP requires SHH
signaling (Placzek and Briscoe, 2005).
The above discussion suggests that defective HH signaling
leads to a disruption of LFP specification or maintenance or
both, but it does not explain why the MFP is relatively spared
in the talpid2 mutant. It is possible that the residual HH
signaling (Figs. 1, 2, 6) that does remain in the talpid2 mutant
is talpid2-independent and is sufficient for the development of
the largely intact MFP. It is equally possible, that as in
anamniotes, HH-independent mechanisms play a role in the
induction of the avian MFP (Strahle et al., 2004). Likely
candidates for this induction are Nodal, other TGFh family
members and their antagonists and the Notch–Delta signaling
cascades (Lopez et al., 2005; Strahle et al., 2004; Placzek and
Briscoe, 2005).
The distinct regional effects of talpid2 may be due to the
differential proximity of the midbrain and spinal cord to the
notochord
What could account for the differential severity of the loss
of talpid2 function in midbrain and spinal cord patterning? One
explanation may be the proximity of each region to the
subjacent notochord and its relative importance in neuronal cell
fate specification. By E3, the notochord has already regressed
from the midbrain, which likely leaves the FP as the principal
ventral source of patterning signals at this AP level. By
contrast, the notochord remains apposed to the spinal cord for a
considerably longer period of time, at least as late as E4 (Figs.
7K, L). Recent observations in the mouse have suggested that
the allocation of cell fates in the spinal cord may well depend
upon notochord-derived signals rather than the FP (Jeong and
McMahon, 2005). Such signals could be HH-dependent, HH-
independent or both. The midbrain, by contrast, cannot depend
upon the notochord due to its early rostral regression and must
rely on patterning signals from the rFP which is severely
disrupted in the talpid2 mutant. As a result, many aspects of
patterning including LFP expansion, cell fate specification,
cell-adhesion, size control and cell survival are affected in the
talpid2 midbrain.
Acknowledgments
We thank R. Bayly, J. Fogel, T. Sanders and J. Wallingford
for critical comments, Jacqueline Pisenti at UC Davis for her
help in supplying us with talpid2 fertilized eggs and P. Beachy,
P. Brickell, C. Cepko, D. Cleveland, G. Eichele, C. Fan, C.
Goridis, M. Goulding, B. Houston, T. Jessell, A. Leutz, A.
McMahon, G. Martin, C. Tabin and M. Wassef for DNA
reagents. This research was supported by grants from the
March of Dimes and the NIH (C.W.R.; J.F.F.) and from UT-
Austin (S.A.).
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