Gene expression pattern

Spatial and temporal expression pattern of Runx3 (Aml2) and Runx1(Aml1) indicates non-redundant functions during mouse embryogenesis

Ditsa Levanona,1, Ori Brennerb,1, Varda Negreanua, David Bettouna, Eilon Woolfa, Raya Eilamb,Joseph Lotema, Uri Gatc, Florian Ottod, Nancy Specke, Yoram Gronera,*

aDepartment of Molecular Genetics, The Weizmann Institute of Science, Rehovot 76100, IsraelbDepartment of Experimental Animals, The Weizmann Institute of Science, Rehovot 76100, Israel

cDepartment of Cell and Animal Biology, The Hebrew University, Jerusalem 9194, IsraeldDepartment of Hematology/Oncology, University of Freiburg Medical Center, 79106 Freiburg, Germany

eDepartment of Biochemistry, Dartmouth Medical School, Hanover, NH 03755, USA

Received 8 August 2001; received in revised form 23 August 2001; accepted 23 August 2001

Abstract

The human RUNX3/AML2 gene belongs to the ‘runt domain’ family of transcription factors that act as gene expression regulators in major

developmental pathways. Here, we describe the expression pattern of Runx3 during mouse embryogenesis compared to the expression

pattern of Runx1. E10.5 and E14.5–E16.5 embryos were analyzed using both immunohistochemistry and b-galactosidase activity of targeted

Runx3 and Runx1 loci. We found that Runx3 expression overlapped with that of Runx1 in the hematopoietic system, whereas in sensory

ganglia, epidermal appendages, and developing skeletal elements, their expression was confined to different compartments. These data

provide new insights into the function of Runx3 and Runx1 in organogenesis and support the possibility that cross-regulation between them

plays a role in embryogenesis. q 2001 Elsevier Science Ireland Ltd. All rights reserved.

Keywords: Runt transcription factors; AML1 and AML2; Spatio-temporal expression; Mouse embryogenesis; Tissue-specific expression; Organogenesis;

Epithelial–mesenchymal interactions; Dorsal root ganglia; Cartilage; Epidermal appendages

1. Results and discussion

The mammalian RUNX3/AML2 gene was cloned and

localized to human and mouse chromosomes 1p36.1 and

4, respectively (Avraham et al., 1995; Bae et al., 1995;

Levanon et al., 1994). Runx3 belongs to a family of three

genes, two of which, Runx1 and Runx2, act as master regu-

lators of gene expression in hematopoiesis and osteogenesis

(Ito, 1999; Karsenty, 2000; Komori and Kishimoto, 1998;

Speck et al., 1999). All RUNX proteins bind to the same

DNA motif and hence their pleiotropic functions are likely

to result from a regulated spatial/temporal expression

pattern (Pozner et al., 2000). Interestingly, Runx1 and

Runx3 genes contain RUNX binding sites in their promoter

region (Bangsow et al., 2001; Ghozi et al., 1996; Levanon et

al., 2001), raising the possibility of cross-regulation, both

positive and negative between them (Levanon et al., 1998).

In adults, RUNX1 and RUNX3 are highly expressed in the

hematopoietic system (Bangsow et al., 2001; Le et al., 1999;

Levanon et al., 1994, 1996; Meyers et al., 1996). In the

mouse, Runx1 is essential for definitive hematopoiesis

(Okuda et al., 1996; Wang et al., 1996) and is also expressed

at numerous other sites (North et al., 1999; Simeone et al.,

1995). However, its expression in non-hematopoietic tissues

has not been thoroughly analyzed. Embryonal expression of

Runx3 has not been documented and its biological function

is largely unknown. We examined Runx3 expression at

E10.5 and E14.5–E16.5 and compared it to the expression

pattern of Runx1. Immunohistochemistry and knock-in

expression of b-galactosidase activity were used in parallel

throughout the analysis to confirm expression patterns.

At E10.5 expression of Runx3 was detected in hemato-

poietic precursors in the liver (Fig. 1a,c) in a cell population

expressing Runx1 (Fig. 1b,d). Expression of Runx3 was also

seen in the cranial (Fig. 1a) and dorsal root ganglia (data not

shown). In the dorsal aorta where Runx1 was expressed

(North et al., 1999) (Fig. 1b), Runx3 expression was not

detected (Fig. 1a).

At E14.5–E16.5 expression of Runx3 was detected only

in organs that also expressed Runx1. In some tissues Runx3

Mechanisms of Development 109 (2001) 413–417

0925-4773/01/$ - see front matter q 2001 Elsevier Science Ireland Ltd. All rights reserved.

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* Corresponding author. Tel.: 1972-8-9343-972; fax: 1972-8-9344-108.

E-mail address: yoram.groner@weizmann.ac.il (Y. Groner).1 These two authors contributed equally to this work.

and Runx1 were apparently present in the same cell types,

including hematopoietic cells in the liver (mononuclear

cells, myeloid precursors and megakaryocytes) (Fig. 2a,b),

the thymus (Fig. 2c,c 0,d,d 0), the eyelid mesenchyme (Fig.

2e,f), the superficial cutaneous mesenchyme and the

mesenchymal element of filiform papillae of the tongue

(data not shown). In all other tissues, expression of Runx3

and Runx1 was confined to distinct compartments (Fig. 3).

In cranial and dorsal root ganglia, Runx3 was expressed in a

small number of large-diameter neurons, whereas Runx1

was expressed in numerous small-diameter neurons (Fig.

3c,d). In cartilage, Runx3 was expressed in prehypertrophic

and hypertrophic chondrocytes (Fig. 3a,g,i), whereas Runx1

was expressed in immature chondrocytes, either of the

permanent or transient type, and in hypertrophic chondro-

cytes (Fig. 3b,h,j). Interestingly, expression of Runx3 in

cartilage appeared to overlap with that of Runx2 (Kim et

al., 1999). In epidermal appendages such as hair follicles

and whiskers, expression of Runx3 was confined to

mesenchymal elements, whereas Runx1 expression was

restricted to epithelium (Fig. 3e,f), suggesting a role for

these two Runx proteins in epithelial–mesenchymal inter-

actions. Epithelial or mesenchymal expression of Runx1

was also seen in other organs that did not express Runx3.

Epithelial expression of Runx1 was detected in salivary

glands (ducts but not alveoli) (Fig. 4a), bronchi (Fig. 4b),

respiratory and olfactory mucosa of the nasal cavity (Fig.

4c), as well as in palatal ridges, mucosa of the esophagus

and stomach, and paramesonephric ducts (data not shown).

Mesenchymal expression of Runx1 was seen in the valvular

region of the heart (Fig. 4d), the periphery of the septum

transversum, corneal stroma, the thoracic and abdominal

midline, the ovarian stroma and external genitalia (data

not shown). Taken together, the results show that Runx3

expression was confined to mesenchymal elements, whereas

Runx1 was expressed in both epithelium and mesenchyme.

Whole mount and coronal sections of E16.5 Runx11/lz

heads, double-stained for b-galactosidase and acetylcholine

esterase, revealed expression in the dorsal vagal nucleus,

hypoglossal nucleus and nucleus ambiguus (Fig. 4e,f), and

a focus in the pontine reticular formation (Fig. 4e). Expres-

sion was also seen in the trigeminal motor nucleus, the facial

nucleus and in bilateral rows of ventral neurons extending

from the caudal hindbrain to the cranial thoracic cord (data

not shown). Staining was also detected in a few anatomi-

cally less defined areas.

The expression pattern of Runx3 and Runx1 provides

important information for phenotypic analysis of knock-

out mice. In most cases, the two Runx genes were expressed

D. Levanon et al. / Mechanisms of Development 109 (2001) 413–417414

Fig. 1. Expression of Runx3 and Runx1 at E10.5. (a) Immunohistochemical

(IHC) staining of Runx3 in the liver (L) and in the IX-X cranial ganglion

complex (IX-X). (b) IHC staining of Runx1 in the liver and dorsal aorta (A,

dorsal aorta). (c,d) Higher magnification of hematopoietic precursors in the

liver. Sagittal paraffin sections were incubated overnight at room tempera-

ture with RUNX3 or RUNX1 antibodies (Ben Aziz-Aloya et al., 1998; Le et

al., 1999) and detected by the avidin-biotin peroxidase technique (ABC,

Vectastain, Vector laboratories). Slides were counterstained with hemat-

oxylin.

Fig. 2. Overlapping expression of Runx3 and Runx1. IHC staining of

Runx3 (a,c,c 0,e) and Runx1 (b,d,d 0,f) in the liver in hematopoietic precur-

sors (a,b) and in the thymus (c,c 0,d,d 0) at E15.5 and in the eyelids (e,f) at

E14.5. Note the weaker expression of Runx3 in the liver and thymus.

D. Levanon et al. / Mechanisms of Development 109 (2001) 413–417 415

Fig. 3. Expression of Runx3 and Runx1 in distinct compartments of the same tissue. (a,b) Lateral view of whole mount with skin removed. Runx31/lz and

Runx11/lz E14.5 embryos stained for b-galactosidase activity (Hames and Higgins, 1993) display complementary expression in the skeleton. (a) Runx3

expression in maturing cartilage of the metaphysis and diaphysis of long bones: scapula (S), humerus (H), radius (R), ulna (U), metacarpals (MC), femur (F),

tibia (T), fibula (Fi) and metatarsals (MT). Note the lack of Runx3 staining in immature costal cartilage (CC). (b) Runx1 expression in immature cartilage in the

proximal scapula (S), the distal acromion (A), the deltoid tuberosity (D) of the humerus, the proximal ulnar epiphysis (O, olecranon), the costal cartilage (CC),

the patella (P), and the proximal tibial epiphysis (TE). (c,d) IHC staining of Runx3 and Runx1 in dorsal root ganglia (DRG) at E15.5. Arrows indicate thoracic

DRG. (e,f) IHC staining in whiskers at E15.5. Runx3 expression is confined to the dermal papilla (DP) and the perifollicular connective tissue sheath (PFS),

whereas Runx1 is expressed in the follicular epithelium (FE). (g–j) Expression of Runx3 and Runx1 in vertebral bodies at E16.5. (g,h) Medial view of thoracic

vertebrae in a sagittally cut whole mount stained for b-galactosidase. (g) Runx3 is expressed in the central portion of each vertebral body. (h) Runx1 is

expressed in the cranial and caudal vertebral tips. Weaker expression is observed in the primary ossification centers of the vertebral bodies. (i,j) IHC staining of

vertebrae. (i) Runx3 in prehypertrophic and hypertrophic chondrocytes. (j) Runx1 in immature and hypertrophic chondrocytes. Arrows in (g–j) indicate

intervertebral spaces and arrowheads indicate the primary ossification center. Runx11/lz mice expressing a knock-in b-galactosidase/Runx1 fused product were

previously reported (North et al., 1999). Runx31/lz mice expressing a knock-in b-galactosidase as part of the Runx3 transcriptional units will be described

elsewhere.

in different cell types, supporting the idea that their function

is not redundant. The biological function of Runx3 is largely

unknown, as Runx3-deficient mice have not been described.

The present data suggest a role for Runx3 in organogenesis

of several tissues including epidermal appendages, the

skeleton and dorsal root ganglia. Of special interest is to

decipher the role of Runx3 and Runx1 in epithelial–

mesenchymal interaction and the function of the three

Runx proteins in bone development.

Acknowledgements

We thank Dorit Nathan, Judith Chermesh and Shoshana

Grossfeld for excellent technical assistance. This work was

supported by grants from the Commission of the EU, the

Israel Science Foundation and Shapell Family Biomedical

Research Foundation at the Weizmann Institute.

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