Gene expression pattern Spatial and temporal expression pattern of Runx3 (Aml2) and Runx1 (Aml1) indicates non-redundant functions during mouse embryogenesis Ditsa Levanon a,1 , Ori Brenner b,1 , Varda Negreanu a , David Bettoun a , Eilon Woolf a , Raya Eilam b , Joseph Lotem a , Uri Gat c , Florian Otto d , Nancy Speck e , Yoram Groner a, * a Department of Molecular Genetics, The Weizmann Institute of Science, Rehovot 76100, Israel b Department of Experimental Animals, The Weizmann Institute of Science, Rehovot 76100, Israel c Department of Cell and Animal Biology, The Hebrew University, Jerusalem 9194, Israel d Department of Hematology/Oncology, University of Freiburg Medical Center, 79106 Freiburg, Germany e Department 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. PII: S0925-4773(01)00537-8 www.elsevier.com/locate/modo * Corresponding author. Tel.: 1972-8-9343-972; fax: 1972-8-9344-108. E-mail address: [email protected] (Y. Groner). 1 These two authors contributed equally to this work.
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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;
E-mail address: [email protected] (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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