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Ets2 is expressed during morphogenesis of the somite and limb in the mouse embryo
Mechanisms of Development 116 (2002) 165–168 www.elsevier.com/locate/modo
Gene expression pattern
Ets2 is expressed during morphogenesis of the somite and limb in the mouse embryo Sika Ristevski a,*, Patrick P.L. Tam b, Paul J. Hertzog a, Ismail Kola a,c a
Centre for Functional Genomics and Human Disease, Monash Institute of Reproduction and Development, Monash University, Clayton, Victoria 3168, Australia b Embryology Unit, Children’s Medical Research Institute, Locked Bag 23, Wentworthville, New South Wales 2145, Australia c Pharmacia and Upjohn, 301 Henrietta St., Kalamazoo, MI 49007, USA Received 4 December 2000; received in revised form 29 March 2002; accepted 7 April 2002
Abstract Ets2 is a member of the ETS family of transcription factors. In order to address the developmental function of Ets2, we have examined its expression pattern in E8.5 to E13.5 embryos using RNA whole-mount in situ hybridization. In the paraxial mesoderm, Ets2 is expressed uniformly in the presomitic mesoderm and then restricted to the cells in the rostral portion of the segmenting and the next two recently formed somites. In the developing limb, Ets2 expression in the mesenchyme reflects the progressive formation of the hand or foot plate and the digital skeleton. In addition, Ets2 is expressed in the otic vesicle and its derivatives, the dorsal (posterior) root ganglia, the neuroepithelium in the dorsal part of the caudal neural tube and the inter-segmental vasculature. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Ets2; Somite segmentation; Limb patterning; Mouse embryo
1. Results and discussion Ets2 is one of the over 40 members of the ETS family of transcription factors that contains a winged helix-turn helix motif that binds to DNA (Graves and Petersen, 1998; Sharrocks et al., 1997). Ets2 was initially identified as a nuclear oncogene (Maroulakou et al., 1994; Dittmer and Nordheim, 1998) but has been shown to play an essential role in embryonic development. Over-expression of Ets2 in the mouse results in dysmorphogenesis of craniofacial structures which phenocopies the abnormalities encountered in the human Down syndrome (Sumarsano et al., 1996). Mutation of Ets2 that deletes the DNA binding domain of the transcription factor leads to early embryonic lethality, primarily due to defects in trophoblast differentiation (Yamamoto et al., 1998). Expression of the Ets2 gene has been examined in the gastrulating mouse embryo at E6.0– E7.5 where it is strongly expressed in the ectoplacental cone, moderately in the extraembryonic ectoderm and not in the epiblast or the visceral endoderm. During gastrulation, Ets2 mRNA is localized in the anterior two-third segment of the primitive streak (Yamamoto et al., 1998). In situ studies of the E14 embryo reveal Ets2 expression in * Corresponding author. Tel.: 161-3-9594-7217; fax: 161-3-9594-7211. E-mail address: [email protected] (S. Ristevski).
all organ systems (Kola et al., 1993; Maroulakou et al., 1994). Here we report the expression of Ets2 during organogenesis of the mouse embryo. When examined by wholemount RNA in situ hybridization of E8.5–E13.5 embryos, Ets2 is expressed in a dynamic pattern during the morphogenesis of the somite and the limb, implicating a possible role of Ets2 which has not been revealed due to the early lethality of the mutant embryo. At the early neural plate stage (E8.0), Ets2 is expressed in the anterior two-third segment of the primitive streak (Fig. 1A), where the progenitors of the lateral and paraxial mesoderm ingress to join the mesodermal tissues (Kinder et al., 1999). At the early somite-stage (E.8.5), Ets2 expression persists in the primitive streak and, in addition, is expressed uniformly in the presomitic mesoderm (the unsegmented paraxial mesoderm that later segments into somites) (Fig. 1B), with some hints of regionalized expression in the anterior part of the newly segmented somite (Fig. 1D). In the rostral end of the presomitic mesoderm of the E9.5 (26–32 somite stage) embryo, Ets2 expression is distinctly confined to two broad transverse domains of the mesenchyme, which may correspond to the anterior half of the two most rostral somitomeres, and to the anterior halves of the two recently segmented somites (Fig. 1C, E). The meristic pattern of Ets2 expression in the paraxial mesoderm remains evident in the E11.5 and E12.5 embryo where strong expression is loca-
0925-4773/02/$ - see front matter q 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0925-477 3(02)00126-0
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Fig. 1. Expression of Ets2 in the paraxial mesoderm revealed by wholemount RNA in situ hybridization. Whole embryo is shown in (A) E8.0 neural plate stage, (B) E8.5 early somite stage, (C) E9.5 early organogenesis stage and the caudal region for embryos is shown in (D) E8.5, (E) E9.5, (F) E11.5 and (G) E12.5. The expression of Ets2 in the inter-segmental vessels is shown in (H) whole-mount and (I) histological section of the trunk somites of the E12.5 embryo. Abbreviations: PS, primitive streak; EC, ectoplacental cone; R, rostral; C, caudal; PSM, presomitic mesoderm; A, anterior; P, posterior; FLB, forelimb bud; HLB, hindlimb bud; NT, neural tube; S, somite; VE, vascular edolthelium. The scale bar represents 100 mm.
lized to the rostral border of the segmenting somitomere and the newly formed somites (Fig. 1F, G). The regionalized pattern of Ets2 expression in the paraxial mesoderm throughout somitogenesis strongly implicates a role for Ets2 in the segmentation and rostro-caudal patterning of the somite. In the more rostral region of the paraxial mesoderm, Ets2 expression is down-regulated in the somite, but is found in the endothelium of the inter-segmental vessels (Fig. 1H, I). Ets2 expression is highly dynamic during the development of the limb buds. In the forelimb, expression is first expressed broadly in the mesenchyme (Fig. 2A) and is then progressively restricted to mesenchyme in the posterior half of the limb bud (Fig. 2B). As the limb bud grows distally, Ets2 expression becomes localized to a crescent band of mesenchyme in the distal part of the limb bud with a diminishing level of expression from the posterior to the anterior region (Fig. 2C, F). An additional site of expression appears after the initial down-regulation in the proximal anterior mesenchyme of the limb bud (Fig. 2C). This Ets2-expressing domain is later localized to the shoulder region of the forelimb (Fig. 2D) and the hip of the hindlimb (Fig. 2I). In the E11.5 embryo Ets2 expression in the mesenchyme is down-regulated, but expression remains strong in the mesenchyme in the anterior and posterior margin (Fig. 2D). The anterior and posterior zones of Ets2 activity demarcate the proximal constriction of the hand and foot
Fig. 2. Expression of Ets2 in the limb bud revealed by whole-mount RNA in situ hybridization in fore limb bud in (A) E9.5, (B,F) E10, (C) E11.5, (E) E12.5 and the hindlimb at (E) E13.5. Limb buds in (A–F) are shown in the same orientation. The hindlimb and ventral body wall of embryos is shown in (G) E10, (H) E11.5 and (I) E12.5. The arrows in (C) and (D) indicate expression in the prospective shoulder and shoulder region, respectively. The arrow in (I) indicates expression in the hip. Abbreviations: R, rostral; P, proximal; D, distal; C, caudal; W, prospective wrist; ID, inter-digital tissues; PC, perichondrium; M, mesenchyme; E, ectoderm. The scale bar represents 100 mm.
plate of the limb (Fig. 2D), at the pre- and post-axial margin of digit I and V, respectively (Fig. 2D, E). As the digits develop, Ets2 is expressed in the cells of the condensing mesenchyme that forms the perichondrium of the phalangeal cartilage (Fig. 2E). Ets2 is not expressed in the surface ectoderm of the limb bud (Fig. 2F). In the hindlimb bud, Ets2 expression is similar to that in the forelimb bud except for the time lag in the development of the hind limb bud (Fig. 3A–D). In contrast to the forelimb bud, the expression domain in the hindlimb extends ventromedially from the limb bud mesenchyme to the ventral body wall but stops short on either side of the tissues in the midline (Fig. 2G–I), which extends from the cloaca to join the ventral ectodermal groove of the tail. Ets2 mRNA is not confined to mesodermal tissues but is also detected in the ectodermal derivatives such as the neuroepithelium of the posterior neuropore (Fig. 1B) and the posterior neural tube (Figs. 1C, F, G and 3F, G), the dorsal epithelium of the otic vesicles and the endolymphatic diverticulum that is derived from this epithelium (Fig. 3A– C, H–I), and in the dorsal (posterior) root ganglia derived from the neural crest cells (Fig. 3B, C, E). In the facial
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Fig. 3. (A–D) A comparison of Ets2 expression in the forelimb and hindlimb buds of (A) E10.5, (B) E11.5, (C) E12.5 and (D) E13.5 embryos. Expression of Ets2 in (E) dorsal (posterior) root ganglia of E12.5 embryo, (F) neural tube in the tail showing uniform expression in the entire neuroepithelium, (G) neural tube at the level of the hindlimb showing expression only in the dorsal neuroepithelium of E10.5 embryos, (H,I) the epithelium of the otic vesicles of E10.5 embryo and (J) the endolymphatic diverticulum of E11.5 embryo. Abbreviations: A, anterior; P, posterior; OV, otic vesicle; E, ectoderm; Vi, vibrissae; R, rostral; C, caudal; DRG, dorsal (posterior) root ganglia; NT, neural tube; D, dorsal; V, ventral; ED, endolymphatic duct. The arrows in (B) and (C) indicate expression in the branchial arch surface ectoderm. The scale bars represent 100 mm.
region, Ets2 is expressed first in the ectoderm in the distal portion (Fig. 3B) and later in the ventral surface ectoderm of the first and second arches (Fig. 3C), and in the vibrissae (Fig. 3D). The dynamic expression of Ets2 in the paraxial mesoderm and the limb bud is consistent with an activity in tissue patterning in these two embryonic structures (Tam et al., 2000; Johnson and Tabin, 1997; Zeller and Duboule, 1997). The meristic pattern of expression in the somitomeres and somites is reminiscent of that displayed by genes such as Jagged1, Dll1, Dll3, Notch1 and Notch2 that encode ligands and receptors of the Notch signaling (Gossler and Hrabe de Angelis, 1998; Tam et al., 2000). Of particular significance is the reciprocal pattern of expression of Dll1, Lnfg and Hes5 and that of Ets2 in different halves of the segment, which raises the possibility that Ets2 is associated with the rostro-caudal partitioning of the somite.
and used to generate digoxigenin-labelled sense and antisense transcripts according to the manufacturer’s instructions (Boehringer Mannheim). Embryos were recovered from F1 (C57Bl6 £ CBA) natural mating as described in Hogan et al. (1994), fixed in 4% paraformaldehyde in phosphate buffered saline (PBS) for 24 h at 48C and dehydrated through a methanol series. Embryos were then bleached in 5% hydrogen peroxide in methanol until no background color remained. Whole-mount RNA in situ hybridization was performed essentially as described in Wilkinson (1992) with the exclusion of embryo powder. Proteinase K treatment was 10 mg/ ml incubated for 15 min at room temperature. 2.2. Histology Embryos were dehydrated, mounted in polyester wax and 8 mm sections counterstained with nuclear fast red. Alternatively, embryos were cryo-embedded in OCT compound and 10 mm cryosections examined without counterstaining.
2. Materials and methods Acknowledgements 2.1. Whole-mount RNA in situ hybridization Mouse Ets2 (Genbank Accession No. NM011809) spanning nucleotide positions 630–1066 (exons VI–VII) was subcloned into pGEM-7Zf(1) (Promega) (Maroulakou et al., 1994; previously demonstrating specificity for Ets2)
We thank Ernst Wolvetang and Tanya Hatzistavrou for helpful discussions and Ann Davies for assistance with histology. Our work was supported by the National Health and Medical Research Council (NHMRC) of Australia. The Monash Institute of Reproduction and Development is
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S. Ristevski et al. / Mechanisms of Development 116 (2002) 165–168
established and supported by Monash University. P.J.H. is a NHMRC Principal Research Fellow and P.P.L.T. is a NHMRC Senior Principal Research Fellow. References Dittmer, J., Nordheim, A., 1998. Ets transcription factors and human disease. Biochim. Biophys. Acta 1377 (2), F1–F11. Gossler, A., Hrabe de Angelis, M., 1998. Curr. Top. Dev. Biol.. Somitogenesis 38, 225–287. Graves, B.J., Petersen, J.M., 1998. Specificity within the ets family of transcription factors. Adv. Cancer Res. 75, 1–55. Hogan, B., Beddington, R., Costantini, F., Lacy, E., 1994. Manipulating the Mouse Embryo: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Johnson, R.L., Tabin, C.J., 1997. Molecular models for vertebrate limb development. Cell 90, 979–990. Kinder, S.J., Tsang, T.E., Quinlan, G.A., Hadjantonakis, A.-K., Nagy, A., Tam, P.P.L., 1999. The orderly allocation of mesodermal cells to the extraembryonic structures and the anteroposterior axis during gastrulation of the mouse embryo. Development 126, 4691–4701. Kola, I., Brookes, S., Green, A.R., Garber, R., Tymms, M., Papas, T.S., Seth, A., 1993. The Ets1 transcription factor is widely expressed during murine embryo development and is associated with mesodermal cells
involved in morphogenetic processes such as organ formation. Proc. Natl Acad. Sci. USA 90, 7588–7592. Maroulakou, I.G., Papas, T.S., Green, J.E., 1994. Differential expression of ets-1 and ets-2 protooncogenes during murine embryogenesis. Oncogene 9, 1551–1565. Sharrocks, A.D., Brown, A.L., Ling, Y., Yates, P.R., 1997. The ETSdomain transcription factor family. Int. J. Biochem. Cell Biol. 29 (12), 1371–1387. Sumarsano, S.H., Wilson, T.J., Tymms, M.J., Venter, D.J., Corrick, C.M., Kola, R., Lahoud, M.H., Papas, T.S., Seth, A., Kola, I., 1996. Down’s syndrome-like skeletal abnormalities in Ets2 transgenic mice. Nature 379, 534–537. Tam, P.P.L., Goldman, D., Camus, A., Schoenwolf, G.C., 2000. Early events of somitogenesis in higher vertebrates: allocation of precursor cells during gastrulation and the organization of a meristic pattern in the paraxial mesoderm. Curr. Top. Dev. Biol. 47, 1–32. Wilkinson, D.G., 1992. Whole mount in situ hybridisation of vertebrate embryos. In: Wilkinson, D. (Ed.). In situ Hybridisation, IRL Press, Oxford, pp. 75–83. Yamamoto, H., Flannery, M.L., Kupriyanov, S., Pearce, J., McKercher, S.R., Henkel, G.W., Maki, R.A., Werb, Z., Oshima, R.G., 1998. Efective trophoblast function in mice with a targeted mutation of Ets2. Genes Dev. 12, 1315–1326. Zeller, R., Duboule, D., 1997. Dorso-ventral limb polarity and origin of the ridge: on the fringe of independence. Bioessays 19, 541–546.
Gene expression pattern
Ets2 is expressed during morphogenesis of the somite and limb in the mouse embryo Sika Ristevski a,*, Patrick P.L. Tam b, Paul J. Hertzog a, Ismail Kola a,c a
Centre for Functional Genomics and Human Disease, Monash Institute of Reproduction and Development, Monash University, Clayton, Victoria 3168, Australia b Embryology Unit, Children’s Medical Research Institute, Locked Bag 23, Wentworthville, New South Wales 2145, Australia c Pharmacia and Upjohn, 301 Henrietta St., Kalamazoo, MI 49007, USA Received 4 December 2000; received in revised form 29 March 2002; accepted 7 April 2002
Abstract Ets2 is a member of the ETS family of transcription factors. In order to address the developmental function of Ets2, we have examined its expression pattern in E8.5 to E13.5 embryos using RNA whole-mount in situ hybridization. In the paraxial mesoderm, Ets2 is expressed uniformly in the presomitic mesoderm and then restricted to the cells in the rostral portion of the segmenting and the next two recently formed somites. In the developing limb, Ets2 expression in the mesenchyme reflects the progressive formation of the hand or foot plate and the digital skeleton. In addition, Ets2 is expressed in the otic vesicle and its derivatives, the dorsal (posterior) root ganglia, the neuroepithelium in the dorsal part of the caudal neural tube and the inter-segmental vasculature. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Ets2; Somite segmentation; Limb patterning; Mouse embryo
1. Results and discussion Ets2 is one of the over 40 members of the ETS family of transcription factors that contains a winged helix-turn helix motif that binds to DNA (Graves and Petersen, 1998; Sharrocks et al., 1997). Ets2 was initially identified as a nuclear oncogene (Maroulakou et al., 1994; Dittmer and Nordheim, 1998) but has been shown to play an essential role in embryonic development. Over-expression of Ets2 in the mouse results in dysmorphogenesis of craniofacial structures which phenocopies the abnormalities encountered in the human Down syndrome (Sumarsano et al., 1996). Mutation of Ets2 that deletes the DNA binding domain of the transcription factor leads to early embryonic lethality, primarily due to defects in trophoblast differentiation (Yamamoto et al., 1998). Expression of the Ets2 gene has been examined in the gastrulating mouse embryo at E6.0– E7.5 where it is strongly expressed in the ectoplacental cone, moderately in the extraembryonic ectoderm and not in the epiblast or the visceral endoderm. During gastrulation, Ets2 mRNA is localized in the anterior two-third segment of the primitive streak (Yamamoto et al., 1998). In situ studies of the E14 embryo reveal Ets2 expression in * Corresponding author. Tel.: 161-3-9594-7217; fax: 161-3-9594-7211. E-mail address: [email protected] (S. Ristevski).
all organ systems (Kola et al., 1993; Maroulakou et al., 1994). Here we report the expression of Ets2 during organogenesis of the mouse embryo. When examined by wholemount RNA in situ hybridization of E8.5–E13.5 embryos, Ets2 is expressed in a dynamic pattern during the morphogenesis of the somite and the limb, implicating a possible role of Ets2 which has not been revealed due to the early lethality of the mutant embryo. At the early neural plate stage (E8.0), Ets2 is expressed in the anterior two-third segment of the primitive streak (Fig. 1A), where the progenitors of the lateral and paraxial mesoderm ingress to join the mesodermal tissues (Kinder et al., 1999). At the early somite-stage (E.8.5), Ets2 expression persists in the primitive streak and, in addition, is expressed uniformly in the presomitic mesoderm (the unsegmented paraxial mesoderm that later segments into somites) (Fig. 1B), with some hints of regionalized expression in the anterior part of the newly segmented somite (Fig. 1D). In the rostral end of the presomitic mesoderm of the E9.5 (26–32 somite stage) embryo, Ets2 expression is distinctly confined to two broad transverse domains of the mesenchyme, which may correspond to the anterior half of the two most rostral somitomeres, and to the anterior halves of the two recently segmented somites (Fig. 1C, E). The meristic pattern of Ets2 expression in the paraxial mesoderm remains evident in the E11.5 and E12.5 embryo where strong expression is loca-
0925-4773/02/$ - see front matter q 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0925-477 3(02)00126-0
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Fig. 1. Expression of Ets2 in the paraxial mesoderm revealed by wholemount RNA in situ hybridization. Whole embryo is shown in (A) E8.0 neural plate stage, (B) E8.5 early somite stage, (C) E9.5 early organogenesis stage and the caudal region for embryos is shown in (D) E8.5, (E) E9.5, (F) E11.5 and (G) E12.5. The expression of Ets2 in the inter-segmental vessels is shown in (H) whole-mount and (I) histological section of the trunk somites of the E12.5 embryo. Abbreviations: PS, primitive streak; EC, ectoplacental cone; R, rostral; C, caudal; PSM, presomitic mesoderm; A, anterior; P, posterior; FLB, forelimb bud; HLB, hindlimb bud; NT, neural tube; S, somite; VE, vascular edolthelium. The scale bar represents 100 mm.
lized to the rostral border of the segmenting somitomere and the newly formed somites (Fig. 1F, G). The regionalized pattern of Ets2 expression in the paraxial mesoderm throughout somitogenesis strongly implicates a role for Ets2 in the segmentation and rostro-caudal patterning of the somite. In the more rostral region of the paraxial mesoderm, Ets2 expression is down-regulated in the somite, but is found in the endothelium of the inter-segmental vessels (Fig. 1H, I). Ets2 expression is highly dynamic during the development of the limb buds. In the forelimb, expression is first expressed broadly in the mesenchyme (Fig. 2A) and is then progressively restricted to mesenchyme in the posterior half of the limb bud (Fig. 2B). As the limb bud grows distally, Ets2 expression becomes localized to a crescent band of mesenchyme in the distal part of the limb bud with a diminishing level of expression from the posterior to the anterior region (Fig. 2C, F). An additional site of expression appears after the initial down-regulation in the proximal anterior mesenchyme of the limb bud (Fig. 2C). This Ets2-expressing domain is later localized to the shoulder region of the forelimb (Fig. 2D) and the hip of the hindlimb (Fig. 2I). In the E11.5 embryo Ets2 expression in the mesenchyme is down-regulated, but expression remains strong in the mesenchyme in the anterior and posterior margin (Fig. 2D). The anterior and posterior zones of Ets2 activity demarcate the proximal constriction of the hand and foot
Fig. 2. Expression of Ets2 in the limb bud revealed by whole-mount RNA in situ hybridization in fore limb bud in (A) E9.5, (B,F) E10, (C) E11.5, (E) E12.5 and the hindlimb at (E) E13.5. Limb buds in (A–F) are shown in the same orientation. The hindlimb and ventral body wall of embryos is shown in (G) E10, (H) E11.5 and (I) E12.5. The arrows in (C) and (D) indicate expression in the prospective shoulder and shoulder region, respectively. The arrow in (I) indicates expression in the hip. Abbreviations: R, rostral; P, proximal; D, distal; C, caudal; W, prospective wrist; ID, inter-digital tissues; PC, perichondrium; M, mesenchyme; E, ectoderm. The scale bar represents 100 mm.
plate of the limb (Fig. 2D), at the pre- and post-axial margin of digit I and V, respectively (Fig. 2D, E). As the digits develop, Ets2 is expressed in the cells of the condensing mesenchyme that forms the perichondrium of the phalangeal cartilage (Fig. 2E). Ets2 is not expressed in the surface ectoderm of the limb bud (Fig. 2F). In the hindlimb bud, Ets2 expression is similar to that in the forelimb bud except for the time lag in the development of the hind limb bud (Fig. 3A–D). In contrast to the forelimb bud, the expression domain in the hindlimb extends ventromedially from the limb bud mesenchyme to the ventral body wall but stops short on either side of the tissues in the midline (Fig. 2G–I), which extends from the cloaca to join the ventral ectodermal groove of the tail. Ets2 mRNA is not confined to mesodermal tissues but is also detected in the ectodermal derivatives such as the neuroepithelium of the posterior neuropore (Fig. 1B) and the posterior neural tube (Figs. 1C, F, G and 3F, G), the dorsal epithelium of the otic vesicles and the endolymphatic diverticulum that is derived from this epithelium (Fig. 3A– C, H–I), and in the dorsal (posterior) root ganglia derived from the neural crest cells (Fig. 3B, C, E). In the facial
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Fig. 3. (A–D) A comparison of Ets2 expression in the forelimb and hindlimb buds of (A) E10.5, (B) E11.5, (C) E12.5 and (D) E13.5 embryos. Expression of Ets2 in (E) dorsal (posterior) root ganglia of E12.5 embryo, (F) neural tube in the tail showing uniform expression in the entire neuroepithelium, (G) neural tube at the level of the hindlimb showing expression only in the dorsal neuroepithelium of E10.5 embryos, (H,I) the epithelium of the otic vesicles of E10.5 embryo and (J) the endolymphatic diverticulum of E11.5 embryo. Abbreviations: A, anterior; P, posterior; OV, otic vesicle; E, ectoderm; Vi, vibrissae; R, rostral; C, caudal; DRG, dorsal (posterior) root ganglia; NT, neural tube; D, dorsal; V, ventral; ED, endolymphatic duct. The arrows in (B) and (C) indicate expression in the branchial arch surface ectoderm. The scale bars represent 100 mm.
region, Ets2 is expressed first in the ectoderm in the distal portion (Fig. 3B) and later in the ventral surface ectoderm of the first and second arches (Fig. 3C), and in the vibrissae (Fig. 3D). The dynamic expression of Ets2 in the paraxial mesoderm and the limb bud is consistent with an activity in tissue patterning in these two embryonic structures (Tam et al., 2000; Johnson and Tabin, 1997; Zeller and Duboule, 1997). The meristic pattern of expression in the somitomeres and somites is reminiscent of that displayed by genes such as Jagged1, Dll1, Dll3, Notch1 and Notch2 that encode ligands and receptors of the Notch signaling (Gossler and Hrabe de Angelis, 1998; Tam et al., 2000). Of particular significance is the reciprocal pattern of expression of Dll1, Lnfg and Hes5 and that of Ets2 in different halves of the segment, which raises the possibility that Ets2 is associated with the rostro-caudal partitioning of the somite.
and used to generate digoxigenin-labelled sense and antisense transcripts according to the manufacturer’s instructions (Boehringer Mannheim). Embryos were recovered from F1 (C57Bl6 £ CBA) natural mating as described in Hogan et al. (1994), fixed in 4% paraformaldehyde in phosphate buffered saline (PBS) for 24 h at 48C and dehydrated through a methanol series. Embryos were then bleached in 5% hydrogen peroxide in methanol until no background color remained. Whole-mount RNA in situ hybridization was performed essentially as described in Wilkinson (1992) with the exclusion of embryo powder. Proteinase K treatment was 10 mg/ ml incubated for 15 min at room temperature. 2.2. Histology Embryos were dehydrated, mounted in polyester wax and 8 mm sections counterstained with nuclear fast red. Alternatively, embryos were cryo-embedded in OCT compound and 10 mm cryosections examined without counterstaining.
2. Materials and methods Acknowledgements 2.1. Whole-mount RNA in situ hybridization Mouse Ets2 (Genbank Accession No. NM011809) spanning nucleotide positions 630–1066 (exons VI–VII) was subcloned into pGEM-7Zf(1) (Promega) (Maroulakou et al., 1994; previously demonstrating specificity for Ets2)
We thank Ernst Wolvetang and Tanya Hatzistavrou for helpful discussions and Ann Davies for assistance with histology. Our work was supported by the National Health and Medical Research Council (NHMRC) of Australia. The Monash Institute of Reproduction and Development is
168
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established and supported by Monash University. P.J.H. is a NHMRC Principal Research Fellow and P.P.L.T. is a NHMRC Senior Principal Research Fellow. References Dittmer, J., Nordheim, A., 1998. Ets transcription factors and human disease. Biochim. Biophys. Acta 1377 (2), F1–F11. Gossler, A., Hrabe de Angelis, M., 1998. Curr. Top. Dev. Biol.. Somitogenesis 38, 225–287. Graves, B.J., Petersen, J.M., 1998. Specificity within the ets family of transcription factors. Adv. Cancer Res. 75, 1–55. Hogan, B., Beddington, R., Costantini, F., Lacy, E., 1994. Manipulating the Mouse Embryo: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Johnson, R.L., Tabin, C.J., 1997. Molecular models for vertebrate limb development. Cell 90, 979–990. Kinder, S.J., Tsang, T.E., Quinlan, G.A., Hadjantonakis, A.-K., Nagy, A., Tam, P.P.L., 1999. The orderly allocation of mesodermal cells to the extraembryonic structures and the anteroposterior axis during gastrulation of the mouse embryo. Development 126, 4691–4701. Kola, I., Brookes, S., Green, A.R., Garber, R., Tymms, M., Papas, T.S., Seth, A., 1993. The Ets1 transcription factor is widely expressed during murine embryo development and is associated with mesodermal cells
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