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Restricted expression of DMRT3 in chicken and mouse embryos
Mechanisms of Development 119S (2002) S73–S76 www.elsevier.com/locate/modo
Restricted expression of DMRT3 in chicken and mouse embryos Craig A. Smith*, Tanya M. Hurley, Peter J. McClive, Andrew H. Sinclair Murdoch Children’s Research Institute and Department of Paediatrics, The University of Melbourne, Royal Children’s Hospital, Parkville, Victoria 3052, Australia Received 28 June 2002; received in revised form 12 September 2002; accepted 16 September 2002
Abstract Vertebrate DM domain genes encode a novel group of proteins related to the Drosophila doublesex and Caenorhabditis elegans mab-3 transcription factors. It is shown here that the recently identified gene, DMRT3, has a restricted embryonic expression profile that is conserved in chicken and mouse embryos. DMRT3 is expressed primarily in the forebrain, neural tube and nasal placode of both species. In the chicken, DMRT3 is also expressed in newly forming tail somites at early developmental stages and, later, in the Mu¨llerian ducts of the urogenital system. q 2003 Elsevier Science Ireland Ltd. All rights reserved. Keywords: DMRT3; DM domain; Cerebral vesicle; Nasal placode; Neural tube; Mu¨llerian duct; Somitogenesis
1. Results and discussion The Drosophila doublesex and Caenorhabditis elegans mab-3 genes encode transcription factors characterized by a conserved zinc finger-like DNA-binding motif, the DM domain. A vertebrate homologue has been identified and named DMRT1 (Doublesex and mab-3-related transcription factor, number 1) (Raymond et al., 1998). DMRT1 is expressed exclusively in the urogenital system and is implicated in vertebrate testis differentiation (Raymond et al., 1998; Smith et al., 1999; Kettlewell et al., 2000; Guan et al., 2000; Moniot et al., 2000). Human DMRT1 is part of a DM gene cluster on chromosome 9p, comprising the series DMRT1-DMRT3-DMRT2 (Brunner et al., 2001; Ottolenghi et al., 2002). These genes share the highly conserved DM domain but have little or no homology outside this region. DMRT2 is expressed primarily in the presomitic mesoderm and in early forming somites of vertebrate embryos (Meng et al., 1999). It is shown here that DMRT3 also has a conserved, restricted expression pattern in vertebrate (chicken and mouse) embryos. This expression pattern differs from that of DMRT1 and DMRT2. In both chicken and mouse embryos, DMRT3 is expressed primarily in the developing forebrain, neural tube and nasal placodes. In the chicken, DMRT3 is also expressed in early forming somites and presomitic mesoderm, and in the Mu¨llerian ducts. * Corresponding author. Tel.: 161-3-9345-6607; fax: 161-3-9345-6000. E-mail address: [email protected] (C.A. Smith).
Embryonic expression of DMRT3 was studied using whole mount in situ hybridization. For both species, embryos were examined during comparable early stages of development. For the chicken, embryos were examined at embryonic day (E)1 (stage 9), E2 (stage 14), E2.5 (stage 16), E3 (stage 18), E4.5 (stage 25) and E7.5 (stage 32). For the mouse, E8.5, 9.5 10.5 and 14.5 embryos were examined. Expression of chicken DMRT3 is shown in Fig. 1. The earliest identifiable site of expression was within newly forming somites and presomitic mesoderm in stage 9 (E1) embryos (Fig. 1A). At stage 14 (E2) expression was maintained in the most posterior, early-forming somites, while some weaker expression was apparent in the prospective forebrain region (prosencephalon) (Fig. 1B). By E2.5 (stage 16) expression had become stronger in the prosencephalon, in the region of the developing telencephalon (Fig. 1C). Strong expression was also now apparent in the nasal (olfactory) placodes (Fig. 1C). In the forebrain region, expression was localized along the midline of the developing telencephalon and also along its dorso-caudal walls (Fig. 1D). By E3 (stage 18), expression persisted within the cerebral vesicles of the telencephalon, the paired nasal placodes and also along the lateral rims of the maxilla. Weaker expression was apparent in the diencephalon (Fig. 1E). Sagittal sections through the head region of E3 embryos revealed restricted expression within the neuroepithelium of the telencephalon (dorsal and dorso-caudal walls) (Fig. 1F). In the facial region, expression was strong within the epithelium of the nasal placodes (Fig. 1G). Expression in the newly forming
0925-4773/03/$ - see front matter q 2003 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0925-477 3(03)00094-7
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Fig. 1. DMRT3 expression in the chicken embryo. (A) Embryonic (E) day 1 (stage 9). Expression in the caudal (youngest) somite pair and presomitic mesoderm (Sm). (B) E2 (stage 14). Expression in the caudal somites/presomitic mesoderm (Sm) and, weakly, in the presumptive forebrain region or prosencephalon (Pros). (C) E2.5 (stage 16). Stronger expression in the caudal somites (Sm) and in the prosencephalon (Pros). Expression is now apparent in the nasal placode (Npl). (D) E2.5 (stage 16). Dorso-frontal view of the head, showing restricted expression in the midline and caudo-lateral regions of the developing Forebrain (arrows), and in nasal placodes (Npl). Mb, midbrain; Hb, hindbrain. (E) E3 (stage 18) Expression in the telencephalon (Tel) and diencephalon (Di) of the forebrain, in nasal placodes (Npl), lateral maxillary process (Mx) and branchial arches (Ba). Staining in the otic vesicle and eye is non-specific. (F) E3 (stage 18). Parasagittal section through the head region, showing expression in the neuroepithelial walls of the dorso-caudal telencephalon (Tel). Mb, midbrain. (G) E3 (stage 18). Parasagittal section through the facial region, showing strong expression in the epithelium of the developing nasal placode (arrow). (H) E3 (stage 18). Expression in the anterior region of the newly forming somites (Sm) and presomitic mesoderm (Psm). Nt, neural tube. (I) E3 (stage 18). Lateral view, with somites/mesoderm removed, revealing a line of expressing cells along the middle region of the neural tube (arrows). (J) E3 (stage 18). Longitudinal section through the neural tube, showing expression among cells along the dorsomedial region (D), but with some expressing cells also scattered ventrally (V). Nt, neural tube; Sm, somites. (K) E3 (stage 18). Transverse section through the neural tube, showing expression within the intermediate (marginal) zone (iz), and not in the ventricular zone (vz). (L) E4.5 (stage 25). Restricted expression persists in the Telencephalon (Tel) and nasal pits (Npi). No somite expression. (M) E4.5 (stage 25). Ventral view of the head, showing strong expression in the epithelium of the nasal pits (Npi) (derived from the nasal placode). Pal, palate. (N) E7.5 (stage 32) female. Dorsal view of the urogenital system, showing strong expression in the paired Mu¨ llerian ducts (Md) but not in the mesonephric kidneys (Ms). (O) E7.5 (stage 32) male. Weaker expression in the Mu¨ llerian ducts. Bars: 0.5 mm in (A–E,H,I,L–O); 100 mm in (F,G,J,K).
C.A. Smith et al. / Mechanisms of Development 119S (2002) S73–S76
(most posterior) somites and presomitic mesoderm continued through stage 18 (E3) (Fig. 1E). In both the somites and presomitic mesoderm, expression was concentrated at the anterior regions, with expression tapering posteriorly (Fig. 1H). Several other genes expressed in the newly forming somites show the same pattern of anterior-posterior expression (e.g. Delta and Notch). During somitogenesis, chicken DMRT3 expression was dynamic, being manifest only in the anterior regions of the newly forming somite pair and presomitic mesoderm. Expression was lost as somites aged, and was extinguished altogether by day 4.5 (stage 25) (Fig. 1L). DMRT3 also showed restricted expression in the developing neural tube, first evident at E2. By E3 (stage 18), a fine line of expressing cells was evident running the length of the neural tube (Fig. 1I). These cells were observed bilaterally along the long axis of the neural tube. Longitudinal sections through the neural tube showed a discrete domain of positive cells arranged along the dorso-medial region of the neural tube, but with some cells scattered ventrally (Fig. 1J). Transverse sections showed that these DMRT3-expressing cells were located within the intermediate or marginal zone of the neural tube (Fig. 1K). This restricted expression pattern of DMRT3 in the chicken embryo persisted through subsequent stages. By E4.5 (stage 25), expression was still evident in the telencephalon (although weaker) and in the nasal placodes (Fig. 1L). The nasal placodes had by this stage given rise to nasal pits, where strong epithelial expression was observed (Fig. 1M). Bilateral expression persisted along the length of the neural tube, but somitic expression had disappeared (not shown). In older embryos (E7.5; stage 32). sexually dimorphic expression was detected in the Mu¨ llerian ducts of the urogenital system, with higher expression in females compared to males (Fig. 1M,N). No expression was seen in the gonads of either sex (not shown). A similar pattern of DMRT3 expression was observed in mouse embryos (Fig. 2). In the mouse, the first detectable site of expression was the cephalic neural fold of E8.5 embryos, in the presumptive forebrain region (Fig. 2A,B). In E9.5 and 10.5 embryos, strong expression was seen in the developing forebrain (cerebral vesicles of the telencephalon) and in the nasal placodes (Fig. 2C). Some expression was also detectable in the post-anal gut and in a small group of cells adjacent to the branchial arches, in the region of the vagal outflow tract (Fig. 2C). Sagittal sections through the head region showed strong DMRT3 expression within the neuroepithelium of the telencephalon, as in the chicken at comparable stages (Fig. 2D). Expression in the nasal placodes was localized within the outer epithelial layers (Fig. 2E,F). As in the chicken, a bilateral line of DMRT3positive cells was detectable running the length of the neural tube in E9.5 and E10.5 embryos. In longitudinal section, these cells were found along the dorso-medial region of the neural tube (Fig. 2G), and in transverse sections, within the intermediate (marginal) zone (but not in the ventricular or ependymal layer) (Fig. 2H).
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As in the chicken, these conserved sites of expression persisted in older embryos. Mouse DMRT3 expression persisted in the forebrain and nasal pits in E12–14.5 embryos (not shown), In addition, in E14.5 embryos, some weak expression was detectable in the developing testes of male embryos (but not in ovaries of females)
Fig. 2. DMRT3 expression in the mouse embryo. (A) E8.5, dorso-lateral view. Whole embryo, showing weak expression in the cephalic neural fold (cnf). (B) E8.5 lateral view, showing localization of expression at the tips of the cephalic neural folds (cnf) in the presumptive forebrain region. (C) E10.5, showing strong expression in the telencephalon (Tel), nasal placodes (Npl), post-anal gut (G) and adjacent to the vagal tract outfow (arrow). Staining in otic vesicle is non-specific. (D) E10.5 Section through the head region, showing strong expression in the neuroepithelial walls of the telencephalon (Tel), and not in the midbrain (Mb). (E) E10.5 Ventral view of the head, with lower jaw removed, showing expression within the nasal placodes (Npl). (F) E10.5 section through the nasal region, showing expression localized in the epithelium of the nasal placode (Npl). (G) E10.5. Longitudinal section through the neural tube, showing expression along the long axis, concentrated within the dorsomedial region. D, dorsal; V, ventral; Nt, nerual tube; Sm, somites. (H) E10.5 Transverse section through the neural tube, showing expression limited to a specific group of cells in the intermediate (marginal) zone (iz; arrows). vz, ventricular zone. (I) E14.5 male urogenital system, showing expression in the testes (Ts). Kd, kidneys. (J) E14.5 female urogenital system, showing absence of expression in ovaries (Ov). Kd, kidneys. Bars: 0.5 mm in (A–C,E); 100 mm in (D,F–H).
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(Fig. 2I,J). This contrasts with the expression profile in chicken embryos, in which DMRT3 transcripts were detectable in the Mu¨ llerian ducts but not in the gonads. Also in contrast to chicken embryos, no somite expression was observed in the mouse. However, overall, DMRT3 shows a restricted expression profile that is largely conserved in the two species. 2. Experimental procedures Specific chicken and mouse DMRT3 DNA fragments were amplified from genomic DNA using degenerate primers. These primers were designed by aligning human and pufferfish DMRT3 with that of a homologous mouse EST (BI904023). The mouse sequence spanned the 3 0 region unique to the DMRTA1-3 subfamily, outside the DM domain (Ottolenghi et al., 2002). Polymerase chain reaction fragments were cloned into PCRscript vector and sequenced to confirm identity (GenBank numbers pending). These sequences were then used as templates to generate antisense and sense RNA probes. Whole-mount in situ hybridization was carried out on paraformaldehyde-fixed embryos or tissues as described previously (Andrews et al., 1997). For sectioning, some tissues were left in chromogen solution for an extended period (2 days), cryoprotected in 20% sucrose/PBS, and snap frozen in OCT embedding compound. Fourteen- to eighteen-micrometer frozen sections were taken. Acknowledgements C.A.S. is supported by an Australian National Health and
Medical Research Council (NH&MRC) R. Douglas Wright Fellowship. The project is supported by an NH&MRC grant to A.H.S. and C.A.S. Dr Bronwyn Morrish and Dr Helen Wilmore provided some of the mouse tissues.
References Andrews, J.E.A., Smith, C.A., Sinclair, A.H., 1997. Sites of estrogen receptor and aromatase expression in the chicken embryo. Gen. Comp. Endocrinol. 108, 182–190. Brunner, B., Hornung, U., Shan, Z., Nanda, I., Kondo, M., Zend-Ajusch, E., Haaf, T., Ropers, H.-H., Shima, A., Schmind, M., Kalscheuer, V.M., Schartl, M., 2001. Genomic organisation and expression of the doublesex-related gene cluster in vertebrates and detection of putative regulatory regions for DMRT1. Genomics 77, 8–17. Guan, G., Kobayashi, T., Nagahama, Y., 2000. Sexually dimoprhic expression of two types of DM (Doublesex-mab-3)-domain genes in a teleost fish, the Tilapia (Oreochromis niloticus). Biochem. Biophys. Res. Commun. 16, 662–666. Kettlewell, J.R., Raymond, C.S., Zarkower, D., 2000. Temperature-dependent expression of turtle Dmrt1 prior to sexual differentiation. Genesis 26, 174–178. Meng, A., Moore, B., Tang, H., Yuan, B., Lin, S., 1999. A doublesexrelated gene, terra, is involved in somitogenesis in vertebrates. Development 126, 1259–1268. Moniot, B., Berta, P., Scherer, G., Su¨ dbeck, P., Poulat, F., 2000. Male specific expression suggests role of DMRT1 in human sex determination. Mech. Dev. 91, 323–325. Ottolenghi, C., Fellous, M., Barbieri, M., McElreavey, K., 2002. Novel paralogy relations among human chromosomes support a link between the phylogeny of doublesex-related genes and the evolution of sex determination. Genomics 79, 333–343. Raymond, C.S., Shamu, C.E., Shen, M.M., Seifert, K.J., Hirsch, B., Hodgkin, J., Zarkower, D., 1998. Evidence for evolutionary conservation of sex-determining genes. Nature 391, 691–695. Smith, C.A., McClive, P.J., Western, P.S., Reed, K.J., Sinclair, A.H., 1999. Conservation of a sex-determining gene. Nature 402, 601–602.
Restricted expression of DMRT3 in chicken and mouse embryos Craig A. Smith*, Tanya M. Hurley, Peter J. McClive, Andrew H. Sinclair Murdoch Children’s Research Institute and Department of Paediatrics, The University of Melbourne, Royal Children’s Hospital, Parkville, Victoria 3052, Australia Received 28 June 2002; received in revised form 12 September 2002; accepted 16 September 2002
Abstract Vertebrate DM domain genes encode a novel group of proteins related to the Drosophila doublesex and Caenorhabditis elegans mab-3 transcription factors. It is shown here that the recently identified gene, DMRT3, has a restricted embryonic expression profile that is conserved in chicken and mouse embryos. DMRT3 is expressed primarily in the forebrain, neural tube and nasal placode of both species. In the chicken, DMRT3 is also expressed in newly forming tail somites at early developmental stages and, later, in the Mu¨llerian ducts of the urogenital system. q 2003 Elsevier Science Ireland Ltd. All rights reserved. Keywords: DMRT3; DM domain; Cerebral vesicle; Nasal placode; Neural tube; Mu¨llerian duct; Somitogenesis
1. Results and discussion The Drosophila doublesex and Caenorhabditis elegans mab-3 genes encode transcription factors characterized by a conserved zinc finger-like DNA-binding motif, the DM domain. A vertebrate homologue has been identified and named DMRT1 (Doublesex and mab-3-related transcription factor, number 1) (Raymond et al., 1998). DMRT1 is expressed exclusively in the urogenital system and is implicated in vertebrate testis differentiation (Raymond et al., 1998; Smith et al., 1999; Kettlewell et al., 2000; Guan et al., 2000; Moniot et al., 2000). Human DMRT1 is part of a DM gene cluster on chromosome 9p, comprising the series DMRT1-DMRT3-DMRT2 (Brunner et al., 2001; Ottolenghi et al., 2002). These genes share the highly conserved DM domain but have little or no homology outside this region. DMRT2 is expressed primarily in the presomitic mesoderm and in early forming somites of vertebrate embryos (Meng et al., 1999). It is shown here that DMRT3 also has a conserved, restricted expression pattern in vertebrate (chicken and mouse) embryos. This expression pattern differs from that of DMRT1 and DMRT2. In both chicken and mouse embryos, DMRT3 is expressed primarily in the developing forebrain, neural tube and nasal placodes. In the chicken, DMRT3 is also expressed in early forming somites and presomitic mesoderm, and in the Mu¨llerian ducts. * Corresponding author. Tel.: 161-3-9345-6607; fax: 161-3-9345-6000. E-mail address: [email protected] (C.A. Smith).
Embryonic expression of DMRT3 was studied using whole mount in situ hybridization. For both species, embryos were examined during comparable early stages of development. For the chicken, embryos were examined at embryonic day (E)1 (stage 9), E2 (stage 14), E2.5 (stage 16), E3 (stage 18), E4.5 (stage 25) and E7.5 (stage 32). For the mouse, E8.5, 9.5 10.5 and 14.5 embryos were examined. Expression of chicken DMRT3 is shown in Fig. 1. The earliest identifiable site of expression was within newly forming somites and presomitic mesoderm in stage 9 (E1) embryos (Fig. 1A). At stage 14 (E2) expression was maintained in the most posterior, early-forming somites, while some weaker expression was apparent in the prospective forebrain region (prosencephalon) (Fig. 1B). By E2.5 (stage 16) expression had become stronger in the prosencephalon, in the region of the developing telencephalon (Fig. 1C). Strong expression was also now apparent in the nasal (olfactory) placodes (Fig. 1C). In the forebrain region, expression was localized along the midline of the developing telencephalon and also along its dorso-caudal walls (Fig. 1D). By E3 (stage 18), expression persisted within the cerebral vesicles of the telencephalon, the paired nasal placodes and also along the lateral rims of the maxilla. Weaker expression was apparent in the diencephalon (Fig. 1E). Sagittal sections through the head region of E3 embryos revealed restricted expression within the neuroepithelium of the telencephalon (dorsal and dorso-caudal walls) (Fig. 1F). In the facial region, expression was strong within the epithelium of the nasal placodes (Fig. 1G). Expression in the newly forming
0925-4773/03/$ - see front matter q 2003 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0925-477 3(03)00094-7
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Fig. 1. DMRT3 expression in the chicken embryo. (A) Embryonic (E) day 1 (stage 9). Expression in the caudal (youngest) somite pair and presomitic mesoderm (Sm). (B) E2 (stage 14). Expression in the caudal somites/presomitic mesoderm (Sm) and, weakly, in the presumptive forebrain region or prosencephalon (Pros). (C) E2.5 (stage 16). Stronger expression in the caudal somites (Sm) and in the prosencephalon (Pros). Expression is now apparent in the nasal placode (Npl). (D) E2.5 (stage 16). Dorso-frontal view of the head, showing restricted expression in the midline and caudo-lateral regions of the developing Forebrain (arrows), and in nasal placodes (Npl). Mb, midbrain; Hb, hindbrain. (E) E3 (stage 18) Expression in the telencephalon (Tel) and diencephalon (Di) of the forebrain, in nasal placodes (Npl), lateral maxillary process (Mx) and branchial arches (Ba). Staining in the otic vesicle and eye is non-specific. (F) E3 (stage 18). Parasagittal section through the head region, showing expression in the neuroepithelial walls of the dorso-caudal telencephalon (Tel). Mb, midbrain. (G) E3 (stage 18). Parasagittal section through the facial region, showing strong expression in the epithelium of the developing nasal placode (arrow). (H) E3 (stage 18). Expression in the anterior region of the newly forming somites (Sm) and presomitic mesoderm (Psm). Nt, neural tube. (I) E3 (stage 18). Lateral view, with somites/mesoderm removed, revealing a line of expressing cells along the middle region of the neural tube (arrows). (J) E3 (stage 18). Longitudinal section through the neural tube, showing expression among cells along the dorsomedial region (D), but with some expressing cells also scattered ventrally (V). Nt, neural tube; Sm, somites. (K) E3 (stage 18). Transverse section through the neural tube, showing expression within the intermediate (marginal) zone (iz), and not in the ventricular zone (vz). (L) E4.5 (stage 25). Restricted expression persists in the Telencephalon (Tel) and nasal pits (Npi). No somite expression. (M) E4.5 (stage 25). Ventral view of the head, showing strong expression in the epithelium of the nasal pits (Npi) (derived from the nasal placode). Pal, palate. (N) E7.5 (stage 32) female. Dorsal view of the urogenital system, showing strong expression in the paired Mu¨ llerian ducts (Md) but not in the mesonephric kidneys (Ms). (O) E7.5 (stage 32) male. Weaker expression in the Mu¨ llerian ducts. Bars: 0.5 mm in (A–E,H,I,L–O); 100 mm in (F,G,J,K).
C.A. Smith et al. / Mechanisms of Development 119S (2002) S73–S76
(most posterior) somites and presomitic mesoderm continued through stage 18 (E3) (Fig. 1E). In both the somites and presomitic mesoderm, expression was concentrated at the anterior regions, with expression tapering posteriorly (Fig. 1H). Several other genes expressed in the newly forming somites show the same pattern of anterior-posterior expression (e.g. Delta and Notch). During somitogenesis, chicken DMRT3 expression was dynamic, being manifest only in the anterior regions of the newly forming somite pair and presomitic mesoderm. Expression was lost as somites aged, and was extinguished altogether by day 4.5 (stage 25) (Fig. 1L). DMRT3 also showed restricted expression in the developing neural tube, first evident at E2. By E3 (stage 18), a fine line of expressing cells was evident running the length of the neural tube (Fig. 1I). These cells were observed bilaterally along the long axis of the neural tube. Longitudinal sections through the neural tube showed a discrete domain of positive cells arranged along the dorso-medial region of the neural tube, but with some cells scattered ventrally (Fig. 1J). Transverse sections showed that these DMRT3-expressing cells were located within the intermediate or marginal zone of the neural tube (Fig. 1K). This restricted expression pattern of DMRT3 in the chicken embryo persisted through subsequent stages. By E4.5 (stage 25), expression was still evident in the telencephalon (although weaker) and in the nasal placodes (Fig. 1L). The nasal placodes had by this stage given rise to nasal pits, where strong epithelial expression was observed (Fig. 1M). Bilateral expression persisted along the length of the neural tube, but somitic expression had disappeared (not shown). In older embryos (E7.5; stage 32). sexually dimorphic expression was detected in the Mu¨ llerian ducts of the urogenital system, with higher expression in females compared to males (Fig. 1M,N). No expression was seen in the gonads of either sex (not shown). A similar pattern of DMRT3 expression was observed in mouse embryos (Fig. 2). In the mouse, the first detectable site of expression was the cephalic neural fold of E8.5 embryos, in the presumptive forebrain region (Fig. 2A,B). In E9.5 and 10.5 embryos, strong expression was seen in the developing forebrain (cerebral vesicles of the telencephalon) and in the nasal placodes (Fig. 2C). Some expression was also detectable in the post-anal gut and in a small group of cells adjacent to the branchial arches, in the region of the vagal outflow tract (Fig. 2C). Sagittal sections through the head region showed strong DMRT3 expression within the neuroepithelium of the telencephalon, as in the chicken at comparable stages (Fig. 2D). Expression in the nasal placodes was localized within the outer epithelial layers (Fig. 2E,F). As in the chicken, a bilateral line of DMRT3positive cells was detectable running the length of the neural tube in E9.5 and E10.5 embryos. In longitudinal section, these cells were found along the dorso-medial region of the neural tube (Fig. 2G), and in transverse sections, within the intermediate (marginal) zone (but not in the ventricular or ependymal layer) (Fig. 2H).
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As in the chicken, these conserved sites of expression persisted in older embryos. Mouse DMRT3 expression persisted in the forebrain and nasal pits in E12–14.5 embryos (not shown), In addition, in E14.5 embryos, some weak expression was detectable in the developing testes of male embryos (but not in ovaries of females)
Fig. 2. DMRT3 expression in the mouse embryo. (A) E8.5, dorso-lateral view. Whole embryo, showing weak expression in the cephalic neural fold (cnf). (B) E8.5 lateral view, showing localization of expression at the tips of the cephalic neural folds (cnf) in the presumptive forebrain region. (C) E10.5, showing strong expression in the telencephalon (Tel), nasal placodes (Npl), post-anal gut (G) and adjacent to the vagal tract outfow (arrow). Staining in otic vesicle is non-specific. (D) E10.5 Section through the head region, showing strong expression in the neuroepithelial walls of the telencephalon (Tel), and not in the midbrain (Mb). (E) E10.5 Ventral view of the head, with lower jaw removed, showing expression within the nasal placodes (Npl). (F) E10.5 section through the nasal region, showing expression localized in the epithelium of the nasal placode (Npl). (G) E10.5. Longitudinal section through the neural tube, showing expression along the long axis, concentrated within the dorsomedial region. D, dorsal; V, ventral; Nt, nerual tube; Sm, somites. (H) E10.5 Transverse section through the neural tube, showing expression limited to a specific group of cells in the intermediate (marginal) zone (iz; arrows). vz, ventricular zone. (I) E14.5 male urogenital system, showing expression in the testes (Ts). Kd, kidneys. (J) E14.5 female urogenital system, showing absence of expression in ovaries (Ov). Kd, kidneys. Bars: 0.5 mm in (A–C,E); 100 mm in (D,F–H).
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(Fig. 2I,J). This contrasts with the expression profile in chicken embryos, in which DMRT3 transcripts were detectable in the Mu¨ llerian ducts but not in the gonads. Also in contrast to chicken embryos, no somite expression was observed in the mouse. However, overall, DMRT3 shows a restricted expression profile that is largely conserved in the two species. 2. Experimental procedures Specific chicken and mouse DMRT3 DNA fragments were amplified from genomic DNA using degenerate primers. These primers were designed by aligning human and pufferfish DMRT3 with that of a homologous mouse EST (BI904023). The mouse sequence spanned the 3 0 region unique to the DMRTA1-3 subfamily, outside the DM domain (Ottolenghi et al., 2002). Polymerase chain reaction fragments were cloned into PCRscript vector and sequenced to confirm identity (GenBank numbers pending). These sequences were then used as templates to generate antisense and sense RNA probes. Whole-mount in situ hybridization was carried out on paraformaldehyde-fixed embryos or tissues as described previously (Andrews et al., 1997). For sectioning, some tissues were left in chromogen solution for an extended period (2 days), cryoprotected in 20% sucrose/PBS, and snap frozen in OCT embedding compound. Fourteen- to eighteen-micrometer frozen sections were taken. Acknowledgements C.A.S. is supported by an Australian National Health and
Medical Research Council (NH&MRC) R. Douglas Wright Fellowship. The project is supported by an NH&MRC grant to A.H.S. and C.A.S. Dr Bronwyn Morrish and Dr Helen Wilmore provided some of the mouse tissues.
References Andrews, J.E.A., Smith, C.A., Sinclair, A.H., 1997. Sites of estrogen receptor and aromatase expression in the chicken embryo. Gen. Comp. Endocrinol. 108, 182–190. Brunner, B., Hornung, U., Shan, Z., Nanda, I., Kondo, M., Zend-Ajusch, E., Haaf, T., Ropers, H.-H., Shima, A., Schmind, M., Kalscheuer, V.M., Schartl, M., 2001. Genomic organisation and expression of the doublesex-related gene cluster in vertebrates and detection of putative regulatory regions for DMRT1. Genomics 77, 8–17. Guan, G., Kobayashi, T., Nagahama, Y., 2000. Sexually dimoprhic expression of two types of DM (Doublesex-mab-3)-domain genes in a teleost fish, the Tilapia (Oreochromis niloticus). Biochem. Biophys. Res. Commun. 16, 662–666. Kettlewell, J.R., Raymond, C.S., Zarkower, D., 2000. Temperature-dependent expression of turtle Dmrt1 prior to sexual differentiation. Genesis 26, 174–178. Meng, A., Moore, B., Tang, H., Yuan, B., Lin, S., 1999. A doublesexrelated gene, terra, is involved in somitogenesis in vertebrates. Development 126, 1259–1268. Moniot, B., Berta, P., Scherer, G., Su¨ dbeck, P., Poulat, F., 2000. Male specific expression suggests role of DMRT1 in human sex determination. Mech. Dev. 91, 323–325. Ottolenghi, C., Fellous, M., Barbieri, M., McElreavey, K., 2002. Novel paralogy relations among human chromosomes support a link between the phylogeny of doublesex-related genes and the evolution of sex determination. Genomics 79, 333–343. Raymond, C.S., Shamu, C.E., Shen, M.M., Seifert, K.J., Hirsch, B., Hodgkin, J., Zarkower, D., 1998. Evidence for evolutionary conservation of sex-determining genes. Nature 391, 691–695. Smith, C.A., McClive, P.J., Western, P.S., Reed, K.J., Sinclair, A.H., 1999. Conservation of a sex-determining gene. Nature 402, 601–602.