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RhoB is expressed in migrating neural crest and endocardial cushions of the developing mouse embryo
Mechanisms of Development 95 (2000) 211±214
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Gene expression pattern
RhoB is expressed in migrating neural crest and endocardial cushions of the developing mouse embryo Deborah J. Henderson 1,*, Patricia Ybot-Gonzalez 1, Andrew J. Copp Neural Development Unit, Institute of Child Health, University College London, 30 Guilford Street, London WC1N 1EH, UK Received 11 February 2000; received in revised form 22 March 2000; accepted 27 March 2000
Abstract RhoB mRNA expression was examined in the developing mouse embryo between E8.5 and E11.5. Speci®c expression was found in migrating neural crest (NC) cells, from the ®rst stages of their migration at E9.5, throughout the migration period. Expression is maintained in NC derivatives for at least one embryonic day after they reach their ®nal destinations, but is then down-regulated. RhoB is also expressed in non NC-derived neural tissues, including motor neurones and the ¯oor plate of the neural tube. RhoB mRNA expression is also found in the developing endocardial cushions of the atrioventricular and out¯ow regions of the developing heart. q 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Neural crest cells; Endocardial cushions; Heart; RhoB; Gene expression; Development; Mouse embryo; Cell migration; Epithelio-mesenchymal transformation
Rho proteins are members of the ras super-family, and are thought to regulate signal transduction pathways linking membrane receptors and the cytoskeleton, thereby transducing signals between the extra-cellular environment and the internal cellular machinery. In this way they may play roles in control of cell adhesion, gene transcription and organization, and assembly of the actin cytoskeleton (Van Aelst and D'Souza-Schorey, 1997; Hall, 1998). A recent study described RhoB expression in the neural crest (NC) cells of the chick embryo, as they ®rst migrate out of the neural tube, but showed down-regulation as NC cells migrated away to the periphery (Liu and Jessell, 1998). We have examined the expression of RhoB in the developing mouse embryo during the period of neural crest cell migration, and ®nd it to be expressed in a different manner from that in the chick. We can detect no expression of RhoB mRNA in pre-migratory NC cells, whereas migrating crest cells express RhoB intensely, providing a valuable gene expression marker for this cell population. RhoB mRNA is also expressed in a number of non-NC related sites, including migrating neurones and endocardial cushion tissue. RhoB is ®rst expressed in the cranial mesoderm at E8.5 * Corresponding author. Tel.: 144-207-905-2326; fax: 144-207-8314366. E-mail address: [email protected] (D.J. Henderson). 1 These authors contributed equally to the work.
(Fig. 1a). By E9.5, RhoB expression is seen in discrete patches of cells within the cranial, branchial arch and paraxial mesoderm, and correlates with the distribution of migrating NC cells (Fig. 1b±f). RhoB is not seen in the pre-migratory NC in the dorsal part of the neural tube at this stage. Although few markers are speci®c for mammalian NC cells, the expression of RhoB closely resembles HNK-1 immuno-staining in the developing chick embryo (Rickman et al., 1985), supporting the idea that RhoB expression is a marker of all migratory (but not pre-migratory) NC cell lineages in the mouse. RhoB is also seen in non NC-related tissues such as the otocyst, the ¯oor plate of the neural tube and the ectoderm of the branchial arches. By E10.5, the expression pattern of RhoB mRNA is less widespread, corresponding mostly to tissues that contain NC derivatives (Fig. 2). These include the trigeminal, glosso-pharyngeal and facio-acoustic cranial ganglia, the dorsal root ganglia of the trunk and the aortic arch arteries. In addition, RhoB expression is seen in the developing sensory nerves projecting from the ventral aspect of the dorsal root ganglia and in the developing motor columns in the ventral neural tube. Expression in peripheral nerves is likely to re¯ect expression by NC-derived Schwann cells. By E11.5 the pattern of RhoB expression no longer marks migrating NC cells, which are now reaching their ®nal destinations in the embryo (Fig. 3). Expression is maintained in the NC-derived cranial and dorsal root ganglia, and in the
0925-4773/00/$ - see front matter q 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0925-477 3(00)00333-6
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D.J. Henderson et al. / Mechanisms of Development 95 (2000) 211±214
Fig. 1. Expression of RhoB mRNA, localized by whole-mount in situ hybridisation at E8.5 (a) and E9.5 (b±f) of mouse embryonic development. (a,b) Whole-mount views with dotted lines to indicate level of sections; (c± f) transverse vibratome sections of whole-mount embryos. (a) RhoB is expressed exclusively in the cranial mesoderm at E8.5, before the start of neural crest cell migration. (b) RhoB expression appears to correlate with migrating neural crest cells at E9.5 of mouse embryogenesis. Expression is seen along the length of the embryo, in streams of cells migrating into the ®rst and second branchial arches (arrowheads), and in a segmental pattern in the paraxial mesoderm of the trunk (arrows). Transcripts also mark the midbrain-hindbrain boundary (large arrow). (c) Section through the brain of the embryo in (b) reveals RhoB expression in the maxillary process (arrowheads), and adjacent to the neural tissue (arrows). This pattern correlates with migrating cranial neural crest cells. RhoB is expressed in the developing neural crest-derived cranial ganglia (V, IX). The dorso-lateral portion of the otocyst also expresses RhoB mRNA (large arrow). (d) Section at the level of the branchial arches reveals intense RhoB expression surrounding the neural tube and in the mesenchyme of the second and third branchial arches (arrowheads), surrounding the branchial arch arteries (arrows). Transcripts are also seen in the presumptive melanocyte precursors underneath the surface ectoderm (large arrow). Expression of RhoB in the ectoderm of the ®rst branchial arch is unlikely to be NC-related (large arrowhead). (e) At the level of the aortic sac, RhoB is expressed in the branchial arch mesenchyme (arrowheads), surrounding the arch arteries (arrow), and within the developing aortic sac. (f) More caudally, at the level of the heart, RhoB is clearly expressed in the forming dorsal root ganglia (arrow), and in the developing sympathetic chain adjacent to the aorta (arrowheads). Isolated patches of cells, positive for RhoB, are also seen in the foregut, suggesting expression by the NC-derived precursors of the enteric nervous system (large arrow). In addition, RhoB transcripts are found in the ¯oorplate of the neural tube (large arrowhead). a, atrium; as, aortic sac; fg, foregut; nt, neural tube; v, ventricles; 1±3, branchial arches 1, 2 and 3; V, trigeminal ganglion; IX, glosso-pharyngeal ganglion. Scale bar in (a,b) 700 mm; (c±f) 175 mm.
Fig. 2. Expression of RhoB mRNA, localized by whole-mount in situ hybridisation at E10.5 of mouse embryonic development. (a) Wholemount view with dotted lines to indicate level of sections; (b±f) vibratome sections of whole-mount embryos. (a) RhoB is expressed in the paraxial mesoderm along the length of the embryo (arrows) and in streams of cells in the branchial arches (arrowheads). (b) A trangential section of the trunk region reveals RhoB expression in cells migrating out of the dorsal part of the neural tube. These cells migrate only through the rostral part of the somite (R is rostral, C is caudal). Somite boundaries are marked by arrows. (c) Transverse section through the cranial region reveals that RhoB is maintained in the trigeminal ganglion (V). (d) At the level of the branchial arches, RhoB can also be seen in the facial component of the facioacoustic ganglion (VII) and in the glossopharyngeal (IX) ganglion. RhoB is expressed intensely in the epithelium of the branchial pouches (arrows). (e) At the level of the aortic sac, RhoB expression is found in the branchial arch arteries as they join the aortic sac (arrowheads), and in the aortic sac itself. Expression is intense in the developing motor columns in the ventral part of the neural tube (see also d and f) (arrows). (f) At the level of the septum transversum, expression can still be seen in the dorsal root ganglia, but with lower intensity than at E9.5 (compare with Fig. 1f). RhoB is now expressed in the developing sensory axons extending from the ventral aspect of the dorsal root ganglia (arrowheads). Expression is maintained in the developing sympathetic chain adjacent to the paired dorsal aortae (arrow). a, dorsal aorta; as, aortic sac; drg, dorsal root ganglia; fg, fore gut; nt, neural tube; sv, septum transversum; 1±3, branchial arches 1, 2 and 3; V, trigeminal ganglion; VII, facio-acoustic ganglion; IX, glosso-pharyngeal ganglion. Scale bar in (a) 1.1 mm; (b±f) 175 mm.
D.J. Henderson et al. / Mechanisms of Development 95 (2000) 211±214
projecting peripheral nerves. By E12.5, RhoB is down-regulated, with expression only in the caudal-most dorsal root ganglia (data not shown). RhoB is expressed in the developing atrioventricular and out¯ow tract endocardial cushions throughout their development (Fig. 4). Transcripts can ®rst be detected in the endocardial cells overlying the cushions, prior to the delamination of cushion mesenchyme. RhoB increases in expression as epithelium to mesenchyme transformation begins, and reaches a peak of expression as the mesenchy-
Fig. 3. Expression of RhoB mRNA, localized by whole-mount in situ hybridisation at E11.5 of mouse embryonic development. (a) Wholemount view with dotted lines to indicate level of sections; (b±f) vibratome sections of whole-mount embryos. (a) As at earlier stages, RhoB is expressed segmentally in the trunk region (arrows) and in the trigeminal (V), facio-acoustic (VII) and glosso-pharyngeal (IX) complexes (arrowheads). (b) Longitudinal sectioning through the developing somites shows expression in the dorsal root ganglia (arrows), and con®rms that the labelled axons (arrowheads) are projecting from the dorsal root ganglia rather than the motor columns. (c) In the cranial region, expression is still seen in the trigeminal ganglion (V), although with lower intensity than at E10.5. (d) Expression can be seen in the branching facial nerve (arrows) and in the branchial clefts (arrowheads). (e) RhoB expression is maintained in the dorsal root ganglia. (f) At the level of the hind limb, peripheral nerves expressing RhoB can be seen projecting into the limb (arrowheads) from both the dorsal root ganglia and the motor columns. drg, dorsal root ganglia; mc, motor columns; nt, neural tube; V, trigeminal ganglia. Scale bar in (a) 2.2 mm; (b±f) 175 mm.
213
mal cells migrate into the cardiac jelly. RhoB expression is down-regulated at E11.5 as the endocardial cushions begin their differentiation to form the valves and septa of the heart.
Fig. 4. Transverse sections of digoxigenin-labelled RhoB whole-mounts, showing expression of RhoB in the developing heart from E9.5 to E11.5. (a) At E9.5, RhoB is expressed with high intensity in the atrioventricular canal and in the endocardium of the proximal out¯ow tract, where it abuts the right ventricle (arrowheads). These are both sites of incipient endocardial cushion development. (b) More rostrally, expression of RhoB is seen in the out¯ow tract and aortic sac endocardium at E9.5 (arrowheads). (c,d) At E10.5, when endocardial cushion formation is underway, RhoB transcripts are now visible within the cushion tissue of the atrioventricular (c) and out¯ow tract (d) regions (arrowheads). (e,f) By E11.5, RhoB levels are declining in the atrioventricular and out¯ow tract (arrowheads in f). Expression is seen in the mesenchymal cap (an endocardial cushion-like structure) of primary atrial septum (arrow). a, atria; as, aortic sac; avc, atrioventricular cushion tissue; nt, neural tube; oftc, out¯ow tract cushions; v, ventricle. Scale bar, 175 mm.
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D.J. Henderson et al. / Mechanisms of Development 95 (2000) 211±214
This study shows that RhoB expression marks migrating NC, endocardial cushion cells and neurones in the developing mouse embryo. NC and endocardial cushion tissues form by analogous processes, involving an epithelium to mesenchyme transformation, followed by a migration phase, as cells move away from their site of origin. Expression of RhoB in migrating cells seems likely to be associated with its role in organization of the actin cytoskeleton. The difference in the pattern of RhoB expression between mouse and chick is marked. RhoB is expressed mainly in pre- and early migratory NC cells in chick (Liu and Jessell, 1998), but only begins to be expressed in mouse NC cells after the onset of migration. This disparity in RhoB expression is mirrored by chick-mouse differences in the expression of several other genes, including slug and cadherin 6 (Nakagawa and Takeichi, 1995; Inoue et al., 1997; Sefton et al., 1998), re¯ecting differences in the molecular mechanisms underlying neural crest development in the two organisms. 1. Materials and methods 1.1. Mouse strains and embryos Timed matings of CD1 mice were used throughout the experiments. Midday on the day of ®nding a copulation plug was designated embryonic day 0.5 (E0.5). The embryos were washed in phosphate-buffered saline (PBS), ®xed for 1±3 days in 4% paraformaldehyde at 48C, then dehydrated through to 100% methanol at 2208C, where they were stored until further use. 1.2. Generation of RhoB probe by RT-PCR Total RNA was isolated from E10.5 CBA/Ca embryos using TRIzol (Gibco BRL). The mouse RhoB sequence was obtained from GenBank (accession number x99963) and a 463 bp fragment was ampli®ed by RT-PCR. This corresponds to the region between positions 4063 and 4526 of the cDNA sequence. The fragment was cloned
into the pGEM-T vector (Promega, UK) and sequenced to con®rm its identity. 1.3. Whole-mount in situ hybridization The methodology of Wilkinson (1992) was followed. Sense controls were carried out with no evidence of nonspeci®c hybridization. Following hybridization, the embryos were photographed and then processed for vibratome embedding (Henderson et al., 1999). Acknowledgements This research was funded by the British Heart Foundation and the Welcome Trust. References Hall, A., 1998. Rho GTPases and the actin cytoskeleton. Science 279, 509± 514. Henderson, D.J., Conway, S.C., Copp, A.J., 1999. Rib truncations and fusions in the Sp 2H mouse reveal a role for Pax3 in the speci®cation of the ventro-lateral and posterior parts of the somite. Dev. Biol. 209, 143±158. Inoue, T., Chisaka, O., Matsunami, H., Takeichi, M., 1997. Cadherin-6 expression transiently delineates speci®c rhombomere, other neural tube subdivisions, and neural crest subpopulations in mouse embryos. Dev. Biol. 183, 183±194. Liu, J.-P., Jessell, T.M., 1998. A role for rhoB in the delamination of neural crest cells from the dorsal neural tube. Development 125, 5055±5067. Nakagawa, S., Takeichi, M., 1995. Neural crest cell-cell adhesion controlled by sequential and subpopulation-speci®c expression of novel cadherins. Development 121, 1321±1332. Rickman, M., Fawcett, J.W., Keynes, R.J., 1985. The migration of neural crest cells and the growth of motor axons through the rostral half of the chick somite. J. Embryol. Exp. Morphol. 90, 437±455. Sefton, M., Sanchez, S., Nieto, M.A., 1998. Conserved and divergent roles for members of the Snail family of transcription factors in the chick and mouse embryo. Development 125, 3111±3121. Van Aelst, L., D'Souza-Schorey, C., 1997. Rho GTPases and signaling networks. Genes Dev. 11, 2295±2322. Wilkinson, D.G., 1992. In Situ Hybridisation: A Practical Approach, IRL Press, Oxford.
www.elsevier.com/locate/modo
Gene expression pattern
RhoB is expressed in migrating neural crest and endocardial cushions of the developing mouse embryo Deborah J. Henderson 1,*, Patricia Ybot-Gonzalez 1, Andrew J. Copp Neural Development Unit, Institute of Child Health, University College London, 30 Guilford Street, London WC1N 1EH, UK Received 11 February 2000; received in revised form 22 March 2000; accepted 27 March 2000
Abstract RhoB mRNA expression was examined in the developing mouse embryo between E8.5 and E11.5. Speci®c expression was found in migrating neural crest (NC) cells, from the ®rst stages of their migration at E9.5, throughout the migration period. Expression is maintained in NC derivatives for at least one embryonic day after they reach their ®nal destinations, but is then down-regulated. RhoB is also expressed in non NC-derived neural tissues, including motor neurones and the ¯oor plate of the neural tube. RhoB mRNA expression is also found in the developing endocardial cushions of the atrioventricular and out¯ow regions of the developing heart. q 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Neural crest cells; Endocardial cushions; Heart; RhoB; Gene expression; Development; Mouse embryo; Cell migration; Epithelio-mesenchymal transformation
Rho proteins are members of the ras super-family, and are thought to regulate signal transduction pathways linking membrane receptors and the cytoskeleton, thereby transducing signals between the extra-cellular environment and the internal cellular machinery. In this way they may play roles in control of cell adhesion, gene transcription and organization, and assembly of the actin cytoskeleton (Van Aelst and D'Souza-Schorey, 1997; Hall, 1998). A recent study described RhoB expression in the neural crest (NC) cells of the chick embryo, as they ®rst migrate out of the neural tube, but showed down-regulation as NC cells migrated away to the periphery (Liu and Jessell, 1998). We have examined the expression of RhoB in the developing mouse embryo during the period of neural crest cell migration, and ®nd it to be expressed in a different manner from that in the chick. We can detect no expression of RhoB mRNA in pre-migratory NC cells, whereas migrating crest cells express RhoB intensely, providing a valuable gene expression marker for this cell population. RhoB mRNA is also expressed in a number of non-NC related sites, including migrating neurones and endocardial cushion tissue. RhoB is ®rst expressed in the cranial mesoderm at E8.5 * Corresponding author. Tel.: 144-207-905-2326; fax: 144-207-8314366. E-mail address: [email protected] (D.J. Henderson). 1 These authors contributed equally to the work.
(Fig. 1a). By E9.5, RhoB expression is seen in discrete patches of cells within the cranial, branchial arch and paraxial mesoderm, and correlates with the distribution of migrating NC cells (Fig. 1b±f). RhoB is not seen in the pre-migratory NC in the dorsal part of the neural tube at this stage. Although few markers are speci®c for mammalian NC cells, the expression of RhoB closely resembles HNK-1 immuno-staining in the developing chick embryo (Rickman et al., 1985), supporting the idea that RhoB expression is a marker of all migratory (but not pre-migratory) NC cell lineages in the mouse. RhoB is also seen in non NC-related tissues such as the otocyst, the ¯oor plate of the neural tube and the ectoderm of the branchial arches. By E10.5, the expression pattern of RhoB mRNA is less widespread, corresponding mostly to tissues that contain NC derivatives (Fig. 2). These include the trigeminal, glosso-pharyngeal and facio-acoustic cranial ganglia, the dorsal root ganglia of the trunk and the aortic arch arteries. In addition, RhoB expression is seen in the developing sensory nerves projecting from the ventral aspect of the dorsal root ganglia and in the developing motor columns in the ventral neural tube. Expression in peripheral nerves is likely to re¯ect expression by NC-derived Schwann cells. By E11.5 the pattern of RhoB expression no longer marks migrating NC cells, which are now reaching their ®nal destinations in the embryo (Fig. 3). Expression is maintained in the NC-derived cranial and dorsal root ganglia, and in the
0925-4773/00/$ - see front matter q 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0925-477 3(00)00333-6
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D.J. Henderson et al. / Mechanisms of Development 95 (2000) 211±214
Fig. 1. Expression of RhoB mRNA, localized by whole-mount in situ hybridisation at E8.5 (a) and E9.5 (b±f) of mouse embryonic development. (a,b) Whole-mount views with dotted lines to indicate level of sections; (c± f) transverse vibratome sections of whole-mount embryos. (a) RhoB is expressed exclusively in the cranial mesoderm at E8.5, before the start of neural crest cell migration. (b) RhoB expression appears to correlate with migrating neural crest cells at E9.5 of mouse embryogenesis. Expression is seen along the length of the embryo, in streams of cells migrating into the ®rst and second branchial arches (arrowheads), and in a segmental pattern in the paraxial mesoderm of the trunk (arrows). Transcripts also mark the midbrain-hindbrain boundary (large arrow). (c) Section through the brain of the embryo in (b) reveals RhoB expression in the maxillary process (arrowheads), and adjacent to the neural tissue (arrows). This pattern correlates with migrating cranial neural crest cells. RhoB is expressed in the developing neural crest-derived cranial ganglia (V, IX). The dorso-lateral portion of the otocyst also expresses RhoB mRNA (large arrow). (d) Section at the level of the branchial arches reveals intense RhoB expression surrounding the neural tube and in the mesenchyme of the second and third branchial arches (arrowheads), surrounding the branchial arch arteries (arrows). Transcripts are also seen in the presumptive melanocyte precursors underneath the surface ectoderm (large arrow). Expression of RhoB in the ectoderm of the ®rst branchial arch is unlikely to be NC-related (large arrowhead). (e) At the level of the aortic sac, RhoB is expressed in the branchial arch mesenchyme (arrowheads), surrounding the arch arteries (arrow), and within the developing aortic sac. (f) More caudally, at the level of the heart, RhoB is clearly expressed in the forming dorsal root ganglia (arrow), and in the developing sympathetic chain adjacent to the aorta (arrowheads). Isolated patches of cells, positive for RhoB, are also seen in the foregut, suggesting expression by the NC-derived precursors of the enteric nervous system (large arrow). In addition, RhoB transcripts are found in the ¯oorplate of the neural tube (large arrowhead). a, atrium; as, aortic sac; fg, foregut; nt, neural tube; v, ventricles; 1±3, branchial arches 1, 2 and 3; V, trigeminal ganglion; IX, glosso-pharyngeal ganglion. Scale bar in (a,b) 700 mm; (c±f) 175 mm.
Fig. 2. Expression of RhoB mRNA, localized by whole-mount in situ hybridisation at E10.5 of mouse embryonic development. (a) Wholemount view with dotted lines to indicate level of sections; (b±f) vibratome sections of whole-mount embryos. (a) RhoB is expressed in the paraxial mesoderm along the length of the embryo (arrows) and in streams of cells in the branchial arches (arrowheads). (b) A trangential section of the trunk region reveals RhoB expression in cells migrating out of the dorsal part of the neural tube. These cells migrate only through the rostral part of the somite (R is rostral, C is caudal). Somite boundaries are marked by arrows. (c) Transverse section through the cranial region reveals that RhoB is maintained in the trigeminal ganglion (V). (d) At the level of the branchial arches, RhoB can also be seen in the facial component of the facioacoustic ganglion (VII) and in the glossopharyngeal (IX) ganglion. RhoB is expressed intensely in the epithelium of the branchial pouches (arrows). (e) At the level of the aortic sac, RhoB expression is found in the branchial arch arteries as they join the aortic sac (arrowheads), and in the aortic sac itself. Expression is intense in the developing motor columns in the ventral part of the neural tube (see also d and f) (arrows). (f) At the level of the septum transversum, expression can still be seen in the dorsal root ganglia, but with lower intensity than at E9.5 (compare with Fig. 1f). RhoB is now expressed in the developing sensory axons extending from the ventral aspect of the dorsal root ganglia (arrowheads). Expression is maintained in the developing sympathetic chain adjacent to the paired dorsal aortae (arrow). a, dorsal aorta; as, aortic sac; drg, dorsal root ganglia; fg, fore gut; nt, neural tube; sv, septum transversum; 1±3, branchial arches 1, 2 and 3; V, trigeminal ganglion; VII, facio-acoustic ganglion; IX, glosso-pharyngeal ganglion. Scale bar in (a) 1.1 mm; (b±f) 175 mm.
D.J. Henderson et al. / Mechanisms of Development 95 (2000) 211±214
projecting peripheral nerves. By E12.5, RhoB is down-regulated, with expression only in the caudal-most dorsal root ganglia (data not shown). RhoB is expressed in the developing atrioventricular and out¯ow tract endocardial cushions throughout their development (Fig. 4). Transcripts can ®rst be detected in the endocardial cells overlying the cushions, prior to the delamination of cushion mesenchyme. RhoB increases in expression as epithelium to mesenchyme transformation begins, and reaches a peak of expression as the mesenchy-
Fig. 3. Expression of RhoB mRNA, localized by whole-mount in situ hybridisation at E11.5 of mouse embryonic development. (a) Wholemount view with dotted lines to indicate level of sections; (b±f) vibratome sections of whole-mount embryos. (a) As at earlier stages, RhoB is expressed segmentally in the trunk region (arrows) and in the trigeminal (V), facio-acoustic (VII) and glosso-pharyngeal (IX) complexes (arrowheads). (b) Longitudinal sectioning through the developing somites shows expression in the dorsal root ganglia (arrows), and con®rms that the labelled axons (arrowheads) are projecting from the dorsal root ganglia rather than the motor columns. (c) In the cranial region, expression is still seen in the trigeminal ganglion (V), although with lower intensity than at E10.5. (d) Expression can be seen in the branching facial nerve (arrows) and in the branchial clefts (arrowheads). (e) RhoB expression is maintained in the dorsal root ganglia. (f) At the level of the hind limb, peripheral nerves expressing RhoB can be seen projecting into the limb (arrowheads) from both the dorsal root ganglia and the motor columns. drg, dorsal root ganglia; mc, motor columns; nt, neural tube; V, trigeminal ganglia. Scale bar in (a) 2.2 mm; (b±f) 175 mm.
213
mal cells migrate into the cardiac jelly. RhoB expression is down-regulated at E11.5 as the endocardial cushions begin their differentiation to form the valves and septa of the heart.
Fig. 4. Transverse sections of digoxigenin-labelled RhoB whole-mounts, showing expression of RhoB in the developing heart from E9.5 to E11.5. (a) At E9.5, RhoB is expressed with high intensity in the atrioventricular canal and in the endocardium of the proximal out¯ow tract, where it abuts the right ventricle (arrowheads). These are both sites of incipient endocardial cushion development. (b) More rostrally, expression of RhoB is seen in the out¯ow tract and aortic sac endocardium at E9.5 (arrowheads). (c,d) At E10.5, when endocardial cushion formation is underway, RhoB transcripts are now visible within the cushion tissue of the atrioventricular (c) and out¯ow tract (d) regions (arrowheads). (e,f) By E11.5, RhoB levels are declining in the atrioventricular and out¯ow tract (arrowheads in f). Expression is seen in the mesenchymal cap (an endocardial cushion-like structure) of primary atrial septum (arrow). a, atria; as, aortic sac; avc, atrioventricular cushion tissue; nt, neural tube; oftc, out¯ow tract cushions; v, ventricle. Scale bar, 175 mm.
214
D.J. Henderson et al. / Mechanisms of Development 95 (2000) 211±214
This study shows that RhoB expression marks migrating NC, endocardial cushion cells and neurones in the developing mouse embryo. NC and endocardial cushion tissues form by analogous processes, involving an epithelium to mesenchyme transformation, followed by a migration phase, as cells move away from their site of origin. Expression of RhoB in migrating cells seems likely to be associated with its role in organization of the actin cytoskeleton. The difference in the pattern of RhoB expression between mouse and chick is marked. RhoB is expressed mainly in pre- and early migratory NC cells in chick (Liu and Jessell, 1998), but only begins to be expressed in mouse NC cells after the onset of migration. This disparity in RhoB expression is mirrored by chick-mouse differences in the expression of several other genes, including slug and cadherin 6 (Nakagawa and Takeichi, 1995; Inoue et al., 1997; Sefton et al., 1998), re¯ecting differences in the molecular mechanisms underlying neural crest development in the two organisms. 1. Materials and methods 1.1. Mouse strains and embryos Timed matings of CD1 mice were used throughout the experiments. Midday on the day of ®nding a copulation plug was designated embryonic day 0.5 (E0.5). The embryos were washed in phosphate-buffered saline (PBS), ®xed for 1±3 days in 4% paraformaldehyde at 48C, then dehydrated through to 100% methanol at 2208C, where they were stored until further use. 1.2. Generation of RhoB probe by RT-PCR Total RNA was isolated from E10.5 CBA/Ca embryos using TRIzol (Gibco BRL). The mouse RhoB sequence was obtained from GenBank (accession number x99963) and a 463 bp fragment was ampli®ed by RT-PCR. This corresponds to the region between positions 4063 and 4526 of the cDNA sequence. The fragment was cloned
into the pGEM-T vector (Promega, UK) and sequenced to con®rm its identity. 1.3. Whole-mount in situ hybridization The methodology of Wilkinson (1992) was followed. Sense controls were carried out with no evidence of nonspeci®c hybridization. Following hybridization, the embryos were photographed and then processed for vibratome embedding (Henderson et al., 1999). Acknowledgements This research was funded by the British Heart Foundation and the Welcome Trust. References Hall, A., 1998. Rho GTPases and the actin cytoskeleton. Science 279, 509± 514. Henderson, D.J., Conway, S.C., Copp, A.J., 1999. Rib truncations and fusions in the Sp 2H mouse reveal a role for Pax3 in the speci®cation of the ventro-lateral and posterior parts of the somite. Dev. Biol. 209, 143±158. Inoue, T., Chisaka, O., Matsunami, H., Takeichi, M., 1997. Cadherin-6 expression transiently delineates speci®c rhombomere, other neural tube subdivisions, and neural crest subpopulations in mouse embryos. Dev. Biol. 183, 183±194. Liu, J.-P., Jessell, T.M., 1998. A role for rhoB in the delamination of neural crest cells from the dorsal neural tube. Development 125, 5055±5067. Nakagawa, S., Takeichi, M., 1995. Neural crest cell-cell adhesion controlled by sequential and subpopulation-speci®c expression of novel cadherins. Development 121, 1321±1332. Rickman, M., Fawcett, J.W., Keynes, R.J., 1985. The migration of neural crest cells and the growth of motor axons through the rostral half of the chick somite. J. Embryol. Exp. Morphol. 90, 437±455. Sefton, M., Sanchez, S., Nieto, M.A., 1998. Conserved and divergent roles for members of the Snail family of transcription factors in the chick and mouse embryo. Development 125, 3111±3121. Van Aelst, L., D'Souza-Schorey, C., 1997. Rho GTPases and signaling networks. Genes Dev. 11, 2295±2322. Wilkinson, D.G., 1992. In Situ Hybridisation: A Practical Approach, IRL Press, Oxford.