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Mespo: a novel basic helix-loop-helix gene expressed in the presomitic mesoderm and posterior tailbud of Xenopus embryos
Mechanisms of Development 82 (1999) 191–194
Gene expression pattern
Mespo: a novel basic helix-loop-helix gene expressed in the presomitic mesoderm and posterior tailbud of Xenopus embryos Elaine M. Joseph*, Luigi A. Cassetta Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138, USA Received 9 November 1998; received in revised form 28 December 1998; accepted 28 December 1998
Abstract We have isolated a novel gene from Xenopus, called Mespo, which encodes a protein containing a basic helix-loop-helix (bHLH) motif characteristic of a family of transcriptional activators. Mespo expression begins at the gastrula stage and continues throughout tailbud stages; expression occurs in the presomitic mesoderm and the posterior tailbud. Mespo has high similarity to a subfamily of bHLH transcription factors involved in segmentation of the presomitic paraxial mesoderm. 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Basic helix-loop-helix transcription factor; Paraxial mesoderm; Segmentation; Xenopus laevis
1. Results Segmentation is an important facet of the patterning events that set up the vertebrate body plan. In all vertebrates, segmentation of the paraxial mesoderm initiates anteriorly and progresses towards the posterior tail region of the embryo. This process of segmentation occurs in two steps (Tam and Trainor, 1994; Jacobson, 1988). The first step involves a prepatterning of the unsegmented mesoderm into groups of cells called somitomeres (Meirer, 1979). This prepatterning appears to be conserved across species (Jen et al., 1997). The second step is the process of somitogenesis whereby the somitomeres become distinct somites. Somitogenesis varies greatly from one species to another, in both the process by which it occurs and the number of somites that result. In Xenopus, the cells of the somitomeres form somites by separating and turning 90 degrees relative to the anterior-posterior axis (Hamilton, 1969; Youn and Malacinski, 1981). Several bHLH transcription factors have been implicated
* Corresponding author. Tel.: +1-617-495-8556; fax: +1-617-495-8557; e-mail: [email protected]
as acting to establish somitomere boundaries (Jen et al., 1997) allowing somitogenesis to proceed properly. In Xenopus, the bHLH transcriptional activator Thylacine 1 is expressed in the anterior region of somitomeres and overexpression of Thylacine 1 disrupts segmentation (Sparrow et al., 1998). In chickens, antisense depletion of cMeso-1 delays or prevents somitogenesis and leads to posterior truncations (Buchberger et al., 1998). In mice, targeted disruption of Mesp1 and Mesp2 leads to an early lethal phenotype and a fused vertebrae phenotype, respectively (Saga et al., 1996; Saga et al., 1997). Extra copies of the Mesp1 gene can rescue the Mesp2 mutant phenotype indicating that both Mesp1 and 2 are involved in the process of segmentation (Saga, 1998). We have cloned a novel bHLH gene, called Mespo, which is expressed in the presomitic mesoderm and the posterior tail region of Xenopus embryos (Fig. 1A). Within the bHLH motif region, Mespo has a high degree of identity with mouse Mesp1 and Mesp2, chicken cMeso-1, and Xenopus Thylacine 1 (54–60% identical) (Fig. 1B). In fact, the basic DNA-binding region of the bHLH motif is identical within Mespo, Mesp1, Mesp2, and cMeso-1 proteins. Despite this high degree of similarity, Mespo does not have high enough homology overall to be the Xenopus orthologue of any known transcription factor and is thus a novel protein.
0925-4773/99/$ - see front matter 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S09 25-4773(99)000 10-6
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E.M. Joseph, L.A. Cassetta / Mechanisms of Development 82 (1999) 191–194
The temporal expression of Mespo was analyzed by RTPCR (Fig. 2). The expression of Mespo begins, after the midblastula transition, during gastrulation and continues strongly throughout the tailbud stages. It is during this period of Xenopus development that the mesoderm forms
Fig. 1. (A) The sequence of Mespo (GenBank accession #AF087650). (B) A comparison of the bHLH region of Mespo with other proteins (Hopwood et al., 1989a,b; Wolf et al., 1991).
Fig. 2. The temporal expression of Mespo was analyzed by RT-PCR. RTPCRs were performed according to Wilson and Melton (1994). The primers used were Mespo: U-GCAGAGTCTTTAGTCATTTTTACCTC/DTCTGACTGACACACCAGGCT (24 cycles) and ODC: U-GATCATGCACATGTCAAGCC/D-CAGGGAGAATGCCATGTTCT (20 cycles) (Bassez et al., 1990).
Fig. 4. Section in situ hybridization analysis. (1) A stage 10.5 section, oriented with the animal pole at the top and the vegetal pole at the bottom, shows Mespo expression in the lateral mesoderm cells at the endomesodermal border. Note that the blastopore lip is out of the plane of this section. (2) A stage 13 section, oriented with dorsal at the top and ventral at the bottom, with anterior to the left and posterior to the right. Mespo expression is excluded from the involuted dorsal mesoderm but dorsallateral, ventral-lateral, and ventral expression is seen in the mesodermal cells around the yolk plug. The cavity at the top is the archenteron and the cavity at the left is the blastocoel. (3) A stage 17 transverse section, oriented with dorsal at the top and ventral at the bottom, shows Mespo expression in the presomitic mesoderm; expression is not seen in the notochord or archenteron roof cells. (4) A stage 22 transverse section shows expression in the tail region. The cavity shown is the hindgut. (5) A sense control of a stage 10.5 embryo section.
E.M. Joseph, L.A. Cassetta / Mechanisms of Development 82 (1999) 191–194
193
Fig. 3. Whole-mount in situ hybridization analysis of Mespo expression. (1) A stage 10.5, vegetal view, shows Mespo expression in the ventral and lateral mesoderm. Note that expression is excluded from the region above the dorsal blastopore lip. (2) Stage 11. (3) Stage 11.5. Expression is seen in the lateral mesoderm extending away from the blastopore lip. (4) Stage 18, a lateral view with anterior oriented towards the left and posterior towards the right, with dorsal at the top and ventral at the bottom. Strong expression is evident in the tailbud and weak expression in the lateral mesoderm. (5) Stage 20. (6) Stage 25. Mespo expression in the tailbud is clearly excluded from the dorsal-most region. (7) Stage 26. Expression is seen in the tail blastema. Expression in the head region is a non-specific artifact in this and the next two panels (7–9). (8) Stage 29. (9) Stage 35/36. Mespo is expressed at the tip of the tail.
somites; somitogenesis begins during the neural fold stages (Nieuwkoop and Faber, 1967). This expression profile, of Mespo, is similar to that of Thylacine 1 (Sparrow et al., 1998). Whole-mount in situ hybridizations illustrate localized Mespo expression in the mesoderm (Fig. 3). Expression continues in the tail blastema. This expression pattern is similar to that of the Xenopus brachyury gene (Xbra), although Mespo expression begins later and is excluded from the dorsal mesoderm (Smith et al., 1991). In situ hybridizations of sectioned embryos show Mespo expression in the ventral and dorsal-lateral types of mesoderm; at the neurula stage, expression is seen in the presomitic mesoderm but not in the more dorsal notochord (Fig. 4). Mespo expression in the tailbud is similar to that of mouse Mesp1, a gene expressed in a pattern shadowing the pathway of primordial germ cells and losing expression in cells as they move into the hindgut endoderm (Saga et al., 1996). In conclusion, Mespo is a gene with localized expression in the presomitic mesoderm and posterior tailbud region which codes for a protein with high homology to a subfamily of basic helix-loop-helix factors involved in segmentation.
2. Materials and methods Mespo was cloned using a subtractive hybridization protocol with RNA isolated from dissected gastrula (tester) and early blastula (driver) vegetal pole region explants (Diatchenko et al., 1996). The full-length Mespo clone was isolated by screening a stage 11 Xenopus cDNA lZAPII library (a gift from Douglas DeSimone). Positive Mespo clones were subcloned into pBluescript SK − using an in vivo excision protocol (Stratagene). Whole-mount in situ hybridizations were performed according to the protocol of Harland (1991), modified to include the use of BM purple substrate (Boehringer). Probes were made from the full-length Mespo clone using Megascript in vitro RNA synthesis kits and digoxigenin-UTP (Ambion).
Acknowledgements We thank Douglas A. Melton for his support and the use of laboratory space, supplies, and reagents. This work was supported by a National Institute of Health grant to Douglas A. Melton.
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E.M. Joseph, L.A. Cassetta / Mechanisms of Development 82 (1999) 191–194
References Bassez, T., Paris, J., Omilli, F., Dorel, C., Osborne, H.B., 1990. Posttranscriptional regulation of ornithine decarboxylase in Xenopus laevis oocytes. Development 110, 955–962. Buchberger, A., Seidl, K., Klein, C., Eberhardt, H., Arnold, H.H., 1998. cMeso-1, a novel bHLH transcription factor, is involved in somite formation in chicken embryos. Dev. Biol. 199, 201–215. Diatchenko, L., Lau, Y.F.C., Campbell, A.P., Chenchik, A., Moqadam, F., Huang, B., Lukyanov, S., Lukyanov, K., Gurskaya, N., Sverdlov, E.D., Siebert, P.D., 1996. Suppression subtractive hybridization: a method for generating differentially regulated or tissue-specific cDNA probes and libraries. Proc. Natl. Acad. Sci. 93, 6025–6030. Hamilton, L., 1969. The formation of somites in Xenopus. J. Embyol. Exp. Morphol. 22, 253–264. Harland, R.M., 1991. In situ hybridization: an improved whole mount method for Xenopus embryos. Methods Cell Biol. 36, 675–685. Hopwood, N.D., Pluck, A., Gurdon, J.B., 1989a. A Xenopus mRNA related to Drosophilia twist is expressed in response to induction in the mesoderm and neural crest. Cell 59, 893–903. Hopwood, N.D., Pluck, A., Gurdon, J.B., 1989b. MyoD expression in the forming somites is an early response to mesoderm induction in Xenopus embryos. EMBO J. 11, 3409–3417. Jacobson, A.G., 1988. Somitomeres: mesodermal segments of vertebrate embryos. Dev. Suppl. 104, 209–220. Jen, W.C., Wettstein, D., Turner, D., Chitnis, A., Kintner, C., 1997. The Notch ligand, X-Delta-2, mediates segmentation of the paraxial mesoderm in Xenopus embryos. Development 124, 1169–1178. Meirer, S., 1979. Development of the chick mesoblast, Formation of embryonic axis and establishment of the metameric pattern. Dev. Biol. 73, 25–45.
Nieuwkoop, P.D., Faber, J., 1967. Normal table of Xenopus laevis. North Holland, Amsterdam. Saga, Y., 1998. Genetic rescue of segmentation defect in Mesp2-deficient mice by Mesp1 gene replacement. Mech. Dev. 75, 53–66. Saga, Y., Hata, N., Kobayashi, S., Magnuson, T., Seldin, M.F., 1996. Mesp1: a novel basic helix-loop-helix protein expressed in the nascent mesodermal cells during mouse gastrulation. Development 122, 2769– 2778. Saga, Y., Hata, N., Koseki, H., Taketo, M.M., 1997. Mesp2: a novel mouse gene expressed in the presegmented mesoderm and essential for segmentation initiation. Genes Dev. 11, 1827–1839. Smith, J.C., Price, B.M.J., Green, J.B.A., Weigel, D., Herrmann, B.G., 1991. Expression of a Xenopus homolog of Brachyury (T) in an immediate-early response to mesoderm induction. Cell 67, 79–87. Sparrow, D.B., Jen, W.C., Kotecha, S., Towers, N., Kintner, C., Mohun, T.J., 1998. Thylacine 1 is expressed segmentally within the paraxial mesoderm of the Xenopus embryo and interacts with the Notch pathway. Development 125, 2041–2051. Tam, P.P., Trainor, P.A., 1994. Specification and segmentation of the paraxial mesoderm. Anat. Embryol. 189, 275–305. Wilson, P.A., Melton, D.A., 1994. Mesodermal patterning by an inducer gradient on secondary cell-cell communication. Curr. Biol. 4, 676–686. Wolf, C., Thisse, C., Stoetzel, C., Thisse, B., Gerlinger, P., Perrin-Schmitt, F., 1991. The M-twist gene of mouse is expressed in subsets of mesodermal cells and is closely related to the Xenopus Xtwi and the Drosophilia twist genes. Dev. Biol. 143, 363–373. Youn, B.W., Malacinski, G.M., 1981. Somitogenesis in the amphibian Xenopus laevis: scanning electron microscopic analysis of intrasomitic cellular arrangements during somite rotation. J. Embyol. Exp. Morphol. 64, 23–43.
Gene expression pattern
Mespo: a novel basic helix-loop-helix gene expressed in the presomitic mesoderm and posterior tailbud of Xenopus embryos Elaine M. Joseph*, Luigi A. Cassetta Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138, USA Received 9 November 1998; received in revised form 28 December 1998; accepted 28 December 1998
Abstract We have isolated a novel gene from Xenopus, called Mespo, which encodes a protein containing a basic helix-loop-helix (bHLH) motif characteristic of a family of transcriptional activators. Mespo expression begins at the gastrula stage and continues throughout tailbud stages; expression occurs in the presomitic mesoderm and the posterior tailbud. Mespo has high similarity to a subfamily of bHLH transcription factors involved in segmentation of the presomitic paraxial mesoderm. 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Basic helix-loop-helix transcription factor; Paraxial mesoderm; Segmentation; Xenopus laevis
1. Results Segmentation is an important facet of the patterning events that set up the vertebrate body plan. In all vertebrates, segmentation of the paraxial mesoderm initiates anteriorly and progresses towards the posterior tail region of the embryo. This process of segmentation occurs in two steps (Tam and Trainor, 1994; Jacobson, 1988). The first step involves a prepatterning of the unsegmented mesoderm into groups of cells called somitomeres (Meirer, 1979). This prepatterning appears to be conserved across species (Jen et al., 1997). The second step is the process of somitogenesis whereby the somitomeres become distinct somites. Somitogenesis varies greatly from one species to another, in both the process by which it occurs and the number of somites that result. In Xenopus, the cells of the somitomeres form somites by separating and turning 90 degrees relative to the anterior-posterior axis (Hamilton, 1969; Youn and Malacinski, 1981). Several bHLH transcription factors have been implicated
* Corresponding author. Tel.: +1-617-495-8556; fax: +1-617-495-8557; e-mail: [email protected]
as acting to establish somitomere boundaries (Jen et al., 1997) allowing somitogenesis to proceed properly. In Xenopus, the bHLH transcriptional activator Thylacine 1 is expressed in the anterior region of somitomeres and overexpression of Thylacine 1 disrupts segmentation (Sparrow et al., 1998). In chickens, antisense depletion of cMeso-1 delays or prevents somitogenesis and leads to posterior truncations (Buchberger et al., 1998). In mice, targeted disruption of Mesp1 and Mesp2 leads to an early lethal phenotype and a fused vertebrae phenotype, respectively (Saga et al., 1996; Saga et al., 1997). Extra copies of the Mesp1 gene can rescue the Mesp2 mutant phenotype indicating that both Mesp1 and 2 are involved in the process of segmentation (Saga, 1998). We have cloned a novel bHLH gene, called Mespo, which is expressed in the presomitic mesoderm and the posterior tail region of Xenopus embryos (Fig. 1A). Within the bHLH motif region, Mespo has a high degree of identity with mouse Mesp1 and Mesp2, chicken cMeso-1, and Xenopus Thylacine 1 (54–60% identical) (Fig. 1B). In fact, the basic DNA-binding region of the bHLH motif is identical within Mespo, Mesp1, Mesp2, and cMeso-1 proteins. Despite this high degree of similarity, Mespo does not have high enough homology overall to be the Xenopus orthologue of any known transcription factor and is thus a novel protein.
0925-4773/99/$ - see front matter 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S09 25-4773(99)000 10-6
192
E.M. Joseph, L.A. Cassetta / Mechanisms of Development 82 (1999) 191–194
The temporal expression of Mespo was analyzed by RTPCR (Fig. 2). The expression of Mespo begins, after the midblastula transition, during gastrulation and continues strongly throughout the tailbud stages. It is during this period of Xenopus development that the mesoderm forms
Fig. 1. (A) The sequence of Mespo (GenBank accession #AF087650). (B) A comparison of the bHLH region of Mespo with other proteins (Hopwood et al., 1989a,b; Wolf et al., 1991).
Fig. 2. The temporal expression of Mespo was analyzed by RT-PCR. RTPCRs were performed according to Wilson and Melton (1994). The primers used were Mespo: U-GCAGAGTCTTTAGTCATTTTTACCTC/DTCTGACTGACACACCAGGCT (24 cycles) and ODC: U-GATCATGCACATGTCAAGCC/D-CAGGGAGAATGCCATGTTCT (20 cycles) (Bassez et al., 1990).
Fig. 4. Section in situ hybridization analysis. (1) A stage 10.5 section, oriented with the animal pole at the top and the vegetal pole at the bottom, shows Mespo expression in the lateral mesoderm cells at the endomesodermal border. Note that the blastopore lip is out of the plane of this section. (2) A stage 13 section, oriented with dorsal at the top and ventral at the bottom, with anterior to the left and posterior to the right. Mespo expression is excluded from the involuted dorsal mesoderm but dorsallateral, ventral-lateral, and ventral expression is seen in the mesodermal cells around the yolk plug. The cavity at the top is the archenteron and the cavity at the left is the blastocoel. (3) A stage 17 transverse section, oriented with dorsal at the top and ventral at the bottom, shows Mespo expression in the presomitic mesoderm; expression is not seen in the notochord or archenteron roof cells. (4) A stage 22 transverse section shows expression in the tail region. The cavity shown is the hindgut. (5) A sense control of a stage 10.5 embryo section.
E.M. Joseph, L.A. Cassetta / Mechanisms of Development 82 (1999) 191–194
193
Fig. 3. Whole-mount in situ hybridization analysis of Mespo expression. (1) A stage 10.5, vegetal view, shows Mespo expression in the ventral and lateral mesoderm. Note that expression is excluded from the region above the dorsal blastopore lip. (2) Stage 11. (3) Stage 11.5. Expression is seen in the lateral mesoderm extending away from the blastopore lip. (4) Stage 18, a lateral view with anterior oriented towards the left and posterior towards the right, with dorsal at the top and ventral at the bottom. Strong expression is evident in the tailbud and weak expression in the lateral mesoderm. (5) Stage 20. (6) Stage 25. Mespo expression in the tailbud is clearly excluded from the dorsal-most region. (7) Stage 26. Expression is seen in the tail blastema. Expression in the head region is a non-specific artifact in this and the next two panels (7–9). (8) Stage 29. (9) Stage 35/36. Mespo is expressed at the tip of the tail.
somites; somitogenesis begins during the neural fold stages (Nieuwkoop and Faber, 1967). This expression profile, of Mespo, is similar to that of Thylacine 1 (Sparrow et al., 1998). Whole-mount in situ hybridizations illustrate localized Mespo expression in the mesoderm (Fig. 3). Expression continues in the tail blastema. This expression pattern is similar to that of the Xenopus brachyury gene (Xbra), although Mespo expression begins later and is excluded from the dorsal mesoderm (Smith et al., 1991). In situ hybridizations of sectioned embryos show Mespo expression in the ventral and dorsal-lateral types of mesoderm; at the neurula stage, expression is seen in the presomitic mesoderm but not in the more dorsal notochord (Fig. 4). Mespo expression in the tailbud is similar to that of mouse Mesp1, a gene expressed in a pattern shadowing the pathway of primordial germ cells and losing expression in cells as they move into the hindgut endoderm (Saga et al., 1996). In conclusion, Mespo is a gene with localized expression in the presomitic mesoderm and posterior tailbud region which codes for a protein with high homology to a subfamily of basic helix-loop-helix factors involved in segmentation.
2. Materials and methods Mespo was cloned using a subtractive hybridization protocol with RNA isolated from dissected gastrula (tester) and early blastula (driver) vegetal pole region explants (Diatchenko et al., 1996). The full-length Mespo clone was isolated by screening a stage 11 Xenopus cDNA lZAPII library (a gift from Douglas DeSimone). Positive Mespo clones were subcloned into pBluescript SK − using an in vivo excision protocol (Stratagene). Whole-mount in situ hybridizations were performed according to the protocol of Harland (1991), modified to include the use of BM purple substrate (Boehringer). Probes were made from the full-length Mespo clone using Megascript in vitro RNA synthesis kits and digoxigenin-UTP (Ambion).
Acknowledgements We thank Douglas A. Melton for his support and the use of laboratory space, supplies, and reagents. This work was supported by a National Institute of Health grant to Douglas A. Melton.
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E.M. Joseph, L.A. Cassetta / Mechanisms of Development 82 (1999) 191–194
References Bassez, T., Paris, J., Omilli, F., Dorel, C., Osborne, H.B., 1990. Posttranscriptional regulation of ornithine decarboxylase in Xenopus laevis oocytes. Development 110, 955–962. Buchberger, A., Seidl, K., Klein, C., Eberhardt, H., Arnold, H.H., 1998. cMeso-1, a novel bHLH transcription factor, is involved in somite formation in chicken embryos. Dev. Biol. 199, 201–215. Diatchenko, L., Lau, Y.F.C., Campbell, A.P., Chenchik, A., Moqadam, F., Huang, B., Lukyanov, S., Lukyanov, K., Gurskaya, N., Sverdlov, E.D., Siebert, P.D., 1996. Suppression subtractive hybridization: a method for generating differentially regulated or tissue-specific cDNA probes and libraries. Proc. Natl. Acad. Sci. 93, 6025–6030. Hamilton, L., 1969. The formation of somites in Xenopus. J. Embyol. Exp. Morphol. 22, 253–264. Harland, R.M., 1991. In situ hybridization: an improved whole mount method for Xenopus embryos. Methods Cell Biol. 36, 675–685. Hopwood, N.D., Pluck, A., Gurdon, J.B., 1989a. A Xenopus mRNA related to Drosophilia twist is expressed in response to induction in the mesoderm and neural crest. Cell 59, 893–903. Hopwood, N.D., Pluck, A., Gurdon, J.B., 1989b. MyoD expression in the forming somites is an early response to mesoderm induction in Xenopus embryos. EMBO J. 11, 3409–3417. Jacobson, A.G., 1988. Somitomeres: mesodermal segments of vertebrate embryos. Dev. Suppl. 104, 209–220. Jen, W.C., Wettstein, D., Turner, D., Chitnis, A., Kintner, C., 1997. The Notch ligand, X-Delta-2, mediates segmentation of the paraxial mesoderm in Xenopus embryos. Development 124, 1169–1178. Meirer, S., 1979. Development of the chick mesoblast, Formation of embryonic axis and establishment of the metameric pattern. Dev. Biol. 73, 25–45.
Nieuwkoop, P.D., Faber, J., 1967. Normal table of Xenopus laevis. North Holland, Amsterdam. Saga, Y., 1998. Genetic rescue of segmentation defect in Mesp2-deficient mice by Mesp1 gene replacement. Mech. Dev. 75, 53–66. Saga, Y., Hata, N., Kobayashi, S., Magnuson, T., Seldin, M.F., 1996. Mesp1: a novel basic helix-loop-helix protein expressed in the nascent mesodermal cells during mouse gastrulation. Development 122, 2769– 2778. Saga, Y., Hata, N., Koseki, H., Taketo, M.M., 1997. Mesp2: a novel mouse gene expressed in the presegmented mesoderm and essential for segmentation initiation. Genes Dev. 11, 1827–1839. Smith, J.C., Price, B.M.J., Green, J.B.A., Weigel, D., Herrmann, B.G., 1991. Expression of a Xenopus homolog of Brachyury (T) in an immediate-early response to mesoderm induction. Cell 67, 79–87. Sparrow, D.B., Jen, W.C., Kotecha, S., Towers, N., Kintner, C., Mohun, T.J., 1998. Thylacine 1 is expressed segmentally within the paraxial mesoderm of the Xenopus embryo and interacts with the Notch pathway. Development 125, 2041–2051. Tam, P.P., Trainor, P.A., 1994. Specification and segmentation of the paraxial mesoderm. Anat. Embryol. 189, 275–305. Wilson, P.A., Melton, D.A., 1994. Mesodermal patterning by an inducer gradient on secondary cell-cell communication. Curr. Biol. 4, 676–686. Wolf, C., Thisse, C., Stoetzel, C., Thisse, B., Gerlinger, P., Perrin-Schmitt, F., 1991. The M-twist gene of mouse is expressed in subsets of mesodermal cells and is closely related to the Xenopus Xtwi and the Drosophilia twist genes. Dev. Biol. 143, 363–373. Youn, B.W., Malacinski, G.M., 1981. Somitogenesis in the amphibian Xenopus laevis: scanning electron microscopic analysis of intrasomitic cellular arrangements during somite rotation. J. Embyol. Exp. Morphol. 64, 23–43.