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Sequence and embryonic expression of deltaC in the zebrafish
Mechanisms of Development 90 (2000) 119±123
Gene expression pattern
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Sequence and embryonic expression of deltaC in the zebra®sh Lucy Smithers 1, Catherine Haddon, Yun-Jin Jiang, Julian Lewis* Vertebrate Development Laboratory, Imperial Cancer Research Fund, 44 Lincoln's Inn Fields, London WC2A 3PX, UK Received 23 July 1999; accepted 7 September 1999
Abstract Four genes ± deltaA, deltaB, deltaC and deltaD ± coding for homologues of the Notch ligand Delta have been discovered in zebra®sh (Haddon et al., 1998b). We report here the cDNA sequence and expression pattern of deltaC. Its closest relatives are deltaB and Xenopus XDelta-2. Unlike deltaA, deltaB, and deltaD, deltaC is not expressed in the majority of nascent primary neurons; but it is strongly expressed in the early retina, where it precedes other delta genes. It is also expressed in cranial ganglia, in sensory epithelia including ear and lateral line, and in scattered epidermal cells. In the mesoderm, expression is visible by 50% epiboly; it is seen subsequently in the tail bud, in stripes in the presomitic mesoderm and in the posterior half of each somite. There is expression also in notochord, blood vessels and pronephros. q 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Zebra®sh; Delta; Notch; X-Delta-2; deltaA; deltaB; deltaC; deltaD; notch1a; pax2.1; vegr-2; Germ ring; Tailbud; Somites; Neural tube; Ear; Lateral line; Cranial ganglia; Retina; Notochord; Aorta; Endothelium; Pronephros; Epidermis
1. Cloning and sequence Proteins of the Delta family, acting as ligands for Notch, are key regulators of cell differentiation (Lewis, 1998; Artavanis-Tsakonas et al., 1999). The zebra®sh has at least four Delta homologues, three of which ± deltaA, deltaB and deltaD ± have been studied in some detail, in the context of neurogenesis (Dornseifer et al., 1997; Appel and Eisen, 1998; Haddon et al., 1998b), somitogenesis (Dornseifer et al., 1997; Takke and Campos-Ortega, 1999; Takke et al., 1999), ear development (Haddon et al., 1998a) and notochord formation (Appel et al., 1999). Here we report on deltaC. deltaC (GenBank accession number AF146429) codes for a protein of 664 amino acids showing the typical Delta family features (Fleming, 1998; Lissemore and Starmer, 1999) ± a DSL domain, 8 EGF repeats, a transmembrane domain, and a conserved C-terminal hydrophobic motif. The closest homologs are zebra®sh deltaB and Xenopus XDelta-2. These show 76% and 72% amino-acid identity, respectively, over the EGF repeats. Homology in the Cterminal region (which is truncated in deltaB) identi®es
* Corresponding author. Tel.: 144-171-269-3510; fax: 144-171-2693417. E-mail address: [email protected] (J. Lewis) 1 Present address: Gene Function and Regulation, Institute of Cancer Research, Chester Beatty Labs, Fulham Road, London SW3 6JB, UK.
deltaC and X-Delta-2 as probable orthologs; overall they are 59% identical. 2. Mesoderm and somites deltaC expression is ®rst observed at shield stage (50% epiboly), in the paraxial germ-ring. A band of expression is seen at the germ ring throughout the rest of epiboly, with additional stripes appearing more anteriorly in the hypoblast on either side of the midline, foreshadowing somitogenesis (Fig. 1A). During somitogenesis, deltaC is expressed in the tailbud, in two or three stripes in the presomitic mesoderm, and in the posterior halves of formed somites (Fig. 1B,C) (Haddon et al., 1998b; Takke and Campos-Ortega, 1999). deltaC is also expressed weakly in the notochord (Fig. 8A). Detailed analysis of the dynamic expression of deltaC in somitogenesis will be given elsewhere (L.S., Y.-J.J., C.H. and J.L., in preparation). 3. Nervous system and sense organs In the CNS, expression of deltaC begins later than that of the other delta genes (Appel and Eisen, 1998; Haddon et al., 1998b), and is con®ned to a smaller subset of cells (Fig. 2). deltaC-expressing cells are also seen in forming cranial ganglia (Fig. 2B±D), and in the ear and lateral line (Fig. 4).
0925-4773/00/$ - see front matter q 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0925-477 3(99)00231-2
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L. Smithers et al. / Mechanisms of Development 90 (2000) 119±123
Fig. 1. Expression of deltaC is seen in presomitic mesoderm and forming somites, from 50% epiboly onwards. (A) By 90% epiboly, bilateral stripes of expression are visible, perpendicular to the midline; (B,C) as somitogenesis proceeds, strong stripes of expression continue to be generated in the presomitic mesoderm, and each newly formed somite expresses deltaC in its posterior half. Anterior is to the left here and in Figs. 2, 5, 6 and 8. Scale bar 100 mm. Fig. 2. (A±D) Dorsal views of the future hindbrain (hb) and spinal cord (sc) at 5±18-somite stages. deltaC expression is ®rst seen in the hindbrain at the 4±5 somite stage, in positions characteristic of reticulospinal neurons, and in the spinal cord soon afterwards (see also Fig. 1C). Scattered cells expressing deltaC ± presumably nascent neurons ± become visible in gradually increasing numbers during subsequent neurogenesis (see also Figs. 4A and 5D). Expression of deltaC is also seen lateral to the neural tube where cranial ganglia (marked VII/VIII and IX) are developing. (E) For comparison, expression of deltaA at the 5somite stage: deltaA, deltaB and deltaD are expressed earlier than deltaC during neurogenesis, and in many more cells. Scale bar 100 mm. Fig. 3. Expression of delta genes and notch1a in the neural retina (nr). (A±E) Expression is at ®rst widespread but then disappears from the central region and becomes restricted to the growing neurogenic margin of the eye cup (arrow), as does the expression of notch1a. (F±L and data not shown) In each region, expression of deltaC precedes expression of deltaA, deltaB and deltaD. The spatio-temporal pattern of deltaC expression in the retina foreshadows the pattern of birthdays of neurons: at 24 h, when many of the cells are expressing deltaC, all the cells are still dividing as judged by [ 3H]thymidine incorporation, and postmitotic cells are not detected until 34 h (Nawrocki, 1985). Scale bar 50 mm.
L. Smithers et al. / Mechanisms of Development 90 (2000) 119±123
121
Fig. 4. (A) Transverse section through ears and hindbrain at 48 h, showing deltaC expression in scattered cells in the sensory epithelium of the ear, as well as in spinal cord. (B) High-magni®cation view of section through a lateral-line neuromast, showing expression in one of its cells. Note that deltaA, deltaB, and deltaD are similarly expressed in ear and lateral line (Haddon et al., 1998a). Scale bar (A) 50 mm (B) 10 mm.\ Fig. 5. deltaC expression in the developing aorta. (A,B) During early somitogenesis, cells ventral to the notochord switch on deltaC near the midline, adjacent to the most anterior somites (arrowhead in (A)). These cells come together to form the dorsal aorta (da), seen in dorsal and lateral views in (C) and (D), respectively. Scale bar 100 mm. Fig. 6. (A,B) deltaC expression in intersomitic vessels (isv) and in the dorsal aorta (da). (C) Cells in these locations also express VEGR-2, a marker for zebra®sh endothelial cells (Sumoy et al., 1997). Scale bar 50 mm. Fig. 7. deltaC expression at the 2-somite stage in scattered cells in a broad band of lateral and ventral surface ectoderm; these lie in the most super®cial layer. Scale bar 100 mm. Fig. 8. deltaC expression in the developing pronephros, lateral to somites two to four. (A) at the 4-somite stage; (B) at the 7-somite stage, counterstained (red) by in situ hybridisation for pax2.1 (a gift from M. Brand), a marker of pronephros 1 pronephric duct (Kimmel et al., 1995; Pfeffer et al., 1998). Note also deltaC expression in the notochord in (A); this persists from about the 4-somite stage to at least the 18-somite stage. Scale bar 100 mm.
Like X-Delta-2 (Jen et al., 1997), deltaC is strongly expressed in the neural retina, and from very early stages: it here precedes the other delta genes, and is already apparent at the 13±14 somite stage (16 h), well before any of the
cells have withdrawn from the division cycle (Nawrocki, 1985). The deltaC-expressing cells are probably the retinoblasts identi®ed by Wetts et al. (1989). The persistence of deltaC expression in the proliferating marginal zone of the
122
L. Smithers et al. / Mechanisms of Development 90 (2000) 119±123
retina up to at least 4±6 d.p.f. (Fig. 3E) is consistent with this (Perron et al., 1998). 4. Vascular system, epidermis, endoderm and pronephros At the 5-somite stage, a few deltaC-expressing cells are seen in the midline, level with the ®rst somite, ventral to the notochord (Fig. 5A). These cells gradually increase in numbers and by the 18-somite stage can be identi®ed as endothelial cells of the dorsal aorta (Fig. 5B,D) (Al-Adhami and Kunz, 1977; Fouquet et al., 1997). deltaC is also expressed in the intersomitic vessels (intercostal arteries) that subsequently form from the dorsal aorta (Sumoy et al., 1997) (Fig. 6A,C), and in the aortic (branchial) arch arteries. Endothelial deltaC expression seems to be con®ned to arteries (cf. Wang et al., 1998). From the 2-somite stage until at least the 18-somite stage, deltaC is expressed in scattered cells in ventral and lateral ectoderm (Fig. 7). In Xenopus, cells with a similar distribution expressing X-Delta1 appear to be precursors of ciliated, and subsequently mucous-secreting, epidermal cells (Jen et al., 1999, p. 1492, and C. Kintner, pers. commun.; Nishikawa et al., 1992). In zebra®sh, they could likewise be precursors of mucous cells or perhaps of solitary chemosensory cells (Kotrschal et al., 1997). We have not observed deltaC expression in embryonic endodermal tissues, although some expression is seen subsequently in the larval gut (M. Skipper, pers. commun.). Lastly, deltaC is expressed in the future pronephros (Fig. 8). 5. Materials and methods Fish rearing and staging were as in Haddon et al. (1998b) and Kimmel et al. (1995). 5.1. Cloning A zebra®sh deltaC partial cDNA was isolated from a 20± 28-h cDNA library (Haddon et al., 1998b) and used to obtain complete sequence data with the help of a second library (15±19-h cDNA; Appel and Eisen, 1998) and 5 0 RACE using total RNA from late gastrula and 24-h zebra®sh. Double-stranded sequencing was performed in both directions. Sequence comparisons were made using Wisconsin GCG software. 5.2. In situ hybridisation and histology Procedures were as in Haddon et al. (1998a,b), with additional riboprobes as follows: VEGR-2 (Sumoy et al., 1997), linearised with SalI and transcribed with SP6; pax2.1 (paxb) (Krauss et al., 1991), linearised with BamHI and transcribed with T7.
Acknowledgements We thank David Grunwald for the 20±28-h zebra®sh cDNA library made by Robert Riggleman and Kathryn Helde; Bruce Appel for the 15±19-h cDNA library; Michael Brand and David Kimelman for reagents; Alastair Morrison and Magdalena Skipper for comments; and the Imperial Cancer Research Fund for support. Y.-J.J. was funded by an EMBO fellowship. References Al-Adhami, M.A., Kunz, Y.W., 1977. Ontogenesis of haematopoietic sites in Brachydanio rerio (Hamilton-Buchanan) (Teleostei). Dev. Growth Diff. 19, 171±179. Appel, B., Eisen, J.S., 1998. Regulation of neuronal speci®cation in the zebra®sh spinal cord by Delta function. Development 125, 371±380. Appel, B., Fritz, A., Wester®eld, M., Grunwald, D.J., Eisen, J.S., Riley, B.B., 1999. Delta-mediated speci®cation of midline cell fates in zebra®sh embryos. Curr. Biol. 9, 247±256. Artavanis-Tsakonas, S., Rand, M.D., Lake, R.J., 1999. Notch signaling: cell fate control and signal integration in development. Science 284, 770± 776. Dornseifer, P., Takke, C., Campos-Ortega, J.A., 1997. Overexpression of a zebra®sh homologue of the Drosophila neurogenic gene Delta perturbs differentiation of primary neurons and somite development. Mech. Dev. 63, 159±171. Fleming, R.J., 1998. Structural conservation of Notch receptors and ligands. Semin. Cell Dev. Biol. 9, 599±607. Fouquet, B., Weinstein, B.M., Serluca, F.C., Fishman, M.C., 1997. Vessel patterning in the embryo of the zebra®sh: guidance by notochord. Dev. Biol. 183, 37±48. Haddon, C., Jiang, Y.-J., Smithers, L., Lewis, J., 1998a. Delta-Notch signalling and the patterning of sensory cell differentiation in the zebra®sh ear: evidence from the mind bomb mutant. Development 125, 4637± 4644. Haddon, C., Smithers, L., Schneider-Maunoury, S., Coche, T., Henrique, D., Lewis, J., 1998b. Multiple delta genes and lateral inhibition in zebra®sh primary neurogenesis. Development 125, 359±370. Jen, W.C., Gawantka, V., Pollet, N., Niehrs, C., Kintner, C., 1999. Periodic repression of notch pathway genes governs the segmentation of Xenopus embryos. Genes Dev 13, 1486±1499. 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. Kimmel, C.B., Ballard, W.W., Kimmel, S.R., Ullmann, B., Schilling, T.F., 1995. Stages of embryonic development of the zebra®sh. Dev. Dyn. 203, 253±310. Kotrschal, K., Krautgartner, W.D., Hansen, A., 1997. Ontogeny of the solitary chemosensory cells in the zebra®sh. Danio rerio. Chem. Senses 22, 111±118. Krauss, S., Johansen, T., Korzh, V., Fjose, A., 1991. Expression pattern of zebra®sh pax genes suggests a role in early brain regionalization. Nature 353, 267±270. Lewis, J., 1998. Notch signalling and the control of cell fate choices in vertebrates. Semin. Cell Dev. Biol. 9, 583±589. Lissemore, J.L., Starmer, W.T., 1999. Phylogenetic analysis of vertebrate and invertebrate Delta/Serrate/LAG- 2 (DSL) proteins. Mol. Phylogenet. Evol. 11, 308±319. Nawrocki, L., 1985. Development of the neural retina in the zebra®sh (Brachydanio rerio), PhD Thesis, University of Oregon. Nishikawa, S., Hirata, J., Sasaki, F., 1992. Fate of ciliated epidermal cells during early development of Xenopus laevis using whole-mount immunostaining with an antibody against chondroitin 6-sulfate proteoglycan
L. Smithers et al. / Mechanisms of Development 90 (2000) 119±123 and anti-tubulin: transdifferentiation or metaplasia of amphibian epidermis. Histochemistry 98, 355±358. Perron, M., Kanekar, S., Vetter, M.L., Harris, W.A., 1998. The genetic sequence of retinal development in the ciliary margin of the Xenopus eye. Dev Biol 199, 185±200. Pfeffer, P.L., Gerster, T., Lun, K., Brand, M., Busslinger, M., 1998. Characterization of three novel members of the zebra®sh Pax2/5/8 family: dependency of Pax5 and Pax8 expression on the Pax2.1 (noi) function. Development 125, 3063±3074. Sumoy, L., Keasey, J.B., Dittman, T.D., Kimelman, D., 1997. A role for notochord in axial vascular development revealed by analysis of phenotype and the expression of VEGR-2 in zebra®sh ¯h and ntl mutant embryos. Mech. Dev. 63, 15±27.
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Takke, C., Campos-Ortega, J.A., 1999. her1, a zebra®sh pair-rule like gene, acts downstream of Notch signalling to control somite development. Development 126, 3005±3014. Takke, C., Dornseifer, P., v Weizsacker, E., Campos-Ortega, J.A., 1999. her4, a zebra®sh homologue of the Drosophila neurogenic gene E(spl), is a target of Notch signalling. Development 126, 1811±1821. Wang, H.U., Chen, Z.F., Anderson, D.J., 1998. Molecular distinction and angiogenic interaction between embryonic arteries and veins revealed by ephrin-B2 and its receptor Eph-B4. Cell 93, 741±753. Wetts, R., Serbedzija, G.N., Fraser, S.E., 1989. Cell lineage analysis reveals multipotent precursors in the ciliary margin of the frog retina. Dev. Biol. 136, 254±263.
Gene expression pattern
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Sequence and embryonic expression of deltaC in the zebra®sh Lucy Smithers 1, Catherine Haddon, Yun-Jin Jiang, Julian Lewis* Vertebrate Development Laboratory, Imperial Cancer Research Fund, 44 Lincoln's Inn Fields, London WC2A 3PX, UK Received 23 July 1999; accepted 7 September 1999
Abstract Four genes ± deltaA, deltaB, deltaC and deltaD ± coding for homologues of the Notch ligand Delta have been discovered in zebra®sh (Haddon et al., 1998b). We report here the cDNA sequence and expression pattern of deltaC. Its closest relatives are deltaB and Xenopus XDelta-2. Unlike deltaA, deltaB, and deltaD, deltaC is not expressed in the majority of nascent primary neurons; but it is strongly expressed in the early retina, where it precedes other delta genes. It is also expressed in cranial ganglia, in sensory epithelia including ear and lateral line, and in scattered epidermal cells. In the mesoderm, expression is visible by 50% epiboly; it is seen subsequently in the tail bud, in stripes in the presomitic mesoderm and in the posterior half of each somite. There is expression also in notochord, blood vessels and pronephros. q 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Zebra®sh; Delta; Notch; X-Delta-2; deltaA; deltaB; deltaC; deltaD; notch1a; pax2.1; vegr-2; Germ ring; Tailbud; Somites; Neural tube; Ear; Lateral line; Cranial ganglia; Retina; Notochord; Aorta; Endothelium; Pronephros; Epidermis
1. Cloning and sequence Proteins of the Delta family, acting as ligands for Notch, are key regulators of cell differentiation (Lewis, 1998; Artavanis-Tsakonas et al., 1999). The zebra®sh has at least four Delta homologues, three of which ± deltaA, deltaB and deltaD ± have been studied in some detail, in the context of neurogenesis (Dornseifer et al., 1997; Appel and Eisen, 1998; Haddon et al., 1998b), somitogenesis (Dornseifer et al., 1997; Takke and Campos-Ortega, 1999; Takke et al., 1999), ear development (Haddon et al., 1998a) and notochord formation (Appel et al., 1999). Here we report on deltaC. deltaC (GenBank accession number AF146429) codes for a protein of 664 amino acids showing the typical Delta family features (Fleming, 1998; Lissemore and Starmer, 1999) ± a DSL domain, 8 EGF repeats, a transmembrane domain, and a conserved C-terminal hydrophobic motif. The closest homologs are zebra®sh deltaB and Xenopus XDelta-2. These show 76% and 72% amino-acid identity, respectively, over the EGF repeats. Homology in the Cterminal region (which is truncated in deltaB) identi®es
* Corresponding author. Tel.: 144-171-269-3510; fax: 144-171-2693417. E-mail address: [email protected] (J. Lewis) 1 Present address: Gene Function and Regulation, Institute of Cancer Research, Chester Beatty Labs, Fulham Road, London SW3 6JB, UK.
deltaC and X-Delta-2 as probable orthologs; overall they are 59% identical. 2. Mesoderm and somites deltaC expression is ®rst observed at shield stage (50% epiboly), in the paraxial germ-ring. A band of expression is seen at the germ ring throughout the rest of epiboly, with additional stripes appearing more anteriorly in the hypoblast on either side of the midline, foreshadowing somitogenesis (Fig. 1A). During somitogenesis, deltaC is expressed in the tailbud, in two or three stripes in the presomitic mesoderm, and in the posterior halves of formed somites (Fig. 1B,C) (Haddon et al., 1998b; Takke and Campos-Ortega, 1999). deltaC is also expressed weakly in the notochord (Fig. 8A). Detailed analysis of the dynamic expression of deltaC in somitogenesis will be given elsewhere (L.S., Y.-J.J., C.H. and J.L., in preparation). 3. Nervous system and sense organs In the CNS, expression of deltaC begins later than that of the other delta genes (Appel and Eisen, 1998; Haddon et al., 1998b), and is con®ned to a smaller subset of cells (Fig. 2). deltaC-expressing cells are also seen in forming cranial ganglia (Fig. 2B±D), and in the ear and lateral line (Fig. 4).
0925-4773/00/$ - see front matter q 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0925-477 3(99)00231-2
120
L. Smithers et al. / Mechanisms of Development 90 (2000) 119±123
Fig. 1. Expression of deltaC is seen in presomitic mesoderm and forming somites, from 50% epiboly onwards. (A) By 90% epiboly, bilateral stripes of expression are visible, perpendicular to the midline; (B,C) as somitogenesis proceeds, strong stripes of expression continue to be generated in the presomitic mesoderm, and each newly formed somite expresses deltaC in its posterior half. Anterior is to the left here and in Figs. 2, 5, 6 and 8. Scale bar 100 mm. Fig. 2. (A±D) Dorsal views of the future hindbrain (hb) and spinal cord (sc) at 5±18-somite stages. deltaC expression is ®rst seen in the hindbrain at the 4±5 somite stage, in positions characteristic of reticulospinal neurons, and in the spinal cord soon afterwards (see also Fig. 1C). Scattered cells expressing deltaC ± presumably nascent neurons ± become visible in gradually increasing numbers during subsequent neurogenesis (see also Figs. 4A and 5D). Expression of deltaC is also seen lateral to the neural tube where cranial ganglia (marked VII/VIII and IX) are developing. (E) For comparison, expression of deltaA at the 5somite stage: deltaA, deltaB and deltaD are expressed earlier than deltaC during neurogenesis, and in many more cells. Scale bar 100 mm. Fig. 3. Expression of delta genes and notch1a in the neural retina (nr). (A±E) Expression is at ®rst widespread but then disappears from the central region and becomes restricted to the growing neurogenic margin of the eye cup (arrow), as does the expression of notch1a. (F±L and data not shown) In each region, expression of deltaC precedes expression of deltaA, deltaB and deltaD. The spatio-temporal pattern of deltaC expression in the retina foreshadows the pattern of birthdays of neurons: at 24 h, when many of the cells are expressing deltaC, all the cells are still dividing as judged by [ 3H]thymidine incorporation, and postmitotic cells are not detected until 34 h (Nawrocki, 1985). Scale bar 50 mm.
L. Smithers et al. / Mechanisms of Development 90 (2000) 119±123
121
Fig. 4. (A) Transverse section through ears and hindbrain at 48 h, showing deltaC expression in scattered cells in the sensory epithelium of the ear, as well as in spinal cord. (B) High-magni®cation view of section through a lateral-line neuromast, showing expression in one of its cells. Note that deltaA, deltaB, and deltaD are similarly expressed in ear and lateral line (Haddon et al., 1998a). Scale bar (A) 50 mm (B) 10 mm.\ Fig. 5. deltaC expression in the developing aorta. (A,B) During early somitogenesis, cells ventral to the notochord switch on deltaC near the midline, adjacent to the most anterior somites (arrowhead in (A)). These cells come together to form the dorsal aorta (da), seen in dorsal and lateral views in (C) and (D), respectively. Scale bar 100 mm. Fig. 6. (A,B) deltaC expression in intersomitic vessels (isv) and in the dorsal aorta (da). (C) Cells in these locations also express VEGR-2, a marker for zebra®sh endothelial cells (Sumoy et al., 1997). Scale bar 50 mm. Fig. 7. deltaC expression at the 2-somite stage in scattered cells in a broad band of lateral and ventral surface ectoderm; these lie in the most super®cial layer. Scale bar 100 mm. Fig. 8. deltaC expression in the developing pronephros, lateral to somites two to four. (A) at the 4-somite stage; (B) at the 7-somite stage, counterstained (red) by in situ hybridisation for pax2.1 (a gift from M. Brand), a marker of pronephros 1 pronephric duct (Kimmel et al., 1995; Pfeffer et al., 1998). Note also deltaC expression in the notochord in (A); this persists from about the 4-somite stage to at least the 18-somite stage. Scale bar 100 mm.
Like X-Delta-2 (Jen et al., 1997), deltaC is strongly expressed in the neural retina, and from very early stages: it here precedes the other delta genes, and is already apparent at the 13±14 somite stage (16 h), well before any of the
cells have withdrawn from the division cycle (Nawrocki, 1985). The deltaC-expressing cells are probably the retinoblasts identi®ed by Wetts et al. (1989). The persistence of deltaC expression in the proliferating marginal zone of the
122
L. Smithers et al. / Mechanisms of Development 90 (2000) 119±123
retina up to at least 4±6 d.p.f. (Fig. 3E) is consistent with this (Perron et al., 1998). 4. Vascular system, epidermis, endoderm and pronephros At the 5-somite stage, a few deltaC-expressing cells are seen in the midline, level with the ®rst somite, ventral to the notochord (Fig. 5A). These cells gradually increase in numbers and by the 18-somite stage can be identi®ed as endothelial cells of the dorsal aorta (Fig. 5B,D) (Al-Adhami and Kunz, 1977; Fouquet et al., 1997). deltaC is also expressed in the intersomitic vessels (intercostal arteries) that subsequently form from the dorsal aorta (Sumoy et al., 1997) (Fig. 6A,C), and in the aortic (branchial) arch arteries. Endothelial deltaC expression seems to be con®ned to arteries (cf. Wang et al., 1998). From the 2-somite stage until at least the 18-somite stage, deltaC is expressed in scattered cells in ventral and lateral ectoderm (Fig. 7). In Xenopus, cells with a similar distribution expressing X-Delta1 appear to be precursors of ciliated, and subsequently mucous-secreting, epidermal cells (Jen et al., 1999, p. 1492, and C. Kintner, pers. commun.; Nishikawa et al., 1992). In zebra®sh, they could likewise be precursors of mucous cells or perhaps of solitary chemosensory cells (Kotrschal et al., 1997). We have not observed deltaC expression in embryonic endodermal tissues, although some expression is seen subsequently in the larval gut (M. Skipper, pers. commun.). Lastly, deltaC is expressed in the future pronephros (Fig. 8). 5. Materials and methods Fish rearing and staging were as in Haddon et al. (1998b) and Kimmel et al. (1995). 5.1. Cloning A zebra®sh deltaC partial cDNA was isolated from a 20± 28-h cDNA library (Haddon et al., 1998b) and used to obtain complete sequence data with the help of a second library (15±19-h cDNA; Appel and Eisen, 1998) and 5 0 RACE using total RNA from late gastrula and 24-h zebra®sh. Double-stranded sequencing was performed in both directions. Sequence comparisons were made using Wisconsin GCG software. 5.2. In situ hybridisation and histology Procedures were as in Haddon et al. (1998a,b), with additional riboprobes as follows: VEGR-2 (Sumoy et al., 1997), linearised with SalI and transcribed with SP6; pax2.1 (paxb) (Krauss et al., 1991), linearised with BamHI and transcribed with T7.
Acknowledgements We thank David Grunwald for the 20±28-h zebra®sh cDNA library made by Robert Riggleman and Kathryn Helde; Bruce Appel for the 15±19-h cDNA library; Michael Brand and David Kimelman for reagents; Alastair Morrison and Magdalena Skipper for comments; and the Imperial Cancer Research Fund for support. Y.-J.J. was funded by an EMBO fellowship. References Al-Adhami, M.A., Kunz, Y.W., 1977. Ontogenesis of haematopoietic sites in Brachydanio rerio (Hamilton-Buchanan) (Teleostei). Dev. Growth Diff. 19, 171±179. Appel, B., Eisen, J.S., 1998. Regulation of neuronal speci®cation in the zebra®sh spinal cord by Delta function. Development 125, 371±380. Appel, B., Fritz, A., Wester®eld, M., Grunwald, D.J., Eisen, J.S., Riley, B.B., 1999. Delta-mediated speci®cation of midline cell fates in zebra®sh embryos. Curr. Biol. 9, 247±256. Artavanis-Tsakonas, S., Rand, M.D., Lake, R.J., 1999. Notch signaling: cell fate control and signal integration in development. Science 284, 770± 776. Dornseifer, P., Takke, C., Campos-Ortega, J.A., 1997. Overexpression of a zebra®sh homologue of the Drosophila neurogenic gene Delta perturbs differentiation of primary neurons and somite development. Mech. Dev. 63, 159±171. Fleming, R.J., 1998. Structural conservation of Notch receptors and ligands. Semin. Cell Dev. Biol. 9, 599±607. Fouquet, B., Weinstein, B.M., Serluca, F.C., Fishman, M.C., 1997. Vessel patterning in the embryo of the zebra®sh: guidance by notochord. Dev. Biol. 183, 37±48. Haddon, C., Jiang, Y.-J., Smithers, L., Lewis, J., 1998a. Delta-Notch signalling and the patterning of sensory cell differentiation in the zebra®sh ear: evidence from the mind bomb mutant. Development 125, 4637± 4644. Haddon, C., Smithers, L., Schneider-Maunoury, S., Coche, T., Henrique, D., Lewis, J., 1998b. Multiple delta genes and lateral inhibition in zebra®sh primary neurogenesis. Development 125, 359±370. Jen, W.C., Gawantka, V., Pollet, N., Niehrs, C., Kintner, C., 1999. Periodic repression of notch pathway genes governs the segmentation of Xenopus embryos. Genes Dev 13, 1486±1499. 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. Kimmel, C.B., Ballard, W.W., Kimmel, S.R., Ullmann, B., Schilling, T.F., 1995. Stages of embryonic development of the zebra®sh. Dev. Dyn. 203, 253±310. Kotrschal, K., Krautgartner, W.D., Hansen, A., 1997. Ontogeny of the solitary chemosensory cells in the zebra®sh. Danio rerio. Chem. Senses 22, 111±118. Krauss, S., Johansen, T., Korzh, V., Fjose, A., 1991. Expression pattern of zebra®sh pax genes suggests a role in early brain regionalization. Nature 353, 267±270. Lewis, J., 1998. Notch signalling and the control of cell fate choices in vertebrates. Semin. Cell Dev. Biol. 9, 583±589. Lissemore, J.L., Starmer, W.T., 1999. Phylogenetic analysis of vertebrate and invertebrate Delta/Serrate/LAG- 2 (DSL) proteins. Mol. Phylogenet. Evol. 11, 308±319. Nawrocki, L., 1985. Development of the neural retina in the zebra®sh (Brachydanio rerio), PhD Thesis, University of Oregon. Nishikawa, S., Hirata, J., Sasaki, F., 1992. Fate of ciliated epidermal cells during early development of Xenopus laevis using whole-mount immunostaining with an antibody against chondroitin 6-sulfate proteoglycan
L. Smithers et al. / Mechanisms of Development 90 (2000) 119±123 and anti-tubulin: transdifferentiation or metaplasia of amphibian epidermis. Histochemistry 98, 355±358. Perron, M., Kanekar, S., Vetter, M.L., Harris, W.A., 1998. The genetic sequence of retinal development in the ciliary margin of the Xenopus eye. Dev Biol 199, 185±200. Pfeffer, P.L., Gerster, T., Lun, K., Brand, M., Busslinger, M., 1998. Characterization of three novel members of the zebra®sh Pax2/5/8 family: dependency of Pax5 and Pax8 expression on the Pax2.1 (noi) function. Development 125, 3063±3074. Sumoy, L., Keasey, J.B., Dittman, T.D., Kimelman, D., 1997. A role for notochord in axial vascular development revealed by analysis of phenotype and the expression of VEGR-2 in zebra®sh ¯h and ntl mutant embryos. Mech. Dev. 63, 15±27.
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Takke, C., Campos-Ortega, J.A., 1999. her1, a zebra®sh pair-rule like gene, acts downstream of Notch signalling to control somite development. Development 126, 3005±3014. Takke, C., Dornseifer, P., v Weizsacker, E., Campos-Ortega, J.A., 1999. her4, a zebra®sh homologue of the Drosophila neurogenic gene E(spl), is a target of Notch signalling. Development 126, 1811±1821. Wang, H.U., Chen, Z.F., Anderson, D.J., 1998. Molecular distinction and angiogenic interaction between embryonic arteries and veins revealed by ephrin-B2 and its receptor Eph-B4. Cell 93, 741±753. Wetts, R., Serbedzija, G.N., Fraser, S.E., 1989. Cell lineage analysis reveals multipotent precursors in the ciliary margin of the frog retina. Dev. Biol. 136, 254±263.