Jen WC et al. (1997), The Notch ligand, X-Delta-2, mediates segmentat...

XB-ART-16816

Development 1997 Mar 01;1246:1169-78. doi: 10.1242/dev.124.6.1169.

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The Notch ligand, X-Delta-2, mediates segmentation of the paraxial mesoderm in Xenopus embryos.

Jen WC , Wettstein D , Turner D , Chitnis A , Kintner C .

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Segmentation of the vertebrate embryo begins when the paraxial mesoderm is subdivided into somites, through a process that remains poorly understood. To study this process, we have characterized X-Delta-2, which encodes the second Xenopus homolog of Drosophila Delta. Strikingly, X-Delta-2 is expressed within the presomitic mesoderm in a set of stripes that corresponds to prospective somitic boundaries, suggesting that Notch signaling within this region establishes a segmental prepattern prior to somitogenesis. To test this idea, we introduced antimorphic forms of X-Delta-2 and Xenopus Suppressor of Hairless (X-Su(H)) into embryos, and assayed the effects of these antimorphs on somite formation. In embryos expressing these antimorphs, the paraxial mesoderm differentiated normally into somitic tissue, but failed to segment properly. Both antimorphs also disrupted the segmental expression of X-Delta-2 and Hairy2A, a basic helix-loop-helix (bHLH) gene, within the presomitic mesoderm. These observations suggest that X-Delta-2, via X-Notch-1, plays a role in segmentation, by mediating cell-cell interactions that underlie the formation of a segmental prepattern prior to somitogenesis.

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Species referenced: Xenopus
Genes referenced: actl6a dlc dll1 hes4 myod1 notch1
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	Fig. 1. Diagram illustrating the segmentation of the paraxial mesoderm in different vertebrate embryos. The paraxial mesoderm on the left side of the diagram represents somitogenesis in Xenopus embryos (Hamilton, 1969) while that on the right represents somitogenesis in higher vertebrates, such as in a chick or mouse embryo. In all vertebrates, anterior region of the presomitic mesoderm appears to be first pre-patterned into somitomeres (Jacobson and Meier, 1986; Meier, 1979). In Xenopus, however, a somite forms when a group of myotomal cells segregates, rotate 90°, and orients parallel to the A-P axis. Each somite consists entirely of mononucleated myotomal cells while the dermatome does not undergo segmentation or rotation (Hamilton, 1969). In mouse or chick embryos, each somite forms when a group of mesenchymal cells forms an epithelial ball, that then undergoes a further subdivision into sclerotome, myotome and dermatome. We have aligned the events of segmentation in the diagram by equating the segregation of myotomal cells in Xenopus to the formation of an epithelial ball in chick or mouse. However, what structures are analogous in the segmentation of different species is not known.
	Fig. 2. The structure of X-Delta-2 from its predicted sequence, compared to that of Delta proteins previously isolated from Drosophila (Delta), mouse (Dll-1), Chick (C-Delta-1) and Xenopus (X-Delta-1). The percentage sequence similarity is for a pairwise comparison between proteins adjacent to each other in the diagram.
	Fig. 3. Expression pattern of X-Delta-2 transcripts in Xenopus embryos. In this and other figures, embryos and sections are oriented with anterior on the left. (A-C) Embryos stained in whole mount for the expression of X-Delta-2 RNA at neural plate stage (A; dorsal view, stage 14), neurulae stage (B; dorsal view, stage 18), and tailbud stage (C; side view, stage 28). Note that X-Delta-2 is expressed within the paraxial mesoderm within stripes of cells that are located progressively more posterior as development progresses (arrows). Note also that X-Delta-2 expression occurs in a large domain in the tailbud. (D) Longitudinal section through neurulae embryos (stage 18), showing just one side of the paraxial mesoderm. The three somites that have formed (labeled a, b and c; demarcated by arrowheads) do not express X-Delta-2 at detectable levels. Within the presomitic mesoderm, however, X-Delta-2 RNA is expressed in stripes of cells, where the spacing between each strip (denoted by arrows) corresponds to about ten cells, and thus the width of a prospective somite, or somitomere (labeled 1-4; Jacobson and Meier, 1986). Note that X-Delta-2 expression in the youngest somitomeres (labeled 3 and 4), is broad but then undergoes a refinement, localizing to the anterior edge of the first somitomere (labeled 1). (E,F) Dorsal view of myogenesis in the paraxial mesoderm of neural tube stage embryos (stage 18) as revealed by the expression of cardiac actin (E) and of MyoD (F). (G) Expression of X-Notch-1 RNA in the presomitic mesoderm (double arrow) in a cleared, early tadpole embryo (stage 24), as shown by whole-mount in situ hybridization. (H) Dorsal view of an early neural plate stage embryo (stage 12.5), double-labeled for the expression of cardiac actin RNA (dark blue) and for X-Delta-2 RNA (light blue). Note that the X-Delta-2 staining (arrow) extends outside the myogenic domain marked by cardiac actin staining.
	Fig. 4. Complementary and segmental expression of X-Delta-2 and Hairy2A in the presomitic mesoderm. Shown are lateral, posterior views of neurulae stage embryos (stage 22) stained by whole-mount in situ hybridization for the expression of (A) X-Delta-2, (B) Hairy2A, or (C) both X-Delta-2 (purple, numbers) and Hairy2A (light blue, arrows). In A, the stripes of X-Delta-2 within somitomeres are numbered as in Fig. 3D. In C, note that Hairy2A is expressed in the posterior portion of the somitomere (arrows) where the expression of X-Delta-2 is lost.
	Fig. 5. Expression of X-Delta-2tr alters the pattern of segmentation in Xenopus embryos. Embryos injected on one side, at the two-cell stage, with (A) nlacZ RNA or (B,C) with a mixture of X-Delta-2tr and nlacZ RNAs. At early tadpole stages, embryos were fixed and reacted in whole-mount with X-gal, which stains the nuclei blue, and with the 12/101 antibody using HRP immunohistochemistry, which stains the muscle cells brown. Shown are representative longitudinal sections. (A) Embryo injected with just the nlacZ tracer. Note that each somite consists of mononucleated muscle cells, whose nuclei line up in a row at the center of each somite. (B) Embryo expressing X-Delta-2tr RNA with a ‘mild’ phenotype. Note that some of the myotomal cells have formed somites which are shorter (arrowhead) or longer than normal (arrow). 12/101 staining on both injected and uninjected sides is incomplete due to penetration artifacts. In subsequent experiments using more stringent staining protocols, the expression of 12/101 was found to be uniform throughout the width of the somite. (C) Embryo expressing X-Delta-2tr RNA with a ‘strong’ phenotype. Arrow marks the formation of intramyotomal junction.
	Fig. 6. Expression of XSu(H)DBM and X-Delta-2 alters the pattern of segmentation in Xenopus embryos. Embryos were injected on one side with (A) nlacZ RNA, (B) a mixture of nlacZ and XSu(H)DBM RNAs, or (C) a mixture of nlacZ and X-Delta-2 RNAs. Embryos were processed for X-gal and 12/101 staining at early tadpole stages as described in the legend of Fig. 5. In each panel, the left side shows the anterior region of the tissue section where mature somites are located, while the right side shows a posterior view where somites begin to form. (A) Negative control showing the pattern of segmentation is not affected by injection of nlacZ RNA. The nuclei within each somite are normally aligned (arrowhead). (B) Embryo injected with XSu(H)DBM RNA. Note that the onset of 12/101 expression is normal, as shown in the right panel (arrow). However, as somites begin to form, the arrangement of somitic tissue is chaotic, failing to segment properly as shown in the left panel. Intramyotomal junctions appear to form (arrow in left panel) as shown by dense 12/101 staining, but the position and number of these junctions are abnormal. (C) Embryo injected with RNA encoding X-Delta-2.
	Fig. 7. The segmental prepattern as marked by the expression of XDelta- 2 is altered by XSu(H)DBM and X-Delta-2tr. Embryos injected with (A) nlacZ RNA, (B) a mixture of nlacZ and XSu(H)DBM RNAs or (C,D) a mixture of nlacZ and X-Delta-2tr RNAs. Embryos were fixed at early neurulae stages, stained for both nlacZ expression with X-gal (light blue), and for X-Delta-2 expression using whole-mount, in situ hybridization (dark blue-purple). (A) Dorsal view of an embryos injected with just nlacZ RNA, with the injected side oriented to the top of the panel. Note the expression pattern of X-Delta-2 demarcated by a double arrow is the same on both sides. (B) Dorsal view of an embryo injected with both nlacZ and XSu(H)DBM RNAs, with the injected side up. Note that on the injected side the expression of XDelta- 2 (demarcated with a double arrow) is not refined into a segmental prepattern, but is uniform within the somitomeric region. (C,D) Embryo injected with a mixture of nlacZ and X-Delta-2tr RNAs. C shows a lateral view of the uninjected side, while D shows a lateral view of the injected side of the same embryo. Double arrow indicates the expression domain of X-Delta-2.
	Fig. 8. The segmental prepattern as marked by the expression of Hairy2A is altered by XSu(H)DBM and X-Delta-2tr. Embryos injected with (A,B) nlacZ RNA, (C,D) a mixture of nlacZ and XSu(H)DBM RNAs, or (E,F) a mixture of nlacZ and X-Delta-2tr RNAs. Embryos were fixed at neurulae stages, stained for both nlacZ expression with X-gal, and for Hairy 2A expression using whole-mount, in situ hybridization. (A,C,E) Lateral views of the uninjected side. (B,D,F) Lateral views of the injected side of the same embryo. Note that nlacZ expression alone has no effect on Hairy2A expression while expression of XSu(H)DBM or X-Delta-2tr suppresses the striped expression of Hairy2A (arrow).