Jen WC , Gawantka V , Pollet N , Niehrs C , Kintner C .

	Structural features of ESR-4 and ESR-5. (A)ESR-4 and ESR-5 encode closely related WRPW–bHLH proteins. The two bottom diagrams illustrate the mutant forms of ESR-5 that were generated by removing the basic domain, or by replacing the WRPW with the activation domain from Gal4. (B) Phylogenetic tree showing the sequence relationships of ESR-4 and ESR-5 to vertebrate WRPW–bHLH proteins recorded in the GenBank database, including those isolated from Xenopus, ESR-1/X–Hairy1/X-Hairy2A; zebrafish, zfHer1/ZfHer6; rat, rHES-1/rHES-2/rHES-3/rHES-5; chick: c-hairy1; and Drosophila, DmHairy1/Espl (m8).
	Expression of ESR-4 and ESR-5 inXenopus embryos. (A–D) Xenopus embryos at different stages were stained for the expression of ESR-4 andESR-5 with whole-mount, in situ hybridization. All embryos are oriented in this figure as in subsequent figures with anterior to the left. (A) Expression of ESR-5 at late gastrulae stage (st. 12, dorsal view), (B) at early neurulae stage (st. 14/15, dorsal view), and (C) at late neurulae stage (st. 22/24, side view). (D) Expression ofESR-4 at late neurulae stage (st. 22/24, side view). Note that the expression patterns of both ESR-4 andESR-5 at these different stages consists of a TBD, and two stripes that lie anterior to the TBD in somitomere 1(S1) and 2(S2). The expression patterns of ESR-4 and ESR-5 differ only in one respect: The TBD staining for ESR-5 is more intense and tends to extend more rostrally than that of ESR-4. (C,D; also see Fig. C,I). (E–G) Three examples ofESR-5 staining taken from a batch of embryos processed at stage 22. Side views of the posterior third of the embryos are shown. The asterisk marks what appears to be the emergence of a gap in the TBD expression of ESR-5 in a region referred to as the TZ. (H–J) Double labeling of embryos with whole-mount in situ hybridization with probes for ESR-5 and X-Delta-2. (H) Embryo stained for X-Delta-2 alone with Magenta–Phos (MP) as the chromogen, (I) ESR-5 RNA alone with BCIP, or (J) both ESR- 5 with BCIP andX-Delta-2 with MP. Note that the ESR-5 staining in S1 overlaps with the S1 staining in X-Delta-2. (K) Diagram showing the position of ESR-4 and ESR-5expression relative to that of other genes known to be expressed segmentally in Xenopus embryos (Jen et al. 1997; Sparrow et al. 1998). Because cells are constantly being added posteriorly to the paraxial mesoderm and somites are constantly forming anteriorly, the stripes of gene expression in the somitomeric region represent transient expression in groups of cells as they pass through this domain. For this reason, the S1-4 notation in this and other figures does not denote a fixed group of cells along the AP axis, but rather to different stages of the segmentation process that cells pass through in the paraxial mesoderm.
	Expression of ESR-4 and ESR-5during segmentation depends on the Notch signaling pathway. Embryos were injected on one side with RNAs encoding X-Delta-2(A,B), X-Su(H)–DBM (C– F), or X-Su(H)–Ank (G–J), and processed for expression of ESR-4(C,D,G,H) or for ESR-5 (A,B,E,F,I,J). A representative embryo from each injection is shown with a view of the uninjected or injected side. (A,B) Note that when embryos ectopically express X-Delta-2, expression of ESR-5 in the TZ increases (B, arrow). X-Su(H)–DBM completely blocksESR-4 expression (D, arrow) while significantly reducing ESR-5 expression in the somitomeric and TZ region (F, arrow). X-Su(H)–Ank expands the expression of bothESR-4 (H) and ESR-5 (J, arrow). The asterisk in H marks ectopic induction of ESR-4expression in the neural tube by X-Su(H)–Ank.
	Segmental expression of ESR-5 is generated by mechanisms intrinsic to TBD. The TBD was dissected from a collection of embryos and either fixed immediately (0 hr) or left for 4 hr in culture and then fixed (4 hr). After staining for ESR-5expression, embryos were scored on the basis of the presence of stripes and gaps (see Table ). Shown are representative samples after clearing in benzyl benzoate. Identical results were obtained forESR-4.
	CHX and TSA treatment rapidly alter the segmental expression of ESR-5, X-Delta-2, andThylacine1. Xenopus embryos at early neurulae stages were left untreated (A,E,I), or treated for 1 hr (B,F,J) or 2 hr (C,G,K) with CHX, or for 2.0 hr with TSA (D,H,L). Embryos were then fixed and stained forESR-5 expression (A–D), X-Delta-2expression (E–H) or for Thylacine1 expression (I–L). A side, posterior view of the stained embryos is shown. Note that following 1 hr of CHX treatment, ESR-5,X-Delta-2, and Thylacine1 expression changes by upregulating in the TZ (asterisks), whereas the striped expression ofX-Delta-2, Thylacine1, and ESR-5 in the somitomeric region is relatively unaffected. Similar effects on the expression of these genes in the TZ also occur after 2.0 hr of TSA treatment.
	Altered activity of ESR-5 produces somite defects in Xenopus embryos. Embryos were injected withlacZ RNA alone (A,B) or along with RNAs encoding ESR-5 (C,D), ESR-5ΔBD (E,F), Gal-4 (G,H), or ESR-5–Gal4 (I,J). Embryos were processed at tadpoles stages by fixation, then staining with X-gal and 12/101. Shown are longitudinal sections of the processed embryos, in which the right panels are taken from the region of the paraxial mesoderm where somites are just forming, whereas theleft panels are taken from a region more anterior. Note that in embryos injected with lacZ alone (A,B) or with Gal4 as a control (G,H), expression of 12/101 is initiated prior to somite formation and, after somites form, the myotomal cells line up along the AP axis with their nuclei aligned along the middle of each somitic unit. In contrast, embryos injected with ESR-5 (C,D), ESR-5ΔBD(E,F), or ESR-5–Gal4 RNA (I,J), 12/101 expression is initiated on schedule, but the myotomal cells fail to organize into a segmental pattern. In the more extreme cases, such as ESR-5–Gal4, the myotomal cells do not show signs of initiating somite formation (J).
	Negative feedback represses Notch pathway genes in the TZ. Embryos at the two-cell stage were injected on one side with RNAs encoding ESR-5ΔBD (A,B, G,H), ESR-5–Gal4 (C,D,I,J), or X-Su(H)–DBM (E,F,K,L), and then processed at stage 22/26 for the expression ofX-Delta-2 (A–F) or Thylacine1(G–L). A representative embryo from each injection is shown with a view of the uninjected (uninj) or injected side (inj). (Som) somitomeric.
	ESR-5 activity alters segmental gene expression. Embryos at the two-cell stage were injected on one side with RNAs encoding ESR- ESR-4 (A–D), X-Delta-2(E,F), or Thylacine1 (G,H). A representative embryo from each injection is shown with a view of the uninjected or injected side.
	Model for Xenopus vertebrate segmentation. Segmental regulation of Notch pathway genes in the paraxial mesoderm ofXenopus embryos. The presomitic mesoderm of Xenopusembryos can be divided into three regions, TBD (red shading), TZ (blue shading), and somitomeric region (green shading) in which the Notch pathway is regulated by different factors. The Notch pathway is active when paraxial cells are in the TBD but is repressed in posterior half- segments when cells enter the TZ. This repression is likely to be due to several factors including negative feedback via ESR-5, as well as segmental repressors such as the Hairy-oscillator. At this point in the process, Notch signaling acts to establish segment size by restricting the domain in which the Notch pathway genes are active. In the somitomeric region, Notch signaling appears to take on another role in which ESR-5 appears to promote the expression of Thylacine1and X-Delta-2 in a positive feedback loop. Positive feedback would act to maintain segmental identity by preserving the domains in which Notch signaling occurs.

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