Vignali R et al. (2000), Xotx5b, a new member of the Otx gene family, ma...

XB-ART-10513

Mech Dev 2000 Aug 01;961:3-13. doi: 10.1016/s0925-4773(00)00367-1.

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Xotx5b, a new member of the Otx gene family, may be involved in anterior and eye development in Xenopus laevis.

Vignali R , Colombetti S , Lupo G , Zhang W , Stachel S , Harland RM , Barsacchi G .

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We describe the cloning, expression pattern and functional overexpression analysis of Xotx5b, a new member of the Otx gene family in Xenopus laevis. Early expression of Xotx5b resembles that of Xotx2, being detected in the organizer region at early gastrula stage, and, shortly after, also in anterior neuroectoderm. During neurula stages Xotx5b exhibits a changing and dynamic pattern of expression. After neural tube closure, Xotx5b is expressed in the eye and pineal gland, both involved in photoreception. Overexpression of Xotx5b has a similar effect to that of Xotx2, producing posterior truncations and inducing ectopic cement gland and neural tissue in whole embryos. In animal cap assays, Xotx5b and Xotx2 are both able to activate XAG, to strongly suppress the expression of the epidermal marker XK81, and to reciprocally activate each other. Finally, in einsteck transplantation assays, Xotx5b is able to respecify a tail/trunk organizer to a head organizer.

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Species referenced: Xenopus laevis
Genes referenced: ag1 bmp4 crx krt12.4 notch1 nrp1 otx2 sox2

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	Fig. 2. Expression of Xotx5b during gastrula stages, as detected by whole-mount in situ hybridization. Expression is detected in the dorsal blastopore lip at stage 10 (A, vegetal view), stage 10+ (B, vegetal view), stage 10.5 (arrowhead in C, dorsal view) and stage 11 (D, vegetal view). Starting from stage 10.5, expression is also detected dorsally in the anterior neuroectoderm (C, dorsal view; D, vegetal view). At stage 11.5, expression in the dorsal lip is no longer detectable (arrowhead in E, vegetal view); Xotx5b expression in the anterior neuroectoderm is increased at this stage (E, vegetal view) and at stage 12 (F, frontal view).
	Fig. 3. Xotx5b (A) and Xotx2 (C) expression at stage 10.25 are compared on sections after whole-mount in situ hybridization. Xotx5b expression is also shown at stage 10.5 (B), where arrows point at Brachet's cleft, and arrowheads show expression in the dorsal neuroectoderm; at stage 12 Xotx5b expression is only in anterior presumptive neural ectoderm, but not in the anterior mesendoderm (arrowheads).
	Fig. 4. Expression of Xotx5b during neurula stages, as detected by whole-mount in situ hybridization. At stage 12.5 (A), expression is detected in the anterior neuroectoderm corresponding to the fore- and midbrain areas, with the exception of a small dorsal indentation (arrowhead). At stage 13 (B), Xotx5b is downregulated in a central area (arrow) of the anterior neural plate, presumably corresponding to the eye field region. At stage 14 (C), expression also extends anteriorly and ventrally to the cement gland anlage (arrowheads). At stages 17 (D) and 18 (E), expression is detected in the cement gland anlage and in the paired anlage of the pineal gland (arrowheads). At stage 20 (neural tube closure, F), Xotx5b transcripts are present in the cement gland and in the epiphysis. All embryos are shown in frontal view.
	Fig. 5. Expression of Xotx5b during tailbud and tadpole stages, as detected by whole-mount in situ hybridization. At stage 27 (A,B), Xotx5b transcripts are detected in the dorsal part of the eye retina and in the pineal gland. At tadpole stage 35 (C,D) expression is detected in the pineal gland and in the eye. Xotx5b expression in the eye CMZ is shown on sections (D); Xotch expression (E) and BMP-4 expression (F) in the CMZ at the same stage are shown for comparison.
	Fig. Effect of Xotx5b overexpression on Xenopus embryos. At stage 13 (A shows a control embryo), injection of Xotx5b mRNA leads to an abnormal blastopore lip (E). Compared to control stage 28 embryos (B), injection of Xotx5b mRNA causes various grades of posterior defects (C,D). Compared to stage 378 controls (F), injected embryos result in weak phenotypes (G) or strong phenotypes (H), depending on the grade of posterior truncation.
	Fig. 7. Molecular marker analysis of Xotx5b injected embryos. (A) Expression of XAG in injected embryos (A,B) or control embryo (C); arrowheads point towards the primary cement gland, while arrows indicate ectopic cement gland tissue. (D) Expression of the pan-neural marker nrp-1 in injected (D,E) and control (F) embryos; ectopic nrp-1 expression is shown by arrows. (G) Ectopic expression of Xotx2 is detected in injected embryos (G,H, arrows); a control embryo is shown (I).
	Fig. 8. Induction of epidermal, neural and cement gland markers by Xotx5b and Xotx2 in stage 14 (A) and stage 212 animal caps (I). Animal caps were either injected with Xotx5b mRNA (B,F,J,N,R), or with Xotx2 mRNA (C,G,K,O,S), or were uninjected (D,H,L,P,T). Results of in situ hybridization on embryos and animal caps are shown for XK81 (A,I), Sox2 (E,M) and XAG (Q). Embryos shown in (A,E) are dorsal views, those in (I,M,Q) are lateral views.
	Fig. 9. Reciprocal induction of Xotx5b and Xotx2 assayed in stage 14 and stage 212 animal caps. Xotx5b injected (B,D), Xotx2 injected (G,I) or uninjected control caps (E,J) were grown up to stage 14 (B,G) or stage 212 (D,E,I,J). Stage 14 embryos (A,F) and stage 212 (C,H) embryos are shown in frontal view. Embryos and explants were hybridized to Xotx2 (A) or to Xotx5b (F) probes.
	Fig. 10.esults of einsteck transplantation assay with the dorsal blastopore lip of stage 12.5 Xenopus embryos. (A) Xotx5b injected stage 12.5 dorsal lip induces a head structure; (B) uninjected stage 12.5 dorsal lip induces a tail.