Kuroda H et al. (2000), Cloning a novel developmental regulating gene, ...

XB-ART-10932

Dev Growth Differ 2000 Apr 01;422:87-93. doi: 10.1046/j.1440-169x.2000.00491.x.

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Cloning a novel developmental regulating gene, Xotx5: its potential role in anterior formation in Xenopus laevis.

Kuroda H , Hayata T , Eisaki A , Asashima M .

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The vertebrate Otx gene family is related to otd, a gene contributing to head development in Drosophila. In Xenopus, Xotx1, Xotx2, and Xotx4 have already been isolated and analyzed. Here the cloning, developmental expression and functions of the additional Otx Xenopus gene, Xotx5 are reported. This latter gene shows a greater degree of homology to Xotx2 than Xotx1 and Xotx4. Xotx5 was initially expressed in Spemann's organizer and later in the anterior region. Ectopic expression of Xotx5 had similar effects to other Xotx genes in impairing trunk and tail development, and especially similar effects to Xotx2 in causing secondary cement glands. Taken together, these findings suggest that Xotx5 stimulates the formation of the anterior regions and represses the formation of posterior structures similar to Xotx2.

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Species referenced: Xenopus laevis
Genes referenced: ag1 crx inhba muc2 otx1 otx2

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	Fig. 1. (A) Sequence of the Xenopus Xotx5 coding region. The deduced peptide sequence of the Xotx2 gene product is also shown using the one-letter amino acid code. The homeodomain is underlined. (B) Peptide sequence similarity between Xotx5 and the other Xotxs. HD, homeodomain; aa, amino acids.
	Fig. 2. Temporal expression pattern of Xotx5. Reverse transcription–polymerase chain reaction (RT-PCR) analysis was performed using early Xenopus embryos. The number indicates the developmental stage of the embryos. Xotx5 expressed maternally, increased at the early gastrula stage (stage 10), and decreased at stage 45. The EF1 a is the internal positive control and RT2 is the negative control.
	Fig. 3. Localized Xotx5 expression. (A) Whole-mount in situ hybridization, dorsal view at stage 11. An arrowhead indicates blastopore. (B) Dorsal view at stage 11. (C) Anterior view at stage 19. (D) Anterior view at stage 25. (E) Head region view at stage 28. fb, forebrain; ov, optic vesicle; ot, otocyst.
	Fig. 4. Phenotypes of embryos injected with Xotx5. (A) Normal embryo at stage 36. (B) The embryos shown were injected with 1.6 ng of Xotx5 at the animal pole of 1-cell embryos. White lines indicate section places. (C) The embryo injected with 0.8 ng of Xotx5 at two ventral vegetal blastomeres at the 8-cell stage had partial additional axis formation. (D) Transverse section through the trunk of an Xotx5-injected embryo. (E) Transverse section through the head of an Xotx5-injected embryo. Note that the normal cement gland shows characteristic columnar cells with dark pigmentation. CG, normal cement gland; CG’, additional cement gland.
	Fig. 5. Reverse transcription–polymerase chain reaction (RT-PCR) analysis of Xotx5 upstream and downstream. (A) Xotx5 induced cement gland marker expression, XAG and XCG, in isolated animal caps. Animal caps isolated from Xotx5-injected embryos, and developed until sibling embryos reached stage 20. (B) Xotx5 induced large cement glands (arrowhead) in animal caps. Animal caps isolated from Xotx5-injected embryos developed until sibling embryos reached stage 30. (C) Activin A induced Xotx5 expression in animal caps and reaggregates. Animal caps treated with 5–100 ng/mL of activin A or untreated developed until sibling embryos reached stage 12.
	Fig. 6. Relationships between 13 chordate otx family genes. The tree is inferred from the 39-domain peptide sequence. Bf, Branchiostoma floridae; Lj, Lampetra japonica; Pm, Petromyzon marinus.