Barbieri AM et al. (1999), A homeobox gene, vax2, controls the patterning ...

XB-ART-12343

Proc Natl Acad Sci U S A 1999 Sep 14;9619:10729-34. doi: 10.1073/pnas.96.19.10729.

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A homeobox gene, vax2, controls the patterning of the eye dorsoventral axis.

Barbieri AM , Lupo G , Bulfone A , Andreazzoli M , Mariani M , Fougerousse F , Consalez GG , Borsani G , Beckmann JS , Barsacchi G , Ballabio A , Banfi S .

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We have identified a transcription factor specifically expressed in the developing vertebrate eye. We named this gene vax2 because of the high degree of sequence similarity to the recently described vax1. Both in the human and mouse genomes, vax2 is localized in the vicinity of the emx1 gene. This mapping assignment, together with the previously reported colocalization of Vax1 and Emx2 in mouse, indicates that the vax and the emx genes may be organized in clusters. vax2 has a remarkable expression domain confined to the ventral portion of the prospective neural retina in mouse, human, and Xenopus. The overexpression of either the frog Xvax2 or the human VAX2 in Xenopus embryos leads to an aberrant eye phenotype and, in particular, determines a ventralizing effect on the developing eye. The expression domain of the transcription factor Xpax2, normally confined to the ventral developing retina, extends to the dorsal region of the retina after overexpression of vax2. On the other hand, the expression of Xvent2, a molecular marker of the dorsal retina, is strongly reduced. Furthermore, vax2 overexpression induces a striking expansion of the optic stalk, a structure deriving from the ventralmost region of the eye vesicle. Altogether, these data indicate that vax2 plays a crucial role in eye development and, in particular, in the specification of the ventral optic vesicle.

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Species referenced: Xenopus
Genes referenced: emx1 emx1l emx2 pax2 rax slc7a5 tcf3 vax1 vax2 ventx2.2

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	Figure 1 Sequence analysis of the vax2 cDNA. (A) Sequence alignment of the human VAX2 predicted protein with the mouse Vax2 protein (mVax2), the murine Vax1 protein (mVax1), and the Xenopus Vax1 and Vax2 proteins (XVax1 and XVax2, respectively). Amino acids that are identical in two or more proteins are shown in black. The homeodomain is underlined. (B) Amino acid sequence alignment of the VAX2 homeodomain and other related homeodomains. Residues identical to VAX2 protein are indicated by dots.
	Figure 2 Mapping of Vax2 and Emx1 in the mouse genome. (A) Haplotype and linkage analysis of Vax2 and flanking loci on mouse chromosome 6 through the analysis of the (C57BL/6j × SPRET/Ei)F1 × SPRET/Ei (BSS) backcross (The Jackson Laboratory). Empty squares indicate the Mus spretus allele; solid squares indicate the C57BL/6J allele; shaded squares refer to a genotypes that are not determined. Numbers to the right, between rows, indicate recombination fractions ±SEM and logarithm of odds scores. Columns represent different haplotypes observed on chromosome 6. Numbers below columns define the number of individuals sharing each haplotype. (B) Position of Vax2 on chromosome 6 with respect to nearby markers independently mapped by others on the BSS backcross. Numbers on the left represent approximate genetic distances (in centimorgans, cM) from the most centromeric chromosome 6 marker in this cross. Chromosomal locations of four syntenic human genes, including VAX2 and EMX1, are also indicated.
	Figure 3 Expression pattern of vax2 in mouse (A–E), human (F), and Xenopus embryos (G–J) as determined by in situ hybridization. Vax2 expression in mouse embryos is restricted to the entire inferior neural retina, starting from E9 (A), when it is expressed in the ventral half of the optic cup, to E12.5 (B) and E16.5 (C). Eye expression in mouse at E12.5 is shown in D–E. Panels are arranged from anterior (D) to posterior (E) in coronal sections. The axis orientation of the developing eye is indicated in D: dorsal (D), ventral (V), lateral (L), and medial (M). Expression is restricted to the entire inferior neural retina, with no significant difference between inner (INL) and outer (ONL) neuroblast layers in E. (F) Sagittal section of the developing eye of a 7-week-old human embryo hybridized with the human VAX2 cDNA showing a similar pattern of expression in the ventral retina. (G–J) Whole-mount in situ hybridization showing expression of Xvax2 during Xenopus development. (G) st. 18, frontal view: the gene is expressed in the anterior neural plate. (H) st. 23, frontal view: Xvax2 is expressed in ventral regions of the forebrain vesicle and in a ventral portion of the optic vesicle. (I) st. 23, lateral view: expression as in H. (J) st. 33, lateral view: expression of Xvax2 is maintained in the ventral regions of both the telencephalon and diencephalon, as well as the retina. Other abbreviations: Cx, cerebral cortex; L, lens (except in D); M, mesencephalon (except in D); Rh, rhombencephalon; NR, neural retina; OC, optic cup; T, telencephalon; and TV, telencephalic vesicle.
	Figure 4 Effects of vax2 overexpression on swimming tadpole embryos after injection of 75 pg of Xenopus coding-region RNA and 250 pg of β-galactosidase RNA (B, D, F–H, and J–L) or 150 pg of full-length human RNA (E, I, and M) into the left dorsal animal blastomere at the 8 cell stage, as compared to the injection of 250 pg of β-galactosidase RNA alone (A and C). (A) A st. 46 embryo injected with β-galactosidase RNA, dorsal view. On the injected side (left), the optic cup and the forebrain are connected by the thin optic nerve only (arrowhead). (B) A st. 46 embryo coinjected with Xvax2 and β-galactosidase RNAs, dorsal view. The optic cup and the forebrain are abnormally connected by a giant optic stalk (arrow). (C) The same embryo shown in A, laterally viewed from the β-galactosidase-injected side. Eye morphogenesis is complete. (D) The same embryo shown in B, laterally viewed from the vax2-injected side. The optic fissure remains wide open. (E) Ventral view of the injected side of a st. 46 embryo microinjected with VAX2 RNA. The optic fissure is open. (F–H) Transversal sections of st. 46 embryos injected with Xvax2 RNA. (J–L) Higher magnifications of F–H, respectively. On the injected side, the right side in F–H, a wide optic stalk-like structure abnormally joins the optic cup to the brain (arrows in J and K). At anterior levels (F and J), a continuous lumen can be detected connecting the brain ventricle and the eye (arrowhead in J). The defective invagination of the ventral optic cup is also evident (G, H, K, and L). (I and M) Transversal sections of a st. 46 embryo injected with VAX2 RNA showing that the aberrantly folded neural retina presents a plexiform layer (pl) and is dorsally covered by a photoreceptor layer (ph).
	Figure 5 Expression of Xpax2 (A–C, J, and K), Xvax2 (D–F), and Xvent2 (G–I) genes in tail-bud (A–I) and tadpole (J and K) embryos injected with VAX2 RNA unilaterally. (A, D, and G) st. 23 injected embryos, frontal view. On the injected side (right), the Xpax2 and Xvax2 expression domains, normally confined to the ventral developing eye, are expanded dorsally (A and D), whereas the Xvent2 expression, which is dorsal in the control side, is strongly reduced (G). (B, E, and H) st. 23 injected embryos laterally viewed from the injected side. (C, F, and I) The same embryos shown in B, E, and H laterally viewed from the control side. (J and K) st. 33 injected embryos, laterally viewed from the injected (J) or the control (K) side. On the control side, Xpax2 expression in the optic cup is exclusively restricted to the lips of the optic fissure (arrowhead in K), whereas, on the injected side, Xpax2 expression expands both in ventral and anterodorsal regions of the eye cup (arrowheads in J).
	Figure 6 Expression of the Xrx1 (A–C) and Xpax2 (D–F) genes in tadpole embryos injected with VAX2 RNA unilaterally, as detected in sections after whole-mount in situ hybridization. Transversal (A and D) and frontal (B and E) sections of st. 33 injected embryos, cut at the level of the optic stalk. Both the morphology and Xpax2 expression indicate the presence of an enlarged optic stalk on the injected side (arrowhead). The extent of the Xrx1 expression domain is similar in both the injected (right) and control (left) sides. Remarkably, dorsal expansion of Xpax2 expression is detectable in the retinal tissue contiguous with the enlarged optic stalk (D). (C and D) Transversal sections of st. 33 injected embryos, cut at the level of the lens. (C) On the injected side (right), surrounding of the lens by the optic cup is abnormally ventral (arrow); the Xrx1 RNA distribution is more uniform along the ventrodorsal axis compared to the control side. (F) Ectopic Xpax2 transcription is evident in the ventral retina of the injected side (right).

References [+] :

Andreazzoli, Activating and repressing signals in head development: the role of Xotx1 and Xotx2. 1997, Pubmed, Xenbase