Vax1, a novel homeobox-containing gene, directs development of the basal forebrain and visual system

Abstract

The novel homeobox-containing gene Vax1, a member of the Emx/Not gene family, is specifically expressed in the developing basal forebrain and optic nerve. Here, we show that Vax1 is essential for normal development of these structures. Mice carrying a targeted mutation of Vax1 show dysgenesis of the optic nerve, coloboma, defects in the basal telencephalon, and lobar holoprosencephaly. With the help of molecular markers we determined that in the developing visual system, the absence of Vax1 results in a proximal expansion of the activity of Pax6 and Rx. This observation suggests that Vax1 may interfere negatively with the expression of Pax6 and Rx. In reciprocal gain-of-function experiments, injection of Xvax1 mRNA or Shh into Xenopus embryos primarily affects the brain at the level of the eye primordium. Consistent with the loss-of-function results, the injection of Xvax1 results in a down-regulation of Rx. Similarly, Shh injection expands the Vax1 and Pax2 territory at the expense of the Pax6 and Rx region. On the basis of these results, we propose a model for a molecular cascade involved in the establishment of structures of the visual system.

Figure 1

Disruption of the Vax1 gene by targeted recombination. (a) Maps of the wild-type Vax1 locus, the targeting vector, and the recombined allele. Relative positions of the exons coding for the two first helices (5′HB) and last helix (3′HB) of the homeobox are indicated by vertical bars. The map of the wild-type locus shows the deleted region in red, consisting of the start codon together with the exon coding for the first two helixes and amino-terminal part of the third helix of the homeobox and flanking intronic sequences. The map of the targeting vector shows the replacement of the deleted region by the β-galactosidase–neomycin cassette pGNA (green) (Le Mouellic et al. 1990). Arrowheads indicate the positions of the primers used for PCR genotyping. The dark line below the recombined locus denote the position of the genomic probe, external to the targeting vector, used to distinguish the EcoRV-generated 20- and 9-kb hybridizing bands on Southern gel of the wild type and recombined allele, respectively (b). Arrowheads in a denote the relative position of the primers used for PCR analysis and generating 300- and 600-bp amplification products for wild-type and recombined DNA, respectively (c). (E) EcoRV; (B) BamHI; (K) KpnI.

Figure 2

Growth and cell differentiation are deficient in the medioventral forebrain of homozygous Vax1 mutants. (a,b,e,f) 16.5-dpc embryos, hematoxylin–eosin staining; (c,d) P15 brain, cresyl violet staining. The optic chiasm (a, OCh) was systematically absent from homozygous animals so that the mutant optic nerves (arrowheads in b) entered the brain at a lateral hypothalamic level. The telencephalic phenotype of Vax1 homozygous mutants ranged from a total absence of growth of medioventral structures, including the septum (Sep) and preoptic area (POA) (c,d) to a growth–recovery of dorsolateral structures fusing medially (e,f). In this latter case only, fibers crossing the midline may be observed at the anterior commissure level (arrows in c,e,f). The medioventral growth defects resulted in lobar holoprosencephaly (d,f). Bar, 0.5 mm.

Figure 3

Despite morphological alterations, the patterning of the ventral telencephalon is apparently normal in Vax1 mutants. Successive pictures are bright field and dark field of the same view to illustrate the morphological defects observed in the mutants. (a–h,i–t) In situ hybridization on sagittal sections of 11.5-dpc embryos, and on transverse sections of 13.5-dpc embryos, respectively. At 11.5 dpc, the anterior preoptic area (POA) is underdeveloped in the mutant (cf. a and e to c and g). At 13.5 dpc, deficient growth of the mutant ventral forebrain is observed in the septum (Sep) and the medial ganglionic eminence (MGE) (cf. i to k and m to o) and results in holoprosencephaly (see k and o). The pattern of expression of Shh (b,d) and Nkx2.1 (f,h) at 11.5 dpc and that of Dlx1 (j,l) or of Pax6 (n,p) at 13.5 dpc are apparently not modified in homozygous mutants, suggesting that the patterning of the forebrain is not affected by the Vax1 mutation. In addition, the Nkx2.1 signal observed in differentiated or differentiating cells in wild-type embryos in the mantle layer over the subventricular zone of the MGE (asterisks in r) was greatly diminished or absent in Vax1 homozygous animals (t), suggesting that the Vax1 mutation perturbes the regulation of the cell proliferation or differentiation at least at that level. (DB) Diagonal band; (LGE) lateral ganglionic eminence; (MGE) medial ganglionic eminence; (POA) preoptic area; (Sep) septum. Bar, 0.5 mm.

Figure 4

Vax1 homozygous mutants show coloboma and dysgenesis of the optic nerve. (a,b) External view of 12.5-dpc eyes. (c,d) Hematoxylin and eosin staining of transverse section of the optic nerve of 16.5-dpc albino embryos. At 12.5 dpc, the optic fissure is closed in the wild-type eye (a) but remains opened in the mutant eye (b, white arrowhead). At 16.5 dpc, in contrast to the wild-type optic nerve (c), a thick epithelium is observed over the mutant optic nerve fibers (d) running over the open optic fissure (white arrowhead). The arrow in d points to the optic recess. Bar, 0.125 mm.

Figure 5

The Vax1 mutation affects the development of the optic nerve in homozygous mutants. (a–h) In situ hybridization with antisense probes as indicated on the figure, 13.5-dpc embryos; (i,j) β-galactosidase staining, 12.5-dpc embryos; (k,l) Pax2 (green) and Pax6 (red) immunocytochemistry, homozygous Vax1 mutant optic nerve at the temporal level, 13.5-dpc embryo; (l) confocal optical section through the z-axis of the section shown in k between the empty arrowheads. (c,d,g,j,k) Transverse sections of the mutant optic nerve. At 13.5 dpc, Pax6 expression is confined to the retina of the wild-type animal (a), whereas it remains expressed in the mutant optic nerve (b,c). Similarly, Rx is expressed ectopically in the mutant optic nerve (d). At 13.5 dpc, Pax2 expression is confined in the optic disk and optic nerve of wild-type animals (e) and remains expressed in these structures in Vax1 mutants (f,g). Similarly, Vax1 is expressed in the optic disk and optic nerve of wild-type animals (h) and the lacZ reporter gene is observed in these structures in Vax1 mutants (i,j). The expression of Pax2 and of lacZ indicates that the induction of the optic stalk occurred in Vax1 mutants. The thick epithelium observed ventrally (c,g,j) and the pigmented epithelium observed dorsally (j) in the mutant optic nerve therefore, could result from an abnormal development of the optic stalk rather than from an elongation of the retina. Pax2 and Pax6 are both expressed in the mutant optic nerve (k). However, protein expression is mostly exclusive at the cellular level. Coexpression is only detected in few cells expressing both proteins at low level (white arrowheads in k and l) suggesting a reciprocal inhibition of Pax6 and Pax2 and a participation of Vax1 in the down-regulation of Pax6. Dorsal is up in all pictures; rostral is left in c,d,g,j,k. The arrow in j points to the optic recess. Bar, 0.125 mm.

Figure 6

Effect of ectopic Xvax1 overexpression on the transcription of Xrx. Normal expression pattern of Xrx at stages 17 (a) and stage 34 (g). A horizontal vibratome section of a stage 17 embryo is shown in d (anterior is up). Embryos were injected with 250 pg (b,e,h) or 500 pg [c,d(is),f,i] of Xvax1 encoding mRNA into both blastomeres at the two-cell stage. The resulting effects were dose dependent. At stage 17, repression of Xrx is either moderate (b,e) or strong [c,d(is),f], resulting in a mild (h) or strong phenotype (i) at stage 34. (nis) Noninjected side; (is) injected side.

Figure 7

Xvax1 expression is positively regulated by X-bhh. Xenopus embryos were injected into one cell (c–g) or into both cells (h) at the two-cell stage with synthetic mRNA encoding X-bhh. (a) Control embryos (stage 34) express Xvax1 in the optic stalk and disc, as well as in the ventral forebrain. (b) Transverse section from a stage 34 control embryo at the level of the eye define differentially regulated Xvax1 (purple) and Xpax6 (red) expression domains. Xvax1 is expressed in the ventral hypothalamus and in the eye disc, whereas Xpax6 is strongly expressed in the neural retina and in the lens, as well as in the dorsal midbrain. (c–h) Injection of X-bhh leads to a strong induction of Xvax1 (purple) expression (d,e,g,h) and to a severe inhibition of Xpax6 (red) expression (g,h) in the optic primordium. (c) Uninjected side of the embryo shown in d. (e) Transverse section of the embryo in c and d. Xvax1 (purple) is ectopically expressed in the whole remaining eye vesicle. (f) The uninjected side of the same embryo in g reveals almost normal Xpax6 expression (red) in the eye. (h) Transverse section of an embryo injected into both cells at the two-cell stage. Xvax1 is strongly expressed in both eye vesicles. (le) Lens; (od) optic disc; (os) optic stalk; (ov) optic vesicle; (vh) ventral hypothalamus.

Figure 8

Molecular cascade involved in the partitioning of the visual system in eye and optic nerve and implicating Vax1. Pax2 and Vax1 are induced in the optic stalk by midline signals, such as shh, and confine Pax6 and Rx expression in the optic cup.