Homozygous Vax2 mutant mice show coloboma in a significant percentage of cases. (A,B) Whole-mount eyes dissected from E16.5 foetuses. (A,B) Frontal views of wild-type (A) and mutant (B) eyes. The open optic fissure in the mutant is indicated by arrows in B. (C,D) Detection of laminin by immunohistochemistry in order to identify the basal lamina in frontal sections of wild-type (C) and Vax2^–/– (D) E12.5 embryos. Note how the basal lamina still persists in the mutant eye in the contact regions of the converging lips of the optic fissure (arrowheads), while in the wild-type eye it has completely dissolved. (E-H) Frontal sections, stained with Haematoxylin and Eosin, of wild-type (E,G) and Vax2 mutant (F, H) eyes at E16.5 (E,F) and P7 (G,H). No differences, apart from the presence of coloboma (arrowheads) can be detected in Vax2 mutant mice compared with wild-type animals in the organisation of retinal cell layers. re, retina; le, lens.

Effects of Vax2 inactivation on the expression of markers of DV and of temporo-nasal asymmetry in the eye, as determined by RNA in situ hybridisation. Pax2 expression at E12.5, which is normally restricted (within the optic cup) to the lips of the optic fissure, was the same in wild-type (A) and Vax2 mutant (B) mice. The white arrow in B indicates the persistence of the optic fissure in Vax2^–/– mice. Similarly, the expression of Tbx5, a marker of the dorsal retina, was comparable between normal mice at E12.5 (C) and E16.5 (E), and mutant animals at the same stages (D,F, respectively). (G,H) Frontal sections of a wild-type (G) and of a Vax2^–/– (H) mouse embryos at E16.5 hybridised with the probe for Efnb2 (ephrin B2) probe. Expression of Efnb2, which is normally restricted to the dorsal retina (G), is extended to the ventral retina of Vax2^–/– mice. (I,J) Frontal sections of a wild-type (I) and of a Vax2^–/– (J) mouse embryos at E16.5 hybridised with the probe for Ephb2. The Ephb2 mRNA at E16.5 is expressed with a high ventral to low dorsal gradient in wild-type mice, while in Vax2^–/– mice, it is expressed uniformly, and at lower levels, throughout the retina. (K-N) Sagittal sections of wild-type (K,M) and of Vax2^–/– (L,N) mouse embryos at E16.5 hybridised with the probes for Epha5 (K,L) and Efna5 probes (M,N). No differences could be detected in the expression of the latter marker between wild-type and Vax2^–/– mice. The white arrows in F,H indicate the presence of coloboma in Vax2^–/– mice. le, lens.

Retinal ganglion cell axon pathway and projections are altered in Vax2 mutant mice. The ventral retina (C,D) or the right optic nerve head (A,B; E,H) of new-born wild-type (WT) or Vax2 mutant mice were filled with DiI to label the retinal ganglion cell axon trajectory. (A-D) Ventral views of the brain at the chiasm region (dotted lines indicate the midline). (E,H) Coronal sections through the contralateral (E,F) and ipsilateral superior colliculus (G,H). All sections are oriented rostral (R) towards the top and lateral (L) towards the right, as indicated in E. Note how retinal ganglion cell axons normally projecting in the ipsilateral optic tract (arrows in A,C) are totally absent in the mutant mice (B,D). Furthermore, note how, in mutant mice, projections to ipsilateral superior colliculus are absent (compare G with H), while those to the contralateral side terminate predominantly in the lateral region (compare E with F), where normally the majority of axons arising from the dorsal retina project.