Fig. 4. Flattened gradients of axon guidance cues and maintained gradients of transcription factors in the retinae of Vax2 mutants at birth. (A) Ephb2 mRNA (visualized as pink/red silver grains) is expressed in a high ventral (V)/low dorsal (D) gradient in both the wild-type and heterozygous retina (+/–, lower and upper left panels, respectively), and is lost in the ventral retina of the mutants (–/–). The positions of the retinal ganglion cell and pigment epithelial layers are denoted by filled and open arrowheads, respectively, in the upper left panel. (The intense signal seen in the pigment epithelial layer of nearly all panels is an artifact caused by refraction of pigment when viewed with dark field optics. The image used for C was, by chance, generated from an unpigmented mutant.) Essentially identical results were obtained for mRNA encoding the closely related and similarly graded receptor EphB3. (B) mRNA encoding ephrin-B1, a ligand for EphB2/B3, is expressed in a high dorsal (D)/low ventral (V) gradient, the inverse of EphB2/B3, in the heterozygous retina (+/–, left panels), and this mRNA is upregulated in the ventral retina of the mutants (–/–, right panels). Essentially identical results were obtained for mRNA encoding the closely related and similarly graded ligand ephrin-B2. (C) Epha5 mRNA is expressed in a high temporal (T)/low nasal (N) gradient in the heterozygous retina (+/–, left panels), and is upregulated in the ventral nasal mutant retina of the mutant (–/–, right panels). (D) mRNA encoding the transcription factor Tbx5 exhibits a pronounced high-dorsal-to-low-ventral gradient of expression in wild-type P0 mouse retinae (Koshiba-Takeuchi et al., 2000), and this gradient is not changed in the Vax2 mutants (–/–). (E) Expression of Bf1 mRNA (dark purple reaction product) at E9.5 in wild type is most pronounced in the ventral nasal quadrant (arrowhead) of the optic vesicle (circled). Anterior is towards the left. (F) Expression of Six6 mRNA at E9.5 in wild type is most pronounced in the ventral temporal quadrant (arrowhead) of the optic vesicle (circled). Anterior is towards the left. (G) Radioactive in situ hybridization to transverse sections of heterozygous (+/–) and mutant (–/–) retinae at E16.5 demonstrate unaltered expression of Bf1 mRNA, which remains preferentially expressed in the nasal (N) as opposed to temporal (T) side of the eye. Scale bar: 0.1 mm.
