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The homeodomain protein Vax2 patterns the dorsoventral and nasotemporal axes of the eye

Stina H. Mui1,2, Robert Hindges1, Dennis D. M. O’Leary1, Greg Lemke1,* and Stefano Bertuzzi1,{dagger}

1 Molecular Neurobiology Laboratory, The Salk Institute, La Jolla, CA 92037 USA
2 Department of Neurosciences, University of California San Diego, La Jolla, CA 92093 USA
{dagger} Present address: Telethon Foundation at CNR Istituto Tecnologie Biomediche, 20090 Segrate (Milan), Italy



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  		Fig. 1. Vax2 mRNA expression in the mouse and inactivation of the mouse Vax2 gene. (A) Whole-mount in situ hybridization for Vax2 mRNA in an E9 mouse embryo. (B) High-power view of an E11.5 mouse eye, illustrating the pronounced dorsal-ventral (DV) Vax2 gradient. D, V, N, and T indicate dorsal, ventral, nasal and temporal poles of the retina. (C,D) Front (C) and back (D) views of a whole-mount in situ hybridization for Vax2 mRNA (purple reaction product) in a dissected mouse retina at E14. Arrowhead denotes a ventral cut made before the eye was dissected from the embryo. (E) In situ hybridization of a coronal section through a wild-type P0 mouse eye, demonstrating high expression of Vax2 mRNA in the retinal ganglion cells (rgc) of the ventral (V) but not the dorsal (D) retina. (F) In situ hybridization of a transverse section through a wild-type P0 mouse eye, demonstrating slightly higher expression of Vax2 mRNA in the retinal ganglion cells (rgc) of the nasal (N) than the temporal (T) retina. (G) Targeting construct for inactivation of the mouse Vax2 gene, and structure of the inactivated allele after homologous recombination in mouse embryonic stem cells. A BamHI/EcoRV genomic fragment containing exon 2 of the Vax2 gene was replaced by a PGK-neo cassette (see Materials and Methods). Exon 2 encodes amino acid residues 83-145; these residues include the first two {alpha} helices of the Vax2 homeodomain, which are essential to the function of all known homeodomain transcription factors. The BamHI/EcoRV deletion also introduces a shift in the Vax2 reading frame. (H) Southern blot of XbaI and SalI-digested genomic DNA from wild-type (+/+) and heterozygous (+/–) ES cell clones probed with the external 3' probe indicated in G and described in Materials and Methods. (I) RT-PCR analysis of mRNA isolated from wild-type (+/+) and Vax2 mutant (–/–) retinae at P0. The primers for the PCR reaction are located in exons 1 and 3, and correspond to nucleotides 127-148 and 586-606 of the mouse Vax2 cDNA sequence (Genbank Accession Number, Y17792). The size of the –/– fragment is consistent with the clean excision of exon 2, which was confirmed by DNA sequence analysis of the 468 and 261bp bands, and by the loss of Vax2 immunoreactivity in the homozygous mutant retina (data not shown). Scale bar: 0.1mm in E,F.

 


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  		Fig. 2. Aberrant projections of retinal ganglion cell (RGC) axons from the retina to the superior colliculus (SC) in Vax2 mutants, as assessed by anterograde axonal tracing with DiI. (A) A focal DiI injection into the ventral temporal (VT) quadrant of the retina (indicated by the circle on the upper panel retinal flatmount schematic) labels RGC axons that terminate at the medial-rostral (MR) border of the SC in wild-type and heterozygous mice [+/–, middle panel, arrow indicates termination zone (TZ)], but at the rostral-lateral border of the SC in the mutant (–/–, lower panel, arrow). Note that for all anterograde labeling we analyzed, the projections of heterozygous Vax2+/– axons were indistinguishable from the previously described projections of wild-type RGCs. (B) A mid-ventral injection (split circle on schematic) labels RGC axons that terminate near the medial border of the SC at the midpoint of the collicular RC axis in both heterozygous (+/–, middle panel, arrow) and mutant mice (–/–, lower panel, arrow). Arrowheads indicate additional multiple ectopic TZs in the mutant, some of which are lateralized. (C) A focal injection into the ventral nasal quadrant of the retina (circle, upper panel) labels RGC axons that terminate in the medial-caudal SC in both heterozygous (+/–, middle panel) and Vax2 mutant mice (–/–, lower panel). (D-W) SC diagrams depicting expected (squares) and observed collicular TZs (gray) from focal DiI injections into the ventral retinae of Vax2 mutants, illustrating the progressive change in misprojection phenotype from extremely strong for ventral temporal injections (D-F) to extremely weak for ventral nasal injections (U-W). The position of the expected TZ in the SC is plotted from the observed position of the retinal injection site, assuming a linear map from extreme ventral retina=extreme medial SC to extreme dorsal retina=extreme lateral SC. Squares and gray shapes are scaled to indicate relative sizes of retinal injection sites and collicular TZs, respectively. With the exception of S, all panels illustrate results from ventral retinal injection between 60-85% of the DV axis, with extreme dorsal defined as 0%. c, caudal; l, lateral; m, medial; r, rostral.

 


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  		Fig. 3. Maintained ipsilateral projections in the Vax2 mutants. Whole single eye injections with fluorescent cholera toxin B subunit label abundant contralateral (contra) and rare ipsilateral (ipsi) RGC projections in both Vax2+/– (upper panels) and Vax2–/– (lower panels) mice at P7. Arrowheads indicate ipsilateral termination sites of RGCs in successive rostral to caudal serial coronal sections, spaced at 200 µm, which traverse the thalamus into the SC. The ventral midline is indicated by the vertical lines.

 


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  		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., 2000Go), 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.

 


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  		Fig. 5. Two models for the concerted combinatorial action of Vax2 and other transcriptional regulators along the nasal-temporal (NT) axis of the ventral retina. (A) Ventral regulators are normally expressed at higher levels in the nasal than the temporal ventral retina, and a minimum aggregate level of the regulators (indicated by the striped bar) is necessary for proper retinocollicular mapping. When one of the regulators (e.g. Vax2) is removed by mutation, the aggregate level becomes limiting (falls below the threshold bar) only in the ventral temporal retina. (B) Vax2 (left discs), which is normally steeply graded along the DV axis, genetically interacts with one or more genes that are graded along the NT axis (middle discs), to form a composite wild-type map of axon guidance regulators (right discs). With the loss of Vax2, the map reverts to that of the NT genes alone, which again most severely affects ventral temporal RGCs. See text for details.

 

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© The Company of Biologists Ltd 2002