Several eye-field transcription factors (EFTFs) are expressed in the anterior region of the vertebrate neural plate and are essential for eye formation. The Xenopus EFTFs ET, Rx1, Pax6, Six3, Lhx2, tlland Optx2 are expressed in a dynamic, overlapping pattern in the presumptive eye field. Expression of an EFTF cocktail with Otx2 is sufficient to induce ectopic eyes outside the nervous system at high frequency. Using both cocktail subsets and functional (inductive) analysis of individual EFTFs, we have revealed a genetic network regulating vertebrate eye field specification. Our results support a model of progressive tissue specification in which neural induction then Otx2-driven neural patterning primes the anterior neural plate for eye field formation. Next, the EFTFs form a self-regulating feedback network that specifies the vertebrate eye field. We find striking similarities and differences to the network of homologous Drosophila genes that specify the eye imaginal disc, a finding that is consistent with the idea of a partial evolutionary conservation of eye formation.

Overexpression of XOptx2, a homeodomain-containing transcription factor expressed in the Xenopus embryonic eye field, results in a dramatic increase in eye size. An XOptx2-Engrailed repressor gives a similar phenotype, while an XOptx2-VP16 activator reduces eye size. XOptx2 stimulates bromodeoxyuridine incorporation, and XOptx2-induced eye enlargement is dependent on cellular proliferation. Moreover, retinoblasts transfected with XOptx2 produce clones of cells approximately twice as large as control clones. Pax6, which does not increase eye size alone, acts synergistically with XOptx2. Our results suggest that XOptx2, in combination with other genes expressed in the eye field, is crucially involved in the proliferative state of retinoblasts and thereby the size of the eye.

Photoreceptor and bipolar cells are molecularly related cell types in the vertebrate retina. XOtx5b is expressed in both photoreceptors and bipolars, while a closely related member of the same family of transcription factors, XOtx2, is expressed in bipolar cells only. Lipofection of retinal precursors with XOtx5b biases them toward photoreceptor fates whereas a similar experiment with XOtx2 promotes bipolar cell fates. Domain swap experiments show that the ability to specify different cell fates is largely contained in the divergent sequence C-terminal to the homeodomain, while the more homologous N-terminal and homeodomain regions of both genes, when fused to VP16 activators, promote only photoreceptor fates. XOtx5b is closely related to Crx and like Crx it drives expression from an opsin reporter in vivo. XOtx2 suppresses this XOtx5b-driven reporter activity providing a possible explanation for why bipolars do not express opsin. Similarly, co-lipofection of XOtx2 with XOtx5b overrides the latter's ability to promote photoreceptor fates and the combination drives bipolar fates. The results suggest that the shared and divergent parts of these homologous genes may be involved in specifying the shared and distinct characters of related cell types in the vertebrate retina.

The fibroblast growth factor (FGF) family consists of seven members whose activities are thought to be mediated by multiple receptors. Here we describe the cDNA cloning, expression, and characterization of a cysteine-rich FGF receptor (CFR) that is distinct from previously identified FGF receptors. The deduced amino acid sequence for CFR suggests that it is an integral membrane protein containing a large extracellular domain comprising 16 cysteine-rich repeated units and an intracellular domain of 13 amino acids. No reported sequences exhibit significant homologies to either the repeated extracellular motif or to the entire CFR amino acid sequence. Several CFR transcripts are present in embryonic chick tissue, suggesting that CFR undergoes alternate mRNA splicing or that related genes are present. Chinese hamster ovary cells transfected with the CFR cDNA express a 150.kDa polypeptide that binds FGF-1, FGF-2, and FGF4 but does not bind several non-FGF family members. The high degree of evolutionary conservation among vertebrate CFRs and its ability to bind three different FGFs with high affinity suggest that this unique receptor plays an important role in FGF biology.

The directed differentiation of pluripotent cells into specific cell-types is a major hurdle in regenerative medicine. This study shows the eye field transcription factor factors can direct pluripotent cells into functioning frog eyes.