We are exploring the mechanisms of photoreceptor patterning from two directions: from a knowledge of the upstream patterning genes and from our studies of the determination of photoreceptor fate. Our work over the last 15 years on the development of photoreceptors has focussed on the determination and differentiation of rod photoreceptors (Morrow et al. 1999). We thus have some hypotheses of how an area of the retina may fail to have rod photoreceptors. We have been looking for genes that control retinal patterning early in development. Several genes that are expressed asymmetrically across the early retina provide a starting point for an understanding of how the central zone is designated to be the rod-free zone (Figure 2). These genes include the mVax2 gene of mice and the cVax gene of chicks, which are expressed only ventrally (Figure 3), the Tbx5 and BMP4 genes, which are expressed only dorsally, and Soho-1, GH6, and BF1 genes of chick, which are expressed only in the anterior portion, and the BF2 gene expressed only in the posterior region.
In addition to the transcription factors listed above, there are small molecules that may pattern the retina through interactions with their receptors. Retinoic acid (RA) synthetic and degrading enzymes are patterned along the dorsoventral axis, and have been shown to play a role in retinal patterning in fish and frogs. Thyroid hormone synthetic and degradative enzymes similarly are patterned in the retina. Work in mice and humans suggest a role for TH in retinal development, and more broadly in many developing tissues. We have been investigating the roles of TH and RA in formation of retinal pattern. As well, we have investigated the roles of several transcription factors in retinal patterning. The data so far suggest that a complex set of interactions direct the formation of photoreceptor pattern. Vax appears to affect the distribution of rod photoreceptors, as does RA and TH. Effects on the cone pattern can also be seen with TH. Our work now focuses on how these genes work in what may be a complex network where they can effect each other’s level of expression. Microarrays are being used to find the genes affected by manipulation of Vax, RA or TH. RNAi vectors that are effective against the TH synthetic and degradative enzymes, as well as the TH receptors, have been made and are being scored for their effects on retinal development. The same has been done for the RA pathway and Vax. We have made reporter constructs for signal transduction by TH and RA and these are being used to reveal where in vivo the TH and RA signals are being read. Bioinformatic analyses of genes whose levels change following such manipulations are also being carried out to find potential cis-acting regulatory sequences for action by TH and RA.
Figure 1. A chick retina from embryonic day 18 is shown. It is flattened to reveal the entire retina following hybridization with a rhodopsin probe. There is a rod-free central zone (arrow) and a rod-sparse horizontal stripe (arrowheads). The human fovea comprises a central rod-free zone.
Figure 2. The chick retina exhibits both anterior-posterior (AP) and dorsal-ventral (DV) gene expression patterns early in development. At the optic vesicle stage, at about stage 10 in the chick, the AP axis is set and the winged helix gene, BF-1, is expressed in the posterior vesicle. When invagination to form an optic cup occurs, the DV axis becomes set. Several genes then appear and are patterned along the AP or DV axis. Soho-1, GH6, and BF-1 are in the anterior retina, while EphA3 is in the posterior retina. Vax is in the ventral domain, while Tbx5 is dorsal.
Figure 3. Vax is expressed in the ventral retina. A murine gene, mVax2, and chick gene, cVax, are expressed early in development in the ventral region. CVax is shown here, at HH stage 14 (A), a section from the retina at E4 (B), and a whole mount retina at E6 (C). Misexpression of cVax or mVax2 throughout the retina causes a ventralization of gene expression patterns and prevents dorsal ganglion axons, but not ventral ganglion axons, from proper targeting in the tectum.
Figure 4. RA synthetic enzymes, RALDH1 and RALDH3, are expressed in the dorsal and ventral domains of the early chick retina, respectively.
Secreted proteins of the Wnt family initiate signal transduction pathways that regulate multiple aspects of central nervous system (CNS) development. We examined the expression of Wnt signaling pathway members in the development of the eye. Wnt2b is expressed very early in the dorsal surface ectoderm, as well as slightly later in the pigmented epithelium and peripheral portion of the optic cup (Figure 6). We investingated the role of Wnt signaling in the developing vertebrate eye using the chick retina. We found that Wnt2b or constitutively active b-catenin (CA-b-catenin) can pattern the optic vesicle, leading to induction of the peripheral fates of the optic cup: the ciliary body and iris. As the Drosophila Wnt, wingless (wg), controls the patterning of peripheral eye structures in Drosophila, these findings strengthen the model of a common origin for the eye of vertebrates and invertebrates.
Figure 5. Expression of Wnt signaling genes and retinal progenitor genes during early chick eye development.
(A to D) In situ hybridization (ISH) for chick Wnt2b. Wnt2b signal was observed exclusively in the surface ectoderm (SE) at the OV stage (A and B), and in the RPE and the tip of the retina, in addition to SE, at the OC stage (C and D). (E to L) ISH for Lef1 (E and I), Frizzled 4 (F and J), Chx10 (G and K) and Cyclin D1 (H and L) at the OV stage (E to H) and the OC stage (I to L). Arrows in E and F, and in I and J mark the expression in the dorsal OV and in the peripheral OC, respectively. Arrowheads in G, and in K and L indicate the absence of Chx10 expression in the dorsal invaginating OV and the absence of Chx10 and Cyclin D1 expression in the peripheral OC. The line in (E and G) demarcates the margin of the OV. (M) Summary of the expression of Wnt signaling (blue) and retinal progenitor (yellow) genes from OV to OC stages. L, lens, Di, diencephalon, and SE, surface ectoderm. Dorsal side is up. Scale bars, 150 mm.