PATTERNING OF THE RETINA

Nathan Billings (Graduate Student)

Sunjay Harpavat, (Former Graduate Student)

Jonaki Sen, (Former Postdoctoral Fellow)

Dorothea Schulte (Former Postdoctoral Fellow)

The retina exhibits several aspects of patterning within the two dimensional sheet of cells of the retina. The most well-studied aspect of this type of patterning is the retinotectal map, the topographic map formed by the retinal ganglion axons with their target, the tectum. Several other aspects of the retina are also patterned. In most species, the photoreceptors are not uniformly distributed across the retina. In humans and birds of prey, there is an area of high acuity vision in the center of the retina. This area, the fovea, has only red and green cones in humans. Just outside of this area are the blue cones and all other cells that normally process the signals from photoreceptors. Very little is known about the mechanisms that direct the distribution of photoreceptors. We are investigating this at the molecular level using the chick and mouse as model systems. The chick has rod-free central spot (Bruhn and Cepko, 1996) (Figure 1), much like the human fovea, and the mouse has an asymmetric distribution of short and long wavelength cones across the dorsoventral axis.

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.

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, and that of others, on the development of photoreceptors has focussed on the determination and differentiation of rod photoreceptors. 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.

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.

We have carried out misexpression studies in the chick with cVax. When cVax is expressed throughout the retina, the rod pattern is lost (Figure 3). In addition, we used ablations of early optic vesicle tissue, and found that the rod-free zone must have both dorsal and ventral tissue present in order to develop. From these studies, we hypothesize that the dorsal-ventral border, in part defined by cVax expression, is required to set up the rod-free zone and other aspects of the rod pattern in chick (Schulte et al. 2005).

Figure 3. Photoreceptor distribution following forced expression of cVax. Panel 3A shows the distribution and spacing of rods in an uninfected control retina in low magnification. Arrows mark the regions shown in higher magnification in panels 3B-D: the rod free zone (Figure 3B), a dorsal-temporal (Figure 3C) and a ventral-temporal area (Figure 3D). Panels 3E-H show representative results obtained after retroviral misexpression of cVax: the rod free zone does not form, the overall rod distribution is disturbed, patches of high rod density are scattered across the retina. Panels 3F-3H show the randomization of the rod distribution in higher magnification. The regions, from which the pictures are taken, are indicated by arrows in panels 3E. Cones expressing green opsin are normally evenly distributed along the A-P and D-V axes (uninfected retina in Figure 3I and 3J). Ectopic cVax expression under similar conditions as used for Figure 3E-H did not affect this pattern (Figure 3K and 3L). The higher magnification views of the green cones of an uninfected control (3J) and RCAS-cVax infected experimental retina (3L), show the same regularity and spacing among green cones in both uninfected and infected retinae. [in all panels is dorsal to the top, ventral to the bottom, nasal / anterior to the right and temporal / posterior to the left. Scale bars: 3.75 mm]. From Schulte et al. 2005.

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 (TH) 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. 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. (See Sen et al., 2005 and Trimarchi et al. 2008)

Figure 4. RA synthetic enzymes, RALDH1 and RALDH3, are expressed in the dorsal and ventral domains of the early chick retina, respectively.

Figure 5. Spreading waves of expression of Dio2, the synthetic enzyme for TH, and Dio3, the enzyme that deactivates TH, at the indicated stages in the chick retina, as seen from flat mounted retinae.

Figures 5-7. The synthetic enzyme that produces active thyroid hormone, deiodinase 2, as well as Dio3, the enzyme that destroys active thyroid hormone, are dynamically regulated in the chick retina during development. In situ hybridizations on chick retinas at the indicated stages demonstrate the somewhat non-overlapping patterns of these genes. Figure 5 shows expression in an intact retina, or flat mount. Figures 6 and 7 show expression in retinal cross sections. From Trimarchi et al 2008.