Previous studies have indicated that retinoic acid (RA) promotes rod photoreceptor differentiation in dissociated cultures of rat retina and in zebrafish embryos. To determine whether RA will have the same affect in the mammalian retina in vivo, pregnant rats were given single i.p. injections of RA on the 18th and 20th days of gestation, and the retinas of the pups were analyzed for rods. HPLC showed that i.p. injections of RA substantially increased levels of retinal RA in the embryos. Embryonic exposure to RA caused an increase in the number of cells that differentiated as rod photoreceptors. There was a comparable decrease in the number of cells that differentiated as amacrine cells. These results demonstrate that RA promotes the differentiation of rods in vivo and further support the hypothesis that differentiation of rods is normally controlled partly by the RA concentration in the developing retina or RPE.
The hedgehog gene family encodes secreted proteins important in many developmental patterning events in both vertebrates and invertebrates. In the Drosophila eye disk, hedgehog controls the progression of photoreceptor differentiation in the morphogenetic furrow. To investigate whether hedgehog proteins are also involved in the development of the vertebrate retina at stages of photoreceptor differentiation, we analyzed expression of the three known vertebrate hedgehog genes. We found that Sonic hedgehog and Desert hedgehog are expressed in the developing retina, albeit at very low levels, whereas Indian hedgehog (Ihh) is expressed in the developing and mature retinal pigmented epithelium, beginning at embryonic day 13. To determine whether hedgehog proteins have activities on developing retinal cells, we used an in vitro system in which much of retinal histogenesis is recapitulated. N-terminal recombinant Sonic Hedgehog protein (SHH-N) was added to rat retinal cultures for 3-12 d, and the numbers of retinal cells of various phenotypes were analyzed by immunohistochemistry. We found that SHH-N caused a transient increase in the number of retinal progenitor cells, and a 2-to 10-fold increase in the number of photoreceptors differentiating in the cultures when analyzed with three different photoreceptor-specific antigens. In contrast, the numbers of retinal ganglion cells and amacrine cells were similar to those in control cultures. These results show that Hedgehog proteins can regulate mitogenesis and photoreceptor differentiation in the vertebrate retina, and Ihh is a candidate factor from the pigmented epithelium to promote retinal progenitor proliferation and photoreceptor differentiation.
The mechanisms by which multipotent progenitor cells are directed to alternative cell identities during the histogenesis of the vertebrate central nervous system are likely to involve several different types of signaling systems. Recent evidence indicates that 9-cis retinoic acid, which acts through members of the steroid/thyroid superfamily of receptors, directs progenitor cells to the rod photoreceptor cell fate. We now report that another effector of this family of receptors, thyroid hormone, induces an increase in the number of cone photoreceptors that develop in embryonic rat retinal cultures, and that combinations of 9-cis retinoic acid and triiodothyronine cause isolated progenitor cells to differentiate as either rods or cones, depending on the relative concentrations of the ligands. These results implicate thyroid hormone in CNS cell fate determination, and suggest that different photoreceptor phenotypes may be modulated through the formation of thyroid/retinoid receptor heterodimers.
The results of several recent studies have demonstrated that cell commitment and differentiation in the developing vertebrate retina are influenced by cell-cell interactions within the microenvironment. Retinoic acid has been shown to influence cell fates during development of the nervous system, and retinoic acid has been detected in the embryonic retina. To determine whether retinoic acid mediates the differentiation of specific neuronal phenotypes during retinal histogenesis, we treated dissociated cell cultures of embryonic and neonatal rat retina with varying concentrations of all-trans or 9-cis retinoic acid and analyzed the effects on cell fate using neuron and photoreceptor-specific antibodies. Addition of exogenous retinoic acid caused a dose-dependent, specific increase in the number of cells that developed as photoreceptors in culture throughout the period of retinal neurogenesis. In the same cultures, retinoic acid also caused a dose-dependent decrease in the number of cells that developed as amacrine cells. Also, results of double-labeled immunohistochemical studies using bromodeoxyuridine demonstrated that the primary effect of retinoic acid was to influence progenitor cells to develop as newly generated rod photoreceptors. Since retinoic acid and at least one of the retinoic acid receptors (RAR alpha) have been localized to the developing neural retina, these results suggest that retinoic acid may play a role in the normal development of photoreceptor cells in vivo.
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