Differentiation of outer retinal cells

We know more about the differentiation of rod photoreceptors than about any other CNS neuron. The extensive background of molecular information derived from numerous studies of phototransduction has provided an excellent set of markers, and the medical importance of photoreceptor degenerative diseases has spurred extensive studies of this cell type. As indicated earlier, most rods become postmitotic near the end of retinal development, in the first few postnatal days in mice and rats. A few rods can be labeled by thymidine injection as early as E14, although they show further signs of differentiation at the same time as the vast majority of rods born later (Morrow et al., 1998). This suggests that the events occurring during the final mitosis can be separated from those of overt differentiation. Rods are formed from late progenitors, some of which can also form bipolar cells and Müller glial cells.

The visual pigment protein opsin is one of the earliest markers of rod differentiation and, because its early appearance is under transcriptional regulation, it has frequently been used to designate rod formation (Treisman et al., 1988). If retinal cells from postnatal rats or mice are plated in dilute monolayer culture, they form many opsin-expressing rods. If cells from early or midembryonic stages are plated, no opsin-positive rods are detected. On the other hand, if the embryonic tissue is maintained as an explant culture or dissociated and allowed to reaggregate, opsin-positive rods are detected at a time equivalent to the normal time of expression. This suggests that the information required to generate rods is intrinsic to the retina but that it needs cell-cell interactions for expression. Embryonic cells mixed with neonatal retinal cells (that are at their peak of rod differentiation) form more rods but only after the appropriate number of days in culture (Watanabe and Raff, 1992). These and related experiments suggest that there are factors present within retina that can promote formation of rod photoreceptors, but only when the progenitor cells are competent to respond to them. At present, the relative roles of factors located on the cell surface and secreted factors are unknown. Numerous factors including FGFs, retinoids, and even compounds like taurine can increase opsin expression in culture. There is, however, little direct evidence that any of them act directly on cell determination or opsin transcription pathways. It is more likely that they have relatively nonspecific effects that make the cells more healthy and better able to express differentiated functions.

In contrast to the positive effects of the growth factors mentioned above, treatment of retinal explants with cytokines of the interleukin-6 (IL-6) family, such as ciliary neurotrophic factor (CNTF) or leukemia inhibitory factor (LIF), completely inhibits the production of rod photore ceptors, as judged by opsin expression (Ezzeddine et al., 1997; Neophytou et al., 1997). These cytokines are known to bind to multisubunit membrane receptors and activate a tyrosine kinase. The intracellular transduction pathways activated by cytokine binding include those characterized by MAPK and STAT proteins. Activation of either of these can lead to changes in transcription. By using pharmacological blockers of MAPK and viruses expressing either constitu-tively active or dominant negative STAT proteins, we have shown that the MAPK pathway is likely to be involved in Müller glial cell formation and the STAT pathway (specifically STAT3) in rod formation. Activation of STAT3 leads to a change in expression of several key transcription factors. Most relevant are increases in the levels of Otx-2 and HES-1. Otx-2 is thought to play a role in the decision of progenitors to become either bipolar cells or rods. Since reduced levels of Otx-2 lead to production of more rods, it is reasonable to assume that elevated levels will help prevent rod formation. Hes-1 is an important regulator of development in a number of tissues, is a bHLH protein homologous to the hairy and enhancer of split genes of Drosophila, and generally acts as a repressor of transcription. Its expression can be increased by activation of the Notch pathway. We do not yet know whether the cytokine-induced increase in Hes-1 expression is mediated through a Notch signaling pathway or whether this is another example of regulation through two independent pathways.

Positive regulators of rod formation include a number of bHLH transcription factors. The best studied of these is Neuro D, the same gene that influences amacrine cell production. Transfection of Neuro D into retinal cultures can increase the number of rods formed, and blocking of Neuro D can diminish rod production. Hes-1 inhibits Neuro D expression; thus, this pathway may be responsible for the cytokine-induced inhibition of rod formation.

At present, we do not how Neuro D facilitates rod formation. It does not appear to have a direct effect on the opsin gene, suggesting that there must be several steps between Neuro D expression and opsin expression. It has also been found that inhibition of Neuro D function increases the production of Müller glial cells. This suggests that Neuro D functions at the time a retinal progenitor is making a choice of cell differentiation pathway.

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