Retinoic acid promotes differentiation of photoreceptors in vitro

Matthew W. Kelley 0 Jennifer K. Turner 0 Thomas A. Reh 0 0 Department of Biological Structure, SM-20, University of Washington , Seattle, WA 98195 , USA - 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 During the development of the vertebrate central nervous system, the proliferating cells of the germinal neuroepithelium generate a variety of different types of neurons and glia. A number of studies in both the central and peripheral nervous systems have shown that interactions between the progenitor cells and their developing microenvironment play an essential role in the determination of cell phenotype (see McConnell, 1992 and Bronner-Fraser, 1992 for reviews). In an effort to characterize further the nature of these intercellular interactions, we have studied the control of cell fate decisions during histogenesis of the rat neural retina in tissue culture. The rat retina is particularly attractive as a model for the study of the factors that control cell commitment in the nervous system, because the different neuronal cell types are well characterized and are generated in a well-defined sequence during retinal histogenesis (see Reh, 1992a for review). In most mammals, including rats, the generation of the different classes of retinal cells can be roughly divided into early and late phases (Robinson, 1991; LaVail et al., 1991). In the early phase, ganglion cells, horizontal cells, cone photoreceptor cells and most amacrine cells are born, while most rod photoreceptor cells, bipolar cells and Muller glia have birthdates in the later phase of histogenesis. Recently, several laboratories have taken advantage of this defined sequence in retinal histogenesis to devise heterochronic co-culture experiments to partly characterize the nature of the interactions necessary for the differentiation of 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 (RARa ) 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. retinal cells in rodents (Watanabe and Raff, 1990, 1992; Reh, 1992a,b; Altshuler and Cepko, 1992). Using reaggregate cultures of dissociated retinal cells, Watanabe and Raff (1990) found that rat retinal cells from embryonic day 15 (E15) could be induced to form rod photoreceptor cells (a cell type generated primarily during the postnatal period of histogenesis) at a higher frequency when mixed with postnatal retinal cells. In a subsequent study (Watanabe and Raff, 1992), the same authors demonstrated that the rod promoting effect was not inhibited when E15 and postnatal cells were separated by a millipore filter. Based on these results, the authors concluded that the rod phenotype was promoted by a soluble factor. In a similar series of experiments, early embryonic mouse cells (E12) were shown to differentiate into rod photoreceptor cells with a higher frequency when co-cultured with either postnatal rat retinal cells (Reh, 1992a) or with later staged embryonic retinal cells that were competent to generate rod photoreceptors (Reh, 1992b). Overall, the results of these studies suggest that the differentiation of retinal progenitor cells as rod photoreceptors is influenced by locally diffusible signals in the extracellular environment, that are developmentally regulated during the period of retinal histogenesis. Although the specific factors that promote differentiation of cells as rod photoreceptors are not known, several classes of molecules have been shown to influence cell determination in other developing systems and might also play a role in the developing retina. For example, small peptide growth factors have been shown to play a role in the developing nervous system (Anchan et al., 1991; Park and Hollenberg, 1989; Pittack et al., 1991; Hicks and Courtois, 1992). Several laboratories have studied the effects of both epidermal growth factor (EGF) and fibroblast growth factor (FGF) in retinal cultures. Although these growth factors have been shown to promote proliferation of retinal progenitor cells and rod photoreceptor survival, none of the factors that were tested stimulated the specific differentiation of photoreceptor cells from embryonic retinal progenitors (Anchan et al., 1991; Hicks and Courtois, 1992; Lillien and Cepko, 1992). Among the other classes of molecules that have been shown to influence cell fate in the developing nervous system is the vitamin A derivative retinoic acid. In particular, all-trans retinoic acid has been shown to alter cell fate decisions in the developing limb bud, hindbrain and inner ear (Tickle et al., 1982; Thaller and Eichele, 1987; Durston et al., 1989; Represa et al., 1990; Ruiz i Altaba and Jessell, 1991; Kelley et al., 1993). More recently, the 9-cis isomer of retinoic acid has been shown to have biological effects that are apparently mediated through the RXR family of nuclear receptors (Mangelsdorf et al., 1992; Thaller et al., 1993; Kraft and Juchau, 1993). In addition, several studies have demonstrated that both all-trans and 9-cis retinoic acid and at least one of the nuclear retinoic acid receptors (RARa ) are present in the developing retina (McCaffery et al., 1992; Ruberte et al., 1991; Kraft and Juchau, 1993). Moreover, recent studies have found that retinoids have effects prior to, and following, the period of retinal histogenesis; retinoic-acid treatment of frog and zebrafish embryos at gastrulation results in subsequent changes in retinal patterning (Manns and Fritzsch, 1991; Hyatt et al., 1992); retinoids, including all-trans retinoic acid, can influence the survival of cells dissociated from E8 chick retina, a stage when neurogenesis is largely complete (Stenkamp et al., 1993). Thus, although (...truncated)