Ligands of steroid/thyroid receptors induce cone photoreceptors in vertebrate retina

Matthew W. Kelley ) Jennifer K. Turner Thomas A. Reh - 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 The mechanisms that direct neuronal progenitor cells to differentiate into various types of neurons in the vertebrate central nervous system are not well understood. Heterochronic coculture studies and in vivo cell deletion studies have demonstrated that factors in the local microenvironment of retinal progenitor cells can influence their ultimate choice of fate (Reh, 1987, 1992a; Raymond et al., 1988). Recently, a number of laboratories have concentrated on the factors that regulate rod photoreceptor identity and several molecules have now been shown to have effects on rod photoreceptor differentiation, including peptide growth factors, taurine and retinoic acid (Anchan et al., 1991; Hicks and Courtois, 1992; Altshuler et al., 1993; Kelley et al., 1994). However, specific cues that promote or influence the differentiation of other types of retinal neurons have not been identified. Several laboratories have demonstrated that the vitamin A derivative, retinoic acid, may be involved in various aspects of retinal development (McCaffery et al., 1993; Kelley et al., 1994; Marsh-Armstrong et al., 1994). In particular all-trans and 9-cis retinoic acid (RA) induce retinal progenitor cells to differentiate as rod photoreceptors and inhibit progenitor cells from differentiating as amacrine cells in vitro (Kelley et al., 1994). These results suggest that one of the effects of retinoic acid may be to specifically direct progenitor cells towards one cell fate over another. The effects of RA are mediated through the activation of retinoic acid receptors (RARs) and retinoid X receptors (RXRs; Petkovich et al., 1987; Giguere et al., 1987; Mangelsdorf et al., 1990), and at 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. least one member of this family of receptors, RXR-g , is expressed by the progenitor cells in embryonic retina (Kelley, Williams and Reh, unpublished observations). These receptors are members of the steroid/thyroid receptor superfamily of transcription factors, and are believed to bind to DNA response elements only after the formation of either RXR:RXR homodimers, or heterodimers of RXR with other members of this receptor family, including RAR, thyroid receptors (TR) and vitamin D receptors (Yu et al., 1991; Durand et al., 1992; Kleiwer et al., 1992; Zhang et al., 1992; Marks et al., 1992; Bugge et al., 1992; Leid et al., 1992). Previous studies have demonstrated that changes in the levels of thyroid hormones during development can have diverse influences on the nervous system and brain. In particular, the rates of cell proliferation and neuronal differentiation in the cerebral cortex and cerebellum are altered in both hyper- and hypothyroid animals (Nicholson and Altman, 1972; Lauder, 1977; Hetzel et al., 1988; Gould and Butcher, 1989; Pickard et al., 1993). In addition, thyroid receptors are expressed throughout the developing rat brain, including the progenitor zones, beginning as early as embryonic day 11.5 (E11.5; Mellstrom et al., 1991; Bradley et al., 1992). Finally, a novel thyroid hormone receptor isoform has recently been shown to be expressed in embryonic chick retina, at stages when progenitor cells are becoming committed to differentiate as photoreceptors (Sjoberg et al., 1992). Based on these results, we hypothesized that activation of TRs might also influence determination of photoreceptor cells in the developing retina. MATERIALS AND METHODS Cell culture Retinal cells were dissected from embryonic day 18 (E18) Long Evans rat fetuses and established in high density cultures, as described previously (Kelley et al., 1994). At this stage in vivo, approximately 10% of newly generated cells will differentiate as cone photoreceptors, 35% as amacrine cells, and 50% as rod photoreceptors (M. LaVail, personal communication). Since progenitor cells are influenced to differentiate as several different types of retinal cells at this point in development, this is an ideal stage to examine the effects of different factors on determination of cell fate. Immediately after plating, a stock solution of 10 mM triiodothyronine (T3; Sigma), the active form of thyroid hormone, in saline was diluted with the culture medium to specific concentrations. Subsequent media changes were made every 48 hours and cultures were maintained for a total of either 6 or 14 days in vitro (DIV). In cultures that were maintained for 14 DIV, increased cell density prevented accurate identification of individual immunolabeled cells. Therefore, 12 hours prior to the completion of an experiment these cultures were redissociated using a previously published technique (Kelley et al., 1995) and then replated at a lower density to allow identification of single-labeled cells. Briefly, the culture medium was removed from individual culture wells and replaced with calcium- and magnesiumfree saline with 0.15% trypsin. The culture plates were then incubated with gentle agitation for 10 minutes at 37C. Cultured retinal cells were collected by aspiration, concentrated by centrifugation, resuspended in medium, dissociated by trituration, and replated at a lower density onto new Matrigel-polylysine-coated coverslips. At the end of the culture period, cells were fixed with 4% paraformaldehyde for 2 hours at room temperature and then washed with phosphate-buffered saline. Overall cell number was determined using a previously described technique (Kelley et al., 1994). Briefly, each well of the 24-well plate contained a coverslip that covered 64% of the total area of the bottom of the well. After the coverslips were removed and fixed for immunohistochemistry, the remaining cells (36% of the total) were dissociated with trypsin and counted using a hemocytometer. Determination of retinal phenotypes Differentiation of specific retinal phenotypes was determined using immunocytochemical markers. The following criteria were used to classify retinal cell types. (1) Newly differentiated photoreceptors were immunoreacti (...truncated)