Plasticity of photoreceptor-generating retinal progenitors revealed by prolonged retinoic acid exposure

Stevens et al. BMC Developmental Biology 2011, 11:51 http://www.biomedcentral.com/1471-213X/11/51 RESEARCH ARTICLE Open Access Plasticity of photoreceptor-generating retinal progenitors revealed by prolonged retinoic acid exposure Craig B Stevens1, David A Cameron2 and Deborah L Stenkamp1* Abstract Background: Retinoic acid (RA) is important for vertebrate eye morphogenesis and is a regulator of photoreceptor development in the retina. In the zebrafish, RA treatment of postmitotic photoreceptor precursors has been shown to promote the differentiation of rods and red-sensitive cones while inhibiting the differentiation of blue- and UVsensitive cones. The roles played by RA and its receptors in modifying photoreceptor fate remain to be determined. Results: Treatment of zebrafish embryos with RA, beginning at the time of retinal progenitor cell proliferation and prior to photoreceptor terminal mitosis, resulted in a significant alteration of rod and cone mosaic patterns, suggesting an increase in the production of rods at the expense of red cones. Quantitative pattern analyses documented increased density of rod photoreceptors and reduced local spacing between rod cells, suggesting rods were appearing in locations normally occupied by cone photoreceptors. Cone densities were correspondingly reduced and cone photoreceptor mosaics displayed expanded and less regular spacing. These results were consistent with replacement of approximately 25% of positions normally occupied by red-sensitive cones, with additional rods. Analysis of embryos from a RA-signaling reporter line determined that multiple retinal cell types, including mitotic cells and differentiating rods and cones, are capable of directly responding to RA. The RA receptors RXRg and RARab are expressed in patterns consistent with mediating the effects of RA on photoreceptors. Selective knockdown of RARab expression resulted in a reduction in endogenous RA signaling in the retina. Knockdown of RARab also caused a reduced production of rods that was not restored by simultaneous treatments with RA. Conclusions: These data suggest that developing retinal cells have a dynamic sensitivity to RA during retinal neurogenesis. In zebrafish RA may influence the rod vs. cone cell fate decision. The RARab receptor mediates the effects of endogenous, as well as exogenous RA, on rod development. Background The vertebrate retina forms from a neuroepithelium that develops into a complex, layered structure of neurons consisting of the ganglion cell layer (GCL); the inner nuclear layer (INL), composed of the amacrine, horizontal, and bipolar cells; and the outer nuclear layer (ONL), composed of the light-sensing photoreceptors. The retinal photoreceptor layer is apposed by the non-neuronal layer of retinal pigmented epithelial (RPE) cells. Retinal neurogenesis follows a common pattern in most species; in zebrafish the ganglion cells are the first to become postmitotic, * Correspondence: 1 Department of Biological Sciences, University of Idaho, Moscow ID 83844, USA Full list of author information is available at the end of the article followed by the cells of the INL [1]. The last neurons to be generated and then differentiate are the photoreceptors [1]. The photoreceptor mosaic of teleost fish, such as zebrafish, forms a spatially regular pattern of rods and cones [2-5]. The signaling pathways that regulate the production of rod and cone photoreceptors into their regular spatial patterns are not well understood. In the larval and adult teleost, rod and cone neurogenesis are spatially distinct, with new cones generated from stem cells residing in a circumferential germinal zone (CGZ), and new rods arising from a proliferative lineage residing within the INL [6-9]. There is evidence that Müller glia constitute the apex of the rod lineage, remaining proliferative and generating progeny that migrate to the ONL, undergo terminal © 2011 Stevens et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Stevens et al. BMC Developmental Biology 2011, 11:51 http://www.biomedcentral.com/1471-213X/11/51 mitoses, and differentiate as rods [10-12]. Despite apparently distinct lineage histories of rods and cones, the two types of progenitor cells are, at the molecular level, virtually indistinguishable, and express several photoreceptor-specific transcription factors including crx, rx1, neuroD, nrl, and nr2e3 [11]. Furthermore, in zebrafish that are mutant for the tbx2b gene, encoding a transcription factor expressed in early retinal progenitors, the UV cones are conspicuously missing from the larval cone mosaic, their positions instead occupied by supernumerary rod photoreceptors [13], suggesting an alteration in cell fate choice by retinal progenitors. Together these findings suggest some overlap of, or plasticity within, the progenitor cell populations otherwise fated to generate rods or cones. The development of retinal cells, including photoreceptors, is known to be controlled by a variety of secreted signaling factors, including retinoic acid (RA). RA and its receptors are essential for morphogenesis of the vertebrate eye. A deficiency of RA or its precursor Vitamin A leads to ocular defects such as coloboma and retinal dysplasia [14-18]. RA signaling occurs via structural dimers formed by one member each of the Retinoic Acid Receptor (RAR) and Retinoid × Receptor (RXR) subtypes [19-22]. In the chick and mouse, specific RARs and RXRs are expressed in cells of the INL, ONL, and the RPE, in overlapping and non-overlapping patterns [23,24]. Mouse embryos deficient in combinations of RAR/RXR genes exhibit defects in eye morphogenesis, including thinning of the retinal layers, targeted defects in the ventral retina, and absence of an ONL [25,26]. RA synthesis in the retina occurs in specific ventral and dorsal domains, defined by the expression of retinaldehyde dehydrogenases (RALDHs) [27-31] with boundaries formed by expression of cyp26 enzymes involved in RA degradation [32,33]. Studies using reporter lines to monitor RA signaling in the eye have also shown RA signaling to be dynamic, occurring initially in the ventral retina and later spreading to other parts of the retina [31,34,35]. The results from previous in vitro and in vivo studies suggested that RA can control the formation of photoreceptors. RA promotes the formation and survival of rod photoreceptors from cultured retinal progenitors, within the developing rat retina [36-38], and from mammalian embryonic stem cells [39]. RA signaling also controls the expression of photoreceptor-specific genes, such as the transcription factor NRL and opsin genes, the latter involving differential effects upon specific rod and cone opsins [31,36 (...truncated)