Background In the vertebrate retina, six types of neurons and Muller glia originate from common progenitor cells. Extracellular signals and intrinsic regulatory mechanisms dictate cell-type specification in the neural retina. We are using both reductionist and systems approaches to investigate complex molecular pathways and cellular interactions controlling differentiation of retinal neurons. - Regulation of photoreceptor development, maturation and homeostasis Rod photoreceptor fate is largely determined by the basic motif leucine zipper transcription factor NRL. Our main research focus is the NRL-centered gene regulatory network that determines rod photoreceptor development. We use the Nrl-/- and Nrlp-GFP mice as tools. Among the genes down-regulated in Nrl-/- retina and direct targets of NRL, we selected receptor accessory protein 6 (Reep6) which encodes a member of a protein family involved in shaping membrane tubules and transport of G-protein coupled receptors. We demonstrated that a novel Reep6 isoform (Reep6.1) is specifically expressed in rod photoreceptors and is a key functional target of NRL. Reep6-/- mice display visual dysfunction and photoreceptor degeneration accompanied by defects in vesicle trafficking and reduction in the level of outer segment-specific membrane proteins. Interaction of REEP6 with clathrin heavy chain on coated vesicles and with syntaxin 3 on the plasma membrane provides evidence for a role of REEP6 in trafficking and docking of vesicles carrying cargoes to the outer segment of rod photoreceptors (2). NRL controls several rod photoreceptor genes including rhodopsin whose expression and transport are under tight control to maintain photoreceptor homeostasis. While the rhodopsin proximal promoter is regulated by specific transcription factors (e.g., NRL and CRX) to control mainly temporal expression, the upstream rhodopsin enhancer region (RER) controls precise, high-level expression of rhodopsin. Among the RER-bound proteins that we identified by mass spectrometry, we focused on NonO, a protein implicated in coupling transcription to splicing, and the NonO-interacting proteins hnRNP M, Ywhaz and Ppp1ca. All proteins induce rhodopsin expression in HEK293 cells and function synergistically with NRL and CRX. In vivo knockdown and rescue experiments revealed the role of NonO and its interacting proteins in enhancing rod-specific gene expression and controlling rod homeostasis (6). In collaboration with D. Forrest (NIDDK), we identified the retinoid-related orphan receptor Rorb gene upstream of Nrl in the transcriptional cascade regulating photoreceptor development. Of the two isoforms encoded by Rorb and regulated by alternative promoters, RORb1 is expressed in retinal progenitors, photoreceptors and other retinal cells whereas RORb2 is restricted to photoreceptors. Both isoforms participate in the control of Nrl expression. We found that the Rorb promoter regulating RORb2 isoform expression is a target of NRL, suggesting a feed-back loop that might be involved in the complex transcriptional network that establishes rod versus cone fates. While investigating a spontaneous mouse mutant with a frameshift mutation in Crx (CrxRip), we found that the binding of homeobox transcription factor OTX2 at the Nrl promoter was obliterated and that ectopic expression of OTX2 rescued the rod differentiation defect in these mice (see EY000473). This established a novel role for OTX2 in maintaining Nrl expression in developing rods to promote rod fate and maturation (4). Maturation of rod photoreceptors involves the establishment of cell polarity, outer segment formation, axonal extension and synaptogenesis. We identified and investigated genes involved in these processes. The outer segment outgrows from the connecting cilium that, in turn, originates from the mother centriole. By knocking out Cc2d2a, a centrosome-cilia gene that causes Meckel and Joubert syndromes in humans, not only we recapitulated features of Meckel syndrome (see EY000473), but also we established that CC2D2A is essential for assembly of sub distal appendages, which anchor cytoplasmic microtubules and prime the mother centriole for cilium biogenesis (5). Synaptic connectivity is distinct in rod spherules and cone pedicles. Based on the observation that synaptic terminals in Nrl-/- retina switch from spherule to pedicle-like morphology, we focused on downstream targets of NRL that are critical for spherule morphogenesis. Combining RNA-seq data in developing wild type and Nrl-/- retina, NRL ChIP-seq data and analysis of the retinal phenotype after shRNA in vivo electroporation, we identified NRL target genes encoding signaling proteins that appear to be determinants of spherule development, thus locating NRL upstream in the cascade of transcription factors that control specificity of synaptic development. We continue our collaborative efforts with intramural and extramural investigators. With T. Li's group (NNRL, NEI), we showed that Prickle1 is a key component of the Wnt-signaling pathway that regulates establishment of planar cell polarity (PCP) and that it may also be involved in establishing apico-basal polarity (3). Furthermore, we contributed to the understanding of the role of horizontal cells interaction with photoreceptors for their development and function (1) in collaboration with B. Reeses group (UC Santa Barbara). - Systems-level approaches in rod photoreceptor development and disease pathogenesis We are performing gene regulatory network and cluster analyses using RNA-seq data generated from developing rod photoreceptors in wild type and retinal degeneration mouse models, combined with histone modification and DNA methylation profile data. Known disease genes and pathways are superimposed in integrated analysis. We envision this integrated analysis as a milestone for the investigation of photoreceptor and retinal development, retinal degenerative disease pathogenesis and therapy design. We are completing a publicly accessible database that includes gene expressions patterns in different organisms, tissues, and experimental conditions in order to facilitate data sharing and comparative system-level approaches. Our integrated analysis has already revealed important elements underlying photoreceptor development. Through developmental and evolutionary gene expression studies, we showed that developing rod photoreceptors in the mouse retina have footprints of S-cones and that rod maturation is accompanied by the loss of S-cone signature and expression of rod-specific genes. Upon integration of transcriptome and epigenome data, we observed repressive epigenetic signatures that include limited active histone modifications and high degree of DNA methylation associated with phototransduction genes in developing rods, thereby suggesting a transcriptional pause of phototransduction genes, despite the presence of rod-specific transcriptional regulators. During functional maturation, rods undergo chromatin reconfiguration from repressive to active state with concurrent commencement of rod gene expression. Such epigenetic programs could be targeted for experimental manipulation, for example in stem cell based therapy. Significance Our studies provide insights into photoreceptor biology and disease. We are defining the role of specific genes and epigenetic signals in establishing photoreceptor identity and function. Our molecular studies reveal a broad overlap between the role of some genes in photoreceptor development and their involvement in retinal disease pathogenesis, making them potential therapeutic targets.
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