Thursday, November 21, 2024
11/21/2024
Patterning Genes in Retinal Development Project Summary The long-range goal of this grant is to determine the cellular and molecular events that lead to the generation of specific cell types in the vertebrate retina. In this renewal we focus upon endocrine signaling pathways, nuclear hormone receptors, and genetic elements that regulate differential expression of the cone visual pigment genes on tandemly-replicated gene arrays. In humans a tandemly-replicated array on the X chromosome consists of one long wavelength-sensitive (LWS) gene followed by 1-9 medium wavelength- sensitive (MWS) genes, which have diverged spectrally from LWS. This recent replication has provided most humans with trichromatic color vision, because LWS, MWS, and SWS1 opsin proteins are uniquely expressed in separate cone populations. Heritable defects in the LWS/MWS array result in various forms of color blindness, X-linked retinal degenerations, and Bornholm Eye Disease, a cone dysfunction associated with high myopia. Insights into regulation of differential expression of LWS vs. MWS opsins could allow therapeutic manipulation of gene expression to treat these disorders. In addition, future regenerative approaches to the treatment of other retinal disorders that involve the loss of cones (age-related macular degeneration; Stargardt's disease) would ideally include similar in vitro or in vivo manipulations to generate cone phenotypes in ratios that support high-acuity color vision. A decades-old model for human LWS vs. MWS opsin regulation states that a stochastic event favors an association of an upstream regulatory region with the LWS or most proximal MWS opsin promoter or exon. However, topographic patterns of the LWS:MWS ratio suggest that a nonrandom, trans-regulatory mechanism may be involved. Pursuit of regulatory mechanisms has been limited because within mammals, only primate and bat genomes are known to harbor tandem opsin arrays. In contrast, the genomes of teleost fish, including zebrafish, contain numerous tandem arrays of opsins, which are the consequences of independent gene replication events and neofunctionalization. In the previous award period, we demonstrated that in zebrafish, the endocrine signal thyroid hormone (TH) regulates differential expression of the tandem duplicates, lws1 vs. lws2, an array that shares a common ancestral LWS gene with the genes of the human LWS/MWS array. Our preliminary data suggest the hypothesis that the TH receptor, Thrb2 (already known to promote the LWS cone phenotype), may have been evolutionarily co-opted to also regulate differential expression of lws1 vs. lws2, such that unliganded Thrb2 promotes lws2 and suppresses lws1, while liganded Thrb2 promotes lws1. We have identified two predicted, palindromic TH response elements (ppTREs) on the lws locus, and preliminary studies suggest that each element has roles in regulating endogenous patterns of lws1 vs. lws2, and in the response to TH. Additional preliminary data suggest that TH can regulate expression of LWS vs. MWS opsin transcripts in human iPSC-derived 3D retinal organoids. Together these findings represent tremendous breakthroughs in revealing novel pathways for regulation of tandemly-replicated gene arrays in vertebrates. In this renewal we drill down to examine the signaling pathways, receptors, and genetic elements that mediate this regulation, with three Aims: Aim 1. Identify the receptor(s) that mediate the response of lws1 and lws2 to TH. Aim 2. Identify the regulatory elements on the lws locus that mediate the response of lws1 and lws2 to TH. Aim 3. Determine the role of TH signaling in regulating LWS vs. MWS ratios in human cones.
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