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Onecut1 and Otx2 directly regulate multiple genes expressed early in cone and horizontal cell development [scRNA-seq]

ABSTRACT: The development of the retina into a highly organized structure occurs via the production of over 100 cell types from a pool of retinal progenitor cells (RPCs). While some RPCs are capable of making many types of retinal cell types, some terminally dividing RPCs are restricted to the production of specific types of daughter cells. Notably, although such restrictions can be limited to the production of a very specific single cell type, in other cases, production of two very different cell types, such as a photoreceptor and an interneuron, occurs. How specific types of RPCs make specific types of neuron is not fully understood. Combining RNA-seq and ATAC-seq, we compared the transcriptome and chromatin profiles between one type of biased RPC and other types of RPCs. The biased RPC that was studied is one that we previously defined by its expression of a cis-regulatory module (CRM) for the thyroid hormone receptor beta gene (Thrb), the earliest known marker of cone photoreceptors. In this earlier study, we found that Otx2 and Onecut1 drove expression of this Thrb CRM, as well as regulated expression of the Thrb gene. Here we explored the roles of these Otx2 and Oc1 further. We discovered that their roles extend well beyond regulating Thrb. They collaborate to regulate multiple genes important in cone development and/or function, including Rxrg, a known partner of Thrb. We also identified several new enhancers of genes active in developing cones. These data expand our understanding of the gene regulatory network (GRN) for cone development and contribute new CRMs for vectors that are expressed only in cones, as well as enable the genesis of cones from stem cells, for their use in retinal disease cell and gene therapies.

ORGANISM(S): Gallus gallus

PROVIDER: GSE151947 | GEO | 2021/07/01

REPOSITORIES: GEO

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Project description:Background: The vertebrate retina consists of six major classes of neuronal cells. During development, these cells are generated from a pool of multipotent retinal progenitor cells (RPCs) that express the gene Vsx2. Fate-restricted RPCs have recently been identified, with limited mitotic potential and cell fate possibilities compared to multipotent RPCs. One population of fate-restricted RPCs, marked by activity of the regulatory element ThrbCRM1, gives rise to both cone photoreceptors and horizontal cells. These cells do not express Vsx2, but co-express the transcription factors (TFs) Onecut1 and Otx2, which bind to ThrbCRM1. The components of the gene regulatory networks that control the transition from multipotent to fate-restricted gene expression are not known. This work aims to identify and evaluate cis-regulatory elements proximal to Onecut1 to identify the gene regulatory networks involved in RPC fate-restriction. Method: We identified regulatory elements through ATAC-seq and conservation, followed by reporter assays to screen for activity based on temporal and spatial criteria. The regulatory elements of interest were subject to deletion and mutation analysis to identify functional sequences and evaluated by quantitative flow cytometry assays. Finally, we combined the enhancer::reporter assays with candidate TF overexpression to evaluate the relationship between the TFs, the enhancers, and early vertebrate retinal development. Statistical tests included ANOVA, Kruskal-Wallis, or unpaired t-tests.Results: Two regulatory elements, ECR9 and ECR65, were identified to be active in ThrbCRM1(+) restricted RPCs. Candidate bHLH binding sites were identified as critical sequences in both elements. Overexpression of candidate bHLH TFs revealed specific enhancer-bHLH interactions. Nhlh1 overexpression expanded ECR65 activity into the Vsx2(+) RPC population, and overexpression of NeuroD1/NeuroG2/NeuroD4 had a similar effect on ECR9. Furthermore, bHLHs that were able to activate ectopic ECR9 reporter were able to induce endogenous Otx2 expression. Conclusions: This work reports a large-scale screen to identify spatiotemporally specific regulatory elements near the Onecut1 locus. These elements were used to identify distinct populations in the developing retina. In addition, fate-restricted regulatory elements responded differentially to bHLH factors, and suggest a role for retinal bHLHs upstream of the Otx2 and Onecut1 genes during the formation of restricted RPCs from multipotent RPCs.

Project description:The loss of cone photoreceptor cells, which are critical for optimal daylight vision, have the great impact on vision during retinal degenerations. Retinal differentiation of human induced pluripotent stem cell (hiPSC) sources could provide a renewable source of cone photoreceptors towards developing a cone cell replacement therapy to treat blindness. Demonstration of comparable gene expression profiles between human foetal and stem cell-derived cones at equivalent stages is required to progress the cell transplantation approach into the patient, as it is hypothesised stem cell-derived cones are required to show high levels of developmental recapitulation of the in vivo generated cones. In this study, the AAV2/9.pR2.1.GFP reporter was used to specifically label L/M-opsin cone photoreceptors in human foetal retinal samples, obtained from the MRC-Wellcome Trust Human Developmental Biology Resource, at a range of developmental stages. The L/M-opsin cone population represent the majority cone cell types in the adult human retina and are the only photoreceptors present within the fovea. Using fluorescence activated cell sorting, L/M-opsin GFP+ cones and GFP- retinal populations, alongside total foetal retinal samples containing all retinal cell tytpes, were isolated and processed for bulk RNA sequencing and downstream comparative analysis. Using DESeq2 differential gene expression analyses, statistically significant genes enriched within the GFP+ human foetal LM-opsin cone populations were determined which led to the identification of a cone enriched gene signature of human L/M-opsin cone photoreceptors. The AAV2/9.pR2.1.GFP reporter was applied to hiPSC-derived retinal cultures to isolate and process cone-like cell populations for RNA sequencing using the same strategy developed within the human foetal retina. Applying the cone enriched gene signature to the transcriptome of hiPSC-derived GFP+ samples at equivalent developmental stages revealed some expression similarities in genes found to be enriched within the late foetal L/M-opsin cone photoreceptors. This analysis overall revealed an intermediate stage of cone differentiation was achieved within the hiPSC-derived samples and the comparison to human foetal L/M-opsin gene express profiles suggesting further differentiation of hiPSC-derived sample is required.

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Onecut1 and Otx2 directly regulate multiple genes expressed early in cone and horizontal cell development [scRNA-seq]

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