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Retinal progenitor cells can produce restricted subsets of horizontal cells.

ABSTRACT: Retinal progenitor cells have been shown to be multipotent throughout development. Similarly, many other structures of the developing central nervous system have been found to contain multipotent progenitor cells. Previous lineage studies did not address whether these multipotent progenitor cells were biased in their production of neuronal subtypes. This question is of interest because subtypes are the basis of distinct types of circuits. Here, lentivirus-mediated gene transfer was used to mark single retinal progenitor cells in vivo, and the different subtypes of horizontal cells (HCs) in each clone were quantified. Clones with two HCs consistently contained a single HC subtype, a pair of either H1 or H3 cells. This suggests that a multipotent progenitor cell produces a mitotic cell fated to make a terminal division that produces two HCs of only one subtype. This bias in production of one HC subtype suggests a previously undescribed mechanism of cell fate determination in at least a subset of retinal cells that involves decisions made by mitotic cells that are inherited in a symmetric manner by both neuronal daughter cells.

SUBMITTER: Rompani SB

PROVIDER: S-EPMC2224184 | biostudies-literature | 2008 Jan

REPOSITORIES: biostudies-literature

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Retinal progenitor cells can produce restricted subsets of horizontal cells.

Rompani S B SB Cepko C L CL

Proceedings of the National Academy of Sciences of the United States of America 20071227 1

Retinal progenitor cells have been shown to be multipotent throughout development. Similarly, many other structures of the developing central nervous system have been found to contain multipotent progenitor cells. Previous lineage studies did not address whether these multipotent progenitor cells were biased in their production of neuronal subtypes. This question is of interest because subtypes are the basis of distinct types of circuits. Here, lentivirus-mediated gene transfer was used to mark ...[more]

PMID: 18162542

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Project description:BackgroundThe 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.MethodWe 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.ResultsTwo 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.ConclusionsThis 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: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.

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Retinal progenitor cells can produce restricted subsets of horizontal cells.

Publications

Retinal progenitor cells can produce restricted subsets of horizontal cells.

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