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Single cell analyses reveal the transient emergence of retinal progenitor cells in the ciliary margin zone during human retinal development [scRNA-seq]

ABSTRACT: Retina is the innermost, light-sensitive layer that lines the back of the eye and is vital for light sensing and image processing. All the retinal cells types are derived from retinal progenitor cells (RPCs) in an orderly spatio-temporal manner that has been well studied in vertebrates. To better understand the emergence of RPCs, their localisation and differentiation to retinal lineages, we performed single cell (sc) RNA-Seq of 24 samples spanning 13 time points from 10-21 post-conception weeks of human development. Several types of progenitors were identified, including early and late RPCs which in turn gave rise the transient neurogenic progenitors that differentiated to all retinal neurones. Spatial transcriptomic analyses combined with scRNA-Seq revealed localisation of early RPCs in the ciliary margin of developing human retinas up to 10 PCW of development and propagation of neural retina differentiation from the centre to the periphery. Single cell ATAC-Seq analyses of 11 samples spanning 8-21 PCW of human development identified Hippo-Yap signalling as key pathway regulating RPC proliferation and cell cycle exit. These results provide the molecular map of human retinal development which may help guide generation of RPCs from human pluripotent stem cells and the design of cell-based replacement therapies for treating retinal dystrophies

ORGANISM(S): Homo sapiens

PROVIDER: GSE234963 | GEO | 2023/11/29

REPOSITORIES: GEO

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Project description:The development of complex tissues requires that mitotic progenitor cells integrate information from the environment. The highly varied outcomes of such integration processes undoubtedly depend at least in part upon variations among the gene expression programs of individual progenitor cells. To date, there has not been a comprehensive examination of these differences among progenitor cells of a particular tissue. Here, we used comprehensive gene expression profiling to define these differences among individual progenitor cells of the vertebrate retina. Retinal progenitor cells (RPCs) have been shown by lineage analysis to be multipotent throughout development and to produce distinct types of daughter cells in a temporal, conserved order. A total of 42 single RPCs were profiled on Affymetrix arrays. An extensive amount of heterogeneity in gene expression among RPCs, even among cells isolated from the same developmental time point, was observed. While many classes of genes displayed heterogeneity of gene expression, the expression of transcription factors constituted a significant amount of the observed heterogeneity. Additionally, the expression of cell cycle related transcripts showed differences among those associated with G2 and M, versus G1 and S phase, suggesting different levels of regulation for these genes. These data provide insights into the types of processes and genes that are fundamental to cell fate choices, proliferation decisions, and, for cells of the central nervous system, the underpinnings of the formation of complex circuitry. Experiment Overall Design: Single retinal cells were isolated in tubes containing lysis buffer, their mRNAs were reverse transcribed, and the resulting cDNAs were PCR amplified for 35 cycles. Labeled cDNA samples were hybridized to Affymetrix 430 2.0 microarrays and the data was normalized using MAS5.0 software. A total of 42 retinal progenitors were identified post hoc and compared with ganglion, amacrine and photreceptor cells isolated from identical timepoints.

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Project description:The coupling between cell-cycle exit and onset of differentiation is a common feature throughout the developing nervous system, but the mechanisms that link these processes are mostly unknown. Although the transcription factor Pax6 was implicated in both proliferation and differentiation of multiple regions within the CNS, its contribution to the transition between these successive states remains elusive. To gain insight into the role of Pax6 during the transition from proliferating progenitors to differentiating precursors, we investigated cell-cycle and transcriptomic changes occurring in Pax6- retinal progenitor cells (RPCs). Our analyses revealed a unique cell-cycle phenotype of the Pax6-deficient RPCs, which included a reduced number of cells in the S phase, an increased number of cells exiting the cell cycle, and delayed differentiation kinetics of Pax6- precursors. These alterations were accompanied by co-expression of factors that promote (Ccnd1, Ccnd2, Ccnd3) and inhibit (P27kip1 and P27kip2) the cell cycle. Further characterization of the changes in transcription profile of the Pax6-deficient RPCs revealed abrogated expression of multiple factors which are known to be involved in regulating proliferation of RPCs, including the transcription factors Vsx2, Nr2e1, Plagl1 and Hedgehog signaling. These findings provide novel insight into the molecular mechanism mediating the pleiotropic activity of Pax6 in RPCs. The results further suggest that rather than conveying a linear effect on RPCs, such as promoting their proliferation and inhibiting their differentiation, Pax6 regulates multiple transcriptional networks which function simultaneously, thereby conferring the capacity to proliferate, assume multiple cell fates and execute the differentiation program into retinal lineages. The data contains 3 control and 3 Pax6 negative samples representing biological repeats. Each sample was prepared from ~1.8 million cells taken from the distal retina of either E12 alpha-Cre or Pax6 floxed alpha-Cre animals. Distally located retinal progenitors expressing Cre and eGFP were separated using FACS.

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Single cell analyses reveal the transient emergence of retinal progenitor cells in the ciliary margin zone during human retinal development [scRNA-seq]

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