The Chd4 chromatin remodeller controls temporal programs during retinal neurogenesis
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ABSTRACT:
PROVIDER: PRJNA1105560 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| Other | ||||
| SRR28824001_1.fastq.gz | Fastqsanger.gz | |||
| SRR28824001_2.fastq.gz | Fastqsanger.gz | |||
| SRR28824006_1.fastq.gz | Fastqsanger.gz | |||
| SRR28824006_2.fastq.gz | Fastqsanger.gz |
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Project description:During retinal development, neurons and glia are produced in a highly stereotyped and evolutionarily conserved birth order. Landmark studies have demonstrated that the sequence of cell production is temporally encoded. Transcription factors and heterochromatic determinants have been elsewhere implicated in the temporal regulation of progenitor competence states, but the underlying mechanisms are not well understood. Since chromatin remodelling complexes interact with both transcription factors and heterochromatic complexes such as polycomb, we focused on the nucleosome remodeller Chd4. We generated conditional knockouts (cKOs) of Chd4 – a key chromatin remodelling enzyme in neural progenitors. Chd4 cKOs exhibited a marked expansion in early-born retinal ganglion cells. Postnatally, rod photoreceptors were drastically underproduced. As a result, progenitors failed to be exhausted during late phases of development, leading to a striking increase in the production of Müller glia. Clonal retroviral lineaging and histological marker analyses suggest that these effects were independent of alterations in cell death or proliferation. Next, we examined the effect of Chd4 on the genome and transcriptome, focusing on retinal progenitors at P0/P1. Multi-seq single-cell transcriptomics demonstrated that deletion of Chd4 created divergent gene expression profiles and developmental trajectories. ATAC-seq experiments performed on sorted P1 retinal progenitors revealed that chromatin accessibility was significantly increased at ~10 000 enhancer elements and ~4000 genes in the Chd4 cKO, demonstrating that Chd4 is required to repress gene expression and genome accessibility. Finally, to determine whether Chd4 regulates retinal cell-type production by altering competence windows, we performed EdU birthdating. These experiments revealed that cell fates were altered without affecting competence windows. Thus, despite a very strong shift in the production of early-born and late-born cell types, our data suggest that chromatin remodelling does not directly regulate competence windows.
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Project description:Regulation of retinal neurogenesis by somatostatin signaling
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Project description:Purpose: Neuropeptide signaling has been reported to impact neurogenesis in the central nervous system. However, these studies have typically been conducted using pharmacological agents in ex vivo preparations, and in vivo evidence for their developmental function is generally lacking. The goal of this study is to identify the impact of somatostatin signaling on retinal neurogenesis and development both ex vivo and in vivo. Methods: Mouse retinal explants grown ex vivo from E14-P0 and treated with a range of doses of agonist for the somatostatin receptor Sstr2 were multiplexed according to the MULTI-seq protocol and used to generate a single scRNA-seq expression library. Retinas from P0 littermates representing an allelic series (+/+, +/-, -/-) of Sstr2 KO mice were mutliplexed according to the MULTI-seq protocol and used to generate a single scRNA-seq expression library. Retinas from P14 littermates representing an allelic series (+/+, +/-, -/-) of Sstr2 KO mice were mutliplexed according to the MULTI-seq protocol and used to generate a single scRNA-seq expression library. Sequencing was performed on an illumina sequencer. Bcl files were analyzed by Cellranger. Samples were deconvoluted with the deMULTIplex r package and clustering and gene expression analysis were performed with the Seurat and Monocle2 r packages. Results: Cell type proportions were found to be impacted by Sstr2 signaling ex vivo with increasing levels of Sstr2 agonist found to decrease photoreceptor proportion and increase primary progenitor proportion. Sstr2 KO was not found to have an impact on cell type proportion in vivo at either P0 or P14. Sstr2 pharmacologic manipulation and Sstr2 KO did produce complementary gene expression changes in neurogenic progenitors indicating that somatostatin signaling downregulates neurogenesis. Conclusions: Our study demonstrates a role for somatostatin in retinal development by downregulating neurogenesis in neurogenic progenitors. However, it also demonstrates that somatostatin signaling is dispensable for in vivo retinal development and likely redundant with other cell extrinsic signals released during the same period.
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Project description:The RNA-binding protein Ptbp1 has been proposed as a master regulator of neuronal fate, repressing neurogenesis through its effects on alternative splicing and miRNA maturation. While prior studies using RNA interference suggested that Ptbp1 loss promotes neurogenesis, recent genetic studies have failed to replicate glia-to-neuron conversion following Ptbp1 loss of function. To evaluate the role of Ptbp1 in developmental neurogenesis in vivo, we conditionally disrupted Ptbp1 in retinal progenitors. Ptbp1 was robustly expressed in both retinal progenitors and Müller glia but absent from postmitotic neurons, and efficient loss of function in mutant animals was confirmed using immunostaining for Ptbp1. Furthermore, bulk RNA-Seq at E16 revealed accelerated expression of late-stage progenitor and photoreceptor-specific genes and altered splicing patterns in Ptbp1 mutants, including increased inclusion of neuron- and retina-specific exons. However, we observed no defects in retinal lamination, progenitor proliferation, or cell fate specification in mature retina. ScRNA-Seq of mature mutant retinas revealed only modest transcriptional changes limited to Müller glia, recapitulating alterations seen following selective deletion of Ptbp1 in mature glia. Our findings demonstrate that Ptbp1 is fully dispensable for retinal development and suggest that its proposed role as a central repressor of neurogenesis should be reevaluated
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Project description:The RNA-binding protein Ptbp1 has been proposed as a master regulator of neuronal fate, repressing neurogenesis through its effects on alternative splicing and miRNA maturation. While prior studies using RNA interference suggested that Ptbp1 loss promotes neurogenesis, recent genetic studies have failed to replicate glia-to-neuron conversion following Ptbp1 loss of function. To evaluate the role of Ptbp1 in developmental neurogenesis in vivo, we conditionally disrupted Ptbp1 in retinal progenitors. Ptbp1 was robustly expressed in both retinal progenitors and Müller glia but absent from postmitotic neurons, and efficient loss of function in mutant animals was confirmed using immunostaining for Ptbp1. Furthermore, bulk RNA-Seq at E16 revealed accelerated expression of late-stage progenitor and photoreceptor-specific genes and altered splicing patterns in Ptbp1 mutants, including increased inclusion of neuron- and retina-specific exons. However, we observed no defects in retinal lamination, progenitor proliferation, or cell fate specification in mature retina. ScRNA-Seq of mature mutant retinas revealed only modest transcriptional changes limited to Müller glia, recapitulating alterations seen following selective deletion of Ptbp1 in mature glia. Our findings demonstrate that Ptbp1 is fully dispensable for retinal development and suggest that its proposed role as a central repressor of neurogenesis should be reevaluated
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Project description:How various ATP-dependent chromatin remodellers bind to nucleosomes to regulate transcription is not well defined in mammalian cells. Here, we present genome-wide remodeller-interacting nucleosome profiles for Chd1, Chd2, Chd4, Chd6, Chd8, Chd9, Brg1 and Ep400 in mouse embryonic stem (ES) cells. These remodellers bind to nucleosomes at specific positions, either at one or both nucleosomes that flank each side of nucleosome-free promoter regions (NFRs), at enhancer elements, or within gene bodies. At promoters, bidirectional transcription commonly initiates on either side of remodeller-bound nucleosomes. Transcriptome analysis upon remodeller depletion reveals reciprocal mechanisms of transcriptional regulation by remodellers. At active genes, certain remodellers are positive regulators of transcription, whereas others act as repressors. At bivalent genes, which are bound by repressive Polycomb complexes, the same remodellers act in the opposite way. Together, these findings reveal how remodellers integrate promoter nucleosomal architecture to regulate ES cell transcription programs.
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Project description:Gene regulatory networks (GRNs), consisting of transcription factors and their target cis-regulatory sequences, control neurogenesis and cell fate specification in the developing central nervous system, but their organization is poorly characterized. In this study, we performed integrated scRNA-seq and scATAC-seq analysis from both mouse and human retina to profile dynamic changes in gene expression, chromatin accessibility and transcription factor footprinting during retinal neurogenesis and gliogenesis. We identified multiple interconnected, evolutionarily-conserved GRNs consisting of cell type-specific transcription factors that both activate expression of genes within their own network and often inhibit expression of genes in other networks. These GRNs control state transitions within primary retinal progenitors that underlie temporal patterning, regulate the transition from primary to neurogenic progenitors, and drive specification of each major retinal cell type. We confirmed the prediction of this analysis that the NFI transcription factors Nfia, Nfib, and Nfix selectively activate expression of genes that promote late-stage temporal identity in primary retinal progenitors. We also used GRNs to identify additional transcription factors that selectively promote (Insm1/2) and inhibit (Tbx3, Tcf7l1/2) rod photoreceptor specification in postnatal retina. This study provides an inventory of cis- and trans-acting factors that control retinal development, identifies transcription factors that control the temporal identity of retinal progenitors and cell fate specification, and will potentially help guide cell-based therapies aimed at replacing retinal neurons lost due to disease.
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