PURPOSE
A comprehensive understanding of the mechanisms regulating the development of retinal ganglion cells (RGCs) and their neurites carries both theoretical significance and translational implications. The YTHDF family members, comprising Ythdf1, Ythdf2, and Ythdf3, are key readers for N6-methyladenosine (m6A), the most abundant internal modification of mRNA. Mounting evidence has demonstrated essential roles of Ythdf family m6A readers in various biological processes. This study aimed to investigate the roles of Ythdf family m6A readers in RGC development and their neurite outgrowth.
METHOD
An in vitro induced RGC (iRGC) system was employed to generate RGC-like neurons. Short hairpin RNAs (shRNAs) were used for gene knockdown expression, coding sequence (CDS)-containing constructs for gene overexpression, immunofluorescence staining for protein expression detection, patch-clamp recording for assessing neuronal electrophysiological properties, and RNA-seq for transcriptome profiling.
RESULTS
Knockdown of Ythdf1 and Ythdf3 significantly reduced iRGC reprogramming efficiency and axon length, whereas overexpression of these two m6A readers exerted the opposite effects. Ythdf2 knockdown had no impact on iRGC reprogramming or axonogenesis, while Ythdf2 overexpression promoted axon growth.
CONCLUSION
Ythdf1 and Ythdf3 are essential for iRGC fate reprogramming and axon development, whereas Ythdf2 is dispensable for these processes but can promote axon growth when overexpressed. These findings reveal important roles of m6A readers in RGC and their neurite development, which may facilitate future fundamental research and translational applications.
Ting Zhang, Kezhong Zhang, Qinghai He et al.· Experimental Eye Research· 0 citations
Purpose Ascl1 is a transcription factor (TF) that plays key roles in regulating retinal development. Recent studies have further uncovered its intriguing potential to drive retinal neuron regeneration by reprogramming Müller glial cells. However, the efficiency and specificity of Ascl1-mediated regeneration outcomes remain far from optimal, and the molecular mechanisms underlying its dual roles in retinal development and adult Müller reprogramming are not fully elucidated. Given the close developmental relationship between late-stage retinal progenitor cells (RPCs) and Müller glial cells, this study aimed to investigate the functional role and underlying molecular mechanisms of Ascl1 in governing retinal fate specification, using late RPCs as a model system. Methods In vivo electroporation (IVE) of the mouse retinas was used to deliver Ascl1-overexpressing (Ascl1-OE) plasmids into late RPCs. Immunofluorescence (IF) staining was employed to recognize retinal cells and assess cell proliferation status. RNA sequencing (RNA-seq) and single-cell RNA sequencing (scRNA-seq) were conducted to profile the transcriptomes of RPCs, and the assay for transposase-accessible chromatin using sequencing (ATAC-seq) was conducted to characterize the chromatin accessibility landscapes of RPCs. Results Ascl1-OE caused late PRCs to exit the cell cycle prematurely and produce more rods but fewer bipolar and Müller glial cells. Integrative RNA-seq, scRNA-seq, ATAC-seq and chromatin immunoprecipitation (ChIP)-seq analyses of Ascl1-OE late RPCs revealed that Ascl1 promoted the expression of Notch pathway inhibitors and neurogenic genes by binding to and opening up relevant genomic regions. Conclusions Ascl1 suppresses Müller glial and bipolar fates and promotes rod photoreceptor fate in late RPCs. Mechanistically, Ascl1 achieves the effect by repressing the Notch signaling and activating neurogenic genes through reprogramming the epigenetic landscape of the cells. The findings of this study may help elucidate the molecular mechanisms of Ascl1-driven Müller reprogramming in adult retinas and facilitate the development of novel methods to improve the efficiency and specificity of retinal neuron regeneration outcomes.
Can Chen, Zhihan Xing, Huilin Liang et al.· Investigative Ophthalmology...· 0 citations
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