Biology subjects

Silveira, M. S.

Publications and source records attributed to Silveira, M. S..

4 recordsLinked to original sources

bioRxiv2026

Klf4 overexpression remodels chromatin to reprogram late retinal progenitors toward an retinal ganglion cell-like fate

Developing neuron-replacement therapies for retinal ganglion cells (RGCs) lost to injury or disease requires a deeper understanding of how restriction to cell identity acquisition may be overcome. Previously, we showed that overexpression of Klf4 in late retinal progenitor cells (late RPCs), which are normally restricted from RGC production, is sufficient to produce cells that display a subset of canonical RGC properties including RGC-associated gene expression and morphological features. In the present study, we investigated the transcriptional and epigenetic mechanisms by which Klf4 overexpression influences the fate of cell types generated from late RPCs. scRNA-seq analysis revealed that Klf4 induces transcriptional changes, with some cells exhibiting gene expression profiles similar to those of resident RGCs. In addition, we observed widespread changes in chromatin accessibility, suggesting that KLF4 remodels the chromatin of late RPCs and influences their transcriptional profile. Our findings show KLF4-driven reprogramming of late RPCs, providing insight into progenitor competence and fate specification to an RGC-like identity. These results suggest that KLF4 could be a component in regenerative therapies due to its ability to reprogram and induce RGC genes outside of the normal RGC developmental window.

neuroscience↗

bioRxiv2024

Pou4f2/Brn3b Overexpression Promotes The Genesis Of Retinal Ganglion Cell-Like Projection Neurons From Late Progenitors

Retinal ganglion cells (RGCs) are the projection neurons of the retina. In early retinal progenitor cells (RPCs), Atoh7 orchestrates the developmental RGC program and regulates the expression of critical downstream targets, including Pou4f factors. The absence of Pou4f2 or more POU4F family genes results in defects in RGC differentiation, aberrant axonal elaboration and ultimately RGC death, confirming the requirement of POU4F factors for RGC development and survival, with a critical role in regulating RGC axon outgrowth and pathfinding. Here, we investigated in vivo whether ectopic Pou4f2 expression in late retinal progenitor cells (late RPCs) is sufficient to induce the generation of cells with RGC properties, including projecting axons to the brain. Using a strong ubiquitous promoter to induce Pou4f2 overexpression in neonates, we observed a change in targeted cell distribution in the retinal tissue, including the presence of cells in the ganglion cell layer and inner plexiform layer with high density of GFP+ processes along the retina. Similar results on the induction of neuron processes were obtained when we overexpressed Pou4f2 in Atoh7 knockout mice, suggesting that POU4F2 is sufficient to induce them. Single-cell RNA sequencing (scRNA-seq) analysis shows that several RGC-genes (such as Rbpms, Gap-43, Hs6st3, and Foxp2) are upregulated after Pou4f2 overexpression. Additionally, gene ontology analysis indicates the induction of genes related to axonogenesis and neuronal differentiation. Imaging throughout the visual pathway revealed high density of axons projecting toward the optic nerve head and extending to brain regions, such as the superior colliculus and geniculate nucleus. Thus, Pou4f2 induced neurons with specific RGC characteristics that share similarities with resident RGCs and notably project axons that reach brain targets. In conclusion, these results showed that POU4F2 alone was sufficient to promote critical properties of projection neurons from retinal progenitors outside their developmental window.

neuroscience↗

bioRxiv2018

Evidence of Muller glia conversion into retina ganglion cells using Neurogenin2

Macular Degeneration, Glaucoma, and Retinitis Pigmentosa are all leading causes of irreversible visual impairment in the elderly, affecting hundreds of millions of patients. Muller glia cells (MGC), the main type of glia found in the vertebrate retina, can resume proliferation in the adult injured retina and contribute to tissue repair. Also, MGC can be genetically reprogrammed through the expression of the transcription factor (TF) Achaete-scute homolog 1 (ASCL1) into induced neurons (iNs), displaying key hallmarks of photoreceptors, bipolar and amacrine cells, which may contribute to regenerate the damaged retina. Here, we show that the TF neurogenin 2 (NEUROG2) is also sufficient to lineage-reprogram MGC into iNs. The efficiency of MGC lineage conversion by NEUROG2 is similar to that observed after expression of ASCL1. However, reprogramming efficiency is affected by previous exposure to EGF and FGF2 during the expansion of MGC population. Transduction of either Neurog2 or Ascl1 led to the upregulation of key retina neuronal genes in MGC-derived iNs, but only NEUROG2 induced a consistent increase in the expression of putative retinal ganglion cell (RGC) genes. In vivo electroporation of Neurog2 in the neonatal retina also induced a shift in the generation of retinal cell subtypes, favoring the differentiation RGCs at the expense of MGCs. Altogether, our data indicate that Neurog2 induces lineage conversion of MGCs into RGC-like iNs.

neuroscience↗

bioRxiv2018

De novo genesis of retinal ganglion cells by targeted expression of KLF4 in vivo

Retinal ganglion cell (RGC) degeneration is a hallmark of glaucoma, the most prevalent cause of irreversible blindness. Thus, innovative therapeutic strategies are needed to protect and replace these projection neurons. It has been shown that endogenous glial cells of the retina, Muller cells, can be directly reprogrammed into late-born retinal interneurons. However, since RGCs are the first neurons born during development, the replacement of damaged RGCs requires the reprograming to an early neurogenic state. Here, we demonstrate that the pluripotency regulator Klf4 is sufficient to reprogram the potency of lineage-restricted retinal progenitor cells (RPCs) to generate RGCs in vivo. Transcriptome analysis disclosed that the overexpression of Klf4 induces crucial regulators of RGC competence and specification, including Atoh7 and Eya2. In contrast, loss-of-function studies in mice and zebrafish demonstrated that Klf4 is not essential for generation or differentiation of RGCs during retinogenesis. Nevertheless, induced RGCs (iRGCs) generated upon Klf4 overexpression migrate to the proper layer and project axons aligned with endogenous fascicles that reach the optic nerve head. Notably, iRGCs survive for up to 30 days after in vivo reprogramming. Finally, we demonstrate that Klf4 converts Muller cells into neurons that express markers of RGCs. Altogether, we identified Klf4 as a promising tool to reprogram retinal cells and regenerate RGCs in the mature retina.\n\nSignificance StatementCell fate determination is a key process for development, regeneration and for the design of therapeutic strategies that involve cellular reprogramming. This work shows that the manipulation of a single pluripotency regulator (Klf4) is sufficient to reprogram restricted progenitor cells in vivo. These reprogrammed progenitors reacquire the potency to generate retinal ganglion cells. Ganglion cell degeneration is the leading cause of irreversible blindness; therefore, manipulation of ganglion cell competence is of relevance for human health. Our findings point to Klf4 as a promising tool to develop therapeutic strategies for the replacement of damaged ganglion cells.

developmental biology↗