Direct neuronal reprogramming by temporal identity factors

Temporal identity factors are sufficient to reprogram developmental competence of neural progenitors and shift cell fate output, but whether they can also reprogram the identity of terminally differentiated cells is unknown. To address this question, we designed a conditional gene expression system that allows rapid screening of potential reprogramming factors in mouse retinal glial cells combined with genetic lineage tracing. Using this assay, we found that co-expression of the early temporal identity transcription factors Ikzf1 and Ikzf4 is sufficient to directly convert Müller glial cells into cells that translocate to the outer nuclear layer (ONL), where photoreceptor cells normally reside. We name these “induced ONL (iONL)” cells. Using genetic lineage tracing, histological, immunohistochemical, and single-cell transcriptome and multiome analyses, we show that expression of Ikzf1/4 in Müller glia in vivo, without retinal injury, mostly generate iONL cells that share molecular characteristics with bipolar cells, although a fraction of them stain for Rxrg, a cone photoreceptor marker. Furthermore, we show that co-expression of Ikzf1 and Ikzf4 can reprogram mouse embryonic fibroblasts to induced neurons (iN) in culture by rapidly remodeling chromatin and activating a neuronal gene expression program. This work uncovers general neuronal reprogramming properties for temporal identity factors in terminally differentiated cells. Overall design: ScRNA-seq and joint cell scRNA/ATAC-seq analysis of reprogrammed Muller glia and RNA-seq/ATAC-seq analysis of reprogrammed MEFs