Comprehensive analysis of retinal development at single cell resolution identifies NFI factors as essential for mitotic exit and specification of late-born cells

Abstract

Precise temporal control of gene expression in neuronal progenitors is necessary for correct regulation of neurogenesis and cell fate specification. However, the extensive cellular heterogeneity of the developing CNS has posed a major obstacle to identifying the gene regulatory networks that control these processes. To address this, we used single cell RNA-sequencing to profile ten developmental stages encompassing the full course of retinal neurogenesis. This allowed us to comprehensively characterize changes in gene expression that occur during initiation of neurogenesis, changes in developmental competence, and specification and differentiation of each of the major retinal cell types. These data identify transitions in gene expression between early and latestage retinal progenitors, as well as a classification of neurogenic progenitors. We identify here the NFI family of transcription factors (Nfia, Nfib, and Nfix) as genes with enriched expression within late RPCs, and show they are regulators of bipolar interneuron and Müller glia specification and the control of proliferative quiescence.

Identification of genes with enriched expression in early developing mouse cone photoreceptors

“…Another cluster assigned was AC/HCs exhibited markers for both these fates, like TFAP2B and PTF1A. As previously reported (Clark et al, 2018), while ACs and HCs are distinct fates, it is difficult to separate them by their transcriptomes in early development and without an appropriate amount of sequenced cells for proper resolution.…”

“…One other cluster was comprised entirely of cycling cells, exhibiting markers such as OLIG2, NEUROG2, OTX2 and ATOH7, which identify neurogenic RPCs (Cepko, 2014;Clark et al, 2018) as these markers in cycling cells indicate restricted RPCs close to their final division. A second cluster had a fraction of cycling cells, high ATOH7 levels and expression of POU4F2, but no OTX2 or OLIG2, which we assigned to RGC/AC RPCs and Precursors as they exhibit some markers of likely differentiation to RGCs or ACs.…”

Modular and efficient pre-processing of single-cell RNA-seq

“…Our RNA velocity workflow, which is 13 times faster than velocyto 11 , is suitable for analysis of large datasets that were previously challenging to pre-process. To illustrate this we computed RNA velocity vectors for recently published data from the developing mouse retina 30 consisting 113,917 cells ( Figure 3). We found that six pseudotime marker genes highlighted in the Clark et al 2019 paper 31 (Crx, Nrl, Otx2, Pax6, Rbpms, Rlbp1) displayed patterns consistent with the RNA velocity vectors, and with the pseudotime analysis of Clark et al 31 (Supplementary Figure 8).…”

A Casz1 - NuRD complex regulates temporal identity transitions in neural progenitors

“…We have previously shown that Casz1 regulates the mid/late temporal identity in RPCs. During retinal development, the expression of Casz1 mRNA and protein steadily increases in RPCs, peaks at around P0, and declines postnatally ( Fig 1A) [3,15]. We previously generated a Casz1 conditional knockout (cKO) mouse, and found that Casz1 is required in the retina to suppress the production of early-born neurons and late-born Müller glia, while promoting the production of rod photoreceptors [15].…”

“…In regions of the central nervous system (CNS) such as the neocortex and retina, progenitors undergo additional temporal identity transitions in competence to generate specific neuron subtypes at precise stages of development. In the vertebrate retina, retinal progenitor cells (RPCs) have two distinctive phases of multipotency [2][3][4][5]. Within each phase, RPCs can simultaneously produce a number of different neuronal and glial subtypes.…”