Single cell transcriptomics reveals lineage trajectory of the retinal ganglion cells in wild-type andAtoh7-null retinas

Abstract

Formation of retinal ganglion cells (RGCs) is governed by a hierarchical gene regulatory network with key transcription factors such as Atoh7, Pou4f2 and Isl1 functioning at different levels. Past studies concluded that Atoh7 is critical for the emergence of the RGC lineage in the developing retina, whereas Pou4f2 and Isl1 function further downstream. Atoh7 is expressed in a subset of retinal progenitor cells (RPCs) and is considered a competence factor for the RGC fate, but the molecular properties of these RPCs have not been well characterized. In this study, we first used conventional RNAseq to investigate transcriptomic changes in Atoh7-, Pou4f2-, and Isl1-null retinas at embryonic (E) day 14.5 and identified the differentially expressed genes (DEGs), which expanded our understanding of the scope of downstream events regulated by these factors. We then performed single cell RNA-seq (scRNA-seq) on E13.5 wild-type and Atoh7-null retinal cells using the 10X Chromium platform. Clustering analysis not only correctly identified known cell types at this developmental stage, including RPCs, RGCs, cones, and amacrine/horizontal cells, but also revealed a transitional cell state which was marked by Atoh7 and genes for other lineages in a highly overlapping fashion and shared by all early developmental trajectories. These results provide significant insights into the nature of RPC competence for different retinal cell fates and the likely mechanism by which these fates are committed. Further, analysis of the Atoh7-null retina not only identified the affected genes/pathways involved in the different cell states but also revealed that in the absence of Atoh7, the RGC lineage still progressed considerably and a substantial amount of RGC-specific gene expression still occurred. Thus, Atoh7 likely collaborates with other factors to shepherd the transitional RPCs to the RGC lineage by competing with other lineage factors and activating RGC-specific genes. This study thus revises our current view on the emergence of the RGC lineage and shed new light on the general mechanisms governing retinal cell differentiation.

Two new genetically modified mouse alleles labeling distinct phases of retinal ganglion cell development by fluorescent proteins

“…The identities of the clusters were clearly identified by marker genes including Atoh7, Pou4f2, Ptf1a, and Crx ( Figure 5D). Atoh7 was expressed in the vast majority of transitional RPCs and trails into all three lineages, which was consistent with recent single cell transcriptomics studies, 27,28,30,46 particularly the finding that Atoh7 marks all transitional RPCs, which is a shared stage for all retinal cell types being generated at this stage. 30 Cells in the tree lineages but not expressing Atoh7 anymore were still purified likely due to the stability of zsGreen ( Figure 5D).…”

“…Atoh7 was expressed in the vast majority of transitional RPCs and trails into all three lineages, which was consistent with recent single cell transcriptomics studies, 27,28,30,46 particularly the finding that Atoh7 marks all transitional RPCs, which is a shared stage for all retinal cell types being generated at this stage. 30 Cells in the tree lineages but not expressing Atoh7 anymore were still purified likely due to the stability of zsGreen ( Figure 5D). These results indicated cells purified by FACS using a low gating threshold from retinas carrying the Atoh7 zsGreenCreERT2/+ allele covered a large window of retinal cell differentiation, including proliferating transitional RPCs and all their progenies including RGCs.…”

“…Recent single cell transcriptomics studies are beginning to address the diverse cell states and cell lineages in the developing retina. [27][28][29][30] One significant finding of these studies is that Atoh7 marks a transition stage shared by all early retinal fates. 29,30 Genetic tools enabling purification of specific cell populations will significantly facilitate further investigation of the properties of these cell states.…”

“…[27][28][29][30] One significant finding of these studies is that Atoh7 marks a transition stage shared by all early retinal fates. 29,30 Genetic tools enabling purification of specific cell populations will significantly facilitate further investigation of the properties of these cell states. With the advantages of being highly sensitive and non-destructive to the function of target cells and proteins, fluorescent protein tags are widely used in labeling and tracking cells.…”

Asymmetric neurogenic commitment of retinal progenitors involves Notch through the endocytic pathway

“…To our knowledge, the existence of such intermediate progenitors has not been thoroughly investigated in the retina. However, a recent study performing single cell RNA sequencing of mouse retinae ( Wu et al, 2020 ) hints that a transitional state of retinal progenitors that express atoh7 and other neurogenic genes could exist. Therefore, it will be interesting to probe how cytoskeletal changes could be linked to these cell identity changes in the developing retina and beyond.…”