Abstract
The developing retina expresses multiple bHLH transcription factors. Their precise functions and interactions in uncommitted retinal progenitors remain to be fully elucidated. Here, we investigate the roles of bHLH factors ATOH7 and Neurog2 in human ES cell-derived retinal organoids. Single cell transcriptome analyses identify three states of proliferating retinal progenitors: pre-neurogenic, neurogenic, and cell cycle-exiting progenitors. Each shows different expression profile of bHLH factors. The cell cycle-exiting progenitors feed into a postmitotic heterozygous neuroblast pool that gives rise to early born neuronal lineages. Elevating ATOH7 or Neurog2 expression accelerates the transition from the pre-neurogenic to the neurogenic state, and expands the exiting progenitor and neuroblast populations. In addition, ATOH7 and Neurog2 significantly, yet differentially, enhance retinal ganglion cell and cone photoreceptor production. Moreover, single cell transcriptome analyses reveal that ATOH7 and Neurog2 each assert positive autoregulation, and both suppress key bHLH factors associated with the pre-neurogenic and states and elevate bHLH factors expressed by exiting progenitors and differentiating neuroblasts. This study thus provides novel insight regarding how ATOH7 and Neurog2 impact human retinal progenitor behaviors and neuroblast fate choices.
Highlights
As an integral component of the central nervous system, the vertebrate neural retina retains a highly conserved laminar structure that senses, processes, and delivers visual information to the brain
We carried out co-infection of retinal organoids with LV-rtTA and either LV-AEP or LV-NEP at the onset of retinogenesis followed by Dox inductions (Figure 1C)
By 6-days after Dox induction, the majority of LV-AEP and LV-NEP infected cells were located in the inner layer of the organoids (Figures 1D,E), To investigate whether virally expressed basic helix-loop-helix (bHLH) factors indicating that viral mediated ATOH7 and Neurog2 expression affected retinal progenitor proliferation, we performed BrdU
Summary
As an integral component of the central nervous system, the vertebrate neural retina retains a highly conserved laminar structure that senses, processes, and delivers visual information to the brain. Classic cell birth dating and lineage tracing studies have established that the seven major neuronal cell types constituting the retinal network are generated in a temporal order from a common ocular progenitor pool during development (Young, 1985; Livesey and Cepko, 2001; Wong and Rapaport, 2009). Cumulative molecular genetic studies have uncovered important roles of cell intrinsic factors involved in retinal development (Xiang, 2013). Transcription factors containing the basic helix-loop-helix (bHLH) motif have emerged as important players regulating the production and differentiation of various retinal cell types (Akagi et al, 2004). Multiple bHLH factors are expressed during retinal development either in proliferating progenitors or in postmitotic neurons; their dynamic regulation and function in specific cellular contexts remain to be fully elucidated
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