Single Cell Transcriptomic Analyses Reveal the Impact of bHLH Factors on Human Retinal Organoid Development

Xiangmei Zhang; Igor Mandric; Kevin H Nguyen; Thao T T Nguyen; Matteo Pellegrini; James C R Grove; Steven Barnes; Xian-Jie Yang

doi:10.3389/fcell.2021.653305

Single Cell Transcriptomic Analyses Reveal the Impact of bHLH Factors on Human Retinal Organoid Development

Front Cell Dev Biol. 2021 May 13:9:653305. doi: 10.3389/fcell.2021.653305. eCollection 2021.

Authors

Xiangmei Zhang¹, Igor Mandric², Kevin H Nguyen¹, Thao T T Nguyen¹, Matteo Pellegrini², James C R Grove¹, Steven Barnes^{1

3}, Xian-Jie Yang^{1

4}

Affiliations

¹ Department of Ophthalmology, Stein Eye Institute, University of California, Los Angeles, Los Angeles, CA, United States.
² Department of Molecular, Cell, and Developmental Biology, University of California, Los Angeles, Los Angeles, CA, United States.
³ Doheny Eye Institute, University of California, Los Angeles, Los Angeles, CA, United States.
⁴ Molecular Biology Institute, University of California, Los Angeles, Los Angeles, CA, United States.

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.

Keywords: bHLH factors; human ES cells; neuronal differentiation; retinal ganglion cells; retinal organoid; single cell RNA-sequencing.

Abstract

Grants and funding