Abstract 1768 Analysis of molecular mechanisms of transcription factor Nhlh2 in amacrine cell differentiation in the mouse retina

In the vertebrate central nervous system including the retina, neurons and glial cells are differentiated from multipotent common progenitor cells. The retina is composed five types neurons and one type glial cell, including photoreceptor cell, bipolar cell, horizontal cell, amacrine cell, ganglion cell, and Müller glial cell. Amacrine cells are inhibitory interneurons that interact at the second synaptic level of the vertically direct pathways consisting of the photoreceptor-bipolar-ganglion cell chain. Previous reports suggested that amacrine cells are consisted of 60 or more cell subtypes by transcriptome analysis. However, the molecular mechanisms underlying amacrine cell differentiation into each subtype remain unclear. To approach the molecular mechanism of amacrine cell differentiation, we performed transcriptome screening by single cell RNA-seq (scRNA-seq) analysis to find candidate genes involved in amacrine cell differentiation. All animal studies were conducted in accordance with the Animal Care and Use Committee guidelines of the Institute for Protein Research, Osaka University. The scRNA-seq results using embryonic and postrenal mouse retinas allowed us to represent the expression patterns of individual genes along retinal development. Furthermore, we identified that Nhlh2, which encodes a bHLH transcription factor, is expressed specifically in developing and mature amacrine cells. These observations suggested that Nhlh2 is involved in the amacrine cell development. We performed overexpression of Nhlh2 in the mouse retinas by retrovirus mediated gene transfer, to clarify the biological function of Nhlh2in vivo in amacrine cell development. Overexpression of Nhlh2 in retinal progenitor cells significantly increased the percentage of amacrine cells in the mouse retina. These results suggested that Nhlh2 may be involved in amacrine cell development. In addition, we performed a reporter assay screening to identify DNA motifs that Nhlh2 binds and activates transcription. This study provides insight into the effect of Nhlh2 transcription factor on amacrine cell differentiation and DNA binding. This project was funded by Grant-in-Aid for Scientific Research (B) (A21H026570), Grant-in-Aid for Scientific Research (C) (23K06303) from the Japan Society for the Promotion of Science, The Takeda Science Foundation and The Uehara Memorial Foundation.