Dissecting a Gene Regulatory Network That Controls a Binary Cell Fate Specification in the Developing Retina

Abstract

During development of complex tissues such as the mammalian central nervous system (CNS), many genes act in concert to create sophisticated gene regulatory networks (GRNs) to instruct cell fate specification of the many cell types of the CNS. In this work, we have dissected a GRN that regulates a binary cell fate decision in the developing murine retina between a rod photoreceptor and a bipolar interneuron. This network converges upon Blimp1, a key transcription factor in rod versus bipolar cell fate decision. A cis-regulatory module (CRM) of Blimp1, B108, was identified by promoter bashing that faithfully recapitulates endogenous Blimp1 expression. Knock-out of B108 by CRISPR/Cas9 by in vivo electroporation in P0 pups resulted in Blimp1 KO phenotype. Otx2 and RORβ, potent transcription factors in retinal development, are required to activate Blimp1 expression via B108. Otx2 is in return repressed by Blimp1 in a negative feedback loop. Vsx2, a key TF for bipolar cells, was found to suppress Blimp1 via previously uncharacterized binding motif on B108 to ensure the proper ratio of rods to bipolar cells. Finally, we have shown Vsx2 and Blimp1 mRNAs may be asymmetrically segregated in dividing progenitors in the post-natal retina that sets up asymmetric cell fate in the siblings. Collectively these findings led to the identification of the GRN that regulates the decision rod vs bipolar fate. The sibling that retains Vsx2 mRNA suppresses Blimp1, maintains high Otx2 to become a bipolar cell. Whereas the sibling that retains Blimp1 suppresses Otx2 and Vsx2 to obtain a rod fate.