AB040. Pou2f1/2 are required for the specification of cone photoreceptors in the developing retina
Retina and Posterior Segment

AB040. Pou2f1/2 are required for the specification of cone photoreceptors in the developing retina

Awais Javed1,2, Pierre Mattar1, Michel Cayouette1,2,3,4

1Cellular Neurobiology Research Unit, Institut de Recherches Cliniques de Montreal (IRCM), Montreal, QC, Canada; 2Molecular Biology Program, Université de Montréal, Montreal, QC, Canada; 3Department of Medicine, Université de Montréal, Montreal, QC, Canada; 4Division of Experimental Medicine, Department of Anatomy and Cell Biology, McGill University, Montreal, QC, Canada

Background: Rods and cones are critical for light detection. Although there has been considerable work done in elucidating the molecular mechanisms involved in rod development, not much is known about how the cone cell fate decision is made by the multipotent retinal progenitor cells during development. Analysis of the promoter regions of Nrl and trβ2, rod and cone differentiation factors respectively, revealed DNA binding motifs of two POU-domain containing transcription factors, Pou2f1 and Pou2f2. Preliminary experiments showed that Pou2f1/2 are expressed during the peak of cone genesis in the embryonic retina. Therefore, we hypothesize that Pou2f1/2 specify cone cell fate in the developing retina.

Methods: We used immunofluorescence and in situ hybridization to establish the spatiotemporal expression of Pou2f1/2 during retinogenesis. We performed in vivo electroporation in post-natal mice to misexpress Pou2f1/2 and used antibodies specific to proteins expressed in cones such as Rxrγ and S-opsin to count cones. Using ex vivo electroporation of embryonic retinal explants, we knocked out Pou2f1 and Pou2f2 using CRISPR/Cas9 gRNAs at the peak of cone production window. Finally, we transfected post-natal retinal explants with a combination of regulatory elements of Nrl or thrb with control backbone vector, Pou2f1 or Pou2f2 using electroporation.

Results: We found that Pou2f1/2 are expressed in retinal progenitor cells in the developing retina and subsequently in the differentiated cones. Pou2f1/2 misexpression outside the cone genesis window led to an increase in cones at the expense of rods. Pou2f1/2 indel knockouts generated by CRISPR/Cas9 gRNAs led to a decrease in cones and a converse increase in rods. Finally, we found that Pou2f1/2 activate the cis-regulatory module (CRM) of the thrb gene and repress the activity of the CRM of Nrl.

Conclusions: These results uncover novel players that establish the complex gene regulatory network for cone photoreceptor fate specification in the retinal progenitor cells. We anticipate that this work should help us devise improved replacement therapies in the future utilizing stem cells for retinal degenerative diseases such as aged-related macular degeneration (AMD) and Stargardt’s disease.

Keywords: Cones; rods; cell biology; molecular biology; retinogenesis

doi: 10.21037/aes.2018.AB040
Cite this abstract as: Javed A, Mattar P, Cayouette M. Pou2f1/2 are required for the specification of cone photoreceptors in the developing retina. Ann Eye Sci 2018;3:AB040.