Involvement of Bcl2-associated transcription factor 1 in the differentiation of early-born retinal cells

Abstract

Purpose: Cell division and differentiation of retinal progenitors are under the control of endogenous and exogenous factors. We identified Bcl-2 associated transcription factor (Bclaf1) as a candidate. This gene which interacts with Bcl-2 related proteins was also described as a regulator of mRNA processing.

Methods: The expression pattern of Bclaf1 was analyzed by immunostaining during retinal development in the mouse, and the retinal phenotype of Bclaf-1 invalidated (Bclaf1-/-) mice was investigated. Ex vivo electroporation has been used to misexpress wild-type or truncated forms containing either the transcription regulatory domain (DC210) alone or the Bcl-2 binding domain (DN384) alone. The constructs were electroporated in wild-type retinal explants, maintained for 3 to 7 days in vitro (DIV) before dissociation and counting of different retinal cell types by immunostaining.

Results: Immunostaining experiments revealed that Bclaf1 is expressed from embryonic stage 14 (E14) to adulthood in post-mitotic early-born retinal precursors and mature cells (ganglion, amacrine and horizontal cells) but not in cone-photoreceptor cells. Analysis of the retinal phenotype of Bclaf1-/- mice showed a delay in the generation of early-born cells of the inner retinal layer from E14 to E18 that later was partially compensated. Conversely, the number of cone precursor cells decreased at E18. Because of perinatal mortality of Bclaf1-/- pups, we used the ex vivo retinal explant approach at E18 to explore postnatal development. These experiments demonstrated that the number of horizontal cell remains reduced in Bclaf1-/- retina after 7 DIV. In order to specify the molecular mechanism underlying the role of Bclaf1, we misexpressed wild-type or mutant forms of Bclaf1. Ex vivo electroporation of postnatal retinal explants revealed that the overexpression of the wild-type form or the DC210 mutant only led to an increase in the fraction of Pax6-positive cells and a decrease in the fraction of rhodopsin-positive cells.

Conclusions: Our results demonstrate that Bclaf1 is implicated in the timing of early-born cells generation and the commitment of early precursors to amacrine and horizontal lineage versus the cone-photoreceptor cell lineage. Our data also indicated that the effects of Bclaf1 occur independently of its interaction with Bcl-2.

Keywords: 698 retinal development • 546 horizontal cells • 648 photoreceptors