Misexpression of Ptf1a regulates the expression of Atoh7 during chick retinogenesis

Abstract

Purpose: : The pancreas transcription factor 1 subunit a (Ptf1a) is necessary for the specification of horizontal cells and the majority of amacrine cell subtypes in the mouse retina. The molecular basis underlying Ptf1a activity during retinogenesis has remained largely unknown. To decipher theses mechanisms, we analyzed the regulation of the expression of transcription factors involved in retinal differentiation Ptf1a and the regulation of Ptf1a activity by PTF1 complex cofactors.

Methods: : Mutated forms of Ptf1a that are unable to interact with RBPJ and/or RBPL were generated by PCR-based mutagenesis. Similarly mutated forms that cannot heterodimerize with E-proteins were generated. Using a retrovirus-mediated gene transfer approach, the mouse Ptf1a and these mutant forms of Ptf1a were overexpressed during chick retinogenesis. The expression level of a set of transcription factors was evaluated by qPCR. Atoh7 regulation was further assayed by ISH and by coexpression of theses mutants with a reporter of atoh7 promoter activity.

Results: : Ptf1a misexpression was sufficient to promote the fates of amacrine and horizontal cells from retinal progenitors and inhibit retinal ganglion cell and photoreceptor differentiation in the chick retina. Ptf1a overexpression resulted in a rapid downregulation of the transcription of genes involved in the photoreceptor and ganglion cell lineage differentiation. Conversely, the transcription of genes involved in the generation of amacrine and horizontal cells were upregulated. Atoh7, a transcription factor involved in ganglion cell specification was found to be strongly repressed by 6.4-fold by Ptf1a overexpression. This was confirmed by ISH. Consistently, ex vivo, ectopic Ptf1a downregulated the activity of the chick Atoh7 promoter. Using this reporter assay, we further demonstrated that the binding of RBPJ to Ptf1a was necessary to inhibit atoh7 expression.

Conclusions: : Our data provide a novel insight into the molecular basis of Ptf1a activity on early cell specification in the chick retina. Particularly, it supports a molecular model where Ptf1a might enable the recruitment of a pool of Atoh7-positive precursors and drive them towards amacrine and HC fates.

Keywords: retinal development • transcription factors • gene/expression