Regulation of Foxn4 during retina development

Abstract

Transcription factor forkhead box N4(Foxn4) is a key regulator involved in a variety of biologic processes in development and metabolism. In particular, Foxn4 plays an essential role in the genesis of amacrine and horizontal neurons from neural progenitors in the retina. Although the functions of Foxn4 have been well established, the transcriptional regulation of Foxn4 expression during progenitor cell differentiation remains unclear. The goal of this dissertation is to identify regulatory mechanisms that define the expression of Foxn4 during retinogenesis. Four evolutionarily conserved regions (CR1-CR4) from non-coding sequences of Foxn4 gene were computationally predicted as cis-elements. Their gene regulatory potential was individually tested in developing chick and mouse embryonic retina using electroporation transfection technique with a reporter assay system. In this dissertation, I describe that CR4.2 (a 129 bp DNA fragment of CR4, located approximately 50kb upstream of Foxn4 transcription start site) functions as a novel cis-regulator that directs retinal cell type specific gene expression. CR4.2 is preferentially active in the Foxn4 expressing cells, primarily in the differentiating and differentiated horizontal and amacrine cells as shown by reporter assays. Specific trans-acting factors, e.g., Meis1, were found to interact with CR4.2 by electrophoretic mobility shift assays (EMSA). Mutation and/or deletion of the Meis1 binding motif through site-directed mutagenesis diminishes the ability of CR4.2 to drive reporter GFP expression. Furthermore, the role of Meis1 in regulating Foxn4 expression during progenitor cell differentiation was determined using a RNAi-based gene silencing assay. Knockdown of Meis1 by short hairpin RNA (shRNA) specific to Meis1 genes abolishes gene regulatory activity of CR4.2, and further diminishes the endogenous level of Foxn4 expression. In addition, cells with Meis1 knockdown failed to differentiate into horizontal cells. Taken together, I demonstrate that Meis1 transcription factor regulate the expression of Foxn4 expression and horizontal cell lineage development in the vertebrate retina via their interactions with CR4.2. These findings provide new insights into molecular mechanisms that govern gene regulation in retinal progenitors and vertebrate retinal cell development.

Ph. D.

Includes bibliographical references

by Mohammed Monirul Islam