Molecular Cloning of zASIP, a Zebrafish Homolog of ASIP/Bazooka/Par-3, and Characterization of its Roles During Retinal Development

Abstract

Abstract: : Purpose: The vertebrate retina develops from a single sheet of retinal epithelial cells, which later differentiate and reorganize into three major layers during retinal neurogenesis. The mechanism underlying this cellular reorganization remains largely unknown. A previous study demonstrated that the epithelial polarity gene nagie oko plays a critical role during this process. To examine the roles of other functionally related epithelial polarity genes during retinal development, we cloned the zebrafish homolog of ASIP/Bazooka/Par-3 (zASIP) and characterized its role during the retinal development. Methods:A zASIP EST clone was identified by a BLAST search using the rat ASIP amino acid sequence as a query. 5’ and 3’ RACE were performed to isolate the full-length zASIP sequence. The function of zASIP during retinal development was assayed using morpholino oligo knock-down and over-expression approaches followed by histological analysis. Results:The zASIP gene is expressed in the neural tube and the retina. Loss of zASIP function through morpholino knock-down resulted in cell death in the retinal pigmented epithelium (RPE) and the ventral region of the forebrain; moreover, it also caused cyclopia and defects in retinal lamination. Cyclopia is possibly a consequence of the cell death in the ventral region of the forebrain, which normally serves to separate the two eyes. While the generation of retinal layers was disrupted in zASIP morpholino-treated embryos, immuno-histological analysis indicated that retinal cell-type specification was not affected. In contrast to the knock-down results, over-expression of zASIP via embryonic injection of zASIP mRNA resulted in cyclopia, without significantly affecting the retinal organization. Conclusions: These findings demonstrated that the zASIP gene plays a critical role during development of the zebrafish retina. It is required for the survival of the RPE cells and the ventral region of the forebrain. Furthermore, zASIP is essential for the retinal lamination. This study confirms that genes critical for generating and/or maintaining epithelial polarity are also essential for the cellular patterning of the retinal cells during development. Further investigation will elucidate the molecular mechanisms by which the zASIP gene controls the cellular organization during retinal development.

Keywords: anatomy • retinal development • retinal pigment epithelium