Abstract
Abstract: :
Purpose: The atypical Protein Kinase C (PKC) isoforms lamda and zeta are important for many aspects of cell polarity. In zebrafish, mutations in aPKC lamda result in multiple defects during retinal development. These include altered mitotic cell behaviors (spindle disorientation and delocalized M–phase), cell migration mistakes, and photoreceptor dismorphogenesis. However, even the strongest null mutations in aPKC lamda affect retinal development less severely than predicted when considering mutations in other cell polarity genes. To test whether aPKC zeta also functions during retinal development, we have isolated the full length zebrafish cDNA for this gene and studied its relationship to aPKC lamda. Methods: Zebrafish aPKC zeta was isolated by PCR from embryonic cDNA. Protein and mRNA expression analysis was conducted by in situ hybridization and immunohistochemistry. Loss–of–function experiments utilized translation blocking anti–sense oligonucleotides in wild type and aPKC lamda null genotypes. Cell migration was assayed by in vivo confocal time–lapse microscopy with embryos expressing GFP–tagged transgenes. Results: Zebrafish aPKC zeta en codes a predicted 599 amino acid serine/threonine kinase with 80% identity to human aPKC zeta and 71% identity to zebrafish aPKC lamda. By comparison, zebrafish and human aPKC lamdas share 89% identify. The zebrafish aPKC zeta gene is expressed widely until 24 hours post fertilization. At this time, expression becomes restricted to the retina, brain, heart, and gut tissues. Within the retina, aPKC zeta is expressed throughout the neuroepithelium and then becomes restricted to the proliferative marginal zone and retinal ganglion cells. Subcellularly, the protein is localized to the apical surface of neuroepithelial cells and to the membranes and neurites of retinal ganglion cells. Loss of aPKC zeta alone did not produce observable phenotypes during embryogenesis. However, loss of aPKC zeta activity in aPKC lamda mutant backgrounds increased the severity of retinal aberations. Analysis of retinal ganglion cell migration in the absence of both aPKC lamda and zeta showed abnormal cell behaviors including lack of migration away from the ventricular zone and retrograde migration away from the basal surface. In contrast, the timing of cell migration and lack of tangential movements were normal. Conclusions: Zebrafish aPKC zeta functions with aPKC lamda to regulate migration and cell type positioning decisions during retinal development.
Keywords: retinal development • cell adhesions/cell junctions • motion-3D