Abstract
Purpose::
The evolutionarily conserved Par-aPKC complex composed of Par3, Par6 and atypical protein kinase C (aPKC) plays a fundamental role in both the establishment and maintenance of epithelial cell polarity. Additionally, these proteins are required for proper retinal lamination, as disruption of aPKC and Par3 causes defects of lamination in mouse and zebrafish. However, the lamination defect in zebrafish aPKC mutants is cell-non-autonomous as small mutant clones are able to adopt normal cell positioning in wild type retinas. The purpose of this study was to analyze the cell autonomous effects of Par3 knockdown on retinal cells. Using genetic mosaic analysis, we are able to analyze the cell autonomous function of Par3 in cellular differentiation, lamination, adherens junction formation and morphogenesis of photoreceptors.
Methods::
Morpholino antisense oligonucleotides for Par3 were injected into wild type animals. Donor cells from these Par3 morphants were transplanted into wild type host embryos and analyzed at 4 days post fertilization via immunohistochemistry. Markers for rod and cone photoreceptors, retinal ganglion cells, as well as markers for adherens junctions including zonula occludens protein-1 (ZO-1), phalloidin, and aPKC were used to analyze cellular differentiation of retinal cells and determine if loss of Par3 affects the formation of adherens junctions in cell-autonomous manner.
Results::
Par3 morphant clones are able to adopt normal cell positions within the retina, as they occupied positions within the retinal ganglion cell layer, inner nuclear layer and photoreceptor layer. Additionally, all cell lineages analyzed were adopted by Par3 morphant clones including rods, red-green double cones, horizontal cells and retinal ganglion cells. Analysis of adherens junction formation reveals that these structures are formed in morphant photoreceptors, as actin foci localize normally in these cells. The tight junction protein ZO-1, and aPKC correctly localize in morphant photoreceptors and overall photoreceptor morphology appears unaffected by Par3 knockdown.
Conclusions::
Our results indicate that the cellular patterning defect within the retina of Par3 morphant zebrafish is cell-non-autonomous. Additionally, Par3 does not play a role in differentiation of any of the cell types analyzed. Par3 morphant clones were able to form adherens junctions and localize components of this structure correctly, indicating Par3 is not absolutely required for the formation of these structures in photoreceptors.
Keywords: cell adhesions/cell junctions • retinal development • photoreceptors