Revealing Novel Skin Biology Using Protein-Trap Gene-Break Transposon Mutagenesis Technology In The Larval Zebrafish Model

Abstract

Abstract Although skin disorders affect as much as a third of the population at any given time, available treatments are limited. Because a more comprehensive understanding of skin development mechanisms can spur the identification of new treatment targets and techniques, we developed the Zebrafish Integument Project (ZIP), an expression-driven platform for identifying new skin genes and new, revertible phenotypes in the vertebrate model Danio rerio (zebrafish). In vivo selection for skin-specific expression of gene-break transposon (GBT) mutant lines identified eleven new, revertible GBT alleles of genes involved in skin development. Eight of those genes had been described in an integumentary context to varying degrees: fras1, grip1, hmcn1, msxc, col4a4, ahnak, capn12, and nrg2a. Three others—arhgef25b, fkbp10b, and megf6a—emerged as novel skin genes. Embryos homozygous for a GBT insertion in neuregulin 2a (nrg2a) revealed a novel requirement for a Neuregulin 2a (Nrg2a) – ErbB2/3 – AKT signaling pathway governing ridge cell morphogenesis and apicobasal organization during median fin fold (MFF) morphogenesis. In nrg2a mutant larvae, the basal keratinocytes that comprise the apical MFF (ridge cells) displayed reduced pAKT levels as well as reduced apical domains and exaggerated basolateral domains. Those defects prevented proper ridge cell elongation into a flattened epithelial morphology, resulting in thickened MFF edges. Additionally, morpholino knockdown of epithelial polarity regulator and tumor suppressor lgl2 ameliorated the nrg2a mutant phenotype. Identifying Lgl2 as an antagonist of Nrg2a – ErbB signaling revealed a significantly earlier role for Lgl2 during epidermal morphogenesis than has been described to date. Furthermore, our findings demonstrated that ridge cells’ squamous flattening morphogenesis drives apical MFF development. We therefore propose MFF ridge cells as a new model for investigating the regulation of cell polarity and cellular morphogenesis with regard to their roles as crucial mechanisms for epithelial morphogenesis generally, and for flattening morphogenesis in particular.