Protein-Trap Insertional Mutagenesis Uncovers New Genes Involved in Zebrafish Skin Development, Including a Neuregulin 2a-Based ErbB Signaling Pathway Required during Median Fin Fold Morphogenesis

Abstract

Skin disorders are widespread, but available treatments are limited. A more comprehensive understanding of skin development mechanisms will drive identification of new treatment targets and modalities. Here we report the Zebrafish Integument Project (ZIP), an expression-driven platform for identifying new skin genes and 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 genes—fras1, grip1, hmcn1, msxc, col4a4, ahnak, capn12, and nrg2a—had been described in an integumentary context to varying degrees, while arhgef25b, fkbp10b, and megf6a emerged as novel skin genes. Embryos homozygous for a GBT insertion within neuregulin 2a (nrg2a) revealed a novel requirement for a Neuregulin 2a (Nrg2a) – ErbB2/3 – AKT signaling pathway governing the apicobasal organization of a subset of epidermal cells during median fin fold (MFF) morphogenesis. In nrg2a mutant larvae, the basal keratinocytes within the apical MFF, known as ridge cells, displayed reduced pAKT levels as well as reduced apical domains and exaggerated basolateral domains. Those defects compromised proper ridge cell elongation into a flattened epithelial morphology, resulting in thickened MFF edges. Pharmacological inhibition verified that Nrg2a signals through the ErbB receptor tyrosine kinase network. Moreover, knockdown of the 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 successive, coordinated ridge cell shape changes drive apical MFF development, making MFF ridge cells a valuable model for investigating how the coordinated regulation of cell polarity and cell shape changes serves as a crucial mechanism of epithelial morphogenesis.

Pharyngeal morphogenesis requires fras1 - itga8 -dependent epithelial-mesenchymal interaction

“…Consistent with previous reports (Carney et al, 2010; Gautier et al, 2008; Talbot et al, 2012; Westcot et al, 2015), fras1 transcript is prominently expressed in ectoderm and endoderm lining pharyngeal arches (Fig. 4A, B).…”

509 Tumor suppression in basal keratinocytes via dual non-cell-autonomous functions of a Na,K-ATPase beta subunit

“…However, atp1b1a and lgl2 mutants also display differences. Although in conjunction with other mutations, loss of Lgl2 function leads to defects in earlier developmental stages (Westcot et al, 2015; this work), epidermal hyperplasia in lgl2 single mutants begins significantly later (4 days post fertilization; dpf) than that in atp1b1a mutants (2 dpf). An even more pronounced requirement in different time windows has been reported for Na,K-ATPase and Lgl during epithelial polarity regulation in Drosophila embryos.…”

“…In lgl2 mutants, the epidermal defects only become apparent comparably late (from 96 hpf onwards) (Reischauer et al, 2009;Sonawane et al, 2005), but a recent study uncovered a pro-basal effect of lgl2 on basal keratinocytes as early as 30-36 hpf (Westcot et al, 2015). This suggests that Atp1b1a might regulate epithelial polarity and epidermal integrity at least in part by cooperating with Lgl2 and by ensuring its proper localization.…”

“…Cryosections were generated as previously described (Westcot et al, 2015). For TEM analysis, embryos were anesthetized with Tricaine, heads were removed using a scalpel and subjected to genotyping, and tails were processed for ultrathin sectioning and TEM as described (Feitosa et al, 2011).…”

“…Embryos were fixed in 4% PFA for stainings using the following primary antibodies: For stainings with the mouse anti-panKeratin1-8 (Progen Pharmaceuticals, Darra, Australia; 61006, 1:10), embryos were fixed with Dent's fixative (80% Methanol, 20% DMSO, at -20˚C overnight). For pAkt stainings, embryos were fixed in EAF (40% ethanol, 5% acetic acid, 4% formaldehyde in PBS) and either washed with PBS-TritonX, followed by an antigen retrieval in 10 mM Tris, 1 mM EDTA, pH 9.0 for 60 min at 60˚C, blocking in 5% sheep serum and antibody incubation with rabbit antipAkt (S473) (Cell Signaling Technology #4060, 1:50) or processed for cryosectioning as described (Westcot et al, 2015). Secondary antibodies were anti-mouseCy3, anti-rabbitCy3, anti-mouseAlexa488, anti-mouseAlexa647, and anti-chickenAlexa488 (all Invitrogen, Carlsbad, CA; 1:200).…”