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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.

Westcot SE, Hatzold J, Urban MD, Richetti SK, Skuster KJ, Harm RM, Lopez Cervera R, Umemoto N, McNulty MS, Clark KJ, Hammerschmidt M, Ekker SC - PLoS ONE (2015)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis, Minnesota, United States of America; Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, Minnesota, United States of America.

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.

No MeSH data available.


Related in: MedlinePlus

GBT protein trapping generates novel revertible alleles of known MFF loci.(A) By 48 hpf, wild-type MFFs are thin and flat, and the MFF edge appears smooth and regular. (B-C) Two ZIP gene-break alleles, fras1mn0156Gt and hmcn1mn0263Gt have homozygous recessive phenotypes, each of which results in blistered MFFs (arrowheads). (D-E) Both fras1mn0156Gt and hmcn1mn0263Gt behaved as classic revertible GBT mutant alleles in Cre reversion experiments. With both alleles, significantly fewer Cre mRNA-injected embryos were phenotypic, compared with their respective uninjected siblings. (D) For offspring of fras1mn0156Gt heterozygote incrosses, 27% (n = 156) of uninjected embryos (-Cre) were phenotypic; only 5% (n = 140) of Cre mRNA-injected siblings (+Cre) were phenotypic (p < 0.05). (E) For offspring of hmcn1mn0263Gt heterozygote incrosses, 25% (n = 146) of uninjected embryos (-Cre) were phenotypic; only 8% (n = 233) of Cre-injected siblings (+Cre) were phenotypic (p < 0.005). (F) The fras1mn0156Gt GBT allele fails to complement the piftm95b ENU allele of fras1. Crossing piftm95b heterozygotes with fras1mn0156Gt heterozygotes does not reduce the proportion of phenotypic offspring (28%, n = 445) (tm95b/mn0156Gt trans-heterozygotes) compared to either piftm95b/tm95b (tm95b/tm95b, n = 332) or fras1mn0156Gt/mn0156Gt (mn0156Gt/mn0156Gt, n = 206) homozygotes. Percentages represent the mean of means (MOM); error bars represent standard deviations (SD).
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pone.0130688.g004: GBT protein trapping generates novel revertible alleles of known MFF loci.(A) By 48 hpf, wild-type MFFs are thin and flat, and the MFF edge appears smooth and regular. (B-C) Two ZIP gene-break alleles, fras1mn0156Gt and hmcn1mn0263Gt have homozygous recessive phenotypes, each of which results in blistered MFFs (arrowheads). (D-E) Both fras1mn0156Gt and hmcn1mn0263Gt behaved as classic revertible GBT mutant alleles in Cre reversion experiments. With both alleles, significantly fewer Cre mRNA-injected embryos were phenotypic, compared with their respective uninjected siblings. (D) For offspring of fras1mn0156Gt heterozygote incrosses, 27% (n = 156) of uninjected embryos (-Cre) were phenotypic; only 5% (n = 140) of Cre mRNA-injected siblings (+Cre) were phenotypic (p < 0.05). (E) For offspring of hmcn1mn0263Gt heterozygote incrosses, 25% (n = 146) of uninjected embryos (-Cre) were phenotypic; only 8% (n = 233) of Cre-injected siblings (+Cre) were phenotypic (p < 0.005). (F) The fras1mn0156Gt GBT allele fails to complement the piftm95b ENU allele of fras1. Crossing piftm95b heterozygotes with fras1mn0156Gt heterozygotes does not reduce the proportion of phenotypic offspring (28%, n = 445) (tm95b/mn0156Gt trans-heterozygotes) compared to either piftm95b/tm95b (tm95b/tm95b, n = 332) or fras1mn0156Gt/mn0156Gt (mn0156Gt/mn0156Gt, n = 206) homozygotes. Percentages represent the mean of means (MOM); error bars represent standard deviations (SD).

Mentions: To determine which ZIP loci were required for early zebrafish skin or fin fold development, we bred each expressing insertion to homozygosity through a standard inbreeding scheme. For each line, we screened offspring of intercrossed heterozygous parents during the first five days of development (120 hpf) for abnormal skin or fin fold morphology present only in mRFP-positive (GBT-expressing) larvae and absent from their wild-type siblings [17] (an “mRFP-linked” phenotype). Of the eleven lines screened, three displayed recessive, mRFP-linked phenotypes: fras1mn0156Gt (Fig 4B), hmcn1mn0263Gt (Fig 4C), and nrg2amn0237Gt (Fig 5B). Comparisons to published phenotypes of ENU-derived alleles indicated that fras1mn0156Gt and hmcn1mn0263Gt represented novel alleles of known skin blistering mutants pinfin (pif) and nagel (nel) [44, 56], respectively (Table 1, Fig 4A–4C).


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.

Westcot SE, Hatzold J, Urban MD, Richetti SK, Skuster KJ, Harm RM, Lopez Cervera R, Umemoto N, McNulty MS, Clark KJ, Hammerschmidt M, Ekker SC - PLoS ONE (2015)

GBT protein trapping generates novel revertible alleles of known MFF loci.(A) By 48 hpf, wild-type MFFs are thin and flat, and the MFF edge appears smooth and regular. (B-C) Two ZIP gene-break alleles, fras1mn0156Gt and hmcn1mn0263Gt have homozygous recessive phenotypes, each of which results in blistered MFFs (arrowheads). (D-E) Both fras1mn0156Gt and hmcn1mn0263Gt behaved as classic revertible GBT mutant alleles in Cre reversion experiments. With both alleles, significantly fewer Cre mRNA-injected embryos were phenotypic, compared with their respective uninjected siblings. (D) For offspring of fras1mn0156Gt heterozygote incrosses, 27% (n = 156) of uninjected embryos (-Cre) were phenotypic; only 5% (n = 140) of Cre mRNA-injected siblings (+Cre) were phenotypic (p < 0.05). (E) For offspring of hmcn1mn0263Gt heterozygote incrosses, 25% (n = 146) of uninjected embryos (-Cre) were phenotypic; only 8% (n = 233) of Cre-injected siblings (+Cre) were phenotypic (p < 0.005). (F) The fras1mn0156Gt GBT allele fails to complement the piftm95b ENU allele of fras1. Crossing piftm95b heterozygotes with fras1mn0156Gt heterozygotes does not reduce the proportion of phenotypic offspring (28%, n = 445) (tm95b/mn0156Gt trans-heterozygotes) compared to either piftm95b/tm95b (tm95b/tm95b, n = 332) or fras1mn0156Gt/mn0156Gt (mn0156Gt/mn0156Gt, n = 206) homozygotes. Percentages represent the mean of means (MOM); error bars represent standard deviations (SD).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4482254&req=5

pone.0130688.g004: GBT protein trapping generates novel revertible alleles of known MFF loci.(A) By 48 hpf, wild-type MFFs are thin and flat, and the MFF edge appears smooth and regular. (B-C) Two ZIP gene-break alleles, fras1mn0156Gt and hmcn1mn0263Gt have homozygous recessive phenotypes, each of which results in blistered MFFs (arrowheads). (D-E) Both fras1mn0156Gt and hmcn1mn0263Gt behaved as classic revertible GBT mutant alleles in Cre reversion experiments. With both alleles, significantly fewer Cre mRNA-injected embryos were phenotypic, compared with their respective uninjected siblings. (D) For offspring of fras1mn0156Gt heterozygote incrosses, 27% (n = 156) of uninjected embryos (-Cre) were phenotypic; only 5% (n = 140) of Cre mRNA-injected siblings (+Cre) were phenotypic (p < 0.05). (E) For offspring of hmcn1mn0263Gt heterozygote incrosses, 25% (n = 146) of uninjected embryos (-Cre) were phenotypic; only 8% (n = 233) of Cre-injected siblings (+Cre) were phenotypic (p < 0.005). (F) The fras1mn0156Gt GBT allele fails to complement the piftm95b ENU allele of fras1. Crossing piftm95b heterozygotes with fras1mn0156Gt heterozygotes does not reduce the proportion of phenotypic offspring (28%, n = 445) (tm95b/mn0156Gt trans-heterozygotes) compared to either piftm95b/tm95b (tm95b/tm95b, n = 332) or fras1mn0156Gt/mn0156Gt (mn0156Gt/mn0156Gt, n = 206) homozygotes. Percentages represent the mean of means (MOM); error bars represent standard deviations (SD).
Mentions: To determine which ZIP loci were required for early zebrafish skin or fin fold development, we bred each expressing insertion to homozygosity through a standard inbreeding scheme. For each line, we screened offspring of intercrossed heterozygous parents during the first five days of development (120 hpf) for abnormal skin or fin fold morphology present only in mRFP-positive (GBT-expressing) larvae and absent from their wild-type siblings [17] (an “mRFP-linked” phenotype). Of the eleven lines screened, three displayed recessive, mRFP-linked phenotypes: fras1mn0156Gt (Fig 4B), hmcn1mn0263Gt (Fig 4C), and nrg2amn0237Gt (Fig 5B). Comparisons to published phenotypes of ENU-derived alleles indicated that fras1mn0156Gt and hmcn1mn0263Gt represented novel alleles of known skin blistering mutants pinfin (pif) and nagel (nel) [44, 56], respectively (Table 1, Fig 4A–4C).

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis, Minnesota, United States of America; Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, Minnesota, United States of America.

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.

No MeSH data available.


Related in: MedlinePlus