Limits...
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

nrg2a mutants display altered MFF morphology, consistent with the epidermal localization of the Nrg2-mRFP fusion protein and of endogenous nrg2a transcripts.(A, B) By 48 hpf, nrg2amn0237Gt/mn0237Gt mutants (mn0237Gt/mn0237Gt) show altered MFF morphology. (A) Wild-type MFF edges are thin, flat, and continuously curved (arrowhead). (B) mn0237Gt/mn0237Gt mutant MFFs have thickened edges (arrowhead), and one or more pointed protrusions (open arrowhead). (C, D) Collagen II (Col II) immunostaining of actinotrichia support fibers within the MFF shows aberrant collagen accumulation and ectopic actinotrichia within mn0237Gt/mn0237Gt mutant apical MFFs (arrowheads) at 48 hpf. (E, G) At 24 hpf, Nrg2a-mRFP fusion protein is present in MFFs of heterozygous (+/mn0237Gt) embryos (E; view on tail of whole mount) and, at slightly lower levels, throughout the entire epidermis (G; section through tail region; immunostained for RFP and counterstained with DAPI). (F, H, I) Whole-mount in situ hybridization (WISH) demonstrates strong MFF expression of the GBT-generated fusion transcript (mRFP; F) in a representative +/mn0237Gt embryo at 24 hpf. When developed with Boehringer Blocking Reagent, WISH staining for endogenous nrg2a transcripts in 24 hpf wild-type embryos also revealed strong MFF expression of the endogenous gene (H, Boeringer). When developed without Boehringer Blocking Reagent, WISH staining further reveals uniform expression of the endogenous nrg2a gene throughout the entire epidermis (I). For cross-sections, see Honjo et al. (2008), Fig 6C [51]). (J-N) Co-labeling of a transverse section through the dorsal MFF of a +/mn0237Gt embryo at 24 hpf reveals restricted localization of the Nrg2a-mRFP fusion protein (J) in ΔNp63-positive basal keratinocytes (L), whereas the outer enveloping layer, labeled with EGFP (K), lacks the Nrg2a-mRFP protein; (M) DAPI counterstain; (N) merged image of different channels shown in (J-M).
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4482254&req=5

pone.0130688.g005: nrg2a mutants display altered MFF morphology, consistent with the epidermal localization of the Nrg2-mRFP fusion protein and of endogenous nrg2a transcripts.(A, B) By 48 hpf, nrg2amn0237Gt/mn0237Gt mutants (mn0237Gt/mn0237Gt) show altered MFF morphology. (A) Wild-type MFF edges are thin, flat, and continuously curved (arrowhead). (B) mn0237Gt/mn0237Gt mutant MFFs have thickened edges (arrowhead), and one or more pointed protrusions (open arrowhead). (C, D) Collagen II (Col II) immunostaining of actinotrichia support fibers within the MFF shows aberrant collagen accumulation and ectopic actinotrichia within mn0237Gt/mn0237Gt mutant apical MFFs (arrowheads) at 48 hpf. (E, G) At 24 hpf, Nrg2a-mRFP fusion protein is present in MFFs of heterozygous (+/mn0237Gt) embryos (E; view on tail of whole mount) and, at slightly lower levels, throughout the entire epidermis (G; section through tail region; immunostained for RFP and counterstained with DAPI). (F, H, I) Whole-mount in situ hybridization (WISH) demonstrates strong MFF expression of the GBT-generated fusion transcript (mRFP; F) in a representative +/mn0237Gt embryo at 24 hpf. When developed with Boehringer Blocking Reagent, WISH staining for endogenous nrg2a transcripts in 24 hpf wild-type embryos also revealed strong MFF expression of the endogenous gene (H, Boeringer). When developed without Boehringer Blocking Reagent, WISH staining further reveals uniform expression of the endogenous nrg2a gene throughout the entire epidermis (I). For cross-sections, see Honjo et al. (2008), Fig 6C [51]). (J-N) Co-labeling of a transverse section through the dorsal MFF of a +/mn0237Gt embryo at 24 hpf reveals restricted localization of the Nrg2a-mRFP fusion protein (J) in ΔNp63-positive basal keratinocytes (L), whereas the outer enveloping layer, labeled with EGFP (K), lacks the Nrg2a-mRFP protein; (M) DAPI counterstain; (N) merged image of different channels shown in (J-M).

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)

nrg2a mutants display altered MFF morphology, consistent with the epidermal localization of the Nrg2-mRFP fusion protein and of endogenous nrg2a transcripts.(A, B) By 48 hpf, nrg2amn0237Gt/mn0237Gt mutants (mn0237Gt/mn0237Gt) show altered MFF morphology. (A) Wild-type MFF edges are thin, flat, and continuously curved (arrowhead). (B) mn0237Gt/mn0237Gt mutant MFFs have thickened edges (arrowhead), and one or more pointed protrusions (open arrowhead). (C, D) Collagen II (Col II) immunostaining of actinotrichia support fibers within the MFF shows aberrant collagen accumulation and ectopic actinotrichia within mn0237Gt/mn0237Gt mutant apical MFFs (arrowheads) at 48 hpf. (E, G) At 24 hpf, Nrg2a-mRFP fusion protein is present in MFFs of heterozygous (+/mn0237Gt) embryos (E; view on tail of whole mount) and, at slightly lower levels, throughout the entire epidermis (G; section through tail region; immunostained for RFP and counterstained with DAPI). (F, H, I) Whole-mount in situ hybridization (WISH) demonstrates strong MFF expression of the GBT-generated fusion transcript (mRFP; F) in a representative +/mn0237Gt embryo at 24 hpf. When developed with Boehringer Blocking Reagent, WISH staining for endogenous nrg2a transcripts in 24 hpf wild-type embryos also revealed strong MFF expression of the endogenous gene (H, Boeringer). When developed without Boehringer Blocking Reagent, WISH staining further reveals uniform expression of the endogenous nrg2a gene throughout the entire epidermis (I). For cross-sections, see Honjo et al. (2008), Fig 6C [51]). (J-N) Co-labeling of a transverse section through the dorsal MFF of a +/mn0237Gt embryo at 24 hpf reveals restricted localization of the Nrg2a-mRFP fusion protein (J) in ΔNp63-positive basal keratinocytes (L), whereas the outer enveloping layer, labeled with EGFP (K), lacks the Nrg2a-mRFP protein; (M) DAPI counterstain; (N) merged image of different channels shown in (J-M).
© Copyright Policy
Related In: Results  -  Collection

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

pone.0130688.g005: nrg2a mutants display altered MFF morphology, consistent with the epidermal localization of the Nrg2-mRFP fusion protein and of endogenous nrg2a transcripts.(A, B) By 48 hpf, nrg2amn0237Gt/mn0237Gt mutants (mn0237Gt/mn0237Gt) show altered MFF morphology. (A) Wild-type MFF edges are thin, flat, and continuously curved (arrowhead). (B) mn0237Gt/mn0237Gt mutant MFFs have thickened edges (arrowhead), and one or more pointed protrusions (open arrowhead). (C, D) Collagen II (Col II) immunostaining of actinotrichia support fibers within the MFF shows aberrant collagen accumulation and ectopic actinotrichia within mn0237Gt/mn0237Gt mutant apical MFFs (arrowheads) at 48 hpf. (E, G) At 24 hpf, Nrg2a-mRFP fusion protein is present in MFFs of heterozygous (+/mn0237Gt) embryos (E; view on tail of whole mount) and, at slightly lower levels, throughout the entire epidermis (G; section through tail region; immunostained for RFP and counterstained with DAPI). (F, H, I) Whole-mount in situ hybridization (WISH) demonstrates strong MFF expression of the GBT-generated fusion transcript (mRFP; F) in a representative +/mn0237Gt embryo at 24 hpf. When developed with Boehringer Blocking Reagent, WISH staining for endogenous nrg2a transcripts in 24 hpf wild-type embryos also revealed strong MFF expression of the endogenous gene (H, Boeringer). When developed without Boehringer Blocking Reagent, WISH staining further reveals uniform expression of the endogenous nrg2a gene throughout the entire epidermis (I). For cross-sections, see Honjo et al. (2008), Fig 6C [51]). (J-N) Co-labeling of a transverse section through the dorsal MFF of a +/mn0237Gt embryo at 24 hpf reveals restricted localization of the Nrg2a-mRFP fusion protein (J) in ΔNp63-positive basal keratinocytes (L), whereas the outer enveloping layer, labeled with EGFP (K), lacks the Nrg2a-mRFP protein; (M) DAPI counterstain; (N) merged image of different channels shown in (J-M).
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