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

Usage of alternative first exons of nrg2a gene leads to differential cytosolic or nuclear localization of resulting protein isoforms.(A) A schematic of the nrg2a locus on linkage group 21 (LG21), including alternative first exons 1A, 1B and 1C. The GBT insertion is located in the intron separating exon 1C and exon 2. (B) RT-PCR analyses of 48 hpf sibling embryos from heterozygote (+/mn0237Gt) intercross, revealing that endogenous nrg2a 1B and 1C transcripts are only expressed in heterozygous (+/mn0237Gt) and wild-type siblings (+/+). nrg2a 1B-mRFP and 1C-mRFP fusion transcripts are only expressed in homozygous mutants (mn0237Gt/mn0237Gt) and heterozygous siblings (+/mn0237Gt). (C-N) Confocal images of MFF epidermis at 24 hpf detecting Nrg2a-RFP fusion protein (C, G, K; red), cell membranes (D, H, L; labeled with EGFP (green) after injection of egfp-caax mRNA at 1-cell stage), and cell nuclei (E, I, M; labeled with BFP (blue) after injection of nls-BFP mRNA at 1-cell stage). Panels (F, J, N) show merged images. (C-F) +/mn0237Gt embryo displays Nrg2amn0237Gt-RFP fusion protein both in the cytoplasm (empty arrowheads) and in the nuclei (filled arrowheads). (G-J) Wild-type embryo injected with mRNA encoding exon 1B-version of the Nrg2a-RFP fusion protein; the fusion protein is largely absent from the nucleus, but present in cyptoplasmic compartments. (K-N) Wild-type embryo injected with mRNA encoding exon 1C-version of the Nrg2a-RFP fusion protein; the fusion protein is present in the cytoplasm and the nuclei, similar to the distribution of the transgene-encoded protein (C-F).
© Copyright Policy
Related In: Results  -  Collection

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

pone.0130688.g006: Usage of alternative first exons of nrg2a gene leads to differential cytosolic or nuclear localization of resulting protein isoforms.(A) A schematic of the nrg2a locus on linkage group 21 (LG21), including alternative first exons 1A, 1B and 1C. The GBT insertion is located in the intron separating exon 1C and exon 2. (B) RT-PCR analyses of 48 hpf sibling embryos from heterozygote (+/mn0237Gt) intercross, revealing that endogenous nrg2a 1B and 1C transcripts are only expressed in heterozygous (+/mn0237Gt) and wild-type siblings (+/+). nrg2a 1B-mRFP and 1C-mRFP fusion transcripts are only expressed in homozygous mutants (mn0237Gt/mn0237Gt) and heterozygous siblings (+/mn0237Gt). (C-N) Confocal images of MFF epidermis at 24 hpf detecting Nrg2a-RFP fusion protein (C, G, K; red), cell membranes (D, H, L; labeled with EGFP (green) after injection of egfp-caax mRNA at 1-cell stage), and cell nuclei (E, I, M; labeled with BFP (blue) after injection of nls-BFP mRNA at 1-cell stage). Panels (F, J, N) show merged images. (C-F) +/mn0237Gt embryo displays Nrg2amn0237Gt-RFP fusion protein both in the cytoplasm (empty arrowheads) and in the nuclei (filled arrowheads). (G-J) Wild-type embryo injected with mRNA encoding exon 1B-version of the Nrg2a-RFP fusion protein; the fusion protein is largely absent from the nucleus, but present in cyptoplasmic compartments. (K-N) Wild-type embryo injected with mRNA encoding exon 1C-version of the Nrg2a-RFP fusion protein; the fusion protein is present in the cytoplasm and the nuclei, similar to the distribution of the transgene-encoded protein (C-F).

Mentions: (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).


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)

Usage of alternative first exons of nrg2a gene leads to differential cytosolic or nuclear localization of resulting protein isoforms.(A) A schematic of the nrg2a locus on linkage group 21 (LG21), including alternative first exons 1A, 1B and 1C. The GBT insertion is located in the intron separating exon 1C and exon 2. (B) RT-PCR analyses of 48 hpf sibling embryos from heterozygote (+/mn0237Gt) intercross, revealing that endogenous nrg2a 1B and 1C transcripts are only expressed in heterozygous (+/mn0237Gt) and wild-type siblings (+/+). nrg2a 1B-mRFP and 1C-mRFP fusion transcripts are only expressed in homozygous mutants (mn0237Gt/mn0237Gt) and heterozygous siblings (+/mn0237Gt). (C-N) Confocal images of MFF epidermis at 24 hpf detecting Nrg2a-RFP fusion protein (C, G, K; red), cell membranes (D, H, L; labeled with EGFP (green) after injection of egfp-caax mRNA at 1-cell stage), and cell nuclei (E, I, M; labeled with BFP (blue) after injection of nls-BFP mRNA at 1-cell stage). Panels (F, J, N) show merged images. (C-F) +/mn0237Gt embryo displays Nrg2amn0237Gt-RFP fusion protein both in the cytoplasm (empty arrowheads) and in the nuclei (filled arrowheads). (G-J) Wild-type embryo injected with mRNA encoding exon 1B-version of the Nrg2a-RFP fusion protein; the fusion protein is largely absent from the nucleus, but present in cyptoplasmic compartments. (K-N) Wild-type embryo injected with mRNA encoding exon 1C-version of the Nrg2a-RFP fusion protein; the fusion protein is present in the cytoplasm and the nuclei, similar to the distribution of the transgene-encoded protein (C-F).
© Copyright Policy
Related In: Results  -  Collection

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

pone.0130688.g006: Usage of alternative first exons of nrg2a gene leads to differential cytosolic or nuclear localization of resulting protein isoforms.(A) A schematic of the nrg2a locus on linkage group 21 (LG21), including alternative first exons 1A, 1B and 1C. The GBT insertion is located in the intron separating exon 1C and exon 2. (B) RT-PCR analyses of 48 hpf sibling embryos from heterozygote (+/mn0237Gt) intercross, revealing that endogenous nrg2a 1B and 1C transcripts are only expressed in heterozygous (+/mn0237Gt) and wild-type siblings (+/+). nrg2a 1B-mRFP and 1C-mRFP fusion transcripts are only expressed in homozygous mutants (mn0237Gt/mn0237Gt) and heterozygous siblings (+/mn0237Gt). (C-N) Confocal images of MFF epidermis at 24 hpf detecting Nrg2a-RFP fusion protein (C, G, K; red), cell membranes (D, H, L; labeled with EGFP (green) after injection of egfp-caax mRNA at 1-cell stage), and cell nuclei (E, I, M; labeled with BFP (blue) after injection of nls-BFP mRNA at 1-cell stage). Panels (F, J, N) show merged images. (C-F) +/mn0237Gt embryo displays Nrg2amn0237Gt-RFP fusion protein both in the cytoplasm (empty arrowheads) and in the nuclei (filled arrowheads). (G-J) Wild-type embryo injected with mRNA encoding exon 1B-version of the Nrg2a-RFP fusion protein; the fusion protein is largely absent from the nucleus, but present in cyptoplasmic compartments. (K-N) Wild-type embryo injected with mRNA encoding exon 1C-version of the Nrg2a-RFP fusion protein; the fusion protein is present in the cytoplasm and the nuclei, similar to the distribution of the transgene-encoded protein (C-F).
Mentions: (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).

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