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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 identifies integument loci with epidermal or fin mesenchymal expression.As identified by mRFP localization, seven GBT alleles have unique epidermal expression patterns emphasizing epidermal continuity over the body and larval fin folds. They are: (A, B) nrg2amn0237Gt, (C, D) col4a4mn0275Gt, (E, F) arhgef25bmn0175Gt, (G, H) ahnakmn0196Gt, (I, J) capn12mn0261Gt, (K, L) fkbp10bmn0316Gt, and (M, N) msxcmn0245Gt. (B, D, I, J, K) Four lines, nrg2amn0237Gt, col4a4mn0275Gt, capn12mn0261Gt, and fkbp10bmn0316Gt, have “holes” or “gaps” in their epidermal pattern (green arrowheads) where neuromasts embedded in the basal layer exclude the mRFP-positive basal keratinocytes. (A, G, I, K, M) nrg2amn0237Gt (A), ahnakmn0196Gt (G), capn12mn0261Gt (I), fkbp10bmn0316Gt (K), and msxcmn0245Gt (M) are also expressed in the pectoral fin folds. (C-D, J, L, M-N) col4a4mn0275Gt, capn12mn0261Gt, fkbp10bmn0316Gt, and msxcmn0245Gt epidermal expression (blue arrowheads) can be difficult to discern among other expression pattern components. (I-N) capn12mn0261Gt, megf6amn0316Gt, and msxcmn0245Gt also show expression in fin mesenchymal cells (pink arrowheads). (C, D, K, L, M, N) Several lines are also expressed in other tissues. (C, D) col4a4mn0275Gt appears in myotomes and the vascular plexus. (K, L) fkbp10bmn0316Gt is seen in the notochord. (M, N) msxcmn0245Gt is strongly expressed in the brain, spinal cord, and sensory maculae. hpf, hours post-fertilization. Comparisons of the mRFP localization patterns with the expression pattern of the endogenous genes are shown in Supporting Information (S1 Fig)
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pone.0130688.g002: GBT protein trapping identifies integument loci with epidermal or fin mesenchymal expression.As identified by mRFP localization, seven GBT alleles have unique epidermal expression patterns emphasizing epidermal continuity over the body and larval fin folds. They are: (A, B) nrg2amn0237Gt, (C, D) col4a4mn0275Gt, (E, F) arhgef25bmn0175Gt, (G, H) ahnakmn0196Gt, (I, J) capn12mn0261Gt, (K, L) fkbp10bmn0316Gt, and (M, N) msxcmn0245Gt. (B, D, I, J, K) Four lines, nrg2amn0237Gt, col4a4mn0275Gt, capn12mn0261Gt, and fkbp10bmn0316Gt, have “holes” or “gaps” in their epidermal pattern (green arrowheads) where neuromasts embedded in the basal layer exclude the mRFP-positive basal keratinocytes. (A, G, I, K, M) nrg2amn0237Gt (A), ahnakmn0196Gt (G), capn12mn0261Gt (I), fkbp10bmn0316Gt (K), and msxcmn0245Gt (M) are also expressed in the pectoral fin folds. (C-D, J, L, M-N) col4a4mn0275Gt, capn12mn0261Gt, fkbp10bmn0316Gt, and msxcmn0245Gt epidermal expression (blue arrowheads) can be difficult to discern among other expression pattern components. (I-N) capn12mn0261Gt, megf6amn0316Gt, and msxcmn0245Gt also show expression in fin mesenchymal cells (pink arrowheads). (C, D, K, L, M, N) Several lines are also expressed in other tissues. (C, D) col4a4mn0275Gt appears in myotomes and the vascular plexus. (K, L) fkbp10bmn0316Gt is seen in the notochord. (M, N) msxcmn0245Gt is strongly expressed in the brain, spinal cord, and sensory maculae. hpf, hours post-fertilization. Comparisons of the mRFP localization patterns with the expression pattern of the endogenous genes are shown in Supporting Information (S1 Fig)

Mentions: Locating expression within a tissue of interest is a key step toward identifying new genes involved in tissue-specific biology. Using the online GBT database and image catalog zfishbook.org (http://www.zfishbook.org) [55] as an initial selection tool, we screened 350 GBT insertional alleles [17] for mRFP expression within the skin of the MFF (Fig 1B–1E; Fig 2B, 2D, 2F, 2H, 2J, 2L, and 2N) and/or covering the larval body (Fig 2A, 2C, 2E, 2G, 2I and 2K) [37, 38, 56]. That expression prioritization screen identified eleven gene-break alleles (GBT lines) with mRFP localization in the integument (Fig 1B–1E; Fig 2). We refer to these as the initial Zebrafish Integument Project (ZIP) GBT lines. The ZIP lines’ RFP localization patterns [17] fell into three broad categories. The first was expression in MFF edges (Fig 1B–1E); the second was expression throughout some or all of the epidermis (Fig 2A–2N); and the third was expression in FMCs (Fig 2I–2N; Fig 3). Because we identified these eleven ZIP lines among the initial cohort of GBT insertional alleles, the GBT-disrupted genes in several ZIP lines had been identified during our initial GBT mutagenesis study [17]. We completed sequence-based identification of ZIP genes not identified in the previous study (Table 1).


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 identifies integument loci with epidermal or fin mesenchymal expression.As identified by mRFP localization, seven GBT alleles have unique epidermal expression patterns emphasizing epidermal continuity over the body and larval fin folds. They are: (A, B) nrg2amn0237Gt, (C, D) col4a4mn0275Gt, (E, F) arhgef25bmn0175Gt, (G, H) ahnakmn0196Gt, (I, J) capn12mn0261Gt, (K, L) fkbp10bmn0316Gt, and (M, N) msxcmn0245Gt. (B, D, I, J, K) Four lines, nrg2amn0237Gt, col4a4mn0275Gt, capn12mn0261Gt, and fkbp10bmn0316Gt, have “holes” or “gaps” in their epidermal pattern (green arrowheads) where neuromasts embedded in the basal layer exclude the mRFP-positive basal keratinocytes. (A, G, I, K, M) nrg2amn0237Gt (A), ahnakmn0196Gt (G), capn12mn0261Gt (I), fkbp10bmn0316Gt (K), and msxcmn0245Gt (M) are also expressed in the pectoral fin folds. (C-D, J, L, M-N) col4a4mn0275Gt, capn12mn0261Gt, fkbp10bmn0316Gt, and msxcmn0245Gt epidermal expression (blue arrowheads) can be difficult to discern among other expression pattern components. (I-N) capn12mn0261Gt, megf6amn0316Gt, and msxcmn0245Gt also show expression in fin mesenchymal cells (pink arrowheads). (C, D, K, L, M, N) Several lines are also expressed in other tissues. (C, D) col4a4mn0275Gt appears in myotomes and the vascular plexus. (K, L) fkbp10bmn0316Gt is seen in the notochord. (M, N) msxcmn0245Gt is strongly expressed in the brain, spinal cord, and sensory maculae. hpf, hours post-fertilization. Comparisons of the mRFP localization patterns with the expression pattern of the endogenous genes are shown in Supporting Information (S1 Fig)
© Copyright Policy
Related In: Results  -  Collection

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

pone.0130688.g002: GBT protein trapping identifies integument loci with epidermal or fin mesenchymal expression.As identified by mRFP localization, seven GBT alleles have unique epidermal expression patterns emphasizing epidermal continuity over the body and larval fin folds. They are: (A, B) nrg2amn0237Gt, (C, D) col4a4mn0275Gt, (E, F) arhgef25bmn0175Gt, (G, H) ahnakmn0196Gt, (I, J) capn12mn0261Gt, (K, L) fkbp10bmn0316Gt, and (M, N) msxcmn0245Gt. (B, D, I, J, K) Four lines, nrg2amn0237Gt, col4a4mn0275Gt, capn12mn0261Gt, and fkbp10bmn0316Gt, have “holes” or “gaps” in their epidermal pattern (green arrowheads) where neuromasts embedded in the basal layer exclude the mRFP-positive basal keratinocytes. (A, G, I, K, M) nrg2amn0237Gt (A), ahnakmn0196Gt (G), capn12mn0261Gt (I), fkbp10bmn0316Gt (K), and msxcmn0245Gt (M) are also expressed in the pectoral fin folds. (C-D, J, L, M-N) col4a4mn0275Gt, capn12mn0261Gt, fkbp10bmn0316Gt, and msxcmn0245Gt epidermal expression (blue arrowheads) can be difficult to discern among other expression pattern components. (I-N) capn12mn0261Gt, megf6amn0316Gt, and msxcmn0245Gt also show expression in fin mesenchymal cells (pink arrowheads). (C, D, K, L, M, N) Several lines are also expressed in other tissues. (C, D) col4a4mn0275Gt appears in myotomes and the vascular plexus. (K, L) fkbp10bmn0316Gt is seen in the notochord. (M, N) msxcmn0245Gt is strongly expressed in the brain, spinal cord, and sensory maculae. hpf, hours post-fertilization. Comparisons of the mRFP localization patterns with the expression pattern of the endogenous genes are shown in Supporting Information (S1 Fig)
Mentions: Locating expression within a tissue of interest is a key step toward identifying new genes involved in tissue-specific biology. Using the online GBT database and image catalog zfishbook.org (http://www.zfishbook.org) [55] as an initial selection tool, we screened 350 GBT insertional alleles [17] for mRFP expression within the skin of the MFF (Fig 1B–1E; Fig 2B, 2D, 2F, 2H, 2J, 2L, and 2N) and/or covering the larval body (Fig 2A, 2C, 2E, 2G, 2I and 2K) [37, 38, 56]. That expression prioritization screen identified eleven gene-break alleles (GBT lines) with mRFP localization in the integument (Fig 1B–1E; Fig 2). We refer to these as the initial Zebrafish Integument Project (ZIP) GBT lines. The ZIP lines’ RFP localization patterns [17] fell into three broad categories. The first was expression in MFF edges (Fig 1B–1E); the second was expression throughout some or all of the epidermis (Fig 2A–2N); and the third was expression in FMCs (Fig 2I–2N; Fig 3). Because we identified these eleven ZIP lines among the initial cohort of GBT insertional alleles, the GBT-disrupted genes in several ZIP lines had been identified during our initial GBT mutagenesis study [17]. We completed sequence-based identification of ZIP genes not identified in the previous study (Table 1).

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