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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

MFF ridge cell in nrg2a mutants display alterations in basolateral versus apical dimensions.(A-H) Transmission electron micrographs (TEM) of the distal-most region within wild-type and nrg2a mutant MFFs at 36 hpf (A, B, E-H) or 52 hpf (C,D). (A) By 36 hpf, wild-type ridge cells (arrowhead) have begun elongating laterally. Their apical and basal surfaces are parallel to each other and to the dermal space. (C) By 52 hpf, wild-ridge cells have continued elongating and have maintained their arrangements. (B, D) In nrg2a mutants (mn0237Gt/mn0237Gt), ridge cells (arrowheads) are morphologically distorted, fail to stay aligned parallel to the direction of the fin, and bulge into the dermal space, giving it a serpentine-like appearance. (E-H) Relative basolateral and apical dimensions in nrg2a mutants are distorted compared to their wild-type counterparts. To illustrate the changes, identical images are shown side by side with and without marked ridge cell borders. (E, F) By 36 hpf, apical (red) and basal (blue) borders of wild-type ridge cells are roughly parallel and of comparable lengths; lateral borders with neighboring basal keratinocytes are in white. (G, H) An example of mn0237Gt/mn0237Gt mutant ridge cells bulging into the dermal space. The pictured bulge consists of two adjacent ridge cells sharing an exaggeratedly lengthened lateral border (white) and with enlarged basal (blue) borders, but strongly reduced apical borders (red). Scale bars: 2 μm. Abbreviations: ds, dermal space; e, EV; cc, cleft cell; arrowheads point to ridge cells.
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pone.0130688.g008: MFF ridge cell in nrg2a mutants display alterations in basolateral versus apical dimensions.(A-H) Transmission electron micrographs (TEM) of the distal-most region within wild-type and nrg2a mutant MFFs at 36 hpf (A, B, E-H) or 52 hpf (C,D). (A) By 36 hpf, wild-type ridge cells (arrowhead) have begun elongating laterally. Their apical and basal surfaces are parallel to each other and to the dermal space. (C) By 52 hpf, wild-ridge cells have continued elongating and have maintained their arrangements. (B, D) In nrg2a mutants (mn0237Gt/mn0237Gt), ridge cells (arrowheads) are morphologically distorted, fail to stay aligned parallel to the direction of the fin, and bulge into the dermal space, giving it a serpentine-like appearance. (E-H) Relative basolateral and apical dimensions in nrg2a mutants are distorted compared to their wild-type counterparts. To illustrate the changes, identical images are shown side by side with and without marked ridge cell borders. (E, F) By 36 hpf, apical (red) and basal (blue) borders of wild-type ridge cells are roughly parallel and of comparable lengths; lateral borders with neighboring basal keratinocytes are in white. (G, H) An example of mn0237Gt/mn0237Gt mutant ridge cells bulging into the dermal space. The pictured bulge consists of two adjacent ridge cells sharing an exaggeratedly lengthened lateral border (white) and with enlarged basal (blue) borders, but strongly reduced apical borders (red). Scale bars: 2 μm. Abbreviations: ds, dermal space; e, EV; cc, cleft cell; arrowheads point to ridge cells.

Mentions: To further characterize the MFF defect in nrg2amn0237Gt/mn0237Gt mutants, we compared transverse sections of median epidermal ridges (MER) from nrg2amn0237Gt/mn0237Gt mutant larvae with those of wild-type siblings. As described above, the MER consists of a central cleft cell, characterized by a cleft-like invagination of the dermal space, and ridge cells to either side of the cleft cell. To visualize the shapes of individual cleft and ridge cells, we crossed nrg2amn0237Gt into a Tg(Ola.Actb:Hsa.hras-egfp)vu119 (hras-egfp) ubiquitously expressed membrane-bound EGFP background (Fig 7A–7C). Furthermore, to specifically label cleft cells, we crossed nrg2amn0237Gt into an ET37 background, in which EGFP is specifically expressed in FMCs and cleft cells (Fig 7D and 7E). We also performed transmission electron microscopy (TEM; Fig 8; S4 Fig) to examine the cleft and ridge cells in finer detail. Because the gross morphological changes characterizing nrg2amn0237Gt/mn0237Gt mutant MFFs were visible by the second day of development (Fig 5B), we conducted our analyses between 30 and 52 hpf.


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)

MFF ridge cell in nrg2a mutants display alterations in basolateral versus apical dimensions.(A-H) Transmission electron micrographs (TEM) of the distal-most region within wild-type and nrg2a mutant MFFs at 36 hpf (A, B, E-H) or 52 hpf (C,D). (A) By 36 hpf, wild-type ridge cells (arrowhead) have begun elongating laterally. Their apical and basal surfaces are parallel to each other and to the dermal space. (C) By 52 hpf, wild-ridge cells have continued elongating and have maintained their arrangements. (B, D) In nrg2a mutants (mn0237Gt/mn0237Gt), ridge cells (arrowheads) are morphologically distorted, fail to stay aligned parallel to the direction of the fin, and bulge into the dermal space, giving it a serpentine-like appearance. (E-H) Relative basolateral and apical dimensions in nrg2a mutants are distorted compared to their wild-type counterparts. To illustrate the changes, identical images are shown side by side with and without marked ridge cell borders. (E, F) By 36 hpf, apical (red) and basal (blue) borders of wild-type ridge cells are roughly parallel and of comparable lengths; lateral borders with neighboring basal keratinocytes are in white. (G, H) An example of mn0237Gt/mn0237Gt mutant ridge cells bulging into the dermal space. The pictured bulge consists of two adjacent ridge cells sharing an exaggeratedly lengthened lateral border (white) and with enlarged basal (blue) borders, but strongly reduced apical borders (red). Scale bars: 2 μm. Abbreviations: ds, dermal space; e, EV; cc, cleft cell; arrowheads point to ridge cells.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0130688.g008: MFF ridge cell in nrg2a mutants display alterations in basolateral versus apical dimensions.(A-H) Transmission electron micrographs (TEM) of the distal-most region within wild-type and nrg2a mutant MFFs at 36 hpf (A, B, E-H) or 52 hpf (C,D). (A) By 36 hpf, wild-type ridge cells (arrowhead) have begun elongating laterally. Their apical and basal surfaces are parallel to each other and to the dermal space. (C) By 52 hpf, wild-ridge cells have continued elongating and have maintained their arrangements. (B, D) In nrg2a mutants (mn0237Gt/mn0237Gt), ridge cells (arrowheads) are morphologically distorted, fail to stay aligned parallel to the direction of the fin, and bulge into the dermal space, giving it a serpentine-like appearance. (E-H) Relative basolateral and apical dimensions in nrg2a mutants are distorted compared to their wild-type counterparts. To illustrate the changes, identical images are shown side by side with and without marked ridge cell borders. (E, F) By 36 hpf, apical (red) and basal (blue) borders of wild-type ridge cells are roughly parallel and of comparable lengths; lateral borders with neighboring basal keratinocytes are in white. (G, H) An example of mn0237Gt/mn0237Gt mutant ridge cells bulging into the dermal space. The pictured bulge consists of two adjacent ridge cells sharing an exaggeratedly lengthened lateral border (white) and with enlarged basal (blue) borders, but strongly reduced apical borders (red). Scale bars: 2 μm. Abbreviations: ds, dermal space; e, EV; cc, cleft cell; arrowheads point to ridge cells.
Mentions: To further characterize the MFF defect in nrg2amn0237Gt/mn0237Gt mutants, we compared transverse sections of median epidermal ridges (MER) from nrg2amn0237Gt/mn0237Gt mutant larvae with those of wild-type siblings. As described above, the MER consists of a central cleft cell, characterized by a cleft-like invagination of the dermal space, and ridge cells to either side of the cleft cell. To visualize the shapes of individual cleft and ridge cells, we crossed nrg2amn0237Gt into a Tg(Ola.Actb:Hsa.hras-egfp)vu119 (hras-egfp) ubiquitously expressed membrane-bound EGFP background (Fig 7A–7C). Furthermore, to specifically label cleft cells, we crossed nrg2amn0237Gt into an ET37 background, in which EGFP is specifically expressed in FMCs and cleft cells (Fig 7D and 7E). We also performed transmission electron microscopy (TEM; Fig 8; S4 Fig) to examine the cleft and ridge cells in finer detail. Because the gross morphological changes characterizing nrg2amn0237Gt/mn0237Gt mutant MFFs were visible by the second day of development (Fig 5B), we conducted our analyses between 30 and 52 hpf.

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