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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 cleft cells of nrg2a mutants are largely unaffected, but ridge cells display altered morphology.(A-C) Transverse sections through MFFs of Tg(Ola.Actb:Hsa.hras-egfp)vu119-expressing (hras-egfp) wild-type and nrg2a mutant embryos. Green: membrane-bound EGFP, blue: DAPI. (D-E) ET37 EGFP is expressed in MFF cleft cells (cc). ET37-EGFP: green, CellMask: red, DAPI: blue. (A) At 30 hpf, wild-type ridge cells (rc) are roughly cuboidal, with parallel apical and basal domains. The dermal space (ds) has not yet straightened, especially within the apical MFF terminus bounded by the cleft cell (cc). (B, D) By 52 hpf, the dermal space of wild-type embryos has straightened (arrowheads) and has invaginated into the basal side of the cleft cell. Ridge cells have elongated laterally, adopting a flat, planar, epithelial morphology. Their apical and basal domains are essentially parallel. (C, E) In nrg2a mutants, the cleft cell (cc) is present and contains the basal invagination of the dermal space (cleft; E) as in wild-type siblings. In contrast, nrg2a mutant ridge cells have elongated incorrectly and display an abnormal morphology, bulging basally into the dermal space, which acquires a serpentine-like appearance. Scale bar: 10 μm. Abbreviations: cc, cleft cell; rc, ridge cell; arrowheads point to dermal space.
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pone.0130688.g007: MFF cleft cells of nrg2a mutants are largely unaffected, but ridge cells display altered morphology.(A-C) Transverse sections through MFFs of Tg(Ola.Actb:Hsa.hras-egfp)vu119-expressing (hras-egfp) wild-type and nrg2a mutant embryos. Green: membrane-bound EGFP, blue: DAPI. (D-E) ET37 EGFP is expressed in MFF cleft cells (cc). ET37-EGFP: green, CellMask: red, DAPI: blue. (A) At 30 hpf, wild-type ridge cells (rc) are roughly cuboidal, with parallel apical and basal domains. The dermal space (ds) has not yet straightened, especially within the apical MFF terminus bounded by the cleft cell (cc). (B, D) By 52 hpf, the dermal space of wild-type embryos has straightened (arrowheads) and has invaginated into the basal side of the cleft cell. Ridge cells have elongated laterally, adopting a flat, planar, epithelial morphology. Their apical and basal domains are essentially parallel. (C, E) In nrg2a mutants, the cleft cell (cc) is present and contains the basal invagination of the dermal space (cleft; E) as in wild-type siblings. In contrast, nrg2a mutant ridge cells have elongated incorrectly and display an abnormal morphology, bulging basally into the dermal space, which acquires a serpentine-like appearance. Scale bar: 10 μm. Abbreviations: cc, cleft cell; rc, ridge cell; arrowheads point to dermal space.

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 cleft cells of nrg2a mutants are largely unaffected, but ridge cells display altered morphology.(A-C) Transverse sections through MFFs of Tg(Ola.Actb:Hsa.hras-egfp)vu119-expressing (hras-egfp) wild-type and nrg2a mutant embryos. Green: membrane-bound EGFP, blue: DAPI. (D-E) ET37 EGFP is expressed in MFF cleft cells (cc). ET37-EGFP: green, CellMask: red, DAPI: blue. (A) At 30 hpf, wild-type ridge cells (rc) are roughly cuboidal, with parallel apical and basal domains. The dermal space (ds) has not yet straightened, especially within the apical MFF terminus bounded by the cleft cell (cc). (B, D) By 52 hpf, the dermal space of wild-type embryos has straightened (arrowheads) and has invaginated into the basal side of the cleft cell. Ridge cells have elongated laterally, adopting a flat, planar, epithelial morphology. Their apical and basal domains are essentially parallel. (C, E) In nrg2a mutants, the cleft cell (cc) is present and contains the basal invagination of the dermal space (cleft; E) as in wild-type siblings. In contrast, nrg2a mutant ridge cells have elongated incorrectly and display an abnormal morphology, bulging basally into the dermal space, which acquires a serpentine-like appearance. Scale bar: 10 μm. Abbreviations: cc, cleft cell; rc, ridge cell; arrowheads point to dermal space.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4482254&req=5

pone.0130688.g007: MFF cleft cells of nrg2a mutants are largely unaffected, but ridge cells display altered morphology.(A-C) Transverse sections through MFFs of Tg(Ola.Actb:Hsa.hras-egfp)vu119-expressing (hras-egfp) wild-type and nrg2a mutant embryos. Green: membrane-bound EGFP, blue: DAPI. (D-E) ET37 EGFP is expressed in MFF cleft cells (cc). ET37-EGFP: green, CellMask: red, DAPI: blue. (A) At 30 hpf, wild-type ridge cells (rc) are roughly cuboidal, with parallel apical and basal domains. The dermal space (ds) has not yet straightened, especially within the apical MFF terminus bounded by the cleft cell (cc). (B, D) By 52 hpf, the dermal space of wild-type embryos has straightened (arrowheads) and has invaginated into the basal side of the cleft cell. Ridge cells have elongated laterally, adopting a flat, planar, epithelial morphology. Their apical and basal domains are essentially parallel. (C, E) In nrg2a mutants, the cleft cell (cc) is present and contains the basal invagination of the dermal space (cleft; E) as in wild-type siblings. In contrast, nrg2a mutant ridge cells have elongated incorrectly and display an abnormal morphology, bulging basally into the dermal space, which acquires a serpentine-like appearance. Scale bar: 10 μm. Abbreviations: cc, cleft cell; rc, ridge cell; arrowheads point to dermal space.
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