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

Gene-break transposon—based protein trapping identifies known and new epidermal median fin fold loci.(A) A schematic of the RP2.1 gene-break transposon (GBT) vector used in this study. Gene-breaking activity occurs when an endogenous locus with a GBT insertion is transcribed. The vector-supplied splice acceptor (SA) in the 5’ protein trap cassette intercepts the endogenous splicing machinery and transcript, redirecting them to read directly into an AUG-free mRFP sequence (*mRFP). That event generates a fusion transcript by tagging the 5’ portion of the endogenous transcript with mRFP. When translated, the mRFP fusion transcript will produce a potentially mutagenic truncated protein product. Simultaneously, the 3’ exon trap cassette uses the vector-supplied splice donor (SD) to create a GFP fusion transcript with the remaining downstream endogenous transcript. GBT alleles are revertible because loxP sites (blue diamonds) flank the cassettes, allowing the mutagenic elements to be excised in the presence of Cre recombinase. (B-E’) At 24 hours post-fertilization (24 hpf), GBT-generated mRFP fusion proteins from megf6amn0325Gt (B), grip1mn0078Gt (C), fras1mn0156Gt (D), and hmcn1mn0263Gt (E) localize along MFF edges. (B’, E’) Both megf6amn0325Gt (B’) and hmcn1mn0263Gt (E’) localize within a narrow region along the MFF edge (blue arrowheads). (C’, D’) grip1mn0078Gt (C’) and fras1mn0156Gt (D’) localization also follows the fin fold edge (blue arrowheads), though they are distributed somewhat more diffusely than are megf6amn0325Gt (B’) and hmcn1mn0263Gt (E’). (F-H) Whole-mount in situ hybridization (WISH) in 24 hpf wild-type embryos reveals similar MFF expression patterns of endogenous grip1 (F), fras1 (G), and hmcn1 (H) genes. The mRFP fusion protein localization patterns observed in the respective GBT lines recapitulate endogenous gene expression (C, D, E).
© Copyright Policy
Related In: Results  -  Collection

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

pone.0130688.g001: Gene-break transposon—based protein trapping identifies known and new epidermal median fin fold loci.(A) A schematic of the RP2.1 gene-break transposon (GBT) vector used in this study. Gene-breaking activity occurs when an endogenous locus with a GBT insertion is transcribed. The vector-supplied splice acceptor (SA) in the 5’ protein trap cassette intercepts the endogenous splicing machinery and transcript, redirecting them to read directly into an AUG-free mRFP sequence (*mRFP). That event generates a fusion transcript by tagging the 5’ portion of the endogenous transcript with mRFP. When translated, the mRFP fusion transcript will produce a potentially mutagenic truncated protein product. Simultaneously, the 3’ exon trap cassette uses the vector-supplied splice donor (SD) to create a GFP fusion transcript with the remaining downstream endogenous transcript. GBT alleles are revertible because loxP sites (blue diamonds) flank the cassettes, allowing the mutagenic elements to be excised in the presence of Cre recombinase. (B-E’) At 24 hours post-fertilization (24 hpf), GBT-generated mRFP fusion proteins from megf6amn0325Gt (B), grip1mn0078Gt (C), fras1mn0156Gt (D), and hmcn1mn0263Gt (E) localize along MFF edges. (B’, E’) Both megf6amn0325Gt (B’) and hmcn1mn0263Gt (E’) localize within a narrow region along the MFF edge (blue arrowheads). (C’, D’) grip1mn0078Gt (C’) and fras1mn0156Gt (D’) localization also follows the fin fold edge (blue arrowheads), though they are distributed somewhat more diffusely than are megf6amn0325Gt (B’) and hmcn1mn0263Gt (E’). (F-H) Whole-mount in situ hybridization (WISH) in 24 hpf wild-type embryos reveals similar MFF expression patterns of endogenous grip1 (F), fras1 (G), and hmcn1 (H) genes. The mRFP fusion protein localization patterns observed in the respective GBT lines recapitulate endogenous gene expression (C, D, E).

Mentions: The zebrafish (Danio rerio) is an excellent vertebrate model for understanding development and disease. Zebrafish not only share significant genomic similarity with humans [9, 10], they also generate numerous transparent, externally fertilized embryos ideal for in vivo imaging and for phenotype-based gene discovery (“forward genetics”) [11, 12]. In addition to traditional chemical mutagenesis [13, 14], forward genetic screening uses insertional mutagenesis methods, including retroviruses [15, 16] and the more recently developed gene-breaking transposon (GBT) technology (Fig 1A) [17]. GBT mutagenesis generates mRFP-tagged, Cre recombinase-revertible insertional alleles with ≥ 97% knockdown of endogenous transcript levels [17]. Zebrafish GBT insertional mutagenesis has already identified and characterized new genes, expression patterns, and phenotypes in the heart [18, 19], vasculature [20], and muscle [21]. GBT insertional mutagenesis has also been used to dissect genetic links between brain and behavior [22]. However, it had not previously been applied to studying skin biology.


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)

Gene-break transposon—based protein trapping identifies known and new epidermal median fin fold loci.(A) A schematic of the RP2.1 gene-break transposon (GBT) vector used in this study. Gene-breaking activity occurs when an endogenous locus with a GBT insertion is transcribed. The vector-supplied splice acceptor (SA) in the 5’ protein trap cassette intercepts the endogenous splicing machinery and transcript, redirecting them to read directly into an AUG-free mRFP sequence (*mRFP). That event generates a fusion transcript by tagging the 5’ portion of the endogenous transcript with mRFP. When translated, the mRFP fusion transcript will produce a potentially mutagenic truncated protein product. Simultaneously, the 3’ exon trap cassette uses the vector-supplied splice donor (SD) to create a GFP fusion transcript with the remaining downstream endogenous transcript. GBT alleles are revertible because loxP sites (blue diamonds) flank the cassettes, allowing the mutagenic elements to be excised in the presence of Cre recombinase. (B-E’) At 24 hours post-fertilization (24 hpf), GBT-generated mRFP fusion proteins from megf6amn0325Gt (B), grip1mn0078Gt (C), fras1mn0156Gt (D), and hmcn1mn0263Gt (E) localize along MFF edges. (B’, E’) Both megf6amn0325Gt (B’) and hmcn1mn0263Gt (E’) localize within a narrow region along the MFF edge (blue arrowheads). (C’, D’) grip1mn0078Gt (C’) and fras1mn0156Gt (D’) localization also follows the fin fold edge (blue arrowheads), though they are distributed somewhat more diffusely than are megf6amn0325Gt (B’) and hmcn1mn0263Gt (E’). (F-H) Whole-mount in situ hybridization (WISH) in 24 hpf wild-type embryos reveals similar MFF expression patterns of endogenous grip1 (F), fras1 (G), and hmcn1 (H) genes. The mRFP fusion protein localization patterns observed in the respective GBT lines recapitulate endogenous gene expression (C, D, E).
© Copyright Policy
Related In: Results  -  Collection

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

pone.0130688.g001: Gene-break transposon—based protein trapping identifies known and new epidermal median fin fold loci.(A) A schematic of the RP2.1 gene-break transposon (GBT) vector used in this study. Gene-breaking activity occurs when an endogenous locus with a GBT insertion is transcribed. The vector-supplied splice acceptor (SA) in the 5’ protein trap cassette intercepts the endogenous splicing machinery and transcript, redirecting them to read directly into an AUG-free mRFP sequence (*mRFP). That event generates a fusion transcript by tagging the 5’ portion of the endogenous transcript with mRFP. When translated, the mRFP fusion transcript will produce a potentially mutagenic truncated protein product. Simultaneously, the 3’ exon trap cassette uses the vector-supplied splice donor (SD) to create a GFP fusion transcript with the remaining downstream endogenous transcript. GBT alleles are revertible because loxP sites (blue diamonds) flank the cassettes, allowing the mutagenic elements to be excised in the presence of Cre recombinase. (B-E’) At 24 hours post-fertilization (24 hpf), GBT-generated mRFP fusion proteins from megf6amn0325Gt (B), grip1mn0078Gt (C), fras1mn0156Gt (D), and hmcn1mn0263Gt (E) localize along MFF edges. (B’, E’) Both megf6amn0325Gt (B’) and hmcn1mn0263Gt (E’) localize within a narrow region along the MFF edge (blue arrowheads). (C’, D’) grip1mn0078Gt (C’) and fras1mn0156Gt (D’) localization also follows the fin fold edge (blue arrowheads), though they are distributed somewhat more diffusely than are megf6amn0325Gt (B’) and hmcn1mn0263Gt (E’). (F-H) Whole-mount in situ hybridization (WISH) in 24 hpf wild-type embryos reveals similar MFF expression patterns of endogenous grip1 (F), fras1 (G), and hmcn1 (H) genes. The mRFP fusion protein localization patterns observed in the respective GBT lines recapitulate endogenous gene expression (C, D, E).
Mentions: The zebrafish (Danio rerio) is an excellent vertebrate model for understanding development and disease. Zebrafish not only share significant genomic similarity with humans [9, 10], they also generate numerous transparent, externally fertilized embryos ideal for in vivo imaging and for phenotype-based gene discovery (“forward genetics”) [11, 12]. In addition to traditional chemical mutagenesis [13, 14], forward genetic screening uses insertional mutagenesis methods, including retroviruses [15, 16] and the more recently developed gene-breaking transposon (GBT) technology (Fig 1A) [17]. GBT mutagenesis generates mRFP-tagged, Cre recombinase-revertible insertional alleles with ≥ 97% knockdown of endogenous transcript levels [17]. Zebrafish GBT insertional mutagenesis has already identified and characterized new genes, expression patterns, and phenotypes in the heart [18, 19], vasculature [20], and muscle [21]. GBT insertional mutagenesis has also been used to dissect genetic links between brain and behavior [22]. However, it had not previously been applied to studying skin biology.

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