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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

The MFF phenotype of nrg2a mutants is rescued upon concomitant loss of Lgl2 function.(A) At 52 hpf, morpholino (MO)-mediated knockdown of lgl2 significantly ameliorated the nrg2a mutant phenotype (12%, n = 639, p < 0.0001) relative to uninjected embryos of an nrg2a -/+ intercross (25%, n = 943). Percentages represent the mean of means (MOM); error bars represent the standard deviations (SD). (B) Percentages of genotyped nrg2a-/- mutants with a strong, medium, weak, or wild-type MFF phenotype, classified by morphological criteria at 52 hpf. While uninjected nrg2a mutants (n = 20) all display a strong phenotype, lgl2 MO-injected mutants (n = 26) show medium, weak or no MFF defects. (C) PCR products obtained via nrg2a genotyping of representative nrg2a -/+, +/+ and-/- embryos at 52 hpf (see Materials and Methods). (D-H) Tail fins of representative live embryos at 52 hpf, as used for quantitative classification in panel B: wild-type (D), uninjected nrg2a-/- mutant with strong MFF phenotype (E), and lgl2 MO-injected nrg2a -/- mutant embryos with medium (F), weak (G) or wild-type (H) phenotype. (I, J) Tail fins of genotyped uninjected (I) and lgl2 MO-injected (J) nrg2a -/- mutant embryo at 48 hpf. Col II immunostaining reveals a normalized organization of collagenous actinotrichia within the dermal space of the Nrg2a/Lgl2-double-deficient embryo (J; compare with Fig 5C for wild-type condition). (K, L) Transverse sections through the dorsal MFF of a genotyped uninjected (K) and an lgl2 MO-injected (L) nrg2a -/- mutant embryo at 52 hpf; CellMask (red) and DAPI (blue) staining reveals a rescue of the dermal space (indicated by arrowheads) from a serpentine-like organization (K) to a straight organization (L) in the Nrg2a/Lgl2-double-deficient embryo (L; compare with Fig 7D for wild-type condition).
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pone.0130688.g010: The MFF phenotype of nrg2a mutants is rescued upon concomitant loss of Lgl2 function.(A) At 52 hpf, morpholino (MO)-mediated knockdown of lgl2 significantly ameliorated the nrg2a mutant phenotype (12%, n = 639, p < 0.0001) relative to uninjected embryos of an nrg2a -/+ intercross (25%, n = 943). Percentages represent the mean of means (MOM); error bars represent the standard deviations (SD). (B) Percentages of genotyped nrg2a-/- mutants with a strong, medium, weak, or wild-type MFF phenotype, classified by morphological criteria at 52 hpf. While uninjected nrg2a mutants (n = 20) all display a strong phenotype, lgl2 MO-injected mutants (n = 26) show medium, weak or no MFF defects. (C) PCR products obtained via nrg2a genotyping of representative nrg2a -/+, +/+ and-/- embryos at 52 hpf (see Materials and Methods). (D-H) Tail fins of representative live embryos at 52 hpf, as used for quantitative classification in panel B: wild-type (D), uninjected nrg2a-/- mutant with strong MFF phenotype (E), and lgl2 MO-injected nrg2a -/- mutant embryos with medium (F), weak (G) or wild-type (H) phenotype. (I, J) Tail fins of genotyped uninjected (I) and lgl2 MO-injected (J) nrg2a -/- mutant embryo at 48 hpf. Col II immunostaining reveals a normalized organization of collagenous actinotrichia within the dermal space of the Nrg2a/Lgl2-double-deficient embryo (J; compare with Fig 5C for wild-type condition). (K, L) Transverse sections through the dorsal MFF of a genotyped uninjected (K) and an lgl2 MO-injected (L) nrg2a -/- mutant embryo at 52 hpf; CellMask (red) and DAPI (blue) staining reveals a rescue of the dermal space (indicated by arrowheads) from a serpentine-like organization (K) to a straight organization (L) in the Nrg2a/Lgl2-double-deficient embryo (L; compare with Fig 7D for wild-type condition).

Mentions: To date, ErbB activity in zebrafish epidermis has only been described in the context of the tumor suppressor lethal giant larvae 2 (lgl2), which blocks ErbB signaling and epithelial-to-mesenchymal transitions (EMT) to safeguard epidermal integrity during late larval stages (120 hpf) [52]. Yet even though lgl2 is epidermally expressed by 24 hpf [54], it has no identified developmental role during that early time frame. However, previous characterizations of Lgl2 as an epithelial polarity regulator that promotes basal fate [54, 89, 90], along with the basolateral domain expansion observed in nrg2amn0237Gt/mn0237Gt mutants’ MFF ridge cells and the temporal overlap among lgl2 [54], nrg2a (Fig 5H and 5I), and erbb3a expression patterns (Fig 9L), led us to speculate that Lgl2 might play an earlier role in epidermal development. We hypothesized that that earlier Lgl2 role involved opposing Nrg2 –ErbB3 signaling during MFF morphogenesis. To test that hypothesis, we suppressed lgl2 in the nrg2amn0237Gt/mn0237Gt background. Morpholino (MO) knockdown [11] of lgl2 significantly restored nrg2amn0237Gt/mn0237Gt mutants’ MFF morphology at 52 hpf (Fig 10A–10H) relative to both uninjected controls and tp53 MO-injected controls (Fig 10A). Indeed, lgl2 MO-injected and genotyped nrg2amn0237Gt/mn0237Gt embryos displayed external MFF morphology indistinguishable from that of wild-type siblings (compare Fig 10H with Fig 10D). Furthermore, MFF internal organization was also ameliorated in lgl2 MO-injected nrg2amn0237Gt/mn0237Gt embryos. Dermal actinotrichia organization, (Fig 10I and 10J; 48 hpf), ridge cells’ apicobasal organization, and MFF dermal space bending (Fig 10K and 10L; 52 hpf) were all dramatically normalized, leading to embryos indistinguishable from wild-type (compare Fig 10J with Fig 5C, and Fig 10L with Fig 7D). These results indicated that loss of Lgl2 activity suppressed the nrg2a phenotype, revealing a new and significantly earlier role for lgl2 in fin fold development, distinct from its established tumor-suppressing and hemidesomosome (basal)-promoting functions in the later body epidermis [52, 54].


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)

The MFF phenotype of nrg2a mutants is rescued upon concomitant loss of Lgl2 function.(A) At 52 hpf, morpholino (MO)-mediated knockdown of lgl2 significantly ameliorated the nrg2a mutant phenotype (12%, n = 639, p < 0.0001) relative to uninjected embryos of an nrg2a -/+ intercross (25%, n = 943). Percentages represent the mean of means (MOM); error bars represent the standard deviations (SD). (B) Percentages of genotyped nrg2a-/- mutants with a strong, medium, weak, or wild-type MFF phenotype, classified by morphological criteria at 52 hpf. While uninjected nrg2a mutants (n = 20) all display a strong phenotype, lgl2 MO-injected mutants (n = 26) show medium, weak or no MFF defects. (C) PCR products obtained via nrg2a genotyping of representative nrg2a -/+, +/+ and-/- embryos at 52 hpf (see Materials and Methods). (D-H) Tail fins of representative live embryos at 52 hpf, as used for quantitative classification in panel B: wild-type (D), uninjected nrg2a-/- mutant with strong MFF phenotype (E), and lgl2 MO-injected nrg2a -/- mutant embryos with medium (F), weak (G) or wild-type (H) phenotype. (I, J) Tail fins of genotyped uninjected (I) and lgl2 MO-injected (J) nrg2a -/- mutant embryo at 48 hpf. Col II immunostaining reveals a normalized organization of collagenous actinotrichia within the dermal space of the Nrg2a/Lgl2-double-deficient embryo (J; compare with Fig 5C for wild-type condition). (K, L) Transverse sections through the dorsal MFF of a genotyped uninjected (K) and an lgl2 MO-injected (L) nrg2a -/- mutant embryo at 52 hpf; CellMask (red) and DAPI (blue) staining reveals a rescue of the dermal space (indicated by arrowheads) from a serpentine-like organization (K) to a straight organization (L) in the Nrg2a/Lgl2-double-deficient embryo (L; compare with Fig 7D for wild-type condition).
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Show All Figures
getmorefigures.php?uid=PMC4482254&req=5

pone.0130688.g010: The MFF phenotype of nrg2a mutants is rescued upon concomitant loss of Lgl2 function.(A) At 52 hpf, morpholino (MO)-mediated knockdown of lgl2 significantly ameliorated the nrg2a mutant phenotype (12%, n = 639, p < 0.0001) relative to uninjected embryos of an nrg2a -/+ intercross (25%, n = 943). Percentages represent the mean of means (MOM); error bars represent the standard deviations (SD). (B) Percentages of genotyped nrg2a-/- mutants with a strong, medium, weak, or wild-type MFF phenotype, classified by morphological criteria at 52 hpf. While uninjected nrg2a mutants (n = 20) all display a strong phenotype, lgl2 MO-injected mutants (n = 26) show medium, weak or no MFF defects. (C) PCR products obtained via nrg2a genotyping of representative nrg2a -/+, +/+ and-/- embryos at 52 hpf (see Materials and Methods). (D-H) Tail fins of representative live embryos at 52 hpf, as used for quantitative classification in panel B: wild-type (D), uninjected nrg2a-/- mutant with strong MFF phenotype (E), and lgl2 MO-injected nrg2a -/- mutant embryos with medium (F), weak (G) or wild-type (H) phenotype. (I, J) Tail fins of genotyped uninjected (I) and lgl2 MO-injected (J) nrg2a -/- mutant embryo at 48 hpf. Col II immunostaining reveals a normalized organization of collagenous actinotrichia within the dermal space of the Nrg2a/Lgl2-double-deficient embryo (J; compare with Fig 5C for wild-type condition). (K, L) Transverse sections through the dorsal MFF of a genotyped uninjected (K) and an lgl2 MO-injected (L) nrg2a -/- mutant embryo at 52 hpf; CellMask (red) and DAPI (blue) staining reveals a rescue of the dermal space (indicated by arrowheads) from a serpentine-like organization (K) to a straight organization (L) in the Nrg2a/Lgl2-double-deficient embryo (L; compare with Fig 7D for wild-type condition).
Mentions: To date, ErbB activity in zebrafish epidermis has only been described in the context of the tumor suppressor lethal giant larvae 2 (lgl2), which blocks ErbB signaling and epithelial-to-mesenchymal transitions (EMT) to safeguard epidermal integrity during late larval stages (120 hpf) [52]. Yet even though lgl2 is epidermally expressed by 24 hpf [54], it has no identified developmental role during that early time frame. However, previous characterizations of Lgl2 as an epithelial polarity regulator that promotes basal fate [54, 89, 90], along with the basolateral domain expansion observed in nrg2amn0237Gt/mn0237Gt mutants’ MFF ridge cells and the temporal overlap among lgl2 [54], nrg2a (Fig 5H and 5I), and erbb3a expression patterns (Fig 9L), led us to speculate that Lgl2 might play an earlier role in epidermal development. We hypothesized that that earlier Lgl2 role involved opposing Nrg2 –ErbB3 signaling during MFF morphogenesis. To test that hypothesis, we suppressed lgl2 in the nrg2amn0237Gt/mn0237Gt background. Morpholino (MO) knockdown [11] of lgl2 significantly restored nrg2amn0237Gt/mn0237Gt mutants’ MFF morphology at 52 hpf (Fig 10A–10H) relative to both uninjected controls and tp53 MO-injected controls (Fig 10A). Indeed, lgl2 MO-injected and genotyped nrg2amn0237Gt/mn0237Gt embryos displayed external MFF morphology indistinguishable from that of wild-type siblings (compare Fig 10H with Fig 10D). Furthermore, MFF internal organization was also ameliorated in lgl2 MO-injected nrg2amn0237Gt/mn0237Gt embryos. Dermal actinotrichia organization, (Fig 10I and 10J; 48 hpf), ridge cells’ apicobasal organization, and MFF dermal space bending (Fig 10K and 10L; 52 hpf) were all dramatically normalized, leading to embryos indistinguishable from wild-type (compare Fig 10J with Fig 5C, and Fig 10L with Fig 7D). These results indicated that loss of Lgl2 activity suppressed the nrg2a phenotype, revealing a new and significantly earlier role for lgl2 in fin fold development, distinct from its established tumor-suppressing and hemidesomosome (basal)-promoting functions in the later body epidermis [52, 54].

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