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

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.

Fig 1. 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 megf6a ^mn0325Gt (B), grip1 ^mn0078Gt (C), fras1 ^mn0156Gt (D), and hmcn1 ^mn0263Gt (E) localize along MFF edges. (B’, E’) Both megf6a ^mn0325Gt (B’) and hmcn1 ^mn0263Gt (E’) localize within a narrow region along the MFF edge (blue arrowheads). (C’, D’) grip1 ^mn0078Gt (C’) and fras1 ^mn0156Gt (D’) localization also follows the fin fold edge (blue arrowheads), though they are distributed somewhat more diffusely than are megf6a ^mn0325Gt (B’) and hmcn1 ^mn0263Gt (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).

Fig 2. 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) nrg2a ^mn0237Gt, (C, D) col4a4 ^mn0275Gt, (E, F) arhgef25b ^mn0175Gt, (G, H) ahnak ^mn0196Gt, (I, J) capn12 ^mn0261Gt, (K, L) fkbp10b ^mn0316Gt, and (M, N) msxc ^mn0245Gt. (B, D, I, J, K) Four lines, nrg2a ^mn0237Gt, col4a4 ^mn0275Gt, capn12 ^mn0261Gt, and fkbp10b ^mn0316Gt, 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) nrg2a ^mn0237Gt (A), ahnak ^mn0196Gt (G), capn12 ^mn0261Gt (I), fkbp10b ^mn0316Gt (K), and msxc ^mn0245Gt (M) are also expressed in the pectoral fin folds. (C-D, J, L, M-N) col4a4 ^mn0275Gt, capn12 ^mn0261Gt, fkbp10b ^mn0316Gt, and msxc ^mn0245Gt epidermal expression (blue arrowheads) can be difficult to discern among other expression pattern components. (I-N) capn12 ^mn0261Gt, megf6a ^mn0316Gt, and msxc ^mn0245Gt 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) col4a4 ^mn0275Gt appears in myotomes and the vascular plexus. (K, L) fkbp10b ^mn0316Gt is seen in the notochord. (M, N) msxc ^mn0245Gt 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)

Fig 3. capn12 and msxc transgenes are co-expressed with a fin mesenchymal cell marker.

(A, E) When capn12 ^mn0261Gt and msxc ^mn0245Gt are crossed into an ET37 background, both Capn12^mn0261Gt-mRFP and MsxC^mn0245Gt-mRFP co-localize with ET37 EGFP-positive fin mesenchymal cells (FMCs) (arrowheads). ET37 also labels the cleft cells along the MFF edge (open arrowheads). Neither Capn12^mn0261Gt-mRFP nor MsxC^mn0245Gt-mRFP localizes to the cleft cells. (B-H) ET37 EGFP labels all FMCs within the fin (arrowheads), both proximal (upper arrowheads) and distal (lower arrowheads), and is present throughout FMC cell bodies and in the extended “arbors” of distal FMCs (lower arrowheads). Both Capn12^mn0261Gt-mRFP and MsxC^mn0245Gt-mRFP localization show similarities with and differences from ET37 EGFP. (B-D) Both ET37 EGFP and Capn12^mn0261Gt-mRFP are expressed in proximal and distal FMCs (arrowheads) but unlike ET37 EGFP, Capn12^mn0261Gt-mRFP localizes to the periphery of FMCs (arrowheads). (F-H) MsxC^mn0245Gt-mRFP co-localizes with ET37 EGFP, but differs from Capn12^mn0261Gt-mRFP. MsxC^mn0245Gt-mRFP is much weaker in proximal FMCs (upper arrowheads) than in distal FMCs (lower arrowheads). Unlike Capn12^mn0261Gt-mRFP, MsxC^mn0245Gt-mRFP subcellular localization is not noticeably distinct from that of ET37 EGFP.

Fig 4. GBT protein trapping generates novel revertible alleles of known MFF loci.

(A) By 48 hpf, wild-type MFFs are thin and flat, and the MFF edge appears smooth and regular. (B-C) Two ZIP gene-break alleles, fras1 ^mn0156Gt and hmcn1 ^mn0263Gt have homozygous recessive phenotypes, each of which results in blistered MFFs (arrowheads). (D-E) Both fras1 ^mn0156Gt and hmcn1 ^mn0263Gt behaved as classic revertible GBT mutant alleles in Cre reversion experiments. With both alleles, significantly fewer Cre mRNA-injected embryos were phenotypic, compared with their respective uninjected siblings. (D) For offspring of fras1 ^mn0156Gt heterozygote incrosses, 27% (n = 156) of uninjected embryos (-Cre) were phenotypic; only 5% (n = 140) of Cre mRNA-injected siblings (+Cre) were phenotypic (p < 0.05). (E) For offspring of hmcn1 ^mn0263Gt heterozygote incrosses, 25% (n = 146) of uninjected embryos (-Cre) were phenotypic; only 8% (n = 233) of Cre-injected siblings (+Cre) were phenotypic (p < 0.005). (F) The fras1 ^mn0156Gt GBT allele fails to complement the pif ^tm95b ENU allele of fras1. Crossing pif ^tm95b heterozygotes with fras1 ^mn0156Gt heterozygotes does not reduce the proportion of phenotypic offspring (28%, n = 445) (tm95b/mn0156Gt trans-heterozygotes) compared to either pif ^tm95b/tm95b (tm95b/tm95b, n = 332) or fras1 ^{mn0156Gt/mn0156Gt} (mn0156Gt/mn0156Gt, n = 206) homozygotes. Percentages represent the mean of means (MOM); error bars represent standard deviations (SD).

Fig 5. nrg2a mutants display altered MFF morphology, consistent with the epidermal localization of the Nrg2-mRFP fusion protein and of endogenous nrg2a transcripts.

(A, B) By 48 hpf, nrg2a ^{mn0237Gt/mn0237Gt} mutants (mn0237Gt/mn0237Gt) show altered MFF morphology. (A) Wild-type MFF edges are thin, flat, and continuously curved (arrowhead). (B) mn0237Gt/mn0237Gt mutant MFFs have thickened edges (arrowhead), and one or more pointed protrusions (open arrowhead). (C, D) Collagen II (Col II) immunostaining of actinotrichia support fibers within the MFF shows aberrant collagen accumulation and ectopic actinotrichia within mn0237Gt/mn0237Gt mutant apical MFFs (arrowheads) at 48 hpf. (E, G) At 24 hpf, Nrg2a-mRFP fusion protein is present in MFFs of heterozygous (+/mn0237Gt) embryos (E; view on tail of whole mount) and, at slightly lower levels, throughout the entire epidermis (G; section through tail region; immunostained for RFP and counterstained with DAPI). (F, H, I) Whole-mount in situ hybridization (WISH) demonstrates strong MFF expression of the GBT-generated fusion transcript (mRFP; F) in a representative +/mn0237Gt embryo at 24 hpf. When developed with Boehringer Blocking Reagent, WISH staining for endogenous nrg2a transcripts in 24 hpf wild-type embryos also revealed strong MFF expression of the endogenous gene (H, Boeringer). When developed without Boehringer Blocking Reagent, WISH staining further reveals uniform expression of the endogenous nrg2a gene throughout the entire epidermis (I). For cross-sections, see Honjo et al. (2008), Fig 6C [51]). (J-N) Co-labeling of a transverse section through the dorsal MFF of a +/mn0237Gt embryo at 24 hpf reveals restricted localization of the Nrg2a-mRFP fusion protein (J) in ΔNp63-positive basal keratinocytes (L), whereas the outer enveloping layer, labeled with EGFP (K), lacks the Nrg2a-mRFP protein; (M) DAPI counterstain; (N) merged image of different channels shown in (J-M).

Fig 6. Usage of alternative first exons of nrg2a gene leads to differential cytosolic or nuclear localization of resulting protein isoforms.

(A) A schematic of the nrg2a locus on linkage group 21 (LG21), including alternative first exons 1A, 1B and 1C. The GBT insertion is located in the intron separating exon 1C and exon 2. (B) RT-PCR analyses of 48 hpf sibling embryos from heterozygote (+/mn0237Gt) intercross, revealing that endogenous nrg2a 1B and 1C transcripts are only expressed in heterozygous (+/mn0237Gt) and wild-type siblings (+/+). nrg2a 1B-mRFP and 1C-mRFP fusion transcripts are only expressed in homozygous mutants (mn0237Gt/mn0237Gt) and heterozygous siblings (+/mn0237Gt). (C-N) Confocal images of MFF epidermis at 24 hpf detecting Nrg2a-RFP fusion protein (C, G, K; red), cell membranes (D, H, L; labeled with EGFP (green) after injection of egfp-caax mRNA at 1-cell stage), and cell nuclei (E, I, M; labeled with BFP (blue) after injection of nls-BFP mRNA at 1-cell stage). Panels (F, J, N) show merged images. (C-F) +/mn0237Gt embryo displays Nrg2a^mn0237Gt-RFP fusion protein both in the cytoplasm (empty arrowheads) and in the nuclei (filled arrowheads). (G-J) Wild-type embryo injected with mRNA encoding exon 1B-version of the Nrg2a-RFP fusion protein; the fusion protein is largely absent from the nucleus, but present in cyptoplasmic compartments. (K-N) Wild-type embryo injected with mRNA encoding exon 1C-version of the Nrg2a-RFP fusion protein; the fusion protein is present in the cytoplasm and the nuclei, similar to the distribution of the transgene-encoded protein (C-F).

Fig 7. 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.

Fig 8. 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.

Fig 9. The nrg2a MFF phenotype can be mimicked and synergistically enhanced via chemical ErbB inhibition, and is characterized by reduced pAKT levels.

(A-J) Pharmacological inhibition of ErbB signaling during MFF morphogenesis (24 through 52 hpf) induces nrg2a ^{mn0237Gt/mn0237Gt}-like effects in wild-type and, with even higher frequencies, nrg2a ^+/mn0237Gt heterozygous embryos. (A) MFFs of wild-type embryos treated both with a low dose (1 μM) (28%; n = 120; p = 0.005) or a high dose (20 μM) (76%; n = 102; p < 0.0001) of PD168393 show an nrg2a ^{mn0237Gt/mn0237Gt}-like MFF morphology at 52 hpf. (B) nrg2a ^+/mn0237Gt heterozygous embryos (red) are significantly more sensitive to low-dose (1 μM) PD168393 treatment, and display nrg2a ^{mn0237Gt/mn0237Gt}-like MFF morphology with a higher frequency (64%; n = 152) than treated nrg2a ^+/+ wild-type siblings (blue) (n = 23%; n = 116; p < 0.0001) or treated unrelated wild-type embryos (grey) (28%; n = 120; p < 0.0001). (D-H) Live images of MFFs of representative examples of wild-type (C, E, G) or nrg2a ^+/mn0237Gt heterozygous (D, F, H) embryos at 52 hpf, after treatment with DMSO (control; C, D), 1 μM PD168393 (E, F) or 20 μM PD168393 (G, H). (I, J) TEM transverse sections of the apical MFF reveal a ridge cell phenotype in PD168393-treated wild-type embryos at 52 hpf. (I) An untreated wild-type embryo has correctly elongated ridge cells (red arrowhead) and a straight dermal space (ds). (J) A sibling embryo treated with 20 μM PD168393 displays basal bulging of MFF ridge cells towards the center the fin fold (red arrowheads) and a corresponding serpentine-like folding of the dermal space (ds), resembling the defects of the nrg2a mutant (compare with Fig 8D). (K, L) Whole-mount in situ hybridization (WISH) for egfra (K) and erbb3a (L) in wild-type embryos at 34 hpf (lateral views of tail) reveals epidermally expressed erbb3a transcripts (L), whereas egfra transcipts are absent in the epidermis, but present in the somites (K). (M-P) Anti-pAKT immunofluorescence of wild-type (M, N) and nrg2a mutant (O, P) embryo at 34 hpf; transverse sections through MER region of MFF, counterstained with DAPI (N, P). The wild-type embryo (M, N) displays pAKT localization in distal epidermal MFF cells (ridge cells and cleft cells; arrowheads), whereas pAKT levels in more proximal epidermal MFF cells are much lower. In addition, pAKT is localized at the tight junctions of the outer EVL (arrows), consistent with previously described roles of pAKT to phosphorylate tight junction proteins ZO-1 and Occludin, and to tighten the junctions [122]. In the nrg2a mutant (O, P), pAKT signals in ridge and cleft cells are strongly reduced (arrowheads), while pAKT signals at EVL tight junctions are unaltered (arrows).

Fig 10. 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).