Figure 1.
The skin phenotype of zebrafish hai1ahi2217 mutants heals spontaneously. (A–D) Bright field images of embryo tails at 48 (A–D) and 96 hpf (A′–D′). Both the hai1ahi2217 and the hai1afr26 alleles show epidermal aggregates at 48 hpf (B and C), but while the hai1ahi2217 phenotype has begun to heal spontaneously by 96 hpf (B′), the hai1afr26 allele continues to worsen (C′). A morpholino targeting mat1a rescues the hai1afr26 phenotype at 48 and 96 hpf (D and D′). See also Fig. S1. (E–G) Representative maximum intensity projection images of proliferation marker BrdU (red) in the caudal fin fold at 48 hpf (E–G) and 96 hpf (E′–G′). See also Fig. S1. (H) Quantification of cell proliferation in the caudal fin fold of embryos at 48 hpf and 96 hpf, in relation to the total number of DAPI-positive nuclei. n = 8–20 embryos per condition. (I) Q-RT-PCR of mmp9 at 48 hpf, indicating an increase in transcript levels in the hai1afr26 allele compared with the hai1ahi2217 allele. n = 40 pooled embryo tails. (J and K) Sanger sequencing chromatograms of WT (J) and hai1afr26 (K) genomic DNA, with the nonsense C > T mutation indicated (*). (L) Schematic of the zebrafish Hai1a protein and the putative truncated protein produced by the hai1afr26 allele. SP, signal peptide; M, MANEC (motif at N terminus with eight cysteines) domain; P, PKD (repeats in polycystic kidney disease 1); KD1, Kunitz domain 1; LDL, low density lipoprotein receptor class A; KD2, Kunitz domain 2; TM, transmembrane domain. Domains were predicted using National Center for Biotechnology Information’s Conserved Domain Database (Marchler-Bauer et al., 2017) and DAS-TM filter (Cserzö et al., 2002). (M) Q-RT-PCR of hai1a at 48 hpf, demonstrating likely nonsense-mediated decay of the transcript in both hai1a mutants. n = 40 pooled embryo tails. (N) Survival curves comparing siblings (black), hai1ahi2217 mutants (blue) and hai1afr26 mutants (red). n = 30 fish. Values represent means ± SD. Significances in (H) were determined via a one-way ANOVA and Tukey’s post hoc test. Different letters indicate statistically significant differences (P < 0.05). Significances in I and M were determined with a two-tailed Student’s t test, *, P < 0.05; **, P < 0.01; ***, P < 0.001. Scale bars, 200 µm in A–D and 50 µm in E–G.

The skin phenotype of zebrafish hai1ahi2217 mutants heals spontaneously. (A–D) Bright field images of embryo tails at 48 (A–D) and 96 hpf (A′–D′). Both the hai1ahi2217 and the hai1afr26 alleles show epidermal aggregates at 48 hpf (B and C), but while the hai1ahi2217 phenotype has begun to heal spontaneously by 96 hpf (B′), the hai1afr26 allele continues to worsen (C′). A morpholino targeting mat1a rescues the hai1afr26 phenotype at 48 and 96 hpf (D and D′). See also Fig. S1. (E–G) Representative maximum intensity projection images of proliferation marker BrdU (red) in the caudal fin fold at 48 hpf (E–G) and 96 hpf (E′–G′). See also Fig. S1. (H) Quantification of cell proliferation in the caudal fin fold of embryos at 48 hpf and 96 hpf, in relation to the total number of DAPI-positive nuclei. n = 8–20 embryos per condition. (I) Q-RT-PCR of mmp9 at 48 hpf, indicating an increase in transcript levels in the hai1afr26 allele compared with the hai1ahi2217 allele. n = 40 pooled embryo tails. (J and K) Sanger sequencing chromatograms of WT (J) and hai1afr26 (K) genomic DNA, with the nonsense C > T mutation indicated (*). (L) Schematic of the zebrafish Hai1a protein and the putative truncated protein produced by the hai1afr26 allele. SP, signal peptide; M, MANEC (motif at N terminus with eight cysteines) domain; P, PKD (repeats in polycystic kidney disease 1); KD1, Kunitz domain 1; LDL, low density lipoprotein receptor class A; KD2, Kunitz domain 2; TM, transmembrane domain. Domains were predicted using National Center for Biotechnology Information’s Conserved Domain Database (Marchler-Bauer et al., 2017) and DAS-TM filter (Cserzö et al., 2002). (M) Q-RT-PCR of hai1a at 48 hpf, demonstrating likely nonsense-mediated decay of the transcript in both hai1a mutants. n = 40 pooled embryo tails. (N) Survival curves comparing siblings (black), hai1ahi2217 mutants (blue) and hai1afr26 mutants (red). n = 30 fish. Values represent means ± SD. Significances in (H) were determined via a one-way ANOVA and Tukey’s post hoc test. Different letters indicate statistically significant differences (P < 0.05). Significances in I and M were determined with a two-tailed Student’s t test, *, P < 0.05; **, P < 0.01; ***, P < 0.001. Scale bars, 200 µm in A–D and 50 µm in E–G.

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