Fig 1

Hypotonic stress and Matriptase-1a function are required for the epidermal phenotype of hai1a and atp1b1a, but not clint1 and epcam mutants.

(A-H) Isotonic medium attenuates the phenotype of hai1a-/- mutants and rescues the phenotype of atp1b1a-/- mutants, but has no effect of clint1-/- or epcam-/- mutants. (A) Quantification of epidermal phenotypes of 48 hpf embryos obtained from parents heterozygous for the mutations clint1^hi1520 [22], epcam^hi2151 [42], atp1b1a^m14 [26], or hai1a^hi2217 [21], raised in E3, E3 + 250 mM Mannitol, or injected with control morpholino or st14a morpholino (n = 59–102 from N = 3 independent clutches per condition, Significances were determined via a Chi-square test, ns, not significantly different (p>0.05); ****, significantly different (p< 0.0001)). (B-E’) Brightfield images of live 48 hpf hai1a morphants raised in E3 (B,B’,C,C’) or E3 + 250 mM Mannitol (D,D’,E,E’) as lateral overviews of entire embroys (A-D) or magnified lateral views of the yolk tube and yolk extension regions of the same embryos (A’-D’); arrows in (B’,C’) point to epidermal aggregates on yolk sac and ventral median fin fold. (F) Quantification of phenotypic classes (n = 27–67) of hai1a morphant embryos raised in E3 or E3 + 250 mM Mannitol, representatives of which are shown in panels (B-E’) Significances were determined via a Chi-square test, ns, not significantly different (p>0.05); ****, significantly different (p< 0.0001). (G) RT-qPCR showing relative quantities of mmp9 transcript of 48 hpf hai1a morphant or 56 hpf atp1b1a mutant embryos raised in E3 or E3 + 250 mM Mannitol, compared to their respective siblings. cDNA was obtained from pools of 15 embryos each, N = 3 for hai1a, N = 3 for atp1b1a. H. Quantification of BrdU-labeled nuclei in defined, equally-sized areas of the fin fold of wild types and hai1a-/- mutants at 48 hpf, raised in E3 or E3 + 250 mM Mannitol, n = 4–6. Significances in G and H were determined via a one-way ANOVA and Tukey’s post hoc test; ns, not significantly different (p>0.05); *,**,***,****, significantly different (p<0.05, 0.01, 0.001, 0.0001, respectively). (A,I–N’) Loss of st14a function rescues epidermal aggregate formation in atp1b1a-/- mutants. Brightfield images of representative live 72 hpf embryos, either as lateral overviews of the entire embryos (I-N), or as magnified lateral views of the tail of the same embryos (I’-N’): wild-type sibling (I,I’), wild-type sibling injected with st14a MO (J,J’), st14a-/- mutant (K,K’), atp1b1a -/- mutant with epidermal aggregates (L,L’), atp1b1a -/- mutant injected with st14a MO (M,M’), and atp1b1a-/-; st14a-/- double mutant (N,N’), both with wild-type-appearing epidermis. For quantifications, see (A) and S1I Fig. Scale bars: 500 μm (B,I), 100 μm (B’,I’).