Zebrafish yap1 plays a role in differentiation of hair cells in posterior lateral line

Abstract

The evolutionarily conserved Hippo signaling pathway controls organ size by regulating cell proliferation and apoptosis and this process involves Yap1. The zebrafish Yap1 acts during neural differentiation, but its function is not fully understood. The detailed analysis of yap1 expression in proliferative regions, revealed it in the otic placode that gives rise to the lateral line system affected by the morpholino-mediated knockdown of Yap1. The comparative microarray analysis of transcriptome of Yap1-deficient embryos demonstrated changes in expression of many genes, including the Wnt signaling pathway and, in particular, prox1a known for its role in development of mechanoreceptors in the lateral line. The knockdown of Yap1 causes a deficiency of differentiation of mechanoreceptors, and this defect can be rescued by prox1a mRNA. Our studies revealed a role of Yap1 in regulation of Wnt signaling pathway and its target Prox1a during differentiation of mechanosensory cells.

Figure 1. Zebrafish yap1 is highly expressed in regions with active cell proliferation and in particular sensory progenitor cells of the inner ear.

(A–C) yap1 is expressed in the brain, somites and tail bud until mid-somitogenesis and its expression in the posterior body declines later on. (D–F) yap1 expression become more restricted to the ventricular zones (proliferative region, arrows) in the brain as the embryo matures. (G–I) Staining of yap1 in pharyngeal arch (arrowhead) at 48–96 hpf. (J), (K) Most proliferative cells detected by anti-pH3 antibody (brown, cell nucleus) are yap1-positive (blue) in cross-section of 24 hpf and (I) sagittal-section of 96 hpf embryos. L-N – cross-sections at the mid-hindbrain (ear) level of 72 hpf SqET33-mi60A transgenic larvae (lnfg, progenitors of sensory cells). (L) yap1 in situ hybridization; (M) GFP expression; (N) composite yap1 in situ hybridization/GFP expression. (A–F) – lateral view, (G), (H) – ventral view. Scale bar = 40 μm. Abbreviations: dc, diencephalon; epi, epiphysis; hyp, hypothalamus; op, otic placode; pa, pharyngeal arches; poa, preoptic area; pt, prethalamus; tb, tail bud; tc, telencephalon; tg, tegmentum.

Figure 2. The number of neuromasts is reduced in yap1 SqET4 morphants.

(A), (C), (F) – controls; (B), (D), (G) – Yap1 morphants. All embryos are shown in lateral view. The table illustrates the rescue effect of yap1 mRNA coinjection. The original data are provided as a supplement. (A), (B) – head and anterior trunk, (C), (D) – intermediate trunk, (F), (G) – tail. Notice a reduction in a number of neuromast cells and increase of background due to longer exposure in (G) comparing to (F). (H), (I) – zath1a expression in the lateral line primordium of controls (H) and morphants (I). Scale bar = 40 μm, (H)&(J) = 20 μm.

Figure 3. prox1a transcript level is yap1-dose-dependent.

Taking un-injected control as 1 fold change, reduced prox1a expression level in yap1 morphants could be rescued with yap1 mRNA. Higher doses of yap1 mRNA led to elevated prox1a expression. Mismatch control embryos showed no significant deviation from un-injected controls. Quantitative PCR results from yap1 morphants, rescued morphants and yap1 gain-of-function were calculated with delta CT method for fold change. The significant difference of the fold change, as compared to control with assumed fold change of one, was analyzed with two-tailed t-test.

Figure 4. The primordium, neuromasts and mechanoreceptors of the lateral line in yap1 morphants.

(A–D) Primordium I (PrimI) in yap1 morphants. (E) Size of PrimI at 36 hpf. (E, F) Mantle cells of neuromast in 72 hpf ET20 yap1 morphants. (G–H) Mechanoreceptors of neuromast (red, DiAsp) in 72 hpf ET33-mi60a embryos, support cells (green). (I–L) hair cells. (J–L) Loss of yap1 abolished the functionality of hair cells in SqET4 transgenics, stained with DiAsp (red); it was rescued with yap1 mRNA injection. (M–O) Mismatch MO, yap1 MO or yap1 MO with prox1 mRNA were co-injected with a marker (Cascade Blue) into single cell of SqET4 embryos at 16-cell stage. (P) The schematic drawing of injected single cell (blue), any one of the middle four cells, at 16-cell stage. Scale bar = 10 μm.