Yes-associated protein (Yap) is necessary for ciliogenesis and morphogenesis during pronephros development in zebrafish (Danio Rerio)

Abstract

The Hippo signaling pathway and its transcriptional co-activator Yap are known as essential regulators for cell proliferation and organ size. However, little is known about their roles in kidney development and ciliogenesis. We examined expression of Yap during zebrafish embryogenesis, and its transcripts were detected in pronephric duct, while Yap protein was found to be localized in the cytoplasm and apical membrane in kidney epithelium cells. By morpholino (MO) knockdown of yap expression in zebrafish, the injected larve exhibits pronephic cysts and many aspects of ciliopathy, which can be rescued by full-length yap mRNA, but not yap (S127A) mRNA. With transgenic Tg(Na(+)/K(+) ATPase:EGFP), we found that lacking Yap led to expansion and discontinuities of pronephric duct, as well as disorganization of cloaca during pronephros morphogenesis. Mis-located Na(+)/K(+) ATPase and ciliary abnormalities are also detected in pronephric duct of yap morphants. In addition, genetic analysis suggests that yap interacts with ift20, ift88 and arl13b in pronephric cyst formation. Taken together, our data reveals that Yap is required for pronephric duct integrity, maintenance of baso-lateral cell polarity, and ciliogenesis during zebrafish kidney development.

Keywords: Cilia; Cyst; Kidney; Pronephros; Yap; Zebrafish.

Figure 1

yap expression and protein sublocalization during zebrafish nephrogenesis. (A) By whole-mount in situ hybridization, yap is detcted in the LPM (arrows) and IM (asterisk) as early as the 6-somite stage. (B-E) At the 19-somite stage, 24 h.p.f, and 3 d.p.f, yap persists in the pronephric ducts, which is confirmed by dorsal view(C; inset), higher magnification view (D') and cross section view (E; dashed red circles). (F) Expression of Yap protein in the trunk of a 2 d.p.f embryo viewed in whole-mount is modest in the pronephric duct (arrowheads). (G-H) A close view of the anterior (G) and posterior (H) segment shows that Yap immunofluorescence is distributed throughout the cytoplasm with a concentration near the apical surface (arrows). (I) Western blot of 1 d.p.f embryos with Yap antibody shows the endogenous Yap at approximately 70 KD, and exogenetic Yap-EGFP at 100 KD, while expression of Yap protein in 1pmol yap MO embryos almost disappear. (J-J') With Tol2 kit, exogenetic EGFP-Yap distributes in the cytoplasm and has co-localization with the apical marker aPKC (arrows). Bar: 10 μm.

Figure 2

yap knockdown by antisense MO results in abnormal phenotypes which can be rescued by full-length and ΔTAD yap mRNA. (A-E) Phenotypes of control embryo and yap morphant at 3 d.p.f. (B) yap morphant exhibit glomerular cyst (inset), heart edema (asterisk), hydrocephalus (arrowhead), smaller eyes (arrow), a short and slightly curved tail. In addition, at the distal end of pronephric duct, the control embryo form obvious outlet at cloaca (C; arrow) while in the morphant, the outlet can't be seen (D; arrow) or is obstructed, promoting dilation or cysts formation (E; arrow). (F-F') Immunostaining shows that exogenetic dominant active form EGFP-S127AYap is translocated into nucleus with the apical staining left (F'; arrow). Bar: 10 μm. (G) Western blot of 2 d.p.f embryos illustrates the protein level of kidney-specifically overexpressed EGFP-Yap and EGFP-S127AYap. (H) Modular structures of zYap1, S127A Yap and ΔTAD Yap. In zYap, the conserved S127 in the TEAD binding domain (aa7-103) is actually located in the aa87. ΔTAD mRNA encodes Yap without the transcription activation domain (aa238-442). aa: amino acids. (I) The pronephric cyst of yap MO embryos can be rescued by co-injecting full-length yap mRNA and ΔTAD yap mRNA (P<0.01), however, co-injecting with yap ^S127A mutant mRNA develops cyst at an average of 38.9%, not significantly different from cyst formation of yap morphants with an average of 44.3%.

Figure 3

Abberant morphology of pronephric duct develops during collective cell migration. (A-I) Morphology of pronephric duct labeled by Tg(Na⁺/K⁺ ATPase:EGFP) from 1 d.p.f to 3 d.p.f. Disorganization of cloaca occurs in about 15% embryos from 1 d.p.f (B; arrow). Discontinuous pronephric duct is found from 2 d.p.f in 6.7% embryos, and the ratio of occurrence increases sharply to 26% at 3 d.p.f (D and F; arrowheads), cldh17 probe shows the similar phenotype (H; arrow and arrowhead). (J) ISH of segment-restricted probe at 3 d.p.f. Pronephric cysts are displayed by wt1b. slc4a4a⁺ cells fail to migrate towards the glomeruli-neck region compared with the control embryos. Discontinuous segments are found in slc4a4a⁺, trpm-7⁺, slc12a1⁺ cells (arrowheads). Decreased or disappeared expression of evx1 is seen. The numbers on the lower left of each figure represent the counted phenotypic and total embryos. All photos are dorsal views except for evx1, which are lateral views.

Figure 4

The Na⁺/K⁺ ATPase is mis-located to the apical surface of pronephric duct in yap morphants. (A-B) Whole-mount staining shows that the inner diameter of the enlarged duct indicated by aPKC is about fourfold of the control, however, the change of the outer diameter indicated with α-6F is not that dramatic. (C-D') The cross section view demonstrates increased cell number surrounding the dilated duct compared with the control duct (white dashed circles represent the cross sections of the ducts), the baso-lateral staining of Na⁺/K⁺ ATPase is not obvious in a part of enlarged tubules (Fig.4D'; red arrow), and even is mis-targeted to the apical surface adjacent to aPKC (D'; arrowhead). (C' and D') Arrows indicate basal surface and asterisks indicate lateral surface. (A-B) Bar: 10 μm. (C-D) Bar: 5 μm.

Figure 5

yap morphants have ciliary defects. (A) In the eye, the outer segment of photoreceptor cell, which is a modified cilium, is hard to detect in the morphants at 3 d.p.f (the left arrow indicates the control, the right arrow shows defect in yap morphant). Bar: 25 μm (B) Compared with the injection control, cilia defects are observed in medial and posterior segment of the pronephric duct in the morphant. Bar: 10 μm. (C) Statistical analysis of the single cilia number per high power field and single cilia length in control and morphant embryos. The cilia are shown by staining with antibody against acetylated α-tublin. α-tub, anti-acetylated α-tublin; GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; MD, medial segment of pronephric duct; PD, posterior segment f pronephric duct; MC, multicilia; SC, single cilia; HPF, high power field.

Figure 6

Yap is required for basal body arrangement and apical docking in pronephric duct. (A) The expression level of foxj1a (cilia master gene) is increased in 24 h.p.f and 3 d.p.f yap morphant. shippo1 (a marker of MCCs) expression is unaffected in 24 h.p.f yap morphant, but is accumulated in the medial pronephric duct in 3 d.p.f morphant. 24 h.p.f embryos are lateral views and 3 d.p.f embryos are dorsal views. (B-C') With anti-γ-tublin staining, basal bodies of MCCs in 3 d.p.f embryos are showed. In control, basal bodies are in a thread-like array at the apical surface (B'; arrowheads), but in morphant, basal bodies are gathered into clusters (C'; arrowheads) and even out of the edge of apical membrane (C; asterisk). Bar: 10 μm. (D-G) Cross sections staining show that basal bodies are unable to migrate to the apical surface in enlarged pronephric duct of both proximal and distal segments (E and G; arrows). Bar: 5 μm. γ-tub, anti-γ-tublin.

Figure 7

yap genetically interacts with ift20, ift88 and arl13b. (A-D) Phenotype of embryos injected with suboptimal yap MO (0.5 pmol) or/and ift20s (0.5 pmol), ift88 (0.05 pmol), arl13b MO (0.2 pmol) at 3 d.p.f. The single MO injection control embryos are almost normal (A) while co-injection embryos exhibit dramatically increased percentage of pronephric cysts, heart edema, tail curvature and smaller eyes (B-D). (E-G) Graphs represent the percentage of embryos that develop pronephric cysts. The single MO injection control is treated with suboptimal single MO and control MO to exclude the effect of mechanical injury to embryos. Each experiment is repeated three times with at least 50 embryos.*: P<0.05;**: P<0.01;***: P<0.001.