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

He L, Xu W, Jing Y, Wu M, Song S, Cao Y, Mei C - Int. J. Biol. Sci. (2015)

Bottom Line: 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.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.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.

View Article: PubMed Central - PubMed

Affiliation: 1. Kidney Institute of CPLA, Division of Nephrology, Changzheng Hospital, Second Military Medical University, NO.415 Fengyang Road, Shanghai 200003, China.

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.

No MeSH data available.


Related in: MedlinePlus

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.
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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.

Mentions: To further investigate the ciliary defects of yap morphants, we performed ISHs for foxj1a and shippo1 (odf3b) (Fig.6A). foxj1a is a cilia master regulator and one target of Hedgehog signaling, promoting ciliary differentiation and function, and its activity is sufficient for ectopic cilia development40. If the ciliary defects in yap morphants are foxj1a dependent, we would expect decreased level of foxj1a. Actually, we found up-regulation of foxj1a in both 24 h.p.f and 3 d.p.f yap morphants (90% and 70.6% respectively; Fig.6A). We additionally over-expressed foxj1a mRNA in wild-type and yap morphant embryos to observe its influence in pronephro ciliogenesis, However, little change had been seen (Supplementary Fig.S4). The results suggest cilia defects in yap morphants are foxj1a independent, and enhance of foxj1a may be a feedback regulation induced by epithelial stretch and cyst formation41. shippo1 is a marker of MCCs42.The expression pattern of shippo1 in 24 h.p.f morphants appeared relatively unaffected (86.7%). But in 3 d.p.f yap morphants, shippo1 labeled MCCs were in a disorganized array and gathered together in the middle region (57.9%), while in control, MCCs placed one by one along the pronephric duct (Fig.6A). Accumulated MCCs could be attributed to defects in lateral inhibition during differentiation of bipotential precursor cells42 or blocked collective cell migration. Unchanged shippo1+ and trpm7+ cells at 24 h.p.f excluded the possibility of biased differentiation towards a MCC fate (Supplementary Fig.S3). Obviously delayed cell migration of yap morphants at 3 d.p.f could explain gathered MCCs in the medial segment (Fig.3J).


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

He L, Xu W, Jing Y, Wu M, Song S, Cao Y, Mei C - Int. J. Biol. Sci. (2015)

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.
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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.
Mentions: To further investigate the ciliary defects of yap morphants, we performed ISHs for foxj1a and shippo1 (odf3b) (Fig.6A). foxj1a is a cilia master regulator and one target of Hedgehog signaling, promoting ciliary differentiation and function, and its activity is sufficient for ectopic cilia development40. If the ciliary defects in yap morphants are foxj1a dependent, we would expect decreased level of foxj1a. Actually, we found up-regulation of foxj1a in both 24 h.p.f and 3 d.p.f yap morphants (90% and 70.6% respectively; Fig.6A). We additionally over-expressed foxj1a mRNA in wild-type and yap morphant embryos to observe its influence in pronephro ciliogenesis, However, little change had been seen (Supplementary Fig.S4). The results suggest cilia defects in yap morphants are foxj1a independent, and enhance of foxj1a may be a feedback regulation induced by epithelial stretch and cyst formation41. shippo1 is a marker of MCCs42.The expression pattern of shippo1 in 24 h.p.f morphants appeared relatively unaffected (86.7%). But in 3 d.p.f yap morphants, shippo1 labeled MCCs were in a disorganized array and gathered together in the middle region (57.9%), while in control, MCCs placed one by one along the pronephric duct (Fig.6A). Accumulated MCCs could be attributed to defects in lateral inhibition during differentiation of bipotential precursor cells42 or blocked collective cell migration. Unchanged shippo1+ and trpm7+ cells at 24 h.p.f excluded the possibility of biased differentiation towards a MCC fate (Supplementary Fig.S3). Obviously delayed cell migration of yap morphants at 3 d.p.f could explain gathered MCCs in the medial segment (Fig.3J).

Bottom Line: 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.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.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.

View Article: PubMed Central - PubMed

Affiliation: 1. Kidney Institute of CPLA, Division of Nephrology, Changzheng Hospital, Second Military Medical University, NO.415 Fengyang Road, Shanghai 200003, China.

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.

No MeSH data available.


Related in: MedlinePlus