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???displayArticle.abstract??? Cilia are essential for embryogenesis and maintenance of homeostasis, but little is known about the signalling pathways that regulate ciliogenesis. Here, we identify ERK7, an atypical mitogen-activated protein kinase, as a key regulator of ciliogenesis. ERK7 is strongly expressed in ciliated tissues of Xenopus embryos. ERK7 knockdown markedly diminishes both the number and the length of cilia in multiciliated cells, and it inhibits the apical migration of basal bodies. Moreover, ERK7 knockdown results in a loss of the apical actin meshwork, which is required for the proper migration of basal bodies. We find that the actin regulator CapZIP, which has been shown to regulate ciliogenesis in a phosphorylation-dependent manner, is an ERK7 substrate, and that Dishevelled, which has also been shown to regulate ciliogenesis, facilitates ERK7 phosphorylation of CapZIP through binding to both ERK7 and CapZIP. Collectively, these results identify an ERK7/Dishevelled/CapZIP axis that regulates ciliogenesis.
Figure1. Expression patterns of ​ERK7 and knockdown phenotypes. (a–d) Whole-mount in situ hybridization analysis of ​ERK7 expression. Magnified images show regions outlined with white dashed lines. (a) The embryos at stage 12 (vegetal view), stage 16 (dorsal view), stage 25 and stage 33/34. Black arrows, cloaca; white arrowheads, ear vesicle; black arrowheads, nephrostomes. (b) The dorsal explant (left panel) and sagittal section (right panel) of the stage 16 embryo. The GRP is outlined by the black dotted line or indicated by the open arrowhead. a, anterior; l, left; p, posterior; r, right. (c) An embryo at stage 13 (lateral view). (d) Whole-mount in situ hybridization of ​ERK7 followed by immunostaining with anti-acetylated ​α-tubulin (Ac.-​tub., green). (e,f) The efficiency of ​ERK7 MO. (e) The indicated sets of MO (10 ng per cell) and mRNA (500 pg per cell) were injected into the two blastomeres at the two-cell stage, and the embryos were harvested at stage 10.5. EGFP mRNA (100 pg per cell) was used as an injection control. (f) Each MO (10 ng per cell) was injected into the animal region of four blastomeres at the four-cell stage. Injected embryos were harvested at stage 33/34. Endogenous ​ERK7 protein was concentrated by immunoprecipitation from embryo lysates. (g,h) Knockdown experiments of ​ERK7. Control MO (20 ng per cell) or ​ERK7 MO (10 or 20 ng per cell) was injected into the dorsal (g) or ventral (h) marginal region of two blastomeres at the four-cell stage. The injected embryos were fixed and observed at stage 33/34. The phenotypes were classified into three groups (severe, mild or normal) according to the extent of the defects in head morphology (g) or body length (h). Scale bars, 300 μm.
mapk15 ( mitogen-activated protein kinase 15) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 12, blastoporal view, dorsal up.
mapk15 ( mitogen-activated protein kinase 15) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 16, dorsal view, anteriorleft.
mapk15 ( mitogen-activated protein kinase 15) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 16, lateral view, anteriorleft,dorsal up ( image on left), and magnification of otic vesicle image on right).
mak15 (mitogen-activated protein kinase 15) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 25, lateral view, anteriorright, dorsal up.
Figure 2: ​ERK7 is required for ciliogenesis. (a) The trajectories of fluorescent beads are shown in a colour gradient (see colour bar). Each MO (10 ng per cell) was injected into the animal region of four blastomeres at the four-cell stage. White lines indicate an outline of the embryo. See Supplementary Movies 3 and 4. Scale bars, 300 μm. (b) The flow velocity of the beads was measured using ImageJ. Error bars represent the s.d. of 90 beads from nine embryo movies for each condition. ***P<0.001 by the t-test. (c–e) Immunostaining with anti-acetylated-​α-tubulin (Ac.-​tub) to visualize cilia (green). As a tracer, membrane-targeted (mem)-mCherry (red) was used. (c,d) Each MO (10 ng per cell) was injected into the animal region of four blastomeres at the four-cell stage. The injected embryos were fixed at stage 35/36. (c) Whole embryos. Scale bars, 300 μm. (d) Multicilia on MCCs. Scale bars, 5 μm. (e) Cilia on the GRP. Each MO (15 ng/cell) was injected into the dorsal marginal region of two blastomeres at the four-cell stage. The right graph shows the average length of cilia on the GRP (control MO, n=62 from five dorsal explants; ​ERK7 MO, n=97 from five dorsal explants). Error bars represent the s.d. Scale bars, 5 μm. ***P<0.001 by the t-test.
Figure 4: Subcellular localization of ​ERK7 in MCCs. a) Subcellular localization of ​ERK7-EGFP in the epidermis. ​ERK7-EGFP mRNA (25 pg per cell) was injected into the animal region of four blastomeres at the four-cell stage, and the injected embryos were observed at stage 33/34. (b) Subcellular localization of ​ERK7-EGFP (green) and ​Centrin2-mCherry (red) in the MCCs. ​ERK7-EGFP mRNA (25 pg per cell) and ​Centrin2-mCherry mRNA (50 pg per cell) were injected into the ventral marginal region of two blastomeres at the four-cell stage, and the injected embryos were observed at stage 35/36. (c) Subcellular localization of ​ERK7-EGFP (green) and ​CLAMP-mCherry (red) in the MCCs. ​ERK7-EGFP mRNA (25 pg per cell) and ​CLAMP-mCherry mRNA (100 pg per cell) were injected into the ventral two blastomeres at the four-cell stage, and the injected embryos were fixed at stage 33/34 and observed. Data are representative of two independent experiments. Scale bars, 5 μm.
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