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Receptor tyrosine kinase EphA7 is specifically expressed in otic region in Xenopus early development. However, its role in otocyst development remains unknown. Knockdown of EphA7 by a specific morpholino oligonucleotide (MO) reduced the size of the otocyst and triggered otic epithelial cell extrusion. Interestingly, EphA7 depletion attenuated the membrane level of the tight junction protein Claudin6 (CLDN6). Utilizing the Cldn6 MO, we further confirmed that CLDN6 attenuation also led to otic epithelial cell extrusion. Our work suggested that EphA7 modulates the otic epithelial homeostasis through stabilizing the CLDN6 membrane level.
Fig. 1. EphA7 knockdown reduces the size of the otocyst. (A) Top, the diagram indicates the MO injection site targeting the presumptive otocyst area in 4-cell stage embryos, with Gfp mRNA as a tracer. Bottom, the targeted otocyst was shown. The dashed lines indicate the outline of the head and the otocyst (ot). (B) Embryos injected with the EphA7 MO developed small otocysts at stage 35, as indicated by in situ hybridization (ISH) for Papc probe. (C) Quantification of the effects of the EphA7 MO on otocyst size. (D) Representative images of immunofluorescence for β-catenin on sections of otocysts of stage 35 embryos injected with EphA7 MO. The box region is shown in enlarged view in D’. Arrowheads indicate that the cells appeared in the otocyst lumen. Control (Con) indicates the un-injected side.
Fig. 2. Cell extrusion of otic epithelium following EphA7 knockdown. (A) Biotin labeling assay showed apical extrusion in the otocyst with EphA7 MO. Sd MO as a control. The box region is shown in enlarged view in A’. The apical sides of otocysts are outlined by white broken circles. The extruded cells are encircled by red dashed lines. MO was injected into one side of the embryos along with Gfp mRNA as a tracer. Scale bar, 10 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Fig. 3. CLDN6 is reduced in the otic epithelium in the EphA7 morphants. (A) Expression of CLDN6 protein at otic epithelium and otocystepithelium in embryos at indicated stages revealed by immunostaining. (B) Representative images of immunofluorescence for CLDN6 on otic sections of the embryos. EphA7 MO was injected into one side of the embryos along with Gfp mRNA as a tracer. The white and yellow dashed lines encircle the control and morphant otic epithelia, respectively. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Fig. 4. CLDN6 attenuation leads to the otocyst epithelial cell extrusion. (A) Injection of the Cldn6 MO reduced the size of the otocyst, as indicated by ISH for Cldn6 probe at stage 33. The control (Con) and morphant otocysts are shown in enlarged views. (B) Representative images of immunofluorescence for β-catenin on sections of otocysts of stage 35 embryos injected with Cldn6 MO. (C) Quantification of the effects of the Cldn6 MO on otocyst size. (D) Biotin labeling assay showing apical extrusion in the otocyst with Cldn6 MO. Sd MO as a control. The box region is shown in enlarged view in D’. The apical sides of otocysts are outlined by white broken circles. The dying cells undergoing extrusion are encircled by yellow dashed lines. Scale bar, 10 μm. (E) Quantification of the numbers of extrude cells inside the otocyst lumen injected with Sd MO, EphA7 MO, and Cldn6 MO. ∗∗P < 0.01; ns, not significant. The targeted areas are labeled green by tracing GFP. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Fig. S1. Cell proliferation and apoptosis of otocystepithelium after EphA7 knockdown. (A) Immunostaining for pH3 on sections of otocysts of stage 35 embryos injected with EphA7 MO. (B) Immunostaining for cleaved Caspase-3 (Casp-3) on sections of otocysts of stage 35 embryos injected with EphA7 MO. (C) apoptosis signals appeared in the EphA7 MO injected otocyst lumens at stage 33. The otocyst is outlined by white broken circles. The apoptotic cell and non-apoptotic cell are encircled by red dashed line and yellow dashed line, respectively. The targeted areas are labeled green by tracing GFP.
Fig. S2. EphA7 knockdown does not affect cell adhesion junction in otocystepithelium. Representative images of immunofluorescence for β-catenin on sections of otocysts of stage 35 embryos injected with EphA7 MO. The targeted areas are labeled green by tracing GFP.
