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Fig. 1. EphA7 is required for pronephros development. (A) Injection of the EphA7-ATG-MO reduced exogenous EphA7 level from stage11 embryos as revealed by western blot. α-tubulin, loading control. (B) Embryos injected bilaterally with the EphA7-ATG-MO developed pericardial edema (arrowhead) at stage 43. (C) Fluorescent dextran excretion assay showing the tubule defects in EphA7-ATG-MO injected embryos. The dashed regions are shown in enlarged views highlighting the reduction in tubular coiling on the injected side. (D–F) A transverse section of a stage 36 embryo showing a single enlarged pronephric tubule on the EphA7-ATG-MO injected side (the arrow in E) in contrast to the 5 normal pronephric tubules cross-sections on the control side (arrowheads in F). (G) Quantification of the effect of the EphA7-ATG-MO on pronephric tubule lumen. (H) Whole-mount in situ hybridization using probes against Lhx1, Pax2 and PDZK1 in stage 36 embryos injected with EphA7-mis-MO, EphA7-ATG-MO, EphA7-ATG-MO plus EphA7 mRNA (175 pg EphA7 rescue) or EphA7 mRNA alone. Control indicates the un-injected side. (I) Statistics of embryos showing deformed nephrostomes. (J) Statistics of embryos showing reduced expression level of PDZK1. |
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Fig. 3. A soluble form of EphA7 is involved in pronephros development. (A) Schematic representation of the structures of full length EphA7 and soluble EphA7 (sEphA7). LBD, ligand binding domain; FN3, fibronectin type III repeat; TM, transmembrane domain; TyrKc, tyrosine kinase domain; SAM, SAM domain. (B) Injection of the EphA7-sp-MO blocked the expression of the full length EphA7 (EphA7-FL). -RT, negative control without reverse transcriptase. H4, internal reference. (C) A transverse section of a stage 36 embryo showing closed pronephric tubules on the EphA7-sp-MO injected side (arrows on the left side) in contrast to the normal pronephric tubules on the control side (arrowheads on the right side). (D) Quantification of the effect of the EphA7-sp-MO on pronephric tubule lumen. (E, F) Whole-mount in situ hybridization using probes against Pax2 and PDZK1 in stage 36 embryos injected with EphA7-sp-MO or EphA7-sp-MO plus EphA7 mRNA (160 pg EphA7 rescue). The statistics are shown in the right panels. (G, G′) Immunostaining of CLDN6 (red) on a section of a pronephric tubule of a stage 35 embryo injected with EphA7-sp-MO. The targeted areas labeled by tracing GFP (green) are outlined. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.) |
