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Fig. 4 Loss of kctd15a/b sways neighboring pronephric fates to express DE signature. (A) Whole-mount in situ hybridization for trpm7 (purple) and slc12a3 (red) in wild-type and kctd15a/b MO pronephric cells at 24 hpf. Blue arrowheads flank the inferred DE footprint; black arrowheads indicate anterior and posterior limits of the PST and DL. Scale bar: 35 μm. (B-D) Absolute length quantifications of the trpm7 expression domain, inferred DE footprint and slc12a3 expression domain. (E) Fluorescent in situ hybridization of kcnj1a.1 (green) and slc12a3 (red) at 24 hpf in pronephric cells in wild type and kctd15a/b MO. Magenta and cyan arrowheads indicate DE and DL boundaries, respectively. Gray box indicates the area shown at higher magnification below. White outlines indicate individual cells dually expressing kcnj1a.1 and slc12a3. Scale bars: 70 μm (top); 5 μm (bottom). (F) Wild-type fluorescent intensity plot of kcnj1a.1 (green) and slc12a3 (red). (G) kctd15a/b MO fluorescent intensity plot of kcnj1a.1 (green) and slc12a3 (red). White arrows indicate ectopic kcnj1a.1 signal invading the slc12a3 domain. (H) Fluorescent in situ hybridization of trpm7 (red) and kcnj1a.1 (green) at 24 hpf in wild-type and kctd15a/b MO pronephric tubules. Cyan and magenta arrowheads indicate PST and DE boundaries, respectively. Gray box indicates area shown at higher magnification below. White outlines indicate individual cells dually expressing kcnj1a.1 and trpm7. Scale bars: 70 μm (top); 5 μm (bottom). (I) Wild-type fluorescent intensity plot of trpm7 (red) and kcnj1a.1 (green). (J) kctd15a/b MO fluorescent intensity plot of trpm7 (red) and kcnj1a.1 (green). White arrows indicate ectopic kcnj1a.1 signal invading the trpm7 domain. n≥3 embryos quantified for each control and experimental group. ***P<0.001. Data are mean±s.d. Absolute lengths were compared using unpaired t-tests.