Fig. 5.

Csde1 is involved in the translational regulation of β-catenin. (A) qPCR analysis of ctnnb1 expression in ECs in control, csde1 morphants and csde1 mutants at 36 hpf. (B) The protein level of β-catenin in control, csde1 morphants and embryos co-injected with csde1 atgMO and hCSDE1 mRNA at 28 hpf. (C) Immunofluorescence showing that nonphosphorylated β-catenin expression in ECs was reduced in csde1 morphants. The white and blue dotted lines mark dorsal aorta and cardinal vein, respectively. The arrowhead denotes nuclear staining in the ECs located in the ventral wall of dorsal aorta. (D) Illustration of the EGFP-ctnnb1 reporter and experimental procedure for reporter assay. (E,F) Representative images (E) and quantification (F) of the relative expression levels of EGFP and EGFP-ctnnb1 with or without csde1 deficiency at the 75% epiboly stage of development. tdTomato mRNA was co-injected into embryos with egfp mRNA or egfp-ctnnb1 mRNA as injection control. (G) qPCR analysis showing the expression of egfp in control embryos and csde1 morphants at the 75% epiboly stage. (H) In vivo transcribed ctnnb1 mRNA pull-down assays followed by immunoblot analysis of anti-Csde1, anti-HA and anti-β-actin in control- and csde1 MO-injected embryos. (I) TOPFlash luciferase reporter assays in ctnnb1 mRNA-injected embryos with or without csde1 atgMO injection. Data are mean±s.d. *P<0.05, ***P<0.001, ****P<0.0001 (two-tailed unpaired Student's t-test). ns, not significant. n≥3 replicates. Numbers indicate the number of embryos with respective phenotype/total number of embryos analyzed in each experiment (E). Scale bars: 15 μm (C); 150 μm (E).