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Figure 6. p62/SQSTM1 is necessary for VANGL2 pathway-mediated in vivo morphogenesis.(a) Two-cell embryos were injected in each blastomere with 12.5 (n=15), 25 (n=13) and 50 (n=104) ng of p62 MO. Morphology was analysed at tailbud stage. (b) Embryos injected with 50 ng of p62 MO were processed for WISH analysis at mid-neurula and late gastrula stages. Convergence extension in the neural tube of late neurula embryos was evaluated by the average length/width ratio of the Sox2 domain (n=17). Convergence extension in the axial mesoderm of late gastrula embryos was evaluated by the average length of the Xbra domain (n=16). Tailbud embryos were stained with the Sox2 probe to highlight neural tube defect (bottom panel, n=30 control, n=104 morphants). (c) Two-cell embryos were injected in each blastomere with 11.5 ng of VANGL2 MO (n=27), 8 ng of p62 MO (n=26) or 11.5 ng of VANGL2+8 ng of p62 MO (n=32). Morphology was analysed at the tailbud stage using criteria of a. These embryos were processed for analysis of Sox2 expression at the tailbud stage (n=3). (d) Ten embryos injected as in b or with 34.5 ng of VANGL2 MO in each blastomere were collected at stage 13 and processed for RT–qPCR. (e) Four-cell embryos injected with Wnt5a mRNA (30 pg per cell) in the animal pole received a second injection of VANGL2 (11.5 ng per cell), or p62 (12.5 ng per cell) in all animal blastomeres at eight-cell stage. Fifteen animal caps per condition were isolated at the blastula stage, cultured for 4 h (at 23 °C) and then processed for RT–qPCR. (f) Two-cell embryos were injected in each blastomere with 4.5 ng of control peptide (n=27) or p62DN peptide (n=26). Morphology was analysed as in a and processed for analysis of Sox2 expression at the tailbud stage. (g) Ten embryos injected as in f were collected at stage 13 and processed for RT–qPCR. For qPCR graphs, error bars represent s.e.m. values of three or more independent experiments with two technical duplicates. Statistical analyses used unpaired Student's t-test, except in e where one-way ANOVA with Dunnett's test (99.9% confidence intervals) were applied. *P≤0.05; **P≤0.005; ***P≤0.0005; Ctrl, control; MO, morpholino; mRNA, messenger RNA; RT–qPCR, reverse transcriptase–quantitative PCR; WISH, whole-mount in situ hybridization. |
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Supplementary Fig. 6 related to Fig. 6: Xenopus p62/SQSTM1 loss of function is rescued by human p62/SQSTM1. (j) Two-cell embryos were injected in each blastomere with 50 ng of p62 MO followed by injection at four-cell stage of synthetic human p62 mRNA (2.5 ng in each blastomere). Morphology was scored at tailbud stage, and embryos were then stained with Sox2 probe to highlight neural tube defects. The number of embryos analysed in each condition is displayed on the graph. (k) Quantification of blastopore rescue. Blastopore closure was estimated using the ratio of blastopore diameter at indicated stages to the mean of control (ctrl) blastopore diameter at st.10.5. Bars represent maximum and minimum values, and lines represent the mean. Approximately 30 embryos per condition were scored. (l) Embryos were processed for WISH analysis at mid-neurula and late gastrula stages. Average neural tube Length/Width ratio for embryos stained with Sox2 probe (n=11) or average notochord length for embryos stained with Xbra probe (n=11) were calculated. Five independent rescue experiments were scored. One-way-ANOVA with Tukey test (95 % confidence intervals) were applied (***P≤0.0005). |
