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. 2018 Aug 6;14(8):e1007551. doi: 10.1371/journal.pgen.1007551

Fig 8. Cooperation of maternal and zygotic Dvl2 and Dvl3a in AP patterning.

Fig 8

Mutant embryos were selected by the extent of CE defects at 12 hpf, and genotyped after in situ hybridization. Immunostaining was performed at shield stage, followed by genotyping. (A-B’) Expression patterns of sp5l and tbx16l in WT embryos. Arrowheads in lateral viewed embryos indicate the anterior end and posterior end. Other embryos are dorsal-posterior view. (C, C’) The expression of sp5l is not affected in Zdvl2;MZdvl3a mutants. (D, D’) Strongly reduced tbx16l expression in Zdvl2;MZdvl3a mutants. (E, E’) Strongly reduced sp5l expression in MZdvl2;MZdvl3a mutants. (F, F’) Residual expression of tbx6l in MZdvl2;MZdvl3a mutants. (G) TOPFlash luciferase reporter activity in Zdvl2;MZdvl3a and MZdvl2;MZdvl3a mutants at 12 hpf. Bars represent the mean ± s.d. from three independent experiments (*, P<0.05; ***, P<0.001). (H) Endogenous ß-catenin nuclear accumulation (arrows) in ventral marginal cells of a WT embryo at shield stage. (I) Absence of ß-catenin nuclear accumulation in ventral marginal cells of an MZdvl2;MZdvl3a mutant at shield stage. (J-P) In situ hybridization analysis of indicated genes in WT and MZdvl2;MZdvl3a embryos at 12 hpf. Dorsal view with anterior to the top (J, K), dorsal view (L-O), and dorsal-posterior view (P). (R-U) Analysis of hoxb1b and zygotic Wnt/ß-catenin target genes by RNA-sequencing at 12 hpf. Note the significant decrease in RPKM (reads per kilobase million) for these genes in MZdvl2;MZdvl3a mutants. Bars represent the mean ± s.d. from three independent samples (*, P<0.05; **, P<0.01). (V, W) Lateral (V) and dorsal-posterior (W) views show rescue of axin2 and tbx16l expression in MZdvl2;MZdvl3a mutants at 12 hpf, following LiCl (0.3 M) treatment for 8 minutes at 5 hpf. op, otic placode; p, pronephric mesoderm; tb, tailbud. Scale bar: (A-F’) 400 μm; (H, I) 20 μm; (J-P) 400 μm; (V, W) 400 μm.