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. 2017 Sep 7;36(20):2987–2997. doi: 10.15252/embj.201796580

Figure EV4. lefty2 deficiency rescues DFC specification defects through restoration of Nodal signaling.

Figure EV4

  1. The expression of lefty2 at 60% epi and 75% epi stage in dnmt1‐deficient embryos was upregulated, compared to control embryos.
  2. Schematic diagram showing the experimental procedure for the isolation of GFP‐positive DFCs from sox17:GFP transgenic zebrafish and using DNA and RNA extracted from these DFCs for bisulfite PCR or qPCR.
  3. lefty2 mRNA overexpression decreased the number of sox17 + DFCs at 75% epi stage. Scale bar, 20 μm.
  4. The reduced expression of foxj1a and sox17 at 60% epi in dnmt1 morphants was restored by lefty2‐MO co‐injection.
  5. lefty2 MO partially rescued the reduced expression of lefty1, gata6 and ephrinb2b in DFCs of dnmt1 morphants. The dotted black outlines denote DFC region.
  6. qPCR analysis of Nodal target genes in control embryos, dnmt1 morphants, and dnmt1 morphants co‐injected with lefty2 MO.
  7. Representative images showing heart pattern labeled by cmlc2 at 30 hpf (upper panel) with quantification (lower panel).
Data information: Error bars, mean ± SD, n ≥ 5 embryos per experiment, n ≥ 2 technical replicates (C) and n = 3 technical replicates (F). *P < 0.05, **P < 0.01, ***P < 0.001. Student's t‐test. (A, D and E) Numbers indicate the number of embryos with the respective phenotype/total number of embryos analyzed in each experiment.