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Fig. S1. Expression of zebrafish tfap2 paralogs. (A-E) Lateral views of fixed 24 hpf embryos processed by in situ hybridization to reveal expression of each of the five zebrafish tfap2 paralogs. Anterior is to the left. Scale bar: 50 µm.
Fig. S2. Additional neural crest derivatives rescued by tfap2 homologs. (A-F) All embryos are viewed dorsally and oriented with anterior to the left. (A) Uninjected embryos show clear foxd3 expression at 28 hpf marking the cranial ganglia (arrowheads, 23/23). (B) This expression is absent in embryos injected with MOs targeting tfap2a and tfap2c (0/24). Embryos injected with these MOs and subsequently injected with mRNA encoding (C) amphioxus tfap2 (29/35), (D) lamprey tfap2 (26/26), (E) zebrafish tfap2b (31/33) and (F) tfap2e (22/22) show restored foxd3 expression within the cranial ganglia on either one or both sides (mRNA was injected mosaically). (G) Graph showing cell counts of tlxa-positive cells at 22 hpf, a marker of Rohon-Beard and dorsal root ganglia sensory neuron precursor cells, found in the trunk of embryos injected similarly to those shown in A-F (n=10/group, *P<0.05 relative to tfap2a/tfap2c MO alone group). Of note, the incomplete absence of tlxa-positive cells in tfap2a/tfap2c MO-injected embryos is a previously described Tfap2-independent population (Li and Cornell, 2007).
Fig. S3. Myc-epitope tagged Tfap2a and Tfap2d show similar stability in zebrafish embryos. Embryos injected with Tfap2a-MT or Tfap2d-MT, or uninjected embryos were harvested at the indicated times, dechorionated and deyolked and subjected to western blot analysis using the indicated antibodies. By ∼15 somites relatively similar amounts of anti-myc signal are present in both Tfap2a-MT- and Tfap2d-MT-injected embryos with no signal detected in uninjected embryos. Alpha-tubulin is used as a loading control.
Fig. S4. Assessment of ability of Drosophila Tfap2 to restore neural crest in tfap2a/c-deficient zebrafish embryos. (A,D,G) Dorsal views of a flat-mounted (A) wild-type zebrafish embryo or (D) an embryo injected with tfap2a and tfap2c targeting MOs alone or (G) with Drosophila tfap2 mRNA, fixed at 11 hpf and processed to reveal foxd3 expression. White asterisks, premigratory neural crest. Red asterisks, non-neural crest-derived tailbud. (B,E,H) Lateral view of (B) wild-type zebrafish embryo or (E) an embryo injected with tfap2a and tfap2c targeting MOs alone or (H) with Drosophila tfap2 mRNA, fixed at 22 hpf and processed to reveal dlx2a expression. White asterisks, migratory neural crest. Yellow asterisks, brain. (C,F,I) Lateral views of live embryos at 48 hpf that are (C) wild-type or tfap2a mutants (F) injected with tfap2c MO alone or (I) in conjunction with Drosophila tfap2 mRNA. (J) Histograms summarizing neural crest rescue results in tfap2a/c-deficient embryos injected with Drosophila (Dm) tfap2 mRNA. Number of embryos (n) per group is displayed below bar. Scale bars: in A, 100 µm for A,D,G; in B, 50 µm for B,E,H; in C, 100 µm; in F, 100 µm for F,I.
Fig. S5. Nuclear anti-Tfap2a immunoreactivity is specific for Tfap2a protein. (A-D) Lateral views of cells of the enveloping layer (EVL), covering the yolk of 24 hpf embryos fixed and processed to reveal anti-Tfap2a immunoreactivity (IR). (A,C) Wild-type and (B,D) presumed tfap2a mutant embryos that are either (A,B) uninjected or (C,D) injected with MO targeting tfap2c. (A,C) In wild-type embryos, anti-Tfap2a IR is visible in nuclei (arrows) and at cell boundaries (red asterisks). (B) Presumed tfap2a mutants lack nuclear staining. Cell boundary staining persists, suggesting that it is non-specific. In (C) wild-type embryos injected with a tfap2c MO, nuclear anti-Tfap2a IR persists, suggesting that anti-Tfap2a is specific to Tfap2a and does not recognize Tfap2c. (D) A presumed tfap2a mutant injected with tfap2c MO.
