DEV033209 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. tfap2a morpholino-injected foxd3^zdf10 mutant embryos precisely phenocopy foxd3^zdf10;tfap2a^low double mutants. Embryos obtained from foxd3^zdf10 heterozygous matings were injected with tfap2a morpholino (tfap2aMO). Wild-type-tfap2aMO and foxd3^zdf10-tfap2aMO phenocopy tfap2a^low and foxd3^zdf10;tfap2a^low mutants, respectively, with 90% efficiency (n>1000). (A) Live larvae at 4 days post-fertilization (dpf), lateral views with either transmitted (left) or reflected (right) light. Compared with uninjected wild type, uninjected foxd3^zdf10 mutant and wild-type tfap2aMO-injected siblings, foxd3^zdf10-tfap2aMO larvae lack melanophores (left, arrows), xanthophores (left, arrowheads) and iridophores (right, white arrows), with the exception of occasional head xanthophores. (B) Alcian Blue staining at 4 dpf, ventral views, anterior to the left. All upper and lower jaw elements, as well as the anterior neurocranium, are absent in foxd3^zdf10-tfap2aMO embryos. (C) 16A11 immunoreactivity, 3 dpf, lateral views with anterior to the left. DRG (arrowheads) and enteric neurons (arrows) are absent in mutant morphants. (D) th expression at 48 hpf, lateral views with anterior to the left. Sympathetic neurons (arrowheads) are absent in foxd3^zdf10 mutants, tfap2a morphants and mutant morphants.

• Supplemental Figure S2 -

  Fig. S2. Neural crest sublineage specification fails to occur in foxd3^zdf10-tfap2aMO embryos. (A-H) Lateral views with anterior to the left. (A) mitf expression at 24 hpf (arrowheads). Just as with foxd3^zdf10;tfap2a^low double mutant embryos, foxd3^zdf10-tfap2aMO embryos completely lack neural crest (NC) mitf expression (asterisks). (B) xanthine dehydrogenase (xdh), diagnostic of xanthophore precursors, at 24 hpf (arrowheads); (C) endothelin receptor B (ednrb1), presumably expressed by all chromatophore precursors, at 30 hpf (arrowheads). NC expression of both genes is absent in mutant morphants (asterisks). (D) dlx2 expression at 24 hpf. dlx2 expression (arrowheads) is absent in the branchial arches of foxd3^zdf10-tfap2aMO embryos (asterisks), but is retained in the forebrain. (E) dlx3 expression at 30 hpf (arrowheads). (F) dhand expression at 30 hpf (arrowheads). NC expression of both genes is absent in mutant morphants (arrow; double asterisks) with dlx3 expression maintained in non-NC-derived otic vesicle (asterisks). (G) tbx1 expression in the branchial arches at 27 hpf is diagnostic of the endodermal component of the arches. tbx1 expression is normal in all experimental embryos. (H) endothelin 1 (edn1) expression in the mesodermal component of the branchial arches at 30 hpf. Branchial mesoderm appears to be develop normally in foxd3^zdf10 single mutant, tfap2aMO morphant and foxd3^zdf10-tfap2aMO mutant-morphant embryos.

• Supplemental Figure S3 -

  Fig. S3. As in foxd3^zdf10;tfap2a^low double mutants, induction of NC SoxE gene expression is severely reduced in foxd3^zdf10-tfap2aMO embryos. (A,C,D) Dorsal views, anterior up. (B) Lateral view, anterior to the left of a 30 hpf embryo. (A) At 4s, NC sox9b expression is virtually absent in mutant morphants. (C,D) At 5s, NC sox10 expression (C) is severely reduced in mutant morphants, whereas NC foxd3 expression (D) is retained. (B) NC sox9a expression in the pharyngeal arches (arrows) is undetectable in mutant morphants.

• Supplemental Figure S4 -

  Fig. S4. The neural plate border develops independent of foxd3 and tfap2a function. (A) pax3 expression at the 3 somite stage, dorsal views with anterior to the top. paired homeobox 3 (pax3) is expressed at the edge of the developing neural plate, and is required for induction of NC-specific gene expression. pax3 is robustly expressed in foxd3^zdf10;tfap2a^low double mutants, as well as in foxd3^zdf10 and tfap2a^low single mutant embryos. (B) msxb expression at the 3 somite stage, dorsal views with anterior to the top. msxb expression is required for proper size and positioning of the neural plate border. msxb expression is normal in foxd3^zdf10;tfap2a^low double mutant, foxd3^zdf10 single mutant and tfap2a^low single mutant embryos compared with wild-type siblings. (C) huC expression in 6 somite stage embryos. At this stage, primary motor neurons (more medial cells) and Rohon-Beard sensory neurons (bilateral stripes of cells just lateral to primary motorneurons) express huC. Both neuronal populations develop normally in both single mutants, as well as in double mutants.