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Fig. S1. Characterization of zebrafish Ovo orthologs. (A) Alignment of zebrafish Ovo1 and Ovo3 sequences with mouse and human Ovo1. The transcriptional repression SNAG domain is boxed (red). The four C2H2 zinc finger and nuclear localization (NLS) motifs are underlined in black and blue, respectively. (B) Phylogenetic tree of the Ovo family of transcription factors. (C,C′) Nuclear localization of Gfp:Ovo1 following injection into 1-cell-stage zebrafish embryos.
Fig. S2. Expression profile of ovo1 and efficacy of Ovo1 MOs. (A) RT-PCR for ovo1 (upper panel) at: 1.5-72 hours post-fertilization (hpf). ef1a (lower panel) is a loading control. (B,C) Live embryos injected with a construct carrying the Ovo1 MO binding site (Ovo1MO 5′UTR) fused to gfp (Ovo1MO 5′UTR:Gfp) showing Gfp expression (B) and loss with co-injection of the 5′UTR Ovo1MO (C).
Fig. S3. Ovo1 disrupts NC-cell-derived pigment development. (A-D) Live images of control uninjected (A,C) and Ovo1 morphants (B,D) at 48 hpf (A,B) and 4 dpf (C,D). Lateral views, anterior to the left. Melanocytes are reduced in Ovo1 morphants (B,D; arrows). e, eye.
Fig. S4. Identification of pacpar2.10 (ncadb>+/b>/b>−) mutants. (A) Schematic of the different ncad mutant isoforms. The wild-type product is 266 bp; mutants contain either a smaller 143 bp form or larger isoforms with as many as 50 additional nucleotides. (B,C) RT-PCR for ncad in pacpar2.10 heterozygous embryos injected with Ovo1 MO (B, all lanes; C, lanes 6-8) or with Rab11fip2 RNA (C, lanes 3-5). All embryos with ectopic dorsal NC cells (B, lanes 7-11; C, lanes 3,4,6,7) are heterozygous for pacpar2.10, whereas embryos whose NC cells migrated normally do not exhibit alternative spliced forms of ncad (B, lanes 2-6; C, lanes 5,8). Uninjected pacpar2.10 heterozygous control (C, lane 2).
Fig. S5. Ncad overexpression does not rescue neural crest defects in Ovo1 morphants. (A) Quantitative PCR shows reduced ncad mRNA levels in Ovo1 morphants (*, P≤0.05). (B-E) In situ hybridization for sox10 in neural crest (NC). Dorsal views, anterior to the top. In contrast to controls (B), NC accumulates (arrows) in the dorsal midline of embryos injected with ncad mRNA (C), Ovo1 morphants (D) or a combination of ncad mRNA and Ovo1 MO (E). ot, otic vesicle.
Fig. S6 Rab11fip2 disrupts the migration of neural crest-derived pigment precursors. Wholemount in situ hybridization for mitfa to label pigment progenitors. These lie lateral to the neural tube in control embryos (A) but cluster over the dorsal midline in embryos overexpressing Rab11fip2 (B,C; arrows). ot, otic vesicle.
Fig. S7. Loss of Rab11fip2 fails to rescue Ovo1 morphants. (A) RT-PCR for rab11fip2 to test morpholino (MO) efficacy (upper panel) using ef1a as a loading control (lower panel). Lanes: 1, control; 2, 0.75 ng Rab11fip2 MO (here referred to as fip2MO); 3, 1.5 ng fip2MO; 4, 3 ng fip2MO; 5, 3 ng Ovo1 MO; 6, 3 ng Ovo1 MO + 0.75 ng fip2MO; 7, 3 ng Ovo1 MO + 1.5 ng fip2MO; 8, 3 ng Ovo1 MO + 3 ng fip2MO. (B) Quantitation of embryos with ectopic NC cells in the dorsal midline (blue) in Ovo1 morphants and Rab11fip2 MO rescued embryos. (C-E) Live images of Gfp expression in NC cells (arrows) in controls (C), Ovo1 single morphants (D) and Ovo1MO, fip2MO double-morphant embryos (E); dorsal views. ot, otic vesicle.
Fig. S8. Cell proliferation, apoptosis and ectodermal specification are unaffected in Ovo1 morphants. (A,B) Wholemount anti-phospho-Histone 3 antibody (PH3) staining at 15 hpf. Dorsal views, anterior to the left. (A′,B′) Insets showing PH3+ mitotic cells in cross-sections through the neural tube of control and Ovo1 morphants at 24 hpf. (C-F) Acridine orange labeling (arrows) reveals elevated apoptosis in Ovo1 morphant embryos compared with controls (C,D), which is eliminated by co-injection of p53 MO (E,F). (G-J) Double in situ hybridization for sox10 in NC and wnt10b in dorsal neural tube. In contrast to controls (G), sox10+ cell aggregates form in the dorsal midline of Ovo1 morphants (H), but not in p53 morphants (I), and injection of p53 MO fails to rescue NC defects in Ovo1 morphants (J). (K,L) Triple in situ hybridization for expression of ap2g in non-neural ectoderm (NNE), hgg1 in prechordal plate mesoderm (PCP) and pax2.1 at the midbrain-hindbrain boundary (MHB) in controls (K) and Ovo1 morphants (L) at 8 hpf. Dorsal views, anterior to the left. Asterisks indicate the borders between the NE and NNE. (M,N) Double labeling for sox10 (red) in NC and krt18 expression in epidermis (dark blue) in control (M) and Ovo1 morphants (N). Dorsal clumps of sox10+ NC cells form in morphants, while epidermis appears unaffected (arrows). e, eye; ot, otic vesicles.
Movie 1. Time-lapse movie using a sox10(7.2):gfp transgenic zebrafish to label migratory NC cells in vivo. Dorsal view, anterior to the left. Gfp-positive NC cells were imaged every 10 minutes for 10 hours beginning at 14 hpf using a 20× objective.
Movie 2. Time-lapse movie of migratory NC cells in sox10(7.2):gfp Ovo1 morphant embryos. Dorsal view, anterior to the left. NC cells accumulate at the midline in Ovo1 morphants over time. Images were taken every 10 minutes starting at 12 hpf and continued for 10 hours using a 20× objective.
