DEV02706 Supplementary Material

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• Supplemental Figure 1 -

  Fig. S1 Zebrafish cyp26c1 (cyp26b1l) is orthologous to human CYP26C1. (A) Phylogenetic analysis of the zebrafish cyp26 genes groups zebrafish cyp26c1 (cyp26b1l) with the human and mouse Cyp26c1 genes. Amino acid sequences were aligned and phylogenetic analysis performed with ClustalX. Numbers at nodes are bootstrap values out of 1000. Species abbreviations: Dr, Danio rerio; Hs, Homo sapiens; Mm, Mus musculus; At, Arabidopsis thaliana. Accession numbers of sequences used for analysis were NP_000774 (Hs CYP26A1), NP_031837 (Mm Cyp26a1), NP_571221 (Dr Cyp26a1), NP_780684 (Mm Cyp26b1), NP_997831 (Dr Cyp26b1), NP_063938 (Hs CYP26B1), NP_899230 (Hs CYP26C1), NP_001025122 (Dr Cyp26b1l), NP_181813 (At CYP718), XP_92032 (Mm Cyp26c1). (B) Conserved syntenies between zebrafish chromosome regions containing cyp26a1 and cyp26c1 (cyp26b1l) and human chromosome 10. B1 shows zebrafish linkage group (LG) 12 (Ensembl Zv6), with all known zebrafish cDNAs immediately surrounding cyp26a1. B3 shows the region of zebrafish LG 17 and cDNAs surrounding cyp26c1 (cyp26b1l). Lines indicate putative orthologs between zebrafish and human genes on human chromosome 10 in B2 (NCBI Build 36). If no line is present, the most similar gene lies on another chromosome, except gnrh3, which appears to be fish-specific. Homologies were determined by NCBI Entrez Homologene entries or reciprocal blast search, and position of the gene is the 5′ end of the gene in megabases (M) according to NCBI map viewer (Build 36) or Ensembl (Zv6 assembly). Chromosomes are not drawn to scale.

• Supplemental Figure 2 -

  Fig. S2. The ventral hindbrain is sensitive to low concentrations of RA. RNA in situ hybridizations showing vhnf1 (blue), and krox20 and pax2a (both in red) expression at the 3-somite stage (11 hpf). In embryos treated with 10 nM RA, which causes very mild, if any, patterning defects, the ventral-most hindbrain is specifically posteriorized, corresponding with the absence of cyp26c1 expression there (arrow in B). Scale bar: 100 μm.

• Supplemental Figure 3 -

  Fig. S3. Effects of RA on cyp26b1 and cyp26c1 expression. RNA in situs at the 3-somite stage showing cyp26bc1 (A-C) and cyp26b1 expressions (D-F). In the presence of 100 nM RA, posterior rhombomeres, including r4, are expanded anteriorly, and the r4 expression of cyp26b1 and cyp26c1 are correspondingly expanded (B,E). In the presence of 10 μM DEAB, which blocks RA synthesis, anterior hindbrain regions, including r2 and r3, are expanded posteriorly, and the r2 and r3 domains of cyp26b1 and cyp26c1 are correspondingly expanded (C,F). Thus, neither gene requires RA for its onset in the hindbrain, although both genes are affected by treatments that alter RA-dependent hindbrain patterning events.

• Supplemental Figure 4 -

  Fig. S4. cyp26 genes function redundantly to protect the anterior hindbrain from endogenous RA. cyp26a1 and cyp26c1 are responsible for setting the r3-r4 limit. (A,C,E,G) Knocking-down cyp26b1 and/or c1 has no effect on hindbrain patterning in wild-type embryos. (B) cyp26a1 mutants exhibit a mild expansion of r4. This effect is enhanced by knocking-down cyp26b1 (D), but hoxb1a expression is shifted to the presumptive cerebellum (En-expressing domains) by knocking-down cyp26c1 (F). Embryos depleted for all three cyp26 proteins exhibit a profound anterior shift of hoxb1 expression to the mid-hindbrain domain (H). Scale bar: 100 μm.