DEV021238 Supplementary Material

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• Supplemental Figure S1 -

  Fig. S1. Similar effects of sa0002 and ti230 mutations and rescue of ti230 mutant by inducible application of wild-type cyp26b1. (A-H) Alcian Blue staining of craniofacial cartilaginous elements at 120 hpf (A-D) and Alizarin Red staining of bone matrix in axial skeleton at 180 hpf (E-H). (A,E) Wild-type siblings; (B,F) ti230g mutants; (C,H) sa0002 mutants. The two alleles show craniofacial and axial skeletal defects of undistinguishable strengths. (D,H) ti230 mutants after reintroduction of wild-type Cyp26b1 at 96 hpf, leading to a loss of vertebral ossification (H), whereas patterning of midline craniofacial cartilage, which occurs earlier (24-50 hpf) (compare with Fig. 7A-D), remains unaltered (D). For cyp26b1 overexpression, the heat-inducible construct pTol2-hse-GTP/cyp26b1 was generated by cloning the cyp26b1 cDNA into the bicistronic vector pSGH2 (Bajoghli et al., 2004), which in addition to cyp26b1 drives expression of GFP under the control of heat-shock elements (hse). Subsequently, the cassette was ligated into vector pT2AL200R150G, which contains Tol2 recognition sites to allow early genomic integration and widespread expression (Urasaki et al., 2006). pTol2-hse-GTP/cyp26b1 plasmid DNA was co-injected with transposase mRNA (Kawakami et al., 2004) into the cytoplasm of one-cell stage embryos from a ti230/+ intercross. For transgene activation, injected embryos were transferred from 28°C to 39°C for 30 minutes at 96 hpf. (I-L) Cyp26b1 carrying the sa0002 or ti230 mutation is biologically inactive. For activity tests, the cDNA of human CYB26B1 was amplified by RT-PCR and cloned into plasmid pCS2+. Nonsense mutations at nucleotides 136 (AAGrTAG) or 697 (TACrTAA) were introduced by PCR-based site-specific in vitro mutagenesis, yielding truncated proteins corresponding to those encoded by the zebrafish sa0002 and ti230g alleles, respectively. Plasmids were linearized with NotI, mRNAs synthesized with the Message Machine Kit (Ambion, TX) and co-injected with fluorescein dextrane (Molecular Probes) into one blastomere of wild-type embryos at the two-cell stage, as described previously (Kudoh et al., 2002). At the 90% epiboly stage (9 hpf), embryos with unilateral fluorescence were selected and fixed for in situ hybridization analysis. Whole-mount in situ hybridization for the anterior neural marker otx2 (Li et al., 1994) and the posterior neural marker hoxb1b (Alexandre et al., 1996); 90% epiboly stage, dorsal views, anterior up. To identify the injected side, some embryos were counterstained with fluorescein-coupled anti-fluorescein antibody (Molecular Probes, 1:200; not shown). (I) Uninjected sibling (n=90/90). (J) Embryo injected with wild-type CYP26B1 mRNA on right side (n=25/27). (K) Embryo injected with ti230 CYP26B1 mRNA (n=30/30). (L) Embryo injected with sa0002 CYP26B1 mRNA (n=35/35). Wild-type CYP26B1 causes loss of hoxb1b expression on the injected side, indicative of an anteriorizing effect caused by Cyp26b1-mediated RA inhibition (Kudoh et al., 2002). By contrast, neither of the mutant versions displayed any effect, suggesting that the encoded truncated proteins are inactive.

  References

  Alexandre, D., Clarke, J. D., Oxtoby, E., Yan, Y. L., Jowett, T. and Holder, N. (1996). Ectopic expression of Hoxa-1 in the zebrafish alters the fate of the mandibular arch neural crest and phenocopies a retinoic acid-induced phenotype. Development 122, 735-746.

  Bajoghli, B., Aghaallaei, N., Heimbucher, T. and Czerny, T. (2004). An artificial promoter construct for heat-inducible misexpression during fish embryogenesis. Dev. Biol. 271, 416-430.

  Kawakami, K., Takeda, H., Kawakami, N., Kobayashi, M., Matsuda, N. and Mishina, M. (2004). A transposon-mediated gene trap approach identifies developmentally regulated genes in zebrafish. Dev. Cell 7, 133-144.

  Li, Y., Allende, M. L., Finkelstein, R. and Weinberg, E. S. (1994). Expression of two zebrafish orthodentical-related genes in the embryonic brain. Mech. Dev. 48, 229-244.

  Urasaki, A., Morvan, G. and Kawakami, K. (2006). Functional dissection of the Tol2 transposable element identified the minimal cis-sequence and a highly repetitive sequence in the subterminal region essential for transposition. Genetics 174, 639-649.

• Supplemental Figure S2 - Fig. S2. Characterization of cyp26b1 MO. (A) RT-PCR of embryos injected with cyp26b1 morpholino (MO) and uninjected controls (WT), amplifying an exon 3-exon 4 cyp26b1 fragment and, as control, an ef1a fragment. For the morphant, only a slightly larger cyp26b1 fragment was amplified. (B) Scheme illustrating the sequence of the exon 3-intron junction of the cyp26b1 precursor mRNA targeted by the morpholino used, with the three relevant splice-donor sites in bold. The GT used in wild-type animals and the next GT used in dol mutants are blocked by the morpholino. In morphants, a further downstream TT is used, causing an insertion of 20 intronic nucleotides. (C) Sequencing profiles of cDNA clones from uninjected (left) and cyp26b1 morphant (right) animals. All (50/50) sequenced cDNA clones contained the underlined 20 bp insert. This insert causes a frame-shift that should lead to a truncation of Cyp26b1 protein similar to that caused by the dol mutation (see Fig. 1G).

• Supplemental Figure S3 - Fig. S3. Expression pattern of cyp26b1 in comparison to cyp26a1, cyp26c1 and aldh1a, and in pectoral fin buds. (A-I) Wild-type zebrafish after whole-mount in situ hybridizations at stages indicated in upper right corners with probes indicated in lower right corners. (A-C,E) Lateral views; (D) ventral view; (F-I) ventrolateral views. (A-C) cyp26a1, cyp26b1 and cyp26c1 show distinct, but partially overlapping expression patterns. In B, unique expression of cyp26b1 in the condensation of the ethmoid plate is indicated by an arrow. (D-E) aldh1a is expressed adjacent to the eye vesicles (red arrows in D,E), lateral of the cyp26b1 expression domain in the midline of the developing neurocranium (blue arrow in D). (F,G) aldh1a is expressed in discrete domains lateral of the cyp26b1 expression domains in the branchial arches; the midline is indicated by arrowheads. Together, this points to the possible existence of a mediolateral RA gradient in the craniofacial system. (H,I) Similar to the earlier exclusion of cyp26b1 expression from chondrogenic neural crest cells of the craniofacial system (see Fig. 2), cyp26b1 is expressed in a distal crescent of the early pectoral fin bud, while the sox9a-positive chondrocyte precursors (Wada et al., 2005) are positioned more proximally (H). A corresponding distribution has been described for mouse Cyp26b1 in the developing limb buds (Yashiro et al., 2004), suggesting that similar proximodistal patterning processes might occur in both zebrafish and mouse, accounting for the profound limb defects in mouse Cyp26b1 mutants (Yashiro et al., 2004), and the moderate pectoral fin defects in zebrafish cyp26b1 mutants (see Fig. 1C,D). e, ethmoid plate; pf, pectoral fin; pp, pharyngeal pouches.

• Supplemental Figure S4 - Fig. S4. Expression pattern of cyp26b1 in craniofacial bone elements in comparison to opn and osx. Stainings of wild-type animals at stages indicated in upper right corners, and with in situ RNA probes indicated in lower right corners. (A-C) cyp26b1 and opn are coexpressed in the opercle. The opn-positive cells in A display weak cyp26b1 expression that is not visible in this image (but compare with Fig. 3A); the opercle visible in B is overstained, showing similar signals in strongly and weakly expressing cells. In C, cells with cyp26b1 and opn coexpression are indicated with black arrow, cells positive for cyp261 or opn mRNA only with blue or red arrows, respectively. (D-G) Confocal sections of double fluorescent in situ hybridizations, counterstained with DAPI to visualize nuclei (blue). In left panels, col10a1 expression is in green; in middle panels, osx, opn or cyp26b1 expression is in red; in right-hand panels, merged green and red channels, superimposed by DAPI staining. (D-F) col101a1, osx and opn are coexpressed in the opercle. At 72 hpf, osteoblasts in central versus peripheral regions of the elements display slightly different signatures. opn expression is highest in regions close to the bony core of the element (E; compare with Fig. 3C), col10a1 expression is strongest in intermediate positions (E), whereas osx is strongest in peripheral regions (D). This suggests that opn is preferentially made by the most mature/most active osteoblasts, whereas osx marks more immature osteoblasts. cyp26b1 seems to be most strongly expressed in even less advanced osteoblasts. At 72 hpf, it displays strong expression at the dorsal base of the element (see Fig. 3A), which at that time is negative for osx, col10a1 and opn (indicated by white arrows in D,E). By contrast, col10a1 and opn are expressed in these dorsal cells at 120 hpf (indicated by white arrow in F). Also note that the location of osteoblasts generally shifts to more peripheral positions as the central bony core of the element grows (compare E and F with Fig. 3C,F). (G) cyp26b1 and col10a1 are coexpressed in cells adjacent to the bony core of the cleithrum. However, cyp26b1 levels in these central cells is lower than in more peripheral, col10a1-negative cells, which most likely represent immature and/or inactive osteoblasts.

• Supplemental Figure S5 - Fig. S5. Brain patterning and cranial neural crest migration is unaffected in cyp26b1 mutants. (A-L) In situ hybridizations of cyp26b1 mutants (dol) and wild-type siblings (WT) at stages indicated in upper right corners, and with probes indicated in lower right corners; dorsal views on head regions. (A,B) Mutant shows normal expression patterns of six3b, a marker for forebrain (Kobayashi et al., 1998), pax2a, a marker for the midbrain-hindbrain boundary region (Krauss et al., 1991), and egr2b (formerly called krox20), a marker for hindbrain rhomobomeres 3 and 5 (Oxtoby and Jowett, 1993), indicating that brain patterning is unaffected, including normal sizing of the forebrain primordium. Similarly, expression patterns of hoxb1a, a marker for rhombomere 4 (Amores et al., 1998; Rohrschneider et al., 2007) (C,D), and hoxb3a, a marker for rhomobomeres 5 and 6 (Amores et al., 1998; Hogan et al., 2004) (E,F) are normal, indicating that cyp26b1 is dispensable for hindbrain patterning. Furthermore, cyp26b1 mutants display normal expression of hoxb3a in r5/r6-derived neural crest, and of dlx2b, a marker for all cranial neural crest cells (Akimenko et al., 1994), indicating that neural crest patterning and migration are unaffected by the mutation. Normal expression pattern of isl1 (Inoue et al., 1994) further indicates that branchiomotor neurons, derivatives of the hindbrain, are specified and patterned normally in cyp26b1 mutants (I,J). IV, trochlear nucleus; V, trigeminal nucleus; VII, facial nucleus; X, vagal nucleus). (K,L) Normal expression of tfap2b (Knight et al., 2005) in tectum, hindbrain and spinal cord of cyp26b1 mutant. fb, forebrain; hb, hindbrain; mhb, midbrain-hindbrain boundary; nc, neural crest; ot, optic tectum; r, rhomobomere; ret, retina; sc, spinal cord.

  References

  Amores, A., Force, A., Yan, Y. L., Joly, L., Amemiya, C., Fritz, A., Ho, R. K., Langeland, J., Prince, V., Wang, Y. L. et al. (1998). Zebrafish hox clusters and vertebrate genome evolution. Science 282, 1711-1714.

  Hogan, B. M., Hunter, M. P., Oates, A. C., Crowhurst, M. O., Hall, N. E., Heath, J. K., Prince, V. E. and Lieschke, G. J. (2004). Zebrafish gcm2 is required for gill filament budding from pharyngeal ectoderm. Dev. Biol. 276, 508-522.

  Inoue, A., Takahashi, M., Hatta, K., Hotta, Y. and Okamoto, H. (1994). Developmental regulation of islet-1 mRNA expression during neuronal differentiation in embryonic zebrafish. Dev. Dyn. 199, 1-11.

  Knight, R. D., Javidan, Y., Zhang, T., Nelson, S. and Schilling, T. F. (2005). AP2-dependent signals from the ectoderm regulate craniofacial development in the zebrafish embryo. Development 132, 3127-3138.

  Kobayashi, M., Toyama, R., Takeda, H., Dawid, I. B. and Kawakami, K. (1998). Overexpression of the forebrain-specific homeobox gene six3 induces rostral forebrain enlargement in zebrafish. Development 125, 2973-2982.

  Krauss, S., Johansen, T., Korzh, V. and Fjose, A. (1991). Expression of the zebrafish paired box gene paxzf-b during early neurogenesis. Development 113, 1193-1206.

  Oxtoby, E. and Jowett, T. (1993). Cloning of the zebrafish Krox-20 (Krx-20) and its expression during hindbrain development. Nucleic Acids Res. 21, 1087-1095.

  Rohrschneider, M. R., Elsen, G. E. and Prince, V. E. (2007). Zebrafish Hoxb1a regulates multiple downstream genes including prickle1b. Dev. Biol. 309, 358-372.

• Supplemental Figure S6 - Fig. S6. RA treatments during late larval stages cause fusions of vertebral bodies. Alizarin Red (alR) stainings of vertebrae at 9 dpf (A,B) or 18 dpf (C-F). (A,C,E) Untreated siblings; numbers indicate anterior-posterior positions of shown vertebrae along the vertebral column. (B,D,E) Corresponding regions of animals treated with RA from 6-9 dpf (B), or from 15-18 dpf (D,F). RA treatments at these late developmental stages cause widespread fusions of centra, including centra 3-6, which ossify first and which have become insensitive to Bmp2 at 4 dpf (compare with Fig. 7). c, centrum; ha, hemal arch; na, neural arch.

• Supplemental Figure S7 -

  Fig. S7. The numbers of cyp26b1- and opn-positive cells in cyp26b1 mutants, while increased overall, change over time with normal relative kinetics. Diagrams illustrating the progressively declining numbers of axial cyp26b1-positive and the progressively increasing numbers of axial opn-positive cells in cyp26b1 mutants and wild-type siblings at different developmental time points. Ten fish were evaluated per condition; standard errors are indicated. In both wild-type and mutant fish, the numbers of cyp26b1-positive cells and opn-positive cells change with the same relative slopes, suggesting that in both cases, cyp26b1-positive cells give rise to mature/active osteoblasts.