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First published online 16 October 2008
doi: 10.1242/dev.021238

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Restriction of retinoic acid activity by Cyp26b1 is required for proper timing and patterning of osteogenesis during zebrafish development

Kathrin Laue^1,2, Martina Jänicke^1,2, Nikki Plaster^1,*, Carmen Sonntag¹ and Matthias Hammerschmidt^1,2,

¹ Max-Planck-Institute of Immunobiology, Stuebeweg 51, D-79108 Freiburg, Germany.
² Institute for Developmental Biology, University of Cologne, D-50923 Cologne, Germany.

View larger version (54K): [in this window] [in a new window]   Fig. 1. dolphin corresponds to cyp26b1. (A-D) Live zebrafish larvae at 120 hpf. Dorsal views of head (A,B) and on pectoral fins (pf; C,D). wt, wild type; dol, dolphin allele ti230g. (E)The dol-bearing interval on chromosome 7. (F) Interval-spanning BAC contig, with indicated recombinations in 4500 meioses and showing the location of cyp26b1 gene. (G) Schematic of wild-type and mutant zebrafish Cyp26b1 proteins. (H) Sequencing profiles of cyp26b1 exon 3-intron junction from genomic DNA of wild type (+/+), heterozygous (+/-) and homozygous (-/-) ti230g mutants. (I) Schematic of exon 3-intron junction of cyp26b1. The mutated G in the splice-donor site of the ti230g allele is in red, the internal GT used in the mutant is in bold, and the 7 bp insert in mutant cDNA is in light gray. (J) Sequencing profiles of cyp26b1 exon 3-exon 4 junction from cDNA of wild type and ti230g mutants. The inserted sequence is underlined.

View larger version (102K): [in this window] [in a new window]   Fig. 2. cyp26b1 is expressed in condensing chondrocytes and perichondrium. Stainings of wild-type zebrafish at the stages indicated in the upper right corners and with the in situ RNA probes or antibodies indicated in the lower right corners. fli1a-positive neural crest cells in D were visualized by anti-GFP immunostaining of a Tg(fli1a:EGFP)y1 transgenic embryo (Isogai et al., 2003). (A,B,D,E,H,I) Lateral views; (C,L-Q) dorsal views; (F,J) longitudinal sections; (G,K) transverse sections. (A-D) cyp26b1 is expressed close to, but not within, postmigratory cranial neural crest (CNC) cells. Arrows in A,B point to dlx2a-positive, cyp26b1-negative cells that according to lineage-tracing data are likely to give rise to the ethmoid plate (e). Arrows in C,D point to two cyp26b1 domains close to the pharyngeal arch-forming CNC. (E-H) cyp26b1 is expressed in chondrogenic mesenchymal condensations. (F,G,H) Magnifications of regions indicated in E. In F,G, pharyngeal endoderm is counterstained with zn5 antibody; in H, neural crest derivatives are stained for sox9a transcripts; double-positive domains in pharyngeal arch (pa) condensations are marked. (I-K) cyp26b1 is expressed in perichondrial cells around the pharyngeal arches (J), the ethmoid plate (K), and along the entire length of the ceratohyal (ch) and the ceratobranchials (cb) (I; inset shows magnification of one cb). (L-Q) By contrast, osx (L), opn (M) and col10a1 (N) are restricted to perichondrial cells around the ossifying, Alizarin Red (alR)-positive region of the ceratohyal (O). In col10a1-positive cells, cyp26b1 levels are lower than in the adjacent col10a1-negative perichondrium (P,Q; arrows point to cells double positive for cyp26b1 and col10a1). spt, subpallial telencephalon; vt, ventral thalamus.

View larger version (114K): [in this window] [in a new window]   Fig. 3. cyp26b1 is expressed in osteoblasts and their precursors. Stainings of wild-type zebrafish at the stages indicated in the upper right corners and with the in situ RNA probes indicated in the lower right corners. (A-F) Confocal sections of double fluorescent in situ hybridizations and Alizarin Red stainings (alR), counterstained with DAPI (blue). Cells with weak cyp26b1 and strong col10a1 expression are indicated with white arrows, cells with strong cyp26b1 but absent col10a1 expression with red arrows, and cells with strong col10a1 but absent cyp26b1 expression, which most likely represent fully mature/active osteoblasts, with green arrows. See text for details. For single-channel images of B,E, see Fig. S4 in the supplementary material. (G-I) cyp26b1 displays uniform expression in perichordal cells in anterior regions of the notochord (n) (G; left panel shows longitudinal section; right panel shows transverse section; counterstained with Eosin), and metameric expression in the trunk (H,I; lateral views). In H, cyp26b1-positive cells are (still) underneath the notochord (arrowhead) and others are (already) in perichordal positions (arrows), whereas in I all cells are perichordal (arrows). Positive cells dorsal of the notochord in H most likely represent ventral spinal cord neurons (scn). (J-O) col10a1 and opn show a similar expression pattern to cyp26b1 (L-N) and transient coexpression with cyp26b1 (J,K) at intersomitic borders, coincident with the anterior borders of the the Alizarin Red-positive vertebral bodies (O). Arrows in L-N point to positive cells, arrowheads to borders of somites 5-8. (P) Numbers of perichordal cyp26b1-, col10a1- and opn-positive cells at different developmental time points. Ten fish were evaluated per condition; standard errors are indicated.

View larger version (116K): [in this window] [in a new window]   Fig. 4. cyp26b1 mutants and morphants display deficiencies in midline cartilages of the neurocranium and visceral skeleton. All panels show ventral views of zebrafish head regions. (A-D) col2a1 in situ hybridization at indicated stages. (E-N) Alcian Blue stainings of cartilaginous craniofacial elements at 120 hpf. Pharyngeal arches are numbered (1, mandibulare; 2, hyoid; 3-7, branchial/gill arches 1-5). (E-G) Overviews of visceral skeleton. (H-K) Magnified views of ceratohyals (H,I) or pharyngeal arches 4-6 (J,K). Arrows in H,I point to ceratohyal (ch) attachment in midline. (L-N) Flat-mounts of neurocranium, revealing the absence of medial ethmoid (e) and anterior basicranial commissure (abc) in mutant and morphant. anc, chondrocytes of anterior neurocranium; bb, basibranchial; bh, basihyal; cb, ceratobranchials; m, Meckel's cartilage; n, notochord; pq, palatoquadrate; t, trabeculae cranii.

View larger version (72K): [in this window] [in a new window]   Fig. 5. cyp26b1 mutants and morphants display increased ossification of endochondral and intramembranous craniofacial bones. (A-C) Lateral views and (D-F) ventral views of zebrafish larval heads after staining of ossified matrix with Alizarin Red at indicated ages. Insets in A-C show hyomandibula (hm; dorsal element of arch 2) of larvae of same genotype stained with Alcian Blue at 120 hpf. Mutant and morphant show an opercle (op) of increased size, whereas the hyomandibula fails to ossify, although its cartilage model is properly formed (insets). A similar combination of gain of opercle and loss of hyomandibula ossification has previously been described for endothelin mutants (Kimmel et al., 2003), possibly reflecting a morphogenetic effect of signaling to pattern ossification along the dorsoventral axis of the second arch and its associated elements. (D-F) Endochondral ossification within the ceratohyal (ch) is much more advanced in the mutant (E), comparable to the situation in a wild-type sibling 2 days later (F).

View larger version (80K): [in this window] [in a new window]   Fig. 6. cyp26b1 mutants and morphants display precocious and increased ossification of vertebral centra in the developing vertebral column. (A-K) Alizarin Red stainings of zebrafish larvae of genotype indicated in the upper right corners and at stages indicated in the lower right corners. (A-F,I-K) Lateral views; (G,H) transverse sections. Arrows in A indicate time course of centra ossification, starting at centra 3/4. In contrast to the segmented ossification of the wild-type larva (A,D,I,K), the cyp26b1 mutant (C,J) and morphant (F) show uniform and caudally extended perichordal ossification. See text for details. bop, basioccipital articulatory process.

View larger version (108K): [in this window] [in a new window]   Fig. 7. Retinoic acid (RA) deficiency reverts, whereas RA excess phenocopies, the skeletal alterations of cyp26b1 mutants, with a pattern of axial hyperossification different from that caused by Bmp2b overexpression. Genotypes are indicated in upper right corners, treatments in lower left corners. (A-D) Flat-mounts of Alcian Blue-stained neurocrania after treatment from 24-50 hpf; 120 hpf, dorsal views. Note the absence of the medial ethmoid plate (e) in the RA-treated wild-type zebrafish (B), and the recovery of this structure in the DEAB-treated cyp26b1 mutant (D). (E-H) Alizarin Red-stained heads after treatment from 24-50 hpf (F) or from 96-180 hpf (G,H); 180 hpf, dorsal views. Late (G), but not early (F), RA treatment phenocopies hyperossification of craniofacial bones, whereas late DEAB treatment reverses the mutant phenotype and causes delayed ossification (H). (I-T) Alizarin Red-stained centra after treatment with RA (J,K,N), R115866 (L), DEAB (Q,R) or heat shock (hs; O,P) at indicated developmental stages, or after aldh1a MO injection (T); 180 hpf(I-L,O-T) or 360 hpf (M,N; vertebrae numbers indicated); lateral views. In I-P, the early-specifiying centra at the level of somites 3-6 are indicated by a bar. Inset in P shows precocious and unsegmented perichordal mineralization at somite levels 18-26 of the same bmp2b transgenic animal. See text for details. cb5, ceratobranchial 5 (with teeth); cl, cleithrum; den, dentary; max, maxilla; ps, parasphenoid; see also Figs 4, 5, 6.

View larger version (114K): [in this window] [in a new window]   Fig. 8. Loss of cyp26b1 and gain of Bmp signaling have different effects on the number and/or activity of osteoblasts. (A-K) In situ hybridization of zebrafish larvae of genotype indicated in upper right corners and with probes and at stages indicated at lower right corners. (A-I) Lateral views; (J,K) dorsal views. (A) Entire head; (B-E) opercle; (F-K) trunk at level of somites 6-10. In A, stronger cyp26b1 expression is seen in all craniofacial skeletogenic elements of the cyp26b1 mutant, but not in the dorsal brain. In G, perichordal opn-positive cells of the cyp26b1 mutant have largely given up their metameric distribution. (L) Average increase in the number of axial cyp26b1-(at 96 hpf), opn- or col10a1-positive cells (at 144 hpf) in the trunk/tail region of cyp26b1 mutants and heat-shocked Tg(hsp70:bmp2b) transgenic fish. Control wild-type (wt) siblings were set to a value of 1. Ten fish were counted per condition; standard errors are indicated. n, notochord.

View larger version (122K): [in this window] [in a new window]   Fig. 9. Treatment of mice with the Cyp26 inhibitor R115866 causes axial hyperossification and fusion of cervical vertebrae. (A-D) Alcian Blue (cartilage) and Alizarin Red (bone matrix) stainings of mouse fetuses at E18.5. Dorsal views. (A,C) Control mice treated with vehicle (PEG 200). (B,D) Mice treated with R115866. (A,B) View of cervical vertebrae (c), with fusions of the neural arches of c3-c5 in an R115866-treated animal. (C,D) View of thoracic and sacral vertebrae, with precocious fusions of centra (ce) and neural arches (na), and broader ribs (rb) in the R115866-treated animal.

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