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jezb22511-sm-0001-SupFig-S1.pdf817KFigure S1. Phylogeny of collagens 8a1, 8a2, and 10a1 in osteichthyans. A: Maximum likelihood (ML) tree showing the relationships between Col8a1, Col8a2, and Col10a1. Col15a1 and Col18a1 sequences are used as outgroups. BE: Phylogeny of Collagens 8a1, 8a2, and 10a1 in osteichthyans using a Bayesian phylogenetic approach. Only posterior probabilities inferior to 1 are shown. Outgroups are composed of members from the Col1a1 (B), Col4a2 (C), Col5a1 (D), and Col15a1 (E) families. Note that Col8a2 and Col10a1 are consistently more closely related to each other than to the Col8a1 paralogues.
jezb22511-sm-0002-SupFig-S2.pdf337KFigure S2. In situ hybridization protocol for paraffin sections of Xenopus tropicalis tadpole skeletal elements.
jezb22511-sm-0003-SupFig-S3.doc252KFigure S3. Exon–intron structure of the Col8a2 gene and splicing divergence between Xenopus tropicalis strains. AD: RNA-seq sequences are shown in pink and RT-PCR sequences are shown in gray. The genomic coordinates of Xenopus tropicalis scaffold 194 are indicated on each side of the sequences. A: Localization of the first Col8a2 exon by RNA-seq and RT-PCR. The positions of the Exon1F and Exon1nestedF primers are indicated (blue). B: RNA-seq and RT-PCR detected different sequences for the Col8a2 second exon. Note that only the exon from the strain tested by RT-PCR harbors a BsrI site (black). C: Localization of the third Col8a2 exon by RNA-seq and RT-PCR. Only two residues at position 336854 and 337017 (white) differ between the RT-PCR sequence and the reference genome. The translation initiation site is indicated in red, and the predicted amino acid sequence is shown below. Residues conserved with mouse are highlighted in green. D: The sequence shows the beginning of the fourth and last Col8a2 exon, until the position of the Exon4R primer used for the RT-PCR (yellow). For the sake of simplicity, the predicted amino acid sequence is not shown. E: Schematic summary of the Xenopus tropicalis Col8a2 structure deduced by RNA-seq (pink) and RT-PCR (gray). The size of exons (pb) and introns (kb), the position of the BsrI restriction site, and of the translation start and stop codons are indicated. F: Nested RT-PCR was performed on hindlimbs and skull both before (NF 51–53) and after (NF58) the differentiation of mature osteoblasts. The primer pairs (Exon1F × Exon4R) and (Exon1nestedF × Exon4R) were used in the first and second PCR reactions, respectively. The PCR product was subjected to restriction digest, revealing the presence of the BsrI site in all cases. Control PCRs were also performed on RNA which was not subjected to reverse transcription (RT−). The fragment sizes of the molecular weight marker are indicated on the left (base pairs).
jezb22511-sm-0004-SupFig-S4.docx1317KFigure S4. Alignment of vertebrate Collagen 8a1, 8a2, and 10a1 protein sequences.
jezb22511-sm-0005-SupFig-S5.pdf135KFigure S5. Chondrocytic expression of Col10a1 in the epiphysis of stage 58 Xenopus tropicalis femoral bones. Arrows point at some of the Col10a1 expressing cells.

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