Hagfish and lancelet fibrillar collagens reveal that type II collagen-based cartilage evolved in stem vertebrates

Abstract

The origin of vertebrates was defined by evolution of a skeleton; however, little is known about the developmental mechanisms responsible for this landmark evolutionary innovation. In jawed vertebrates, cartilage matrix consists predominantly of type II collagen (Col2alpha1), whereas that of jawless fishes has long been thought to be noncollagenous. We recently showed that Col2alpha1 is present in lamprey cartilage, indicating that type II collagen-based cartilage evolved earlier than previously recognized. Here, we investigate the origin of vertebrate cartilage, and we report that hagfishes, the sister group to lampreys, also have Col2alpha1-based cartilage, suggesting its presence in the common ancestor of crown-group vertebrates. We go on to show that lancelets, a sister group to vertebrates, possess an ancestral clade A fibrillar collagen (ColA) gene that is expressed in the notochord. Together, these results suggest that duplication and diversification of ColA genes at the chordate-vertebrate transition may underlie the evolutionary origin of vertebrate skeletal tissues.

Fig. 1.

Extended majority-rule consensus tree for the Bayesian phylogenetic analysis of clade A fibrillar collagen proteins. Numbers at each node indicate posterior probability (pp) values based on one million replicates. Branch lengths are proportional to means of the pp densities for their expected replacements per site. The tree is rooted by tunicate (C. intestinalis) clade B fibrillar collagen (ColB) and sea urchin fibrillar collagen (ColP2α). Hagfish Col2α1 (boxed) is grouped with lamprey Col2α1a and Col2α1b with a pp of 0.99. This cyclostome Col2α1 clade joins to the base of the gnathostome Col2α1 clade with a pp of 0.96. Lancelet clade A fibrillar collagen (AmphiColA; boxed) is joined to the vertebrate clade A collagen family with a pp of 0.99. Minimum evolution and maximum likelihood methods confirm these positions for hagfish Col2α1 and AmphiColA (see also).

Fig. 2.

Col2α1 in cranial and tail fin cartilages of Atlantic hagfish. Transverse sections through adult hagfish stained with hematoxylin/eosin and fast green (A–D) or with Col2α1 antibody (A′–D′) are shown. (A and A′) Soft cranial cartilage stained with hematoxylin contains large hypertrophic chondrocytes surrounded by thin layer extracellular matrix (A), and it is positive for Col2α1 (A′). (B and B′) Section through caudal fin shows cartilaginous fin rays with soft-cartilage characteristics (B) that are rich in Col2α1 (B′). (C and C′) Cranial cartilage element exhibiting a mosaic distribution of soft (blue-stained chondrocytes) and hard (red-stained chondrocytes) cartilage (C). Note that Col2α1 protein is restricted to the soft cartilage region (C′). (D and D′) Hard cranial cartilage stained with eosin but not hematoxylin contains small chondrocytes surrounded by a thick layer of extracellular matrix (D), and it is negative for Col2α1 (D′).

Fig. 3.

Expression of AmphiColA during lancelet development. Whole-mount in situ hybridizations of lancelet embryos with an AmphiColA antisense riboprobe are shown. Anterior is to the left in A–C and to the right in D. Stages shown are 18 h (A), 30 h (B), 36 h (C), and 5 days (D) postfertilization. (A Inset) Transverse section of 18-h embryo. The arrowhead indicates notochord, and the arrows point to the neural tube.

Fig. 4.

Origin of type II collagen-based cartilage. Phylogenetic relationships of tunicates, lancelets, hagfishes, lampreys, and gnathostomes (37, 38) are shown. Dashed lines indicate alternative branch positions (27). Letters point out major steps in Col2α1-based cartilage evolution. The solid rectangle indicates the origin of the Col2α1 gene by ColA gene duplication(s). The hollow rectangle indicates the origin of Col2α1a and Col2α1b by Col2α1 gene duplication. (A) Ancestral clade A collagen gene expression in notochord of stem chordates. (B) Origin of Col2α1-based cartilage in stem vertebrates. (C) Subfunctionalization of Col2α1, in which the expression domain of the ancestral gene is partitioned between the duplicate genes (Col2α1a and Col2α1b); arrows indicate possible positions of this event.