Abstract
A collagen-based extracellular matrix is one defining feature of all Metazoa. The thick sheet-like extracellular matrix (mesoglia) of the diploblast, hydra, has characteristics of both a basement membrane and an interstitial matrix. Several genes associated with mesoglea have been cloned including a basement membrane and fibrillar collagen and an A and B chain of laminin. Here we report the characterization of a further three fibrillar collagen genes (Hcol2, Hcol3, and Hcol5) and the partial sequence of a collagen gene with a unique structural organization consisting of multiple von Willebrand factor A domains interspersed with interrupted collagenous triple helices (Hcol6) from Hydra vulgaris. Hcol2 and -5 have major collagenous domains of classical length ( approximately 1020 amino acid residues), whereas the equivalent domain in Hcol3 is shorter (969 residues). The N-propeptide of Hcol2 contains a whey acid protein four-cysteine repeat (WAP) domain, and the equivalent domain of Hcol3 contains two WAP and two von Willebrand factor A domains. Phylogenetic analyses reveal that the hydra fibrillar collagen genes form a distinct clade that appears related to the protostome/deuterostome A clade of fibrillar collagens. Data base searches reveal Hcol2, -5, and -6 are highly conserved in Hydra magnipapillata, which also provided preliminary evidence for the expression of a B-clade fibrillar collagen. All four of the H. vulgaris collagens are expressed specifically by the ectoderm. The expression pattern for Hcol2 is similar to that previously reported for Hcol1 (Deutzmann, R., Fowler, S., Zhang, X., Boone, K., Dexter, S., Boot-Handford, R. P., Rachel, R., and Sarras, M. P., Jr. (2000) Development 127, 4669-4680) but distinct from the pattern shared by Hcol3 and Hcol5. The characterization of multiple collagen genes in relatively simple diploblastic organisms provides new insights into the molecular evolution of collagens and the origins of the collagen-based extracellular matrix found throughout the multicellular animal kingdom.
Highlights
We have cloned and characterized several matrix genes from hydra associated with extracellular matrices (ECM) including a type IV collagen, Hcol-IV, hereafter referred to as Hcol4 [7], a fibrillar collagen Hcol-I, hereafter referred to as Hcol1 [8], and laminin A and B chains [9, 10]
Four Novel Collagen Genes Identified in H. vulgaris—Differential screening of a cDNA library using a combination of nonstringent and stringent conditions resulted in the identification of four novel collagen genes in addition to the previously described fibrillar Hcol1 [8] and basement membrane Hcol4 genes [5]
The three novel hydra fibrillar collagen genes contain uninterrupted N-terminal minor triple helical domains separated from their respective major collagenous domains by telopeptide-like sequences
Summary
Animal Maintenance—H. vulgaris, L2 stain was used for in situ hybridization experiments. Bayesian tree inference hydra collagen gene (Hcol6) with a unique organization con- values were produced from the MrBayes program [17]. We demonstrate by phylogenetic anal- A and WAP domains were originally identified using the rpsyses that the hydra fibrillar collagen genes form a distinct blast and cdart protocols (www.ncbi.nlm.nih.gov/BLAST). In vitro transcription was carried out using “Maxiscript” kit (Ambion) and Dig labeling mix (Roche Applied Science) according to manufacturer protocols. Both wild type adult polyps and polyps that were decapitated and followed during head regeneration for different times were used for in situ. Some samples were embedded in JB4 after the color reaction and sectioned (20-m thickness) with a glass knife for microscopic examination
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