The Evolution of Collagen IV and Basement Membrane Enabling Metazoan Multicellularity

Multicellular life has independently evolved over twenty times from fungi to plants to animals, yet none were as successful and explosive as that of the animals. A myriad of environmental, ecological, and biological factors enabled this transition. However, the role of the cellular microenvironment in enabling metazoan tissue genesis and evolution remains obscure. A collagen IV scaffold, a major component of the cellular microenvironment, is essential for tissues and dysfunctional in several diseases. This scaffold is stabilized by sulfilimine chemical bonds (S=N), which covalently crosslink methionine and hydroxylysine residues at the interface of adjoining triple-helical protomers, and are formed by peroxidasin via the production of hypohalous acids. Here we characterized the extracellular matrix (ECM), with a focus on collagen IV, of the eight major bilaterian phyla in comparison to the four non-bilaterian animal phyla. Furthermore, we explored whether the sulfilimine crosslink is a fundamental requirement in the genesis and evolution of epithelial tissues by determining its occurrence and evolutionary origin, and its essentiality in early development. The results show that the crosslink is conserved throughout Bilateria and Cnidaria, and arose at the divergence of Porifera and Cnidaria over 500 million years ago. Morpholino knockdown of peroxidasin in zebrafish revealed that the crosslink is essential for organogenesis. Within Ctenophora, we identified basement membrane (BM) and collagen IV, and show that the structural and genomic features of collagen IV are homologous to those of non-bilaterian animal phyla and Bilateria. Yet, ctenophore features are more diverse and distinct, expressing up to twenty genes compared to six in vertebrates. Moreover, collagen IV is absent in unicellular sister-groups. Collectively, these findings establish that the triad—a collagen IV scaffold with sulfilimine crosslinks, peroxidasin, and hypohalous acids—is a primordial innovation of the ECM essential for organogenesis and tissue evolution. Furthermore, we conclude that collagen IV is a primordial component of the extracellular microenvironment that enabled the transition from unicellular organisms to multicellular animals, and as a component of BM, collagen IV enabled the assembly of a fundamental architectural unit for multicellular tissue genesis