Combined extracellular matrix cross-linking activity of the peroxidase MLT-7 and the dual oxidase BLI-3 is critical for post-embryonic viability in Caenorhabditis elegans.

The nematode cuticle is a protective collagenous extracellular matrix that is modified, cross-linked, and processed by a number of key enzymes. This Ecdysozoan-specific structure is synthesized repeatedly and allows growth and development in a linked degradative and biosynthetic process known as molting. A targeted RNA interference screen using a cuticle collagen marker has been employed to identify components of the cuticle biosynthetic pathway. We have characterized an essential peroxidase, MoLT-7 (MLT-7), that is responsible for proper cuticle molting and re-synthesis. MLT-7 is an active, inhibitable peroxidase that is expressed in the cuticle-synthesizing hypodermis coincident with each larval molt. mlt-7 mutants show a range of body morphology defects, most notably molt, dumpy, and early larval stage arrest phenotypes that can all be complemented with a wild type copy of mlt-7. The cuticles of these mutants lacks di-tyrosine cross-links, becomes permeable to dye and accessible to tyrosine iodination, and have aberrant collagen protein expression patterns. Overexpression of MLT-7 causes mutant phenotypes further supporting its proposed enzymatic role. In combination with BLI-3, an H2O2-generating NADPH dual oxidase, MLT-7 is essential for post-embryonic development. Disruption of mlt-7, and particularly bli-3, via RNA interference also causes dramatic changes to the in vivo cross-linking patterns of the cuticle collagens DPY-13 and COL-12. This points toward a functionally cooperative relationship for these two hypodermally expressed proteins that is essential for collagen cross-linking and proper extracellular matrix formation.

Caenorhabditis elegans exoskeleton collagen COL-19: an adult-specific marker for collagen modification and assembly, and the analysis of organismal morphology.

The integral role that collagens play in the morphogenesis of the nematode exoskeleton or cuticle makes them a useful marker in the examination of the collagen synthesizing machinery. In this study, a green fluorescent protein-collagen fusion has been constructed by using the Caenorhabditis elegans adult-specific, hypodermally synthesized collagen COL-19. In wild-type nematodes, this collagen marker localized to the circumferential annular rings and the lateral trilaminar alae of the cuticle. Crosses carried out between a COL-19::GFP integrated strain and several morphologically mutant strains, including blister, dumpy, long, small, squat, and roller revealed significant COL-19 disruption that was predominantly strain-specific and provided a structural basis for the associated phenotypes. Disruption was most notable in the cuticle overlying the lateral seam cell syncytium, and confirmed the presence of two distinct forms of hypodermis, namely the circumferentially contracting lateral seam cells and the laterally contracting ventral-dorsal hypodermis. The effect of a single aberrant collagen being sufficient to mediate widespread collagen disruption was exemplified by the collagen mutant strain dpy-5 and its disrupted COL-19::GFP and DPY-7 collagen expression patterns. Through the disrupted pattern of COL-19 and DPY-7 in a thioredoxin mutant, dpy-11, and through RNA interference of a dual oxidase enzyme and a vesicular transport protein, we also show the efficacy of the COL-19::GFP strain as a marker for aberrant cuticle collagen synthesis and, thus, for the identification of factors involved in the construction of collagenous extracellular matrices.