SUMMARYPeroxidasins form a highly conserved family of extracellular peroxidases of unknown cellular function. We identified the C. elegans peroxidasin PXN-2 in screens for mutants defective in embryonic morphogenesis. We find that PXN-2 is essential for specific stages of embryonic morphogenesis and muscle-epidermal attachment, and is also required postembryonically for basement membrane integrity. The peroxidase catalytic activity of PXN-2 is necessary for these developmental roles. pxn-2 mutants display aberrant ultrastructure of the extracellular matrix, suggesting a role in basement membrane consolidation. PXN-2 affects specific axon guidance choice points in the developing nervous system but is dispensable for maintenance of process positions. In adults, loss of pxn-2 function promotes regrowth of axons after injury, providing the first evidence that C. elegans extracellular matrix can play an inhibitory role in axon regeneration. Loss of function in the closely related C. elegans peroxidasin pxn-1 does not cause overt developmental defects. Unexpectedly, pxn-2 mutant phenotypes are suppressed by loss of function in pxn-1 and exacerbated by overexpression of wild-type pxn-1, indicating that PXN-1 and PXN-2 have antagonistic functions. These results demonstrate that peroxidasins play crucial roles in development and reveal a new role for peroxidasins as extracellular inhibitors of axonal regeneration.

Tetrahydrobiopterin (BH4) is the natural cofactor of several enzymes widely distributed among eukaryotes, including aromatic amino acid hydroxylases (AAAHs), nitric oxide synthases (NOSs), and alkylglycerol monooxygenase (AGMO). We show here that the nematode Caenorhabditis elegans, which has three AAAH genes and one AGMO gene, contains BH4 and has genes that function in BH4 synthesis and regeneration. Knockout mutants for putative BH4 synthetic enzyme genes lack the predicted enzymatic activities, synthesize no BH4, and have indistinguishable behavioral and neurotransmitter phenotypes, including serotonin and dopamine deficiency. The BH4 regeneration enzymes are not required for steady-state levels of biogenic amines, but become rate limiting in conditions of reduced BH4 synthesis. BH4-deficient mutants also have a fragile cuticle and are generally hypersensitive to exogenous agents, a phenotype that is not due to AAAH deficiency, but rather to dysfunction in the lipid metabolic enzyme AGMO, which is expressed in the epidermis. Loss of AGMO or BH4 synthesis also specifically alters the sensitivity of C. elegans to bacterial pathogens, revealing a cuticular function for AGMO-dependent lipid metabolism in host–pathogen interactions.

Basement membranes are extracellular matrices essential for embryonic development in animals. Peroxidasins are extracellular peroxidases implicated in the unique sulfilimine cross-links between type IV basement membrane collagens. Loss of function in the Caenorhabditis elegans peroxidasin PXN-2 results in fully penetrant embryonic or larval lethality. Using genetic suppressor screening, we find that the requirement for PXN-2 in development can be bypassed by gain of function in multiple genes encoding other basement membrane components, or proteins implicated in cell-matrix attachment. We identify multiple alleles of let-805, encoding the transmembrane protein myotactin, which suppress phenotypes of pxn-2 null mutants and of other basement membrane mutants such as F-spondin/spon-1. These let-805 suppressor alleles cause missense alterations in two pairs of FNIII repeats in the extracellular domain; they act dominantly and have no detectable phenotypes alone, suggesting they cause gain of function. We also identify suppressor missense mutations affecting basement membrane components type IV collagen (emb-9, let-2) and perlecan (unc-52), as well as a mutation affecting spectraplakin (vab-10), a component of the epidermal cytoskeleton. These suppressor alleles do not bypass the developmental requirement for core structural proteins of the basement membrane such as laminin or type IV collagen. In conclusion, putative gain-of-function alterations in matrix proteins or in cell-matrix receptors can overcome the requirement for certain basement membrane proteins in embryonic development, revealing previously unknown plasticity in the genetic requirements for the extracellular matrix.