Nanoscale patterning of collagens in C. elegans apical extracellular matrix.

Apical extracellular matrices (aECMs) are complex extracellular compartments that form important interfaces between animals and their environment. In the adult C. elegans cuticle, layers are connected by regularly spaced columnar structures known as struts. Defects in struts result in swelling of the fluid-filled medial cuticle layer ('blistering', Bli). Here we show that three cuticle collagens BLI-1, BLI-2, and BLI-6, play key roles in struts. BLI-1 and BLI-2 are essential for strut formation whereas activating mutations in BLI-6 disrupt strut formation. BLI-1, BLI-2, and BLI-6 precisely colocalize to arrays of puncta in the adult cuticle, corresponding to struts, initially deposited in diffuse stripes adjacent to cuticle furrows. They eventually exhibit tube-like morphology, with the basal ends of BLI-containing struts contact regularly spaced holes in the cuticle. Genetic interaction studies indicate that BLI strut patterning involves interactions with other cuticle components. Our results reveal strut formation as a tractable example of precise aECM patterning at the nanoscale.

The basement membrane components nidogen and type XVIII collagen regulate organization of neuromuscular junctions in Caenorhabditis elegans.

Vertebrate neuromuscular junctions (NMJs) contain specialized basal laminas enriched for proteins not found at high concentrations extrasynaptically. Alterations in NMJ basement membrane components can result in loss of NMJ structural integrity and lead to muscular dystrophies. We demonstrate here that the conserved Caenorhabditis elegans basement membrane-associated molecules nidogen/entactin (NID-1) and type XVIII collagen (CLE-1) are associated with axons and particularly enriched near synaptic contacts. NID-1 is concentrated laterally, between the nerve cord and muscles, whereas CLE-1 is concentrated dorsal to the ventral nerve cord and ventral to the dorsal nerve cord, above the regions where synapses form. Mutations in these molecules cause specific and distinct defects in the organization of neuromuscular junctions. The mutant animals exhibit mild movement defects and altered responses to an inhibitor of acetylcholinesterase and a cholinergic agonist, indicating altered synaptic function. Our results provide the first demonstration that basement membrane molecules are important for NMJ formation and/or maintenance in C. elegans and that collagen XVIII and nidogen can have important roles in synapse organization.

Gene expression markers for Caenorhabditis elegans vulval cells.

The analysis of cell fate patterning during the vulval development of Caenorhabditis elegans has relied mostly on the direct observation of cell divisions and cell movements (cell lineage analysis). However, reconstruction of the developing vulva from EM serial sections has suggested seven different cell types (vulA, vulB1, vulB2, vulC, vulD, vulE, and vulF), many of which cannot be distinguished based on such observations. Here we report the vulval expression of seven genes, egl-17, cdh-3, ceh-2, zmp-1, B0034.1, T04B2.6 and F47B8.6 based on gfp, cfp and yfp (green fluorescent protein and color variants) reporter fusions. Each gene expresses in a specific subset of vulval cells, and is therefore useful as a marker for vulval cell fates. Together, expressions of markers distinguish six cell types, and reveal a strict temporal control of gene expression in the developing vulva.

Gene expression markers for Caenorhabditis elegans vulval cells.

The analysis of cell fate patterning during the vulval development of Caenorhabditis elegans has relied mostly on the direct observation of cell divisions and cell movements (cell lineage analysis). However, reconstruction of the developing vulva from EM serial sections has suggested seven different cell types (vulA, vulB1, vulB2, vulC, vulD, vulE, and vulF), many of which cannot be distinguished based on such observations. Here we report the vulval expression of seven genes, egl-17, cdh-3, ceh-2, zmp-1, B0034.1, T04B2.6 and F47B8.6 based on gfp, cfp and yfp (green fluorescent protein and color variants) reporter fusions. Each gene expresses in a specific subset of vulval cells, and is therefore useful as a marker for vulval cell fates. Together, expressions of markers distinguish six cell types, and reveal a strict temporal control of gene expression in the developing vulva.

C. elegans dystroglycan coordinates responsiveness of follower axons to dorsal/ventral and anterior/posterior guidance cues.

Neural development in metazoans is characterized by the establishment of initial process tracts by pioneer axons and the subsequent extension of follower axons along these pioneer processes. Mechanisms governing the fidelity of follower extension along pioneered routes are largely unknown. In C. elegans, formation of the right angle-shaped lumbar commissure connecting the lumbar and preanal ganglia is an example of pioneer/follower dynamics. We find that the dystroglycan ortholog DGN-1 mediates the fidelity of follower lumbar commissure axon extension along the pioneer axon route. In dgn-1 mutants, the axon of the pioneer PVQ neuron faithfully establishes the lumbar commissure, but axons of follower lumbar neurons, such as PVC, frequently bypass the lumbar commissure and extend along an oblique trajectory directly toward the preanal ganglion. In contrast, disruption of the UNC-6/netrin guidance pathway principally perturbs PVQ ventral guidance to pioneer the lumbar commissure. Loss of DGN-1 in unc-6 mutants has a quantitatively similar effect on follower axon guidance regardless of PVQ axon route, indicating that DGN-1 does not mediate follower/pioneer adhesion. Instead, DGN-1 appears to block premature responsiveness of follower axons to a preanal ganglion-directed guidance cue, which mediates ventral-to-anterior reorientation of lumbar commissure axons. Deletion analysis shows that only the most N-terminal DGN-1 domain is required for these activities. These studies suggest that dystroglycan modulation of growth cone responsiveness to conflicting guidance cues is important for restricting follower axon extension to the tracts laid down by pioneers.

Neural maintenance roles for the matrix receptor dystroglycan and the nuclear anchorage complex in Caenorhabditis elegans.

Recent studies in Caenorhabditis elegans have revealed specific neural maintenance mechanisms that protect soma and neurites against mispositioning due to displacement stresses, such as muscle contraction. We report that C. elegans dystroglycan (DG) DGN-1 functions to maintain the position of lumbar neurons during late embryonic and larval development. In the absence of DGN-1 the cell bodies of multiple lumbar neuron classes are frequently displaced anterior of their normal positions. Early but not later embryonic panneural expression of DGN-1 rescues positional maintenance, suggesting that dystroglycan is required for establishment of a critical maintenance pathway that persists throughout later developmental stages. Lumbar neural maintenance requires only a membrane-tethered N-terminal domain of DGN-1 and may involve a novel extracellular partner for dystroglycan. A genetic screen for similar lumbar maintenance mutants revealed a role for the nesprin/SYNE family protein ANC-1 as well as for the extracellular protein DIG-1, previously implicated in lumbar neuron maintenance. The involvement of ANC-1 reveals a previously unknown role for nucleus-cytoskeleton interactions in neural maintenance. Genetic analysis indicates that lumbar neuron position is maintained in late embryos by parallel DGN-1/DIG-1 and ANC-1-dependent pathways, and in larvae by separate DGN-1 and ANC-1 pathways. The effect of muscle paralysis on late embryonic- or larval-stage maintenance defects in mutants indicates that lumbar neurons are subject to both muscle contraction-dependent and contraction-independent displacement stresses, and that different maintenance pathways may protect against specific types of displacement stress.

Basement membrane sliding and targeted adhesion remodels tissue boundaries during uterine-vulval attachment in Caenorhabditis elegans.

Large gaps in basement membrane occur at sites of cell invasion and tissue remodelling in development and cancer. Though never followed directly in vivo, basement membrane dissolution or reduced synthesis have been postulated to create these gaps. Using landmark photobleaching and optical highlighting of laminin and type IV collagen, we find that a new mechanism, basement membrane sliding, underlies basement membrane gap enlargement during uterine-vulval attachment in Caenorhabditis elegans. Laser ablation and mutant analysis reveal that the invaginating vulval cells promote basement membrane movement. Further, an RNA interference and expression screen identifies the integrin INA-1/PAT-3 and VAB-19, homologue of the tumour suppressor Kank, as regulators of basement membrane opening. Both concentrate within vulval cells at the basement membrane gap boundary and halt expansion of the shifting basement membrane. Basement membrane sliding followed by targeted adhesion represents a new mechanism for creating precise basement membrane breaches that can be used by cells to break down compartment boundaries.

C. elegans dystroglycan DGN-1 functions in epithelia and neurons, but not muscle, and independently of dystrophin.

The C. elegans dystroglycan (DG) homolog DGN-1 is expressed in epithelia and neurons, and localizes to basement membrane (BM) surfaces. Unlike vertebrate DG, DGN-1 is not expressed in muscle or required for muscle function. dgn-1 null mutants are viable but sterile owing to severe disorganization of the somatic gonad epithelium, and show defects in vulval and excretory cell epithelia and in motoneuron axon guidance. The defects resemble those of epi-1 laminin alphaB mutants, suggesting that DGN-1 serves as a receptor for laminin. dgn-1(0)/+ animals are fertile but show gonad migration defects in addition to the defects seen in homozygotes, indicating that DGN-1 function is dosage sensitive. Phenotypic analyses show that DGN-1 and dystrophin-associated protein complex (DAPC) components have distinct and independent functions, in contrast to the situation in vertebrate muscle. The DAPC-independent functions of DGN-1 in epithelia and neurons suggest that vertebrate DG may also act independently of dystrophin/utrophin in non-muscle tissues.

Selective assembly of fibulin-1 splice variants reveals distinct extracellular matrix networks and novel functions for perlecan/UNC-52 splice variants.

Fibulin-1C and fibulin-1D splice variants have been conserved throughout metazoan evolution and have distinct functions in Caenorhabditis elegans development. Both splice variants are required for the assembly of hemidesmosome-mediated mechanosensory neuron and uterine attachments, although the molecular associations that underlie their distinct functions at these locations are not known. Here, we show that the assembly of fibulin-1C and fibulin-1D splice variants at these anchorages is dependent upon distinct components of the extracellular matrix (ECM): Fibulin-1D assembly at uterine and mechanosensory neurons attachments is dependent upon a perlecan/ UNC-52 splice variant that includes alternately spliced IG8-IG10, whereas the assembly of fibulin-1C at mechanosensory neuron attachments is dependent upon laminin/ EPI-1. These data not only indicate that fibulin-1C and fibulin-1D are components of distinct networks of ECM but also demonstrates a novel function for a major class of perlecan splice variants found in C. elegans and mouse. In addition, we demonstrate that overexpression of another ECM protein, collagen XVIII, can suppress gonad morphogenesis defects associated with loss of fibulin-1C, suggesting that some genetic defects that result in a weakened basement membrane can be compensated by overexpression of genes for ECM components that stabilize basement membranes.

Basement membranes.

Basement membranes are thin, specialized extracellular matrices surrounding most tissues in all metazoans. The compositions and functions of basement membranes have generally been well conserved throughout the subkingdom. Genetic analyses of basement membrane components in C. elegans have provided insights into their assembly and functions during development. Immuno- or GFP-tagged localization studies have shown that basement membranes on different tissues, or even sub-regions of tissues, contain different sets of proteins or alternatively spliced isoforms of them. Several components, including laminin, perlecan, type IV collagen and possibly osteonectin/SPARC, are essential for completion of embryogenesis, being necessary for tissue organization and structural integrity. In contrast, type XVIII collagen and nidogen are not required for viability but primarily influence organization of the nervous system. All of these proteins, with the exception of nidogen and the addition of fibulin, have roles of varying degree in morphogenesis of the gonad. A major family of cellular receptors for basement membrane proteins, the integrins, have also been characterized in C. elegans. As one might expect, integrins have been shown to function in many of the same processes as their potential ligands, the basement membrane components. While much remains to be explored, studies of basement membranes in C. elegans have been highly informative and hold great promise for improving our understanding of how these structures are assembled and how they function in development.

FOS-1 promotes basement-membrane removal during anchor-cell invasion in C. elegans.

Cell invasion through basement membranes is crucial during morphogenesis and cancer metastasis. Here, we genetically dissect this process during anchor-cell invasion into the vulval epithelium in C. elegans. We have identified the fos transcription factor ortholog fos-1 as a critical regulator of basement-membrane removal. In fos-1 mutants, the gonadal anchor cell extends cellular processes normally toward vulval cells, but these processes fail to remove the basement membranes separating the gonad from the vulval epithelium. fos-1 is expressed in the anchor cell and controls invasion cell autonomously. We have identified ZMP-1, a membrane-type matrix metalloproteinase, CDH-3, a Fat-like protocadherin, and hemicentin, a fibulin family extracellular matrix protein, as transcriptional targets of FOS-1 that promote invasion. These results reveal a key genetic network that controls basement-membrane removal during cell invasion.