James M. Kramer

Efficient gene transfer in C.elegans: extrachromosomal maintenance and integration of transforming sequences.

We describe a dominant behavioral marker, rol‐6(su‐1006), and an efficient microinjection procedure which facilitate the recovery of Caenorhabditis elegans transformants. We use these tools to study the mechanism of C.elegans DNA transformation. By injecting mixtures of genetically marked DNA molecules, we show that large extrachromosomal arrays assemble directly from the injected molecules and that homologous recombination drives array assembly. Appropriately placed double‐strand breaks stimulated homologous recombination during array formation. Our data indicate that the size of the assembled transgenic structures determines whether or not they will be maintained extrachromosomally or lost. We show that low copy number extrachromosomal transformation can be achieved by adjusting the relative concentration of DNA molecules in the injection mixture. Integration of the injected DNA, though relatively rare, was reproducibly achieved when single‐stranded oligonucleotide was co‐injected with the double‐stranded DNA.

The Caenorhabditis elegans rol-6 gene, which interacts with the sqt-1 collagen gene to determine organismal morphology, encodes a collagen.

The rol-6 gene is one of the more than 40 loci in Caenorhabditis elegans that primarily affect organismal morphology. Certain mutations in the rol-6 gene produce animals that have the right roller phenotype, i.e., they are twisted into a right-handed helix. The rol-6 gene interacts with another gene that affects morphology, sqt-1; a left roller allele of sqt-1 acts as a dominant suppressor of a right roller allele of rol-6. The sqt-1 gene has previously been shown to encode a collagen. We isolated and sequenced the rol-6 gene and found that it also encodes a collagen. The rol-6 gene was identified by physical mapping of overlapping chromosomal deficiencies that cover the gene and by identification of an allele-specific restriction site alteration. The amino acid sequence of the collagen encoded by rol-6 is more similar to that of the sqt-1 collagen than to any of the other ten C. elegans cuticle collagen sequences compared. The locations of cysteine residues flanking the Gly-X-Y repeat regions of rol-6 and sqt-1 are identical, but differ from those in the other collagens. The sequence similarities between rol-6 and sqt-1 indicate that they represent a new collagen subfamily in C. elegans. These findings suggest that these two collagens physically interact, possibly explaining the genetic interaction seen between the rol-6 and sqt-1 genes.

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.

Structures and functions of collagens in Caenorhabditis elegans.

Two types of collagens have been identified in Caenorhabditis elegans corresponding to two types of extracellular matrix, the cuticle and basement membranes. Cuticle collagens are encoded by a developmentally regulated family of ~100 genes. Mutations in cuticle collagens can produce animals that are longer or shorter than normal and/or that are helically twisted. Mutations in different collagens can cause different morphological abnormalities, as can different mutations in the same collagen. Genetic interactions between collagen genes have been described and may identify collagens that interact to form the cuticle. Two basement membrane (type IV) collagen genes have been identified in C. elegans. They encode proteins similar in structure to vertebrate type IV collagen. One of the genes produces two alternatively spliced forms, one predominantly expressed in embryos and the other in larvae and adults, suggesting that embryonic basement membranes may have unique properties. Most mutations in the type IV genes cause embryonic lethality, indicating that normal basement membranes are required for embryogenesis. Temperature‐sensitive mutations have been used to show that type IV collagen function is also required for larval development and adult fertility.—Kramer, J. M. Structures and functions of collagens in Caenorhabditis elegans. FASEB J. 8: 329‐336; 1994.

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.

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.

Number and organization of collagen genes in Caenorhabditis elegans.

We analyzed the number and organization of collagen genes in the nematode Caenorhabditis elegans. Genomic Southern blot hybridization experiments and recombinant phage library screenings indicated that C. elegans has between 40 and 150 distinct collagen genes. A large number of recombinant phages containing collagen genes were isolated from C. elegans DNA libraries. Physical mapping studies indicated that most phage contained a single small collagen gene less than 3 kilobases in size. A few phage contained multiple collagen hybridizing regions and may contain a larger collagen gene or several tightly linked small collagen genes. No overlaps were observed between phages containing different collagen genes, implying that the genes are dispersed in the C. elegans genome. Consistent with the small size of most collagen genes, we found that the predominant class of collagen mRNA in C. elegans is 1.2 to 1.4 kilobases in length. Genomic Southern blot experiments under stringent hybridization conditions revealed considerable sequence diversity among collagen genes. Our data suggest that most collagen genes are unique or are present in only a few copies.

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 αB 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.

In vitro mutagenesis of Caenorhabditis elegans cuticle collagens identifies a potential subtilisin-like protease cleavage site and demonstrates that carboxyl domain disulfide bonding is required for normal function but not assembly.

The importance of conserved amino acids in the amino and carboxyl non-Gly-X-Y domains of Caenorhabditis elegans cuticle collagens was examined by analyzing site-directed mutations of the sqt-1 and rol-6 collagen genes in transgenic animals. Altered collagen genes on transgenic arrays were shown to produce appropriate phenotypes by injecting in vivo cloned mutant alleles. Equivalent alterations in sqt-1 and rol-6 generally produced the same phenotypes, indicating that conserved amino acids in these two collagens have similar functions. Serine substitutions for either of two conserved carboxyl domain cysteines produced LRol phenotypes. Substitution for both cysteines in sqt-1 also resulted in an LRol phenotype, demonstrating that disulfide bonding is important for normal function but not required for assembly. Arg-1 or Arg-4 to Cys mutations in homology block A (HBA; consensus, 1-RXRRQ-5; in the amino non-Gly-X-Y domain) caused RRol phenotypes, while the same alteration at Arg-3 had no effect, indicating that Arg-3 is functionally different from Arg-1 and Arg-4. Substitutions of Arg-4 with Ser, Leu, or Glu also produced the RRol phenotype, while Lys substitutions for Arg-1 or Arg-4 did not generate any abnormal phenotypes. His substitutions for Arg-1 or Arg-4 caused somewhat less severe RRol phenotypes. Therefore, strong positively charged residues, Arg or Lys, are required at positions 1 and 4 for normal function. The conserved pattern of arginines in HBA matches the cleavage sites of the subtilisin-like endoproteinases. HBA may be a cleavage site for a subtilisin-like protease, and cleavage may be important for cuticle collagen processing.

Cuticle chirality and body handedness in Caenorhabditis elegans

Caenorhabditis elegans adult animals exhibit an inherent chirality of fiber orientation in the basal layer of the cuticle, as well as a naturally invariant but experimentally reversible handedness in the left-right (L-R) asymmetry of the body plan. We have examined the relationship between cuticle chirality and body handedness in normal and L-R reversed animals, using Roller (Rol) mutants and transmission electron microscopy to monitor cuticle properties. Rol phenotypes, several of which have been shown to result from mutations in cuticle collagen genes, are characterized by an invariant, allele-specific handedness in their direction of rolling. We show for several alleles that this direction is not affected by L-R reversal of the body plan. We further show, by electron microscopy, that the chiral orientation of cuticle fibers in animals with normal cuticle is not reversed by L-R body-plan reversal. We conclude that cuticle chirality must be established independently of body-plan handedness. The cues that establish cuticle chirality are still unknown, as are the causes for different rolling directions in different Roller mutants. We discuss the question of how cuticle chirality maintains its independence, and how the orientations of the fiber layers may be determined.