Cali BM et al. (1995) International C. elegans Meeting "CHARACTERIZATION OF A NEW smg GENE, smg-7."

Anderson P, Cali BM

[International C. elegans Meeting, 1995]

Nonsense mutant RNAs are generally less stable than WT mRNAs. The six C. elegans smg genes are required for this nonsense-mediated mRNA decay. In smg mutants, otherwise unstable nonsense mRNAs accumulate to approximately WT levels. Nonsense-mediated mRNA decay is not essential. Null mutations in several smg genes eliminate nonsense-mediated mRNA decay, yet these animals are essentially WT. However, several other smg genes may have additional essential functions, as viable alleles of these genes are rare. Screens for essential smg genes are not saturated. Thus, we searched for new smg genes using a previously described screen (1993 Meeting Abstracts, p.255). smg mutants were isolated in a mut-5 background, in which Tc1 is active. One such mutant (r1131) appears to define a new smg gene: (1) r1131 exhibits the allele-specific but not gene-specific suppression pattern of other smg mutations, (2) r1131 complements smg-1 smg-6 alleles, (3) r1131 maps 0.2 m.u. right of smg-3, (4) unc-54 nonsense mRNAs accumulate to WT levels in r1131 animals. We identified a polymorphic Tc1 tightly linked to and inseparable from smg-7(r1131). Analysis of genomic and cDNA clones surrounding this Tc1 shows that the Tc1 is inserted into an exon of a gene encoding a 1.5 kb mRNA. This mRNA is trans-spliced to SL2 and appears to be cotranscribed with the upstream gene raf-1 (Nature, 363:133, 1993 and Andy Golden, pers. comm.). When expressed under control of the hsp16-2 promoter, a full length cDNA clone of this gene exhibits heat-shock dependent rescue of smg-7(r1131), establishing that this gene is smg-7. smg-7 is predicted to encode a novel 50 kDa protein with two notable features: (i.) a protein:protein interaction domain known as a tetratricopeptide repeat and (ii.) a negatively charged carboxy terminus. To test whether smg-7 is essential, we are isolating new alleles using a noncomplementation screen. Of three EMS-induced alleles isolated to date, two are recessive lethal and one is a weak viable allele. One lethal allele is a reciprocal translocation between LGIV and LGV; the other deletes >8 kb left and right of smg-7. Less complex alleles must be isolated to unambiguosly define the smg 7 null phenotype. Characterization of new smg-7 alleles, as well as progress in localizing SMG-7 immunologically, will be presented.

Kelley, Laura C et al. (2013) International Worm Meeting "Understanding the Role of MMPs In Basement Membrane Breaching In Vivo."

Kelley, Laura C, Chi, Qiuyi, Sherwood, David R, Matus, David Q

[International Worm Meeting, 2013]

Basement membrane (BM) is a dense sheet-like extracellular matrix that encapsulates and separates tissue compartments. The invasive ability of cells to cross BM barriers is required for normal and disease processes. Matrix metalloproteinases (MMPs) are overexpressed in cells responsible for tissue remodeling, wound healing, and cancer and are hypothesized to enzymatically facilitate BM removal. Due to the high number of MMPs expressed in vertebrates, the relevance and potential function MMPs in movement through BM is unclear. Our laboratory has combined high-resolution 4D imaging with the visually accessible and genetically tractable model of anchor cell (AC) invasion in C. elegans to analyze BM breaching in vivo. We have previously shown that the c-fos oncogene homologue, FOS-1A, promotes BM penetration during AC invasion (Sherwood and Sternberg, 2005). In fos-1a mutants, the AC extends invasive protrusions that are blocked at the BM, indicating that FOS-1A regulates the transcription of genes that mediate BM removal. Recently, we identified that FOS-1A regulates the expression of three MMPs in the AC during the time of invasion. In addition, two of the three remaining MMPs in the C. elegans genome are expressed in the tissues surrounding the invasive cell. Individual knockdown of these MMPs fails to cause defects in invasion suggesting that they act redundantly to promote invasion. To test this we genetically derived animals that lack all five MMPs expressed within the gonad prior to, and during the invasive process. Unexpectedly, AC invasion persists in the quintuple-MMP mutant animals, indicating that these proteases are not absolutely required for BM removal. Live-cell imaging of the BM, however, revealed distinct differences in the BM dynamics and architecture in MMP-deficient animals. In the MMP (-) mutants BM removal under the AC occurred at a slower rate. Further, BM clearing was disorganized and less uniform in the absence of MMPs. Thus, it appears that MMPs play a modulatory role during the invasive process, functioning to ensure more precise and rapid breaching of BM. These results also suggest that other transcriptional targets of FOS-1A must play a role alongside the MMPs in clearing BM.

McIntyre, Daniel et al. (2019) International Worm Meeting "Somatic gonad precursors assemble a basement membrane that guarantees primordial germ cell quiescence and gonad integrity during embryogenesis."

Nance, Jeremy, McIntyre, Daniel

[International Worm Meeting, 2019]

Somatic niche cells are often critical regulators of germline precursor cells, controlling their survival, proliferation and differentiation. We are investigating how niche cells and germline precursor cells interact with the surrounding basement membrane (BM) in the C. elegans embryo. We find that BM performs two critical roles in development: first, BM helps to establish and maintain niche cell wrapping during embryogenesis; second, BM guarantees primordial germ cell (PGC) quiescence in the embryo. The gonad primordium in C. elegans is a simple, 4-cell structure formed during embryogenesis that will give rise to the entire germ line and somatic gonad in the adult worm. It is comprised of two primordial germ cells (PGC), each of which interacts with a single somatic gonadal precursor cell (SGP). Each SGP extends its cell membrane around the body of a PGC, so as to completely enwrap it. Wrapping establishes a morphology that is carried into larval development, and provides a protective niche for the PGCs in the embryo. The value of this niche is demonstrated by unwrapped PGCs, which die unexpectedly when they are cannibalized by neighboring endodermal cells. We show that the SGPs are required to recruit laminin to the outer surface of the gonad primordium. If the BM is disrupted, SGP wrapping is not maintained and PGCs escape quiescence late in embryogenesis, rather than in fed L1 larva. These failures in development of the gonad primordium lead to the disorganized larval gonad and tumorous, sterile germ line previously reported in BM mutants. Finally, we find that the BM receptor DGN-1/dystroglycan is expressed by the SGPs on surfaces contacting the BM, is required to maintain SGP wrapping, but does not affect PGC quiescence. We conclude that SGPs template a BM that assures the integrity of the niche throughout embryogenesis and that PGCs remain quiescent until after hatching. These results show how a developmentally programed structure, the BM surrounding an organ primordium, plays both structural and signaling roles in the embryonic PGC niche.

SL Kuchma et al. (1999) International C. elegans Meeting "The cloning and characterization of smg-3, a gene required for mrna surveillance in C. elegans"

BM Cali, SL Kuchma, P Anderson

[International C. elegans Meeting, 1999]

Eukaryotic mRNAs containing premature termination codons are rapidly degraded relative to their wild-type counterparts, a phenomenon termed "nonsense-mediated mRNA decay" (NMD). In C. elegans , the functions of seven smg genes, smg-1 through smg-7 , are required for NMD. While nonsense mutant mRNAs are unstable in smg(+) genetic backgrounds, such mRNAs exhibit normal stability in smg(-) mutants. Thus, the smg genes function as a cellular mRNA surveillance system which prevents the accumulation of nonsense mutant messages. Such messages can be generated by somatic or germline mutations, or by errors in transcription, RNA processing, or transport. Nonsense mutant messages encode truncated polypeptides that can be deleterious to the cell. By eliminating such messages, NMD prevents the synthesis of potentially disruptive nonsense fragment polypeptides. We have cloned smg-3 in order to identify its role in NMD. smg-3 localizes to a cosmid gap in the physical map. This gap is spanned by a single 600 kb YAC, Y73B6, that has been shown by DNA transformation experiments to rescue smg-3 mutants. Using DNA sequence from Y73B6, BLAST searches reveal a region of homology to UPF2/NMD2 , a gene required for NMD in Saccharomyces cerevisiae . When used as a probe, DNA from this smg-3 candidate gene detects Southern blot polymorphisms associated with certain smg-3 mutations. For example, genetic evidence suggests that smg-3(r930) is a deletion mutation. In r930 mutants, DNA from the smg-3 region is undetectable on Southern blots. Northern blot analysis detects a message in wildtype that is absent in r930 mutants and altered in other smg-3 mutants. To confirm that this candidate gene is smg-3 and to uncover additional information about this gene, the following experiments are in progress: 1) DNA transformation experiments to complement smg-3 mutants; 2) Sequencing wild-type smg-3 cDNA to obtain the entire predicted protein sequence; and 3) Sequencing DNA from smg-3 mutants to identify causative mutations.

Elliott J. Hagedorn et al. (2007) International Worm Meeting "Time-lapse visualization of anchor cell invasion in C. elegans."

David R. Sherwood, Elliott J. Hagedorn

[International Worm Meeting, 2007]

A lack of in vivo models has hampered insight into the mechanisms driving cell-invasive behavior. The behavior of a single uterine cell in Caenorhabditis elegans called the anchor cell (AC) provides such an in vivo model, allowing for easy visualization and genetic manipulation. During normal development in the C. elegans hermaphrodite, the AC invades through the underlying juxtaposed uterine and ventral epidermal basement membranes (BM) to establish the initial uterine-vulval contact. In addition to the genetic tractability and ease of visualization, the consistency of this event, occurring at the same time in every wild-type hermaphrodite, makes for a powerful model of cell invasion. We have established an in vivo system to visualize anchor cell invasion as it occurs in real time using a laminin-GFP fusion (LAM-1::GFP, Kao and Wadsworth 2006, Dev Biol. 290(1): 211-219) or a hemicentin-GFP fusion (GFP-hemicentin, Vogel and Hedgecock 2001, Development 128(6): 883-894), both markers of the BM, in combination with a PH::mCherry (PH domain from PLC?, a gift from Anjon Audhya) driven by an AC-specific promoter, labeling the invasive membrane (Sherwood et al. 2005, Cell 121(6): 951-962). To investigate the real-time dynamics of invasion, we have begun to characterize AC invasion in wild-type animals. Prior to invasion there is a deposition of GFP-hemicentin and an apparent increase of LAM-1::GFP in the uterine BM directly below the AC, suggesting that the AC deposits or recruits additional BM components, prior to migration through it. Invasion appears to initiate with a single filopod extending through a tiny hole in the BM. Once this filopodial process reaches through the BM, possibly contacting vulval cells below, it begins to widen, often fanning out on the ventral side of the BM. This widening of the AC extension seems to be coincident with widening of the hole in the BM, as visualized by LAM-1::GFP. The initial characterization of wild-type invasion has brought about several questions. 1) How is the initial filopod generated, and how does it penetrate through the BM? 2) What dictates the precise location of the initial protrusive filopod? 3) What happens to the BM under the AC as the filopod widens? 4) Do other BM components similarly increase under the AC, how are they recruited there, and what function does their increase serve? It is our hope that through a detailed cell-biological analysis, in combination with genetic screens and mutant analysis, that we will gain insight into these questions and lead to a better understanding of the mechanisms that endow invasive cells with the ability to traverse basement membranes.

Caceres, R. et al. (2017) International Worm Meeting "Actin cytoskeleton dynamics in anchor cell invasion."

Sherwood, D., Kelly, L., Bojanala, N., Plastino, J., Caceres, R.

[International Worm Meeting, 2017]

Basement membrane (BM) is a dense sheet of specialised extracellular matrix, composed principally of laminin and type IV collagen polymers and glycoproteins that separate epithelia from underlying tissues. The invasion of cells through BM barriers is important during normal tissue development and in cancer metastasis. The breach in the BM is mediated by an actin-rich protrusion known as an invadopodium, and actin assembly dynamics in the invadopodia are essential for invasion. Much has been understood concerning the genetics and signalling of how holes are formed in the BM during invasion. However less is clear about the biophysics that is involved: how do cells exert mechanical forces via their actin cytoskeleton to make holes in BM barriers? To address this question, we study an invasion event in a developmental process, anchor cell (AC) invasion in Caenorhabditis elegans. We use mutants, RNAi treatments and dominant negative approaches to disrupt actin polymerization and interfere with actin binding proteins in the AC. Under these different perturbations, we evaluate the efficiency of invasion and observe the dynamics of the actin cytoskeleton during protrusion across the BM. In addition to the known role of WASP in cancer cell invasion, previous studies on AC invasion have shown the importance of the Arp2/3 complex activator WASP/WSP-1: WASP-null worms, wsp-1 (gm324), display only 18% full invasion at a developmental stage where WT worms show 100% invasion (Lohmer et al. 2016). Alternately, we expressed a dominant-negative form of an Arp2/3 complex activator specifically in the AC, which led to a complete block in invasion, confirming the importance of Arp2/3 complex-based actin polymerization for AC invasion.

C Huang et al. (1999) International C. elegans Meeting "Each C. elegans Laminin a Subunit Mediates Distinct Aspects of Morphogenesis"

WG Wadsworth, C Huang, PD Yurchenco

[International C. elegans Meeting, 1999]

Laminin is a heterotrimeric glycoprotein composed of a , b , and g subunits. The genome sequencing project reveals two a , one b and one g laminin subunit genes. Based on sequence analyses, major features and known binding sites are conserved. The predicted a chains, laminin a A and laminin a B, are most similar to the vertebrate a 1/ a 2 and a 5 chains respectively, while the b and g resemble the b 1/ b 2 and g 1 chains. Thus, there are two predicted laminin trimers, a A bg and a B bg . Mutations in epi-1 , which encodes laminin a B, have been isolated and characterized (abstract ECWM 98). They cause defects that are consistent with the developmental roles for basement membranes (BMs). We report the expression patterns of laminin a A and a B. By immunostaining, both first appear at gastrulation between germ layers. However, laminin a A is heavily deposited anteriorly, surrounding pharygeal precursors, whereas laminin a B is more posteriorly localized. Laminin a B becomes localized to the BMs associated with pharynx, intestine, gonad, body wall, body wall muscle, and muscles throughout development. In contrast, laminin a A accumulates in pharyngeal BM, intestinal BM and body wall muscle BM during elongation and its level in intestinal BM and body wall muscle BM gradually decreases. In larvae and adults, laminin a A is only sometimes detected weakly in pharyngeal BM and body wall muscle BM. It is primarily localized in the spermatheca. Injection of dsRNA directed to epi-1 produces phenotypes similar to those observed in epi-1 alleles and the injection of dsRNA directed to lam3 , which encodes laminin aB, causes L1 larval arrest with severe pharyngeal defects. Double dsRNA-mediated interference directed to both genes produces embryos that arrest at morphogenesis with phenotypes more severe than those caused by the RNAi of either epi-1 or lam3 alone, indicating that both genes contribute to morphogenesis. Together, our RNAi and immunostaining results indicate that laminin a B has a broad role in maintaining the structural integrity of BMs and for regulating many aspects of morphogenesis, while laminin a A is restricted to specialized membranes and is required for the morphogenesis of specific tissues.

G Kao et al. (1999) International C. elegans Meeting "Expression of Dominant Negative Forms of Laminin b: Evidence for a Functional Role for Polymerized Laminin in Basement Membranes."

W Wadaworth, G Kao, X Ren

[International C. elegans Meeting, 1999]

Laminin, a major component of all basement membranes (BM) is a heterotrimer of a , b and g subunits which trimerize along their long arms to form a cruciform with three short arms. There are pleiotropic effects on survival, reproduction and behavior when any laminin subunit is missing. Here we address the issue of BM assembly and function. Our understanding of the roles of the subunits during development, allows us to address a long standing issue of whether polymerized laminin is important for a functional BM. Biochemical studies have shown that trimers can polymerize via short arm interactions to form a 2-dimensional network. This can be blocked by adding trimers lacking short arms or by adding short arm fragments to compete for polymerization sites. It is not clear to what extent polymerization takes place in vivo and how important it is for a functional BM. If important, the biochemical studies predict that transgenes expressing short arm deletions in any subunit or over expressing a short arm ought to act in a dominant negative fashion. We tested this hypothesis by using the hsp16-2 promoter to drive expression of truncated versions of lam-1( b subunit) that encode only the short arm or have deletions in the short arm to varying extents. Transgenic embryos exhibit embryonic and L1 lethality with phenotypes similar to those caused by strong epi-1 ( a subunit) alleles. Heat shocked L1 animals survive and grow, but show defects in gonad migration and germline development. We see gonad arms without sperm or oocytes, with reduced germ cell proliferation, and with sperm but no oocytes. Gonadal sheath cells provide cues for germline development. In laminin mutants they often peel away from the arms due to defects in the BM. It is likely that perturbation of the BMs by these transgenes has the same effect on the sheath and hence on germline development. We are now examining the integrity of the BM in the heat shocked animals by electron microscopy and by a functional b :GFP fusion which labels all the BMs. These results provide the first evidence that laminin polymerizes in vivo and that polymerized laminin is required for BM functions b .

Aboobaker AA et al. (2000) European Worm Meeting "The evolution of the nematode homeoebox gene cluster"

Blaxter ML, Aboobaker AA

[European Worm Meeting, 2000]

Molecular phylogeny data, the structure of the C. elegans Hox cluster and available data of Hox gene function in other nematodes suggests that the nematode Hox cluster is undergoing rapid evolutionary change. How different are the genomic structures of nematode Hox clusters? Are there differences in the gene membership of nematode Hox clusters? How conserved are the Hox genes between nematodes with respect to sequence and function? These questions are being addressed by performing a series of comparisons with other nematode species. The nematode species P. pacificus (in which the study of Hox genes is advanced), B. malayi, T. spiralis and S. ratti represent a spectrum of distant to very close relationships to C. elegans. We have employed a degenerate PCR approach to identify Hox cluster cDNAs from B. malayi. So far we have identified six Brugia Antennapaedia family homeodomains, including Bm-pal-1, Bm-mab-5, Bm-lin-39 and Bm-egl-5. Two B. malayi Hox-like genes have no homologues in the C. elegans genome. Some T. spiralis Hox genes have also been identified. We have also isolated Bm-hox-20, a homolog of Ce-ceh-20 and Drosophila extradenticle, which are Hox cofactors. The B. malayi cluster is being mapped by hybridisation of Hox genomic clones to gridded genomic BAC libraries. B. malayi homologs of other genes within the C. eleganscluster are being used as probes to assess conservation of synteny between nematode Hox clusters. We hope to be able to assess the mode and tempo of evolution in the nematode Hox cluster by elucidating its structure and content in a cross section of the phylum.

Hanako Yashiro et al. (2007) International Worm Meeting "b (PAT-3) Integrin Plays Multiple Roles during Anchor Cell Invasion in C. elegans."

Hanako Yashiro, David R Sherwood

[International Worm Meeting, 2007]

Cell invasion through basement membranes (BM) is required in various developmental processes and is misregulated in diseases such as cancer. We investigate the underlying mechanisms regulating cell-invasive behavior by studying anchor cell (AC) invasion in C. elegans. During development, the gonadal AC invades through the BM to contact the underlying vulval precursor cells, which send a diffusible cue that guides AC invasion. In a whole-genome RNAi screen, we found that RNAi against both the <font face=symbol>b</font> integrin ina-1 and <font face=symbol>b</font>pat-3 caused defects in AC invasion. We also found that a loss-of-function allele in ina-1(gm39) showed a moderately penetrant invasion defect. Furthermore, in wild-type transgenic animals, PAT-3::GFP localization is increased at the basolateral invasive surface of the AC, suggesting a direct role in promoting invasion To further investigate the role of integrin, we have driven a dominant-negative construct of pat-3 (pAC&#62;HA-<font face=symbol>b</font>tail, a gift from Dr. Jean Schwarzbauer) specifically in the AC. In integrated lines of pAC&#62;HA-<font face=symbol>b</font>tail, AC invasion is severely disrupted. Examination of laminin::GFP, a BM marker, showed that the AC fails to breach the BM in pAC&#62;HA-<font face=symbol>b</font>tail worms and instead detaches from the membrane at the time of invasion. Notably, the AC fails to send cellular protrusions toward the vulval cells after detachment, suggesting an inability to respond to the vulval cue. Recent work in our lab has shown that the Rac protein MIG-2, F-actin, and PtdIns(4,5)P₂ localize to the basolateral membrane of the AC where they promote the formation of filapodia in response to the vulval cue. We have found that GFP::MIG-2 and PH::mCherry (a PtdIns(4,5)P₂-specific promoter) polarity at the invasive AC membrane is retained in pAC&#62;HA-<font face=symbol>b</font>tail animals as long as there is contact between the AC and the BM. Once the AC is detached, however, the polarization is significantly reduced, suggesting that integrin-mediated attachment of the AC to the BM is required to maintain polarity along the invasive membrane. Finally, we studied the loss of integrin in the AC on BM dynamics at the site of invasion by examining the localization of hemicentin::GFP. Hemicentin is a conserved extracellular matrix protein that is deposited under the AC at the site of invasion, where it promotes the movement of the AC through the BM. Strikingly, in pAC&#62;HA-<font face=symbol>b</font>tail worms, hemicentin is still made in the AC but is not deposited. Together, these results indicate that integrin function in the AC is crucial for establishing a properly organized invasive cell membrane and regulates the composition of the BM at the site of invasion.