Forces drive basement membrane invasion in Caenorhabditis elegans.

During invasion, cells breach basement membrane (BM) barriers with actin-rich protrusions. It remains unclear, however, whether actin polymerization applies pushing forces to help break through BM, or whether actin filaments play a passive role as scaffolding for targeting invasive machinery. Here, using the developmental event of anchor cell (AC) invasion in Caenorhabditis elegans, we observe that the AC deforms the BM and underlying tissue just before invasion, exerting forces in the tens of nanonewtons range. Deformation is driven by actin polymerization nucleated by the Arp2/3 complex and its activators, whereas formins and cross-linkers are dispensable. Delays in invasion upon actin regulator loss are not caused by defects in AC polarity, trafficking, or secretion, as appropriate markers are correctly localized in the AC even when actin is reduced and invasion is disrupted. Overall force production emerges from this study as one of the main tools that invading cells use to promote BM disruption in C. elegans.

Rotational manipulation of single cells and organisms using acoustic waves.

The precise rotational manipulation of single cells or organisms is invaluable to many applications in biology, chemistry, physics and medicine. In this article, we describe an acoustic-based, on-chip manipulation method that can rotate single microparticles, cells and organisms. To achieve this, we trapped microbubbles within predefined sidewall microcavities inside a microchannel. In an acoustic field, trapped microbubbles were driven into oscillatory motion generating steady microvortices which were utilized to precisely rotate colloids, cells and entire organisms (that is, C. elegans). We have tested the capabilities of our method by analysing reproductive system pathologies and nervous system morphology in C. elegans. Using our device, we revealed the underlying abnormal cell fusion causing defective vulval morphology in mutant worms. Our acoustofluidic rotational manipulation (ARM) technique is an easy-to-use, compact, and biocompatible method, permitting rotation regardless of optical, magnetic or electrical properties of the sample under investigation.

Sumoylated NHR-25/NR5A regulates cell fate during C. elegans vulval development.

Individual metazoan transcription factors (TFs) regulate distinct sets of genes depending on cell type and developmental or physiological context. The precise mechanisms by which regulatory information from ligands, genomic sequence elements, co-factors, and post-translational modifications are integrated by TFs remain challenging questions. Here, we examine how a single regulatory input, sumoylation, differentially modulates the activity of a conserved C. elegans nuclear hormone receptor, NHR-25, in different cell types. Through a combination of yeast two-hybrid analysis and in vitro biochemistry we identified the single C. elegans SUMO (SMO-1) as an NHR-25 interacting protein, and showed that NHR-25 is sumoylated on at least four lysines. Some of the sumoylation acceptor sites are in common with those of the NHR-25 mammalian orthologs SF-1 and LRH-1, demonstrating that sumoylation has been strongly conserved within the NR5A family. We showed that NHR-25 bound canonical SF-1 binding sequences to regulate transcription, and that NHR-25 activity was enhanced in vivo upon loss of sumoylation. Knockdown of smo-1 mimicked NHR-25 overexpression with respect to maintenance of the 3° cell fate in vulval precursor cells (VPCs) during development. Importantly, however, overexpression of unsumoylatable alleles of NHR-25 revealed that NHR-25 sumoylation is critical for maintaining 3° cell fate. Moreover, SUMO also conferred formation of a developmental time-dependent NHR-25 concentration gradient across the VPCs. That is, accumulation of GFP-tagged NHR-25 was uniform across VPCs at the beginning of development, but as cells began dividing, a smo-1-dependent NHR-25 gradient formed with highest levels in 1° fated VPCs, intermediate levels in 2° fated VPCs, and low levels in 3° fated VPCs. We conclude that sumoylation operates at multiple levels to affect NHR-25 activity in a highly coordinated spatial and temporal manner.

Genetic control of vulval development in Caenorhabditis briggsae.

The nematode Caenorhabditis briggsae is an excellent model organism for the comparative analysis of gene function and developmental mechanisms. To study the evolutionary conservation and divergence of genetic pathways mediating vulva formation, we screened for mutations in C. briggsae that cause the egg-laying defective (Egl) phenotype. Here, we report the characterization of 13 genes, including three that are orthologs of Caenorhabditis elegans unc-84 (SUN domain), lin-39 (Dfd/Scr-related homeobox), and lin-11 (LIM homeobox). Based on the morphology and cell fate changes, the mutants were placed into four different categories. Class 1 animals have normal-looking vulva and vulva-uterine connections, indicating defects in other components of the egg-laying system. Class 2 animals frequently lack some or all of the vulval precursor cells (VPCs) due to defects in the migration of P-cell nuclei into the ventral hypodermal region. Class 3 animals show inappropriate fusion of VPCs to the hypodermal syncytium, leading to a reduced number of vulval progeny. Finally, class 4 animals exhibit abnormal vulval invagination and morphology. Interestingly, we did not find mutations that affect VPC induction and fates. Our work is the first study involving the characterization of genes in C. briggsae vulva formation, and it offers a basis for future investigations of these genes in C. elegans.

Conserved mechanism of Wnt signaling function in the specification of vulval precursor fates in C. elegans and C. briggsae.

The C. elegans hermaphrodite vulva serves as a paradigm for understanding how signaling pathways control organ formation. Previous studies have shown that Wnt signaling plays important roles in vulval development. To understand the function and evolution of Wnt signaling in Caenorhabditis nematodes we focused on C. briggsae, a species that is substantially divergent from C. elegans in terms of the evolutionary time scale yet shares almost identical morphology. We isolated mutants in C. briggsae that display multiple pseudo-vulvae resulting from ectopic VPC induction. We cloned one of these loci and found that it encodes an Axin homolog, Cbr-PRY-1. Our genetic studies revealed that Cbr-pry-1 functions upstream of the canonical Wnt pathway components Cbr-bar-1 (beta-catenin) and Cbr-pop-1(tcf/lef) as well as the Hox target Cbr-lin-39 (Dfd/Scr). We further characterized the pry-1 vulval phenotype in C. briggsae and C. elegans using 8 cell fate markers, cell ablation, and genetic interaction approaches. Our results show that ectopically induced VPCs in pry-1 mutants adopt 2° fates independently of the gonad-derived inductive and LIN-12/Notch-mediated lateral signaling pathways. We also found that Cbr-pry-1 mutants frequently show a failure of P7.p induction. A similar, albeit low penetrant, defect is also observed in C. elegans pry-1 mutants. The genetic analysis of the P7.p induction defect revealed that it was caused by altered regulation of lin-12 and its transcriptional target lip-1 (MAP kinase phosphatase). Thus, our results provide evidence for LIN-12/Notch-dependent and independent roles of Wnt signaling in promoting 2 degrees VPC fates in both nematode species.