Joshua W Ziel et al. (2007) International Worm Meeting "UNC-6/Netrin orients the Anchor Cell for Invasion."

David R Sherwood, Joshua W Ziel, Anjon Auydha

[International Worm Meeting, 2007]

Understanding the cellular circuitry governing cell-invasive behavior and basement membrane degradation is critical for designing strategies to delay or prevent tumor cell metastasis in vivo. Analysis of Anchor Cell (AC) invasion into the vulval epithelium allows us to address this question using the high-resolution genetic and cell-biological strengths of C. elegans. Our previous work has shown that AC invasion is correlated with the production of a migratory cue by the primary Vulval Precursor Cells (VPC). To extend these findings, we have characterized the intra-cellular distribution of F-actin and the membrane lipid PI(4,5)P2, known markers of a migratory leading edge, over the time-course of AC invasion. In the AC, both markers are strikingly concentrated along the basal membrane and define an invasive cellular surface prior to frank degradation of the basement membrane. In addition, we find that known regulators of the actin cytoskeleton such as the Rac orthologs MIG-2 and CED-10, as well as UNC-34/Enabled also localize to the invasive membrane, clearly demonstrating that migratory activity is under tight spatial control in wild-type ACs. To better understand the signals that polarize the migratory response within the AC, we have examined mutations in known guidance pathways. Null mutations in unc-6/Netrin or unc-40/DCC result in penetrant AC invasion defects. Genetically these mutations enhance the phenotypes of mutants unable to produce the vulval cue, and thus comprise a second pathway that collaborates with the vulval signal to promote invasion. Intriguingly, the UNC-6 signal and the vulval cue behave differently with respect to the localization leading edge markers, including MIG-2/Rac. We find that UNC-6 is required for early establishment the invasive basal surface, whereas the vulval cue is dispensable, at least during early stages, for leading edge formation. These data suggest that UNC-6/Netrin and the vulval stimulus are qualitatively distinct while informing AC migration. We describe models that may explain directed migration during AC invasion and experiments we hope will distinguish them.

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.