The primary focus of my laboratory is the LIN-12/Notch signaling system. LIN-12/Notch signaling mediates cell-cell interactions that specify cell fate during animal development. Our work on regulatory circuitry underlying paradigmatic cell fate decisions in the nematodeCaenorhabditis elegans has established fundamental principles that are conserved across the animal kingdom. In addition, our work on identifying and characterizing core components and modulators of LIN-12/Notch signaling has helped elucidate mechanisms relevant to cancer and Alzheimer's disease.
Identification of Conserved Modulators of lin-12/Notch Activity
LIN-12/Notch proteins are transmembrane receptors. Binding of a Delta/Serrate–LAG-2 (DSL) ligand triggers proteolytic cleavages within the extracellular domain, mediated by transmembrane metalloprotease, and within the transmembrane domain, mediated by γ-secretase. These cleavages culminate in the release of the intracellular domain, which translocates to the nucleus and forms a complex with CBF1/Suppressor of Hairless/LAG-1 (CSL), a sequence-specific DNA-binding protein, converting CSL from a repressor to an activator of target gene transcription.
Missense mutations resulting in constitutive or elevated LIN-12/Notch signaling cause developmental abnormalities and distinctive mutant phenotypes. In mammals, equivalent missense mutations also cause cancer. For many years, we have isolated suppressors that correct the defects caused by constitutive lin-12activity in C. elegans. Such suppressors have defined core components and modulators of the LIN-12/Notch signaling system; for conserved components, analysis inC. elegans illuminates aspects of Notch signaling that are relevant to mammalian development and cancer.
In addition to suppressors that we have identified using standard mutagenesis screens, we have recently shifted our emphasis to conserved genes identified through analogous RNAi (RNA interference) screens. Such genes are not only potentially involved in Notch signaling per se but also are potential targets for anticancer therapies. In essence, we now use C. elegans principally for gene "discovery" and then test whether the mammalian orthologs facilitate Notch signaling in a mammalian cell culture context. For example, we identified a particular tetraspanin (a member of a large family of proteins thought to organize signaling microdomains) through suppression of an activated form of Notch in C. elegans; then, by extending the analysis to human cells, we found that its human orthologs play a positive role in Notch signaling and act at the γ-secretase cleavage step (collaboration with Maria Luisa Sulis and Adolfo Ferrando, Columbia University).
SEL-10/Fbw7, first identified in a different suppressor screen designed to identify negative regulators of lin-12, targets LIN-12/Notch for proteasome-mediated degradation and acts as a tumor suppressor. SEL-10/Fbw7 is an attractive candidate for mediating crosstalk between LIN-12/Notch and other pathways, a process potentially important in both normal development and disease contexts. We have identified one new substrate of SEL-10 in C. elegans that is involved in signal transduction and cell fate specification; we are currently investigating whether its mammalian ortholog, a known oncogene, is also a substrate of Fbw7, and we are expanding our analysis to additional substrates in both C. elegans and mammals.
Regulatory Circuitry Underlying Cell Fate Decisions and Spatial Patterning
We have concentrated on two simple paradigms for intercellular communication. These paradigms allow us to deduce fundamental logic and describe molecular events underlying cell fate decision making.
One paradigm is a simple lateral specification paradigm, the anchor cell (AC)/ventral uterine precursor cell (VU) decision. In this case, feedback mechanisms influence LIN-12/Notch activity such that signaling that is initially bidirectional becomes unidirectional.
During the AC/VU decision, LIN-12-mediated signaling between two equivalent cells ensures that only one of the two cells becomes the AC while the other becomes a VU. Both cells initially express LIN-12 and LAG-2, the ligand for LIN-12 in this decision. Activation of LIN-12 causes positive autoregulation of lin-12 transcription and post-translational down-regulation of a bHLH (basic helix-loop-helix) transcription factor needed for lag-2transcription. The cell that has the "edge" in LIN-12 activation continues to express lin-12 and becomes the VU, whereas the other cell adopts the "default" fate, to become an AC, and continues to express lag-2. We are continuing to identify and characterize other components of the circuitry, including microRNAs and their targets, with the goal of understanding the feedback amplification mechanism that dictates this decision. Such feedback mechanisms are a fundamental and intrinsic aspect of lateral specification/lateral inhibition in all animals.
In the other paradigm, vulval precursor cell (VPC) patterning, LIN-12/Notch signaling is integrated with other signaling inputs; in this case, the fundamental question we are investigating is how the different signaling events are integrated.
Six VPCs are arranged linearly along the ventral side of the hermaphrodite and in wild type have an invariant pattern of fates: 3°-3°-2°-1°-2°-3°. The descendants of the 1° and 2° VPCs will form the vulva; the daughters of the 3° VPCs fuse with the hypodermal syncytium that constitutes the major epidermis of the worm. The three VPCs that generate the vulva are patterned by an inductive signal from the anchor cell to the VPCs, mediated by a receptor tyrosine kinase (RTK)/Ras/mitogen-activated protein kinase (MAPK) cascade, and a lateral signal between VPCs, mediated by LIN-12.
We have discovered different modes for integrating the activity of the RTK/Ras/MAPK cascade and the LIN-12/Notch pathway that act in the signaling or receiving cells. In the presumptive 1° cell, the RTK/Ras/MAPK pathway controls the transcription of genes encoding ligands for LIN-12 and also directly stimulates endocytosis of LIN-12 to activate the lateral signal. In the presumptive 2° cells, LIN-12 directs transcription of genes that antagonize RTK/Ras/MAPK and also transcription of microRNAs that antagonize negative regulators of LIN-12. These mechanisms ensure that the inductive and lateral signaling events are sequenced properly and spatially restrained so that the correct pattern of cell fates is invariantly specified. As with the AC/VU decision, we are continuing to identify and characterize other components of the circuitry to achieve greater understanding of the molecular mechanisms underlying this decision.
Temporal and Environmental Inputs into VPC Specification
VPCs are born in the first larval stage (L1), yet vulval fates are not specified until L3. Several lines of evidence indicate that there are mechanisms for maintaining competence and preventing premature activation of spatial patterning pathways in L2. Our investigations in this area have included an examination of the contribution of Wnt and EGF (epidermal growth factor) signaling toward maintaining competence in L2 and the finding that the stage-specific regulator LIN-14 specifically antagonizes LIN-12-mediated 2° fate specification in L3. Our interests in this aspect of VPC specification have led us to begin to study how environmental stresses and entry into an alternative L3, the dauer larva, may influence LIN-12/Notch signaling and the "reprogramming" of committed VPC descendants back to a multipotential state.
This work is supported in part by a grant from the National Institutes of Health.
As of May 30, 2012