LIN-12/Notch is the receptor component of one of the major signaling systems for specifying cell fate during animal development. In addition, mutations in core components and modulators of the LIN-12/Notch pathway have been implicated in cancer, Alzheimer's disease, and other diseases and syndromes. Using C. elegans, we study how LIN-12/Notch signaling is modulated during normal development and identify potential ways it may be modulated to combat disease.

In our developmental studies, we aim to understand the fundamental logic and molecular events that govern cell fate decision-making. We mainly study two simple cell fate decision paradigms: the "AC/VU decision" and "VPC fate specification." These simple paradigms offer the opportunity to apply powerful methods of genetic analysis to fundamental mechanisms of cell fate specification that operate in all animals. During these events, different modulatory mechanisms regulate LIN-12/Notch activity and different signaling inputs are integrated so that correct cell fate decisions are made.

We also use sensitive and specific suppressor and enhancer screens in C. elegans for "gene discovery", with the aim of identifying new, conserved modulators of LIN-12/Notch and other signaling pathways.

LIN-12/Notch signaling

Lin-12/Notch signaling

The main events in signal transduction have been elucidated:

  • A ligand presented by a neighboring cell binds to the ectodomain of LIN-12, activating signal transduction by exposing a cleavage site in the ectodomain.
  • An ADAM protease such as SUP-17/ADAM10 mediates cleavage at this site, resulting in shedding of the ectodomain.
  • The resulting transmembrane stub is cleaved within the transmembrane domain by a multiprotein enzyme called "γ-secretase," which contains the catalytic subunit SEL-12/Presenilin.
  • The intracellular domain, thereby released from its tether, translocates to the nucleus.
  • The intracellular domain, in a complex with a sequence-specific DNA binding protein, promotes target gene expression.

Genetic analysis was crucial for elucidating the mechanism of signal tranduction and is now leading to deeper understanding of how signaling is modulated. In addition, this basic signaling mechanism enables strategies for identifying target genes and for identifying and analyzing new factors that influence signal transduction.

Greenwald, I. (2012) Notch and the awesome power of genetics. Genetics 191, 655-669. PMID: 22785620 Greenwald, I. and Kovall, R. (2013) Notch signaling: genetics and structure. WormBook.

AC/VU decision

Lateral specification and feedback mechanisms during gonadogenesis

AC/VU decision
The "α" and "β" cells, marked with nuclear GFP.
AC/VU decision
α cells express lin-12 and lag-2. Feedback causes positive autoregulation of lin-12 transcription and degradation of HLH-2 to downregulate lag-2 transcription in the presumptive VU.

The ventral uterus of hermaphrodites is derived from four cells of the somatic gonad primordium, two "α" cells and their sisters, the "β" cells. The two α cells undergo the "AC/VU decision", a simple paradigm for lateral specification. The β cells are the sisters of the α cells; they lose the potential to be the AC early and generally adopt a VU fate even in a lin-12 null mutant.

During the AC/VU decision, the two α cells communicate so that only one AC is made. Both cells initially express lin-12 and lag-2, the gene encoding its ligand for this decision. As the decision progresses, the transcription of both genes becomes mutually exclusive through feedback mechanisms. The α cell that has the "edge" in LIN-12 activation continues to express lin-12 and becomes the VU; the other α cell becomes the AC and continues to express lag-2.

We are interested in understanding what distinguishes α from β cells, the regulatory circuitry underlying feedback in the AC/VU decision, and how β cells adopt the VU fate in the absence of lin-12. The hlh-2 gene, which encodes the bHLH transcription factor ortholog of mammalian E2A, is critical for these processes. The hlh-2 gene is transcribed in the α and β cells, as well as in the AC and VUs, but HLH-2 protein is post-translationally downregulated in the VUs. We have recently found that HLH-2 undergoes dimerization-dependent degradation in VUs and that lin-12 activity contributes to the robustness of ╬▓VU fate.

Seydoux, G. and Greenwald, I. (1989) Cell autonomy of lin-12 function in a cell fate decision in C. elegans. Cell 57: 1237-1245. PMID: 2736627 Wilkinson, H.A., Fitzgerald, K., and Greenwald, I. (1994) Reciprocal changes in expression of lin-12 (receptor) and lag-2 (ligand) prior to commitment in a C. elegans cell fate decision. Cell 79, 1187-1198 PMID: 8001154 Karp, X. and Greenwald, I. (2003) Post-transcriptional regulation of the E/Daughterless ortholog HLH-2, negative feedback, and birth order bias during the AC/VU decision in C. elegans. Genes Dev. 17, 3100-3111. PMID: 14701877 Karp, X. and Greenwald, I. (2004) Multiple roles for the E/Daughterless ortholog HLH-2 during C. elegans gonadogenesis. Dev Biol. 272, 460-469. PMID: 15282161 Sallee, M.D. and Greenwald, I. (2015) Dimerization-driven degradation of C. elegans and human E proteins. Genes Dev. 29: 1356-1361. PMID: 26159995 Sallee, M.D., Aydin, T. and Greenwald, I. (2015) Influences of LIN-12/Notch and POP-1/TCF on the robustness of ventral uterine cell fate specification in Caenorhabditis elegans gonadogenesis. G3 (Bethesda) 5:2775-2782. PMID: 26483009
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VPC specification

Integrating LIN-12/Notch and EGFR-Ras-ERK signaling
Temporal control and life history

VPC Specification
Spatial patterning of vulval fates.
VPC Specification
The daughters of P5.p, P6.p and P7.p after spatial patterning (red membrane marker). In P6.p daughters, LIN-12::GFP (green) has been endocytosed and degraded in response to the inductive signal.

Six VPCs, named P3.p-P8.p, have the potential to generate vulval cells. In the L3 stage, the anchor cell of the gonad produces an EGF-like inductive signal. The inductive signal activates EGFR-Ras-ERK signaling in P6.p, causing it to adopt the "1o fate" and to produce a "lateral signal". The lateral signal consists of ligands for LIN-12/Notch. The lateral signal therefore activates LIN-12 in the neighboring cells, P5.p and P7.p, specifying them to adopt the "2o fate". The descendants of the 1o and 2o VPCs undergo morphogenesis to become the vulva.

This invariant and correct pattern of vulval fates depends on proper spatial and temporal control of these signaling pathways. We have identified multiple mechanisms that underlie crosstalk between the EGFR and LIN-12/Notch pathways as well as temporal and spatial blockades of signal transduction by activated LIN-12/Notch. During continuous development, EGFR-Ras- ERK pathway and developmental timing regulators can block even potent constitutively active forms of LIN-12. In dauer larvae, a prolonged quiescent state that results when environmental conditions are unfavorable, DAF-16/ FoxO mediates a block to potent constitutively active components of both the EGFR and LIN-12/Notch pathways. Our current work aims to understand the mechanisms by which these blocks are achieved.

Shaye, D.D. and Greenwald, I. (2002) Endocytosis-mediated downregulation of LIN-12/Notch upon Ras activation in C. elegans. Nature 420, 686-690. PMID: 12478297 Yoo, A.S., Bais, C., and Greenwald, I. (2004) Cross-talk between the EGF receptor-MAP kinase and LIN 12/Notch pathways in Caenorhabditis elegans vulval development. Science 303, 663-666. PMID: 14752159 Yoo, A.S. and Greenwald, I. (2005) LIN-12/Notch activation leads to microRNA-mediated downregulation of Vav in C. elegans. Science 310, 1330-1333. PMID: 16239437 Li, J. and Greenwald, I. (2010) Inhibition of lin-12/Notch by LIN-14: precision and timing of lateral signaling in vulval fate patterning. Current Biology 20, 1875-1879. PMID: 20951046 Zhang, X. and Greenwald, I. (2011) Spatial regulation of lag-2 transcription during vulval precursor cell fate patterning in Caenorhabditis elegans. Genetics 188, 847-858. PMID: 21596897 Karp, X. and Greenwald, I. (2013) Control of cell fate plasticity and maintenance of multipotency by DAF-16/FoxO in quiescent C. elegans. Proc. Natl. Acad. Sci. (USA), in press. PMID: 23341633
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Gene Discovery

Identifying novel, conserved modulators of LIN-12/Notch activity

Gene Discovery
2o fate marker expressed in all VPC daughters of lin-12(intra); kin(RNAi).
Gene Discovery
Enhanced activity of a mild lin-12 hypermorph by kin(RNAi).

Several core components of the LIN-12/Notch signaling system were first identified as suppressors of "lin-12(d)" mutations, which cause constitutive LIN-12/Notch activity and distinctive mutant phenotypes. For example, suppression of lin-12(d) phenotypes led to the identification of proteases that cleave in the ectodomain and transmembrane domain.

Other suppressors or enhancers may reveal modulators that affect signal strength or duration. By using RNAi for gene discovery, we aim to identify modulators that are conserved between C. elegans and humans. For example, we recently assessed all 240 predicted conserved protein kinases and found 12 previously unknown negative regulators of lin-12(d) activity.

Since lin-12(d) mutations are similar to mutations of human Notch identified in patients with T-cell Acute Lymphoblastic Leukemia (T-ALL), and alterations in the level of Notch activity is associated with other cancers, we hope that modulators we identify in C. elegans may have clinical relevance. Suppressor and enhancer assays are also being used to evaluate candidate genes obtained from whole genome sequencing of Notch- driven tumors.

Levitan, D. and Greenwald, I. (1995) Facilitation of lin-12-mediated signalling by sel-12, a C. elegans S182 Alzheimer's disease gene. Nature 377, 351-354. PMID: 7566091 Katic, I., Vallier, L. and Greenwald I. (2005) New positive regulators of lin-12 activity in Caenorhabditis elegans include the BRE-5/Brainiac glycosphingolipid biosynthesis enzyme. Genetics 17, 1605-1615. PMID: 16157663 de Souza, N., Vallier, L., Fares, H. and Greenwald, I. (2007) SEL-2, the C. elegans neurobeachin/ LRBA homolog, is a negative regulator of lin-12/Notch activity and affects endosomal traffic in polarized epithelial cells. Development 134, 691-702. PMID: 17215302 Dunn, C.D., Sulis, M.L., Ferrando, A.A. and Greenwald, I. (2010) A conserved tetraspanin subfamily promotes Notch signaling in C. elegans and in human cells. Proc. Natl. Acad. Sci. (USA) 107, 5907-5912. PMID: 20220101
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Other Projects

Other Projects
GFP-labeled excretory cell.
Other Projects
Top, activated LIN-45/Braf has a normal vulva (*). Bottom, phosphodegron mutation of activated LIN-45/Braf abrogates SEL-10/Fbw7-mediated negative feedback and causes ectopic vulval induction (arrows).

After an initial training period involving research in the mainstream of the Greenwald lab, postdoctoral fellows are encouraged to develop independent projects in their own areas of interest. Such projects provide the foundation for a successful transition to a career as an independent investigator.

Dan Shaye is working on tubulogenesis in the excretory cell. In parallel to developing cell markers and genetic screening approaches for mutants in this process, he compiled a list of C. elegans genes with human orthologs to allow any RNAi screen to be focused on conserved genes. In his initial RNAi screen, he focused on conserved kinases that are required for tubule formation, and is currently characterizing a subset of these kinases in detail. He also studied EXC-6, an ortholog of the human formin INF2, and found that it regulates MT and F-actin accumulation at EC tips and dynamics of basolateral-localized MTs during tubulogenesis.

Claire de a Cova is studying SEL-10, the C. elegans ortholog of human Fbw7, an important tumor suppressor. First identified as a modulator of LIN-12/Notch in our lab, SEL-10/Fbw7 is now known to target Notch and other oncogenic substrates for ubiquitin-mediated degradation. Claire devised a simple computational approach to identify novel potential SEL-10/Fbw7substrates and showed that it targets the C. elegans Braf ortholog LIN-45 via a phosphodegron recognition motif conserved in human Braf. She is currently working on mechanistic aspects of LIN-45/Braf activation and analyzing other potential SEL- 10/Fbw7 substrates.

Shaye, D.D. and Greenwald, I. (2011) OrthoList: a compendium of C. elegans genes with human orthologs. PLoS One. 2011;6(5):e20085. doi: 10.1371/journal.pone.0020085. Epub 2011 May 25. PMID: 21647448 de la Cova, C. and Greenwald, I. (2012) SEL-10/Fbw7-dependent negative feedback regulation of LIN-45/Braf signaling in C. elegans via a conserved phosphodegron. Genes Dev. 26, 2524-2535. PMID: 23154983 Shaye, D.D. and Greenwald, I. (2015). The disease-associated formin INF2/EXC-6 organizes lumen and cell outgrowth during tubulogenesis by regulating F-actin and microtubule cytoskeletons. Dev Cell 32: 743-755. PMID: 25771894.
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