Much of our work is concerned with LIN-12/Notch, the receptor component of one of the major signaling systems for specifying cell fate during animal development. 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.

In more recent extensions of our work, our studies of regulatory circuitry in the AC/VU decision have led us to study programming and reprogramming of cell fate in gonadogenesis, and our studies of signaling integration in VPC fate specification have stimulated our interest in biosensors for signaling pathway activity. We are also studying how signal transduction is modulated in dauer larvae, a natural state of prolonged developmental arrest that allows worms to survive harsh environmental conditions.

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
A “bHLH code” for sexual dimorphism of gonad form and function
Temporal control, life history and 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.
Cell fate transformation
The α and β cells are reprogrammed into male Distal Tip Cells (mDTCs) when LIN-32 and HLH-8 are expressed

The ventral uterus of hermaphrodites is derived from four proximal 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.  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.

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.

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 degraded in the VUs via a dimerization-dependent process. We are currently investigating the regulation and mechanism of dimerization-driven HLH-2 degradation and other aspects of transcriptional regulation of lag-2 and lin-12.

The AC is one of three “regulatory cells” of the hermaphrodite (female) somatic gonad primordium, and serves to organize uterine and vulval fate pattern and to forge the connection between the two; the two hermaphrodite Distal Tip Cells (hDTCs) control gonad arm outgrowth, leading to the characteristic U-shape, and serve as the germline niche.   In males, there are also three regulatory cells, which have similar roles apportioned differently among them.  We have identified a “bHLH code” for somatic gonad regulatory cell identity and are studying how upstream inputs connect through bHLH genes to diverse outputs for terminal features and testing if differences in bHLH complements contribute to the evolutionary plasticity of gonad form seen in nematodes.

The somatic gonad primordium forms in the L2 stage, and the other cells present are precursor cells that will divide in the L3 stage, ultimately to generate cells that form the main structures.  We are currently investigating the mechanisms that prevent progression of gonad development in dauer larvae, a prolonged quiescent state that results when environmental conditions are unfavorable. 

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 Sallee, M.D.*, Littleford, H.E.* and Greenwald, I. (2017) A bhlh code for sexually dimorphic form and function of the C. elegans somatic gonad.Curr. Biol. 27, 1853-1860.  PMID: 28602651
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VPC specification

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

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 mechanisms that oppose undesirable temporal or spatial signal transduction by activated LIN-12/Notch. In addition, we have developed a biosensor that allows for investigation of ERK signaling quantitatively and dynamically in vivo, and are attempting to develop a biosensor for LIN-12/Notch for similar purposes.       

During continuous development, EGFR-Ras- ERK pathway and developmental timing regulators oppose potent constitutively active forms of LIN-12. In dauer larvae, a prolonged quiescent state that results when environmental conditions are unfavorable, DAF-16/ FoxO opposes potent constitutively active components of both the EGFR and LIN-12/Notch pathways.

Our current work aims to understand how LIN-12 and other components of its signaling mechanism are regulated to achieve the precise and robust spatial pattern, and how progression of vulval fate specification is halted and even reversed in dauer larvae.

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) 110, 2181-2186. PMID: 23341633  de la Cova, C., Townley, R., Regot, S.* and Greenwald, I.* (2017) A real-time biosensor for ERK activity reveals signaling dynamics during C. elegans cell fate specification. Dev Cell 42, 542-553. PMID: 28826819Underwood, R.S., Deng, Y. and Greenwald, I. (2017) Integration of EGFR and LIN-12/Notch signaling by LIN-1/Elk1, the Cdk8 kinase module, and SUR-2/Med23 in Vulval Precursor Cell fate patterning in Caenorhabditis elegans. Genetics 207, 1473-1488. PMID: 28954762
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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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