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.
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.
Lateral specification and feedback mechanisms during gonadogenesis
A “bHLH code” for sexual dimorphism of gonad form and function
Temporal control, life history and gonadogenesis
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 a 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 have been using high-throughput lineage analysis in a microfluidic device, CRISPR/Cas9-generated, fluorescently-tagged endogenous genes, and a novel Notch activity sensor we have developed to understand how this edge develops. There are two inter-related stochastic elements: one encompasses the relative birth order and the amount of time between the birth of the α cells, which in turn influences the relative order of onset of expression of the transcription factor HLH-2 in the parents of the α cells. Using the biosensor, we have been able to visualize the α cells gaining an edge in LIN-12 activation during cell fate specification in real-time.
Current projects in the lab are continuing to take advantage of advances in genome engineering and microscopy, coupled with the unparalleled advantages of C. elegans for genetic analysis, to achieve a deeper mechanistic understanding of this paradigmatic cell fate decision. Other projects are concerned with sexual dimorphism in gonad development, and the regulation of gonadogenesis in response to temporal and environmental cues.
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.*, 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
Tenen, C.C. and Greenwald, I. (2019) Cell non-autonomous function of daf-18/PTEN in the somatic gonad coordinates somatic gonad and germline development in C. elegans dauer larvae. Curr. Biol. 29, 1064-1072. PMID: 30827916
Attner, M.A., Keil, W., Benavidez, J.M. and Greenwald, I. HLH-2/E2A expression links stochastic and deterministic elements of a cell fate decision during C. elegans gonadogenesis. (2019) Curr Biol. 29, 3094-3100. PMID: 31402303
Integrating LIN-12/Notch and EGFR-Ras-ERK signaling
Temporal control, life history and VPC patterning
Six VPCs, names 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, which activate 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 to achieve the correct pattern of VPC fates. We have also studied how environmental and temporal cues for developmental progression regulate signaling by studying signaling in dauer larvae, a prolonged quiescent state induced by adverse environmental conditions. For these studies we have developed tools for quantitative and dynamic studies of signaling in live worms, including genetically-encoded biosensors for ERK and LIN-12/Notch activity.
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
Identifying novel, conserved modulators on LIN-12/Notch activity
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. We therefore also assay these modulators in simple cell culture assays for effects on human Notch activity to begin to forge such a connection.
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