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Cell-Cell Interactions and Cell Fate Specification in Development
Summary: Iva Greenwald's work is focused on LIN-12/Notch function in cell-cell interactions that specify cell fate during development.
During animal development, intercellular communication plays important roles in specifying cell fates. Work from my laboratory has helped establish that cell-cell interactions in Caenorhabditis elegans involve the conserved LIN-12/Notch signaling system. Human homologs of some of the components of the signaling system have been implicated in devastating and unfortunately all-too-common diseases: cancer and Alzheimer's disease. Our work has ranged from elucidating the role of lin-12 in cell fate decisions to identifying and characterizing factors that influence lin-12 activity.
The AC/VU Decision
We have concentrated on a simple model for intercellular communication that involves signaling between only two cells, Z1.ppp and Z4.aaa. These two cells are in the developing hermaphrodite gonad. Initially each cell has an equal chance of becoming the anchor cell (AC), a terminally differentiated cell type, or a ventral uterine precursor cell (VU), which contributes descendants to the ventral uterus. In any given hermaphrodite, however, only one of these cells will become the AC, while the other becomes a VU: in half of all hermaphrodites, Z1.ppp becomes the AC observed; in the other half, Z4.aaa becomes the AC. This AC/VU decision is always specified correctly, so that only one of the two cells becomes the AC, because Z1.ppp and Z4.aaa communicate with each other.
Our work in the past established that LIN-12 serves as a receptor for signaling between Z1.ppp and Z4.aaa. During the AC/VU decision, LIN-12 and its ligand, LAG-2, are initially expressed in both Z1.ppp and Z4.aaa. A stochastic variation in ligand and/or receptor activity, correlated with birth order, is subsequently amplified by a feedback mechanism that involves differential transcription of lin-12 and lag-2. As a consequence of this feedback mechanism, one cell eventually expresses only lag-2 and becomes the AC, while the other cell expresses only lin-12 and becomes the VU. The feedback mechanism ensures that the AC/VU decision is always specified correctly.
We have found that negative regulation of the basic helix-loop-helix (bHLH) protein HLH-2, a positive factor for lag-2 transcription, is a step in the circuit that leads to negative regulation of lag-2 in the presumptive VU. Our results suggest that activation of LIN-12 leads to post-transcriptional down-regulation of HLH-2, thereby extinguishing lag-2 transcription. We are working to identify cis-acting sequences and trans-acting factors that regulate HLH-2 accumulation. By manipulating these parameters, we hope to understand both the mechanism and role of HLH-2 down-regulation in the AC/VU decision.
VPC Specification
Six vulval precursor cells (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°. This pattern is the outcome of several different intercellular signaling events mediated by different signal transduction pathways. 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.
We have been particularly interested in how two of these events are integrated. 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 (MAP) kinase cascade, and a lateral signal between VPCs, mediated by LIN-12. We have characterized three different modes for crosstalk between the RTK/Ras/MAP kinase cascade and the LIN-12/Notch pathway during vulval development. First, we have shown that the lateral signal is expressed in the presumptive 1° cell under direct transcriptional control of the inductive signaling pathway. Second, we have shown that one consequence of activating Ras is a reduction of LIN-12 protein in the presumptive 1° cell, and that this reduction must occur for the lateral signal to be operational. Third, we found a group of genes that are direct transcriptional targets of LIN-12/Notch function redundantly as negative regulators of the epidermal growth factor receptor–MAP kinase pathway. These mechanisms ensure that the inductive and lateral signaling events are sequenced and spatially restrained so that the correct pattern of cell fates is invariantly specified.
The lateral signal also promotes the 2° fate. In searching for potential LIN-12 target genes with this role, we identified two microRNAs that appear to be expressed in the presumptive 2° cells. When misexpressed in the presumptive 1° cell, these microRNAs cause it to adopt 2° fate characteristics. We have thus far focused on the characterization of the role of the microRNA mir-61 and its target, VAV-1. We have found that mir-61 is transcribed in response to LIN-12 activation and that VAV-1 is a negative regulator of lin-12 activity. Our results suggest that mir-61 leads to post-transcriptional down-regulation of VAV-1, which promotes LIN-12 activity in presumptive 2o VPCs. Preliminary characterization of the second microRNA and its target suggest that they form another negative regulatory circuit of this type.
Genetic studies from several different labs suggest that other signaling events also impact on VPC fates. Our endeavors in this area suggest, however, that there will be major new additions and changes to the VPC specification paradigm. Recently, in collaboration with Min Han (HHMI, University of Colorado at Boulder), we obtained surprising results that question the prevalent view that there is an "inhibitory signal" from the hyp7 hypodermal syncytium. We are pursuing our recent results suggesting that Wnt signaling may be integrated with the canonical inductive and lateral signaling events in unanticipated ways.
Identification of Genes That Modulate lin-12 Activity
LIN-12/Notch is activated by binding of a ligand of the DSL (Delta/Serrate/LAG-2) family. Ligand binding induces proteolytic cleavages of LIN-12/Notch that culminate in the release of the intracellular domain, which translocates to the nucleus. The intracellular domain forms a complex with the sequence-specific DNA-binding protein LAG-1 in C. elegans and activates the transcription of target genes.
For many years, we have used genetic screens to identify both positive and negative influences on lin-12 signaling. Screens for positive factors have yielded components of protease complexes that mediate the proteolytic processing events that constitute signal transduction and components of the nuclear complex that promote target gene expression. Screens for negative factors have principally yielded factors involved in stability or transport of LIN-12. In the past, we used conventional genetic analysis to identify such modulating factors; we are now using RNAi (RNA interference) screens.
Work in my laboratory is also supported by a grant from the National Institutes of Health.
Last updated: May 9, 2006
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