Identification of residues of the Caenorhabditis elegans LIN-1 ETS domain that are necessary for DNA binding and regulation of vulval cell fates

LIN-1 is an ETS domain protein. A receptor tyrosine kinase/Ras/mitogen-activated protein kinase signaling pathway regulates LIN-1 in the P6.p cell to induce the primary vulval cell fate during Caenorhabditis elegans development. We identified 23 lin-1 loss-of-function mutations by conducting several genetic screens. We characterized the molecular lesions in these lin-1 alleles and in several previously identified lin-1 alleles. Nine missense mutations and 10 nonsense mutations were identified. All of these lin-1 missense mutations affect highly conserved residues in the ETS domain. These missense mutations can be arranged in an allelic series; the strongest mutations eliminate most or all lin-1 functions, and the weakest mutation partially reduces lin-1 function. An electrophoretic mobility shift assay was used to demonstrate that purified LIN-1 protein has sequence-specific DNA-binding activity that required the core sequence GGAA. LIN-1 mutant proteins containing the missense substitutions had dramatically reduced DNA binding. These experiments identify eight highly conserved residues of the ETS domain that are necessary for DNA binding. The identification of multiple mutations that reduce the function of lin-1 as an inhibitor of the primary vulval cell fate and also reduce DNA binding suggest that DNA binding is essential for LIN-1 function in an animal.     

LIN-14 inhibition of LIN-12 contributes to precision and timing of C. elegans vulval fate patterning

Studies of C. elegans vulval development have illuminated mechanisms underlying cell fate specification and elucidated intercellular signaling pathways [1]. The vulval precursor cells (VPCs) are spatially patterned during the L3 stage by the EGFR-Ras-MAPK-mediated inductive signal and the LIN-12/Notch-mediated lateral signal. The pattern is both precise and robust [2] because of crosstalk between these pathways [3]. Signaling is also regulated temporally, because constitutive activation of the spatial patterning pathways does not alter the timing of VPC fate specification [4, 5]. The heterochronic genes, including the microRNA lin-4 and its target lin-14, constitute a temporal control mechanism used in different contexts [6-8]. We find that lin-4 specifically controls the activity of LIN-12/Notch through lin-14, but not other known targets, and that persistent lin-14 blocks LIN-12 activity without interfering with the key events of LIN-12/Notch signal transduction. In the L2 stage, there is sufficient lin-14 activity to inhibit constitutive lin-12. Our results suggest that lin-4 and lin-14 contribute to spatial patterning through temporal gating of LIN-12. We propose that in the L2 stage, lin-14 sets a high threshold for LIN-12 activation to help prevent premature activation of LIN-12 by ligands expressed in other cells in the vicinity, thereby contributing to the precision and robustness of VPC fate patterning.     

C. elegans LIN-18 is a Ryk ortholog and functions in parallel to LIN-17/Frizzled in Wnt signaling

Wnt proteins are intercellular signals that regulate various aspects of animal development. In Caenorhabditis elegans, mutations in lin-17, a Frizzled-class Wnt receptor, and in lin-18 affect cell fate patterning in the P7.p vulval lineage. We found that lin-18 encodes a member of the Ryk/Derailed family of tyrosine kinase-related receptors, recently found to function as Wnt receptors. Members of this family have nonactive kinase domains. The LIN-18 kinase domain is dispensable for LIN-18 function, while the Wnt binding WIF domain is required. We also found that Wnt proteins LIN-44, MOM-2, and CWN-2 redundantly regulate P7.p patterning. Genetic interactions indicate that LIN-17 and LIN-18 function independently of each other in parallel pathways, and different ligands display different receptor specificities. Thus, two independent Wnt signaling pathways, one employing a Ryk receptor and the other a Frizzled receptor, function in parallel to regulate cell fate patterning in the C. elegans vulva.     

sem-4 promotes vulval cell-fate determination in Caenorhabditis elegans through regulation of lin-39 Hox

Vulval cell-fate determination in Caenorhabditis elegans requires the action of numerous gene products, including components of the Ras/Raf/MAPK signaling cascade and the hox gene lin-39. sem-4 encodes a zinc finger protein with previously characterized roles in fate specification of sex myoblasts, coelomocytes, and multiple neuronal lineages in C. elegans (M. Basson and R. Horvitz, 1996, Genes Dev. 10, 1953-1965). By characterizing three new alleles of sem-4 that we identified in a screen for vulval-defective mutants, we determined that loss of sem-4 activity results in abnormal specification of the secondary vulval cell lineages. We analyzed sem-4 interactions with other genes involved in vulval differentiation and determined that sem-4 does not function directly in the Ras-mediated signal transduction pathway but acts in close association with and upstream of lin-39 to promote vulval cell fate. We demonstrate that sem-4 regulates lin-39 expression and propose that sem-4 is a regulator of lin-39 in the vulval cell-fate determination pathway that may act to link lin-39 to incoming signals.     

C. elegans EVI1 proto-oncogene, EGL-43, is necessary for Notch-mediated cell fate specification and regulates cell invasion

During C. elegans development, LIN-12 (Notch) signaling specifies the anchor cell (AC) and ventral uterine precursor cell (VU) fates from two equivalent pre-AC/pre-VU cells in the hermaphrodite gonad. Once specified, the AC induces patterned proliferation of vulva via expression of LIN-3 (EGF) and then invades into the vulval epithelium. Although these cellular processes are essential for the proper organogenesis of vulva and appear to be temporally regulated, the mechanisms that coordinate the processes are not well understood. We computationally identified egl-43 as a gene likely to be expressed in the pre-AC/pre-VU cells and the AC, based on the presence of an enhancer element similar to the one that transcribes lin-3 in the same cells. Genetic epistasis analyses reveal that egl-43 acts downstream of or parallel to lin-12 in AC/VU cell fate specification at an early developmental stage, and functions downstream of fos-1 as well as upstream of zmp-1 and him-4 to regulate AC invasion at a later developmental stage. Characterization of the egl-43 regulatory region suggests that EGL-43 is a direct target of LIN-12 and HLH-2 (E12/47), which is required for the specification of the VU fate during AC/VU specification. EGL-43 also regulates basement membrane breakdown during AC invasion through a FOS-1-responsive regulatory element that drives EGL-43 expression in the AC and VU cells at the later stage. Thus, egl-43 integrates temporally distinct upstream regulatory events and helps program cell fate specification and cell invasion.     

Structural requirements for the tissue-specific and tissue-general functions of the Caenorhabditis elegans epidermal growth factor LIN-3.

Caenorhabditis elegans lin-3 encodes a homolog of the epidermal growth factor (EGF) family of growth factors. LIN-3 is the inductive signal for hermaphrodite vulval differentiation, and it is required for animal viability, hermaphrodite fertility, and the specification of anterior cell fates in the male B cell lineage. We describe the cloning of a lin-3 homolog from C. briggsae, sequence comparison of C. elegans lin-3 with C. briggsae lin-3, and the determination of molecular lesions in alleles of C. elegans lin-3, including three new alleles. We also analyzed the severity of phenotypes caused by the new and existing alleles of lin-3. Correlation of mutant phenotypes and their molecular lesions, as well as sequence comparison between two species, reveal that the EGF motif and the N-terminal portion of the cytoplasmic domain are important for the functions of LIN-3 in all tissues, while the C-terminal portion of the cytoplasmic domain is involved in the tissue-specific functions of lin-3. We discuss how the structure of lin-3 contributes to its functions in multiple developmental processes.     

ced-10 Rac and mig-2 function redundantly and act with unc-73 trio to control the orientation of vulval cell divisions and migrations in Caenorhabditis elegans.

Vulval development in the nematode Caenorhabditis elegans can be divided into a fate specification phase controlled in part by let-60 Ras, and a fate execution phase involving stereotypical patterns of cell division and migration controlled in part by lin-17 Frizzled. Since the small GTPase Rac has been implicated as a downstream target of both Ras and Frizzled and influences cytoskeletal dynamics, we investigated the role of Rac signaling during each phase of vulval development. We show that the Rac gene ced-10 and the Rac-related gene mig-2 are redundantly required for the proper orientation of certain vulval cell divisions, suggesting a role in spindle positioning. ced-10 Rac and mig-2 are also redundantly required for vulval cell migrations and play a minor role in vulval fate specification. Constitutively active and dominant-negative mutant forms of mig-2 cause vulval defects that are very similar to those seen in ced-10;mig-2 double loss-of-function mutants, indicating that they interfere with the functions of both ced-10 Rac and mig-2. Mutations in unc-73 (a Trio-like guanine nucleotide exchange factor) cause similar vulval defects, suggesting that UNC-73 is an exchange factor for both CED-10 and MIG-2. We discuss the similarities and differences between the cellular defects seen in Rac mutants and let-60 Ras or lin-17 Frizzled mutants.     

sel-11 and cdc-42, Two Negative Modulators of LIN-12/Notch Activity in C. elegans

Background

LIN-12/Notch signaling is important for cell-cell interactions during development, and mutations resulting in constitutive LIN-12/Notch signaling can cause cancer. Loss of negative regulators of lin-12/Notch activity has the potential for influencing cell fate decisions during development and the genesis or aggressiveness of cancer.

Methodology/Principal Findings

We describe two negative modulators of lin-12 activity in C. elegans. One gene, sel-11, was initially defined as a suppressor of a lin-12 hypomorphic allele; the other gene, cdc-42, is a well-studied Rho GTPase. Here, we show that SEL-11 corresponds to yeast Hrd1p and mammalian Synoviolin. We also show that cdc-42 has the genetic properties consistent with negative regulation of lin-12 activity during vulval precursor cell fate specification.

Conclusions/Significance

Our results underscore the multiplicity of negative regulatory mechanisms that impact on lin-12/Notch activity and suggest novel mechanisms by which constitutive lin-12/Notch activity might be exacerbated in cancer.     

Control and integration of cell signaling pathways during C. Elegans vulval development

Vulval development in the Caenorhabditis elegans hermaphrodite represents a simple, genetically tractable system for studying how cell signaling events control cell fata decisions. Current models suggest that proper specification of vulval cell fates relies on the integration of multiple signaling systems, including one that involves a receptor tyrosine kinase (RTK)→Ras→mitogen activated protein kinase (MAPK) cascade and one that involves a LIN-12/Notch family receptor. In this review, we first discuss how genetic strategies are being used to identify and analyze components that control vulval cell fate decisions. We then describe the different signaling systems that have been elucidated and how they relate to one another. Finally, we highlight several recently characterized genes that encode positive regulators, negative regulators or potential targets of the RTK→Ras→MAPK cascade involved in vulval induction.