Cell Fate Control
A major ongoing focus has been the role of peptide growth factors in controlling cell fate patterns. We have analyzed the roles of LIN-3, a nematode homolog of human epidermal growth factor (EGF), and LET-23, its receptor, a homolog of human EGF receptor. We studied several roles for this ligand-receptor pair including induction of the hermaphrodite vulva, induction of the P12 neuroblast fate, male spicule induction, hermaphrodite ovulation, and induction of the uterine uv1 cells by the vulva. For all but ovulation, LET-23 signals via a pathway utilizing the C. elegans homolog of the RAS proto-oncogene. Hermaphrodite ovulation is of interest because it is stimulated by LET-23 via a signaling pathway distinct from that mediated by RAS. This RAS-independent pathway involves regulation of inositol trisphosphate and presumably intracellular calcium. We have found that two enzymes that decrease inositol trisphosphate levels act as negative regulators of this pathway during ovulation; elimination of one gene leads to ovulation of two or more oocytes instead of one, as in the wild type.
Expression of LIN-3 in the anchor cell of the gonad induces the vulva. We have found a small region of the lin-3 gene that directs its expression specifically in the anchor cell; by studying this element, we are learning how the state of anchor cell differentiation is programmed. After vulval induction, the vulval precursor cells generate cells that differentiate as vulval cells (of which there are seven types) and undergo morphogenesis to form the mature vulva. We have developed a panel of yellow and cyan fluorescent protein markers for these terminally differentiated cells and are elucidating how multiple signaling pathways and a number of transcriptional regulatory proteins interact to control expression of these genes. Two WNT signaling pathways act together to control the polarity of one of the vulval precursor cells. One of these signaling pathways involves a receptor-type tyrosine kinase-related protein; another pathway involves a classical WNT receptor. After these interactions, particular differentiated vulval cells connect to the anchor cell and the uterus. As part of this process, the anchor cell breaks down the basement membrane separating the gonad and vulva, and invades the vulval epithelium; this process guides the ultimate attachment of the uterus and vulva. We have found several genes that are involved in these processes. Analysis of these genes suggests that distinct transcriptional programs regulate the inductive and invasive functions of the anchor cell.
Another intense focus has been on negative regulators of the LET-23-mediated signaling pathway. The types of mutations that activate LET-23-mediated signaling are analogous to mutations that contribute to human tumors by activating proto-oncogenes. Thus mutations that abolish gene function and result in increased LET-23-mediated signaling will define negative regulators and are analogous to tumor-suppressor genes. These genes include ones that encode regulators of membrane trafficking, transmembrane tyrosine phosphatases, and nuclear factors. Our observation that activated Gq increases vulval differentiation led to our discovery that under certain growth conditions, excitable cells signal to the developing vulva and can increase the extent of vulval differentiation; this signal appears to act in parallel to LET-23.
We have continued to analyze the mating behavior of the C. elegans male to understand how genes control neuronal function and to identify new proteins involved in neuronal function. By ablating cells and observing mating behavior, we dissected the behavior into several steps, and we are identifying genes used at many of these steps. For example, some mutants fail to turn at the end of the hermaphrodite; others fail to transfer sperm. We have developed assays for male attraction to hermaphrodites and found that hermaphrodites produce a diffusible signal detected by males.
By studying how males sense the hermaphrodite, we discovered the lov-1 gene, necessary for two aspects of male sensory function, and found that it encodes a homolog of polycystin-1, a human protein that when mutated leads to polycystic kidney disease. We found that mutation of pkd-2, a homolog of human polycystin-2, has the same phenotypes as mutation of lov-1, thereby validating this model for polycystin genetics. LOV-1 and PKD-2 are both expressed in three classes of male-specific sensory neurons, consistent with these genes acting together to mediate male sensory function. We are extending this analysis to find genes involved with LOV-1 and PKD-2. We have found additional mutants with dual sensory defects characteristic of lov-1 mutants; two of these are alleles of pkd-2. (The National Institutes of Health has provided support to continue this study in collaboration with former postdoc Maureen Barr.) The polycystins, divergent members of the transient receptor potential (TRP) family of calcium channels, piqued our interest in the classic members of this family, the TRPC proteins. We have screened for deletions of all three TRPC genes in C. elegans and have started to analyze their functions. We have found that one member of the TRPC family of calcium channels, TRP-3, is necessary for fertilization; this channel is active only in mature sperm.
Male Tail Development
To examine how broadly used regulatory pathways combine to specify cell fates, and how the specificity of LET-23 signaling is determined, we have initiated analyses of neuroblast P12 development and male hook development. As part of this effort, we are using mutations in existing genes and screening for mutations that perturb P12 or hook development to compare the genetic requirements for vulval induction, P12 specification, and hook patterning. The male sensory neuron HOB is part of the hook, and in a screen for mutants with defective HOB development, we found a transcriptional regulator that controls HOB-specific gene expression, namely the lov-1 and pkd-2 genes. This ciliated sensory neuron also requires a general ciliated-neuron pattern of gene expression, and the HOB-specific gene expression depends on the cilia-specific regulatory pathway.
By studying how neurons and muscles control the twitching and protraction of the copulatory spicules, we are defining a molecular pathway that controls one important motor output for mating behavior. Genes that control spicule protraction have been identified by screens for mutant males that either cannot protract their spicules, or protract them in the absence of mating partners, and we are identifying the proteins affected. One of these proteins is the C. elegans version of a human potassium channel involved in hereditary cardiac arrhythmia.
We have also used our knowledge of nematode development to address issues in the evolution of development: How do control circuits change during evolution to cause observed differences between species in morphology and behavior? We have initiated developmental genetic studies of C. briggsae, a close relative of C. elegans whose genome has just been sequenced. We are focusing on two processes-vulval development and dauer formation-for which there are known, albeit subtle, differences between the two species. We have identified hundreds of C. briggsae mutants and have a draft genetic map. In collaboration with Barbara Wold (California Institute of Technology), we have started a pilot project to analyze cis-regulatory sequences in two other Caenorhabditis species and to determine what one learns from having multiple genomic sequences to align. (This project is funded in part by the Department of Energy.) Knowledge of gene structure in C. elegans is fairly advanced, but there is little experimental evidence in C. briggsae. We have devised a method for rapidly identifying the positions of trans-splicing events at the 5' ends of nematode mRNAs. We have applied this to the SL1 and SL2 spliced leaders of C. elegans and C. briggsae and have found new genes and alternate 5' ends in C. elegans. We are extending this to C. briggsae.
We participate in an international effort to organize information about C. elegans genomics, genetics, and biology and present this information in an Internet-accessible database, WormBase. Our main contribution is to extract information from the literature, focusing on gene, protein, and cell function. To help speed up this process, we have developed a useful search engine for the C. elegans literature, Textpresso. (This effort is funded by the National Human Genome Research Institute.) In collaboration with Robert Stirbl (Jet Propulsion Laboratory) and Jehoshua Bruck (California Institute of Technology), we are developed tools for automated analysis of behavior (this was supported by a grant from the Defense Advanced Research Projects Agency).