Cell differentiation/signal transductionCell-cell interactions play key roles in directing developmental pattern formation. Vulval development provided us with both an excellent model system to elucidate the mechanism of cell differentiation and pattern formation, and a sensitive assay for analyzing the in vivo functions of many known cell signaling molecules and for identifying new components in a defined signaling pathway. During the development of the nematode C. elegans hermaphrodite, six equipotent precursor cells differentiate to generate two different tissues: three cells are induced to generate a vulva, and three become part of the epidermis. This process is controlled by an inductive signal from the anchor cell of the gonad and an inhibitory signal from the epidermis. The transduction of the inductive signal is controlled by a common signal transduction pathway mediated by a receptor tyrosine kinase and the Ras protein. The research in our laboratory has been focusing on the roles of Ras, Raf and many other molecules in the vulval signal transduction pathway.
To identify new genes that act downstream of Ras in the signaling pathway, we have isolated more than 100 mutations (in >15 genes) that suppress either the Multivulva phenotype of an activated let-60 ras mutation or the Vulvaless phenotype of a dominant negative let-60 ras mutation. We have cloned five of these genes (mek-2, sur-1, sur-2, sur-5, and ksr-1) that appear to act downstream of Ras. The mek-2 gene encodes a MAP kinase kinase and the sur-1 gene encodes a MAP kinase. Loss-of- function mutations in both genes cause a Vulvaless phenotype, while gain-of-function mutations of mek-2 result in a Multivulva phenotype. The sur-2 gene encodes a novel protein that is essential for vulval cell fate specification and appears to act downstream of sur-1 /mpk in the pathway. The ksr-1 gene encodes a novel protein kinase that is likely to act downstream of Ras but upstream or in parallel to Raf in the signaling pathway. The sur-5 gene encodes a novel protein that act negatively on the Ras pathway. We are also mapping and analyzing a number of suppressors that appear to define new genes. ksr-1, sur-5 and several other new genes are likely to function as modifiers and regulators of the pathway. By characterizing these genes, we hope to understand the regulation the Ras pathway and the connection between the common signal transduction pathway with specific developmental function such as vulval cell differentiation.
Mutations in many genes of the vulval signaling pathway cause pleiotropic defects, suggesting that the Ras-mediated signal transduction pathway plays a role in many other aspects of C. elegans development. We are interested to analyze the function of the Ras pathway in other developmental events and also hope to elucidate the specificity of this comonly used the pathway in specific developmental events.
Cell migration sex-myoblast (SM) migration. We have discovered that let-60 ras and several other genes are required for proper migration of sex-myoblasts (SMs). During L2 larval stage, two SMs migrate anteriorly from the tail region to the middle of the gonad which is thought to provide signals for positioning the cells during the migration. We have found that loss of function mutations in ksr-1, let-60 ras, lin-45 raf, mek-2 and sur-1 cause defects in SM final positions that resemble those seen in gonad-ablated animals, while constitutively active let-60 ras (G13E) transgenes allow precise SM positioning to occur in the absence of the gonad. Our results suggest that gonadal signals normally stimulate let-60 ras activity, and that signaling through let-60 ras is required for SMs to become competent to sense or respond to positional cues that determine the precise endpoint of their migration. We have also investigated genetic interactions between let-60 ras and egl-15 FGFR which play important roles in SM migration. Our results suggest that let-60 ras and egl-15 FGFR do not act together in a single linear pathway. We are currently carrying out mosaic analysis of let-60 ras to study its roles in various developmental processes.
hypodermal P cell migration
We have begun to study the function of the unc-83 and unc-84 genes. The study of these genes was initiated in R. Horvitz's laboratory (MIT).Our study has started by cloning both genes and by analyzing them using molecular and cytological means. Mutations in either gene disrupt nuclear migration of P cells causing severe Unc and Vul phenotypes due to missing neurons and vulval cells. We have already cloned the unc-84 gene and are close to cloning unc-83. The study of these two genes should reveal interesting aspects of cell and nuclei migration.
lrp-1 gene. The lrp-1 gene encodes a homolog of human LDL receptor-Related Protein or LRP. John Yochem initiated the study of this gene when he was a postdoctoral fellow in Iva Greenwald_s lab (Princeton) and has been studying its role in C. elegans. Little is known about the cellular function of LRP, though knocking out LRP in mice results in dramatical mutant phenotypes. LRP may also be linked to Alzheimer's disease. Using various methods, Dr. Yochem has been trying to isolate and characterize mutations in the lrp gene.
rrp-2 gene. We cloned by PCR screening the rrp-1 and rrp-2 genes, both of which are very similar to both Ras and Rap-1. The rrp-2 gene is particularly interesting because it has an effector domain nearly identical to that of Ras or Rap-1 (one aa difference), and it is expressed exclusively in vulval cells after L3 larval stage.
Phosphatase genes. In collaboration with K. L. Guan (Michigan), we are studying the function of two phosphatase genes, one homologous to the mammalian PTP1 and Drosophila csw proteins, and another homologous to MAP kinase phosphatase.
A human gene. We are collaborating with Dr. K. Zhang to clone an inherited human macular dystrophy gene.