Worm Breeder's Gazette 15(4): 47 (October 1, 1998)
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
Massachusetts General Hospital, Jackson 14, Boston, MA 02114
The human Neurofibromatosis 2 gene is a tumor suppressor gene whose germline loss of one allele predisposes individuals to certain nervous system tumors, which are initiated by the somatic loss of the other normal allele. The identification of the human gene as a homolog of the ezrin-radixin-moesin family of cytoskeletal linker proteins provided evidence for the role of the cytoskeleton in tumor formation. In order to investigate this putative role of the cytoskeleton in growth regulation, we had performed extensive screens in C. elegans by PCR and low stringency hybridization in a search for homologs of this class of cytoskeletal genes. We found two genes, one with closest homology to the Neurofibromatosis 2 disease gene (nf2) and the other with equal homology to ezrin, radixin and moesin (erm) (originally called radixin - WBG 13 ). No additional genes of this family have subsequently been identified by the sequencing project. The splicing pattern in the C. elegans gene designated nf2 is closely conserved to the human gene while the genomic structure of C. elegans erm bears no similarity. NF2 expression is first observed during gastrulation in the intestinal precursor cells and subsequently in all developing intestinal cells, the pharynx and the intestinal-rectal valve and can be detected throughout embryonic development. Additionally, the organ borders of the adult reproductive system (vulva and uterus) express high levels of NF2. Finally, a punctate staining pattern is observed within the proximal and distal gonad arms. The protein is localized around the cell borders of the intestinal cells and the pharynx. In double staining experiments with the apical adherens junction antibody MH27, NF2 was found to overlap and encompass the MH27 apical expression pattern and additionally stain the lateral and basal sides of the membrane. We have carried out a direct PCR deletion screen on mutagenized pools of worms kindly provided by Carl Johnson. An animal containing a 1.4 kb germline deletion in nf2 was isolated using primer pairs in the 5' region of the gene. The deletion starts 18 bases after the start codon and removes 263 out of the 650 amino acids in the NF2 protein and additionally results in the creation of a stop codon right at the start of the predicted truncated protein. Homozygous nf2 deletion animals die as late embryos, most with an eversion of the head at the level of the base of the pharynx. The shape of the embryos is distorted with an enlarged head and a crooked tail. There is no gross observable defect in the development of the pharynx or intestine and the apical junctions appear to be correctly placed, as judged by MH27 staining. We have used this deletion allele in an F1 screen for sublethal alleles in nf2. Visible phenotypes found to arise in the F1 generation which were picked for further study were primarily: protruding vulvas, egl, abnormal bends in the gonad and hyperproliferation in the tail We are currently in the process of further analyzing the phenotypes and characterizing the corresponding defect in the primary sequence. erm RNA interference induces an early larval lethal phenotype with high penetrance. L1s display vacuoles of various sizes in the areas of the head and throughout the body in or along the digestive tract. Most commonly, a large vacuole is present on one side of the pharynx, at the location of the pharyngeal-intestinal valve, and in the tail. Animals additionally have a distorted rounded head. A PCR based deletion screen for erm is in progress.