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.

The Neurofibromatosis 2 homolog (nf2) and its ezrin-radixin-moesin family member (erm) are essential genes in C. elegans

Verena Gobel, John Fleming

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
[4]).  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.