Program of the 2001 International Worm Meeting - Sternberg Lab ...

144
144. Putative matrix proteins with
thrombospondin type I repeats and
serine protease inhibitor domains are
involved in guidance of the distal tip
cell
Takehiro Kawano, Hong Zheng,
Joseph G. Culotti
Samuel Lunenfeld Research Institute of Mt.
Sinai Hospital, Toronto, Ontario, Canada M5G
1X5
In C. elegans hermaphrodites, the gonad
acquires a U-shape by the directed migration of
distal tip cells (DTCs). They start migration at
late L2 stage from the ventral mid body of the
animal, and by late L4 stage they finish
migration. The first phase of DTC migration is
longitudinal migration along the ventral muscle
bands. The second phase is a circumferential
migration, where DTCs migrate from ventral to
dorsal along the epidermis, then finally they
migrate retrogradely along the dorsal muscle
band back to mid-body.
We have characterized two phenotypic classes
of mig-6 mutants. In class 1 mig-6 mutant,
originally described by Hedgecock et al. (1), the
first longitudinal migration is defective but
ventral to dorsal migration is normal, resulting
in short and immature gonad, which is sterile.
By a large scale F1 screen for DTC migration
mutants, we have obtained a new class of mig-6
mutant (class 2). The heterozygotes of class 2
display ventral reflection of gonad, which
indicates that the circumferential migration of
DTCs is defective but longitudinal migration is
normal, similar to the defects seen in unc-5, -6,
-40, and -130 (2, 3). Interestingly, homozygotes
of this class 2 show leaky embryonic/early
larval lethality, with escapers (average 17)
growing to adulthood and their gonads
displaying supernumerary turns, indicating
disruption of both longitudinal and
circumferential guidance of DTC migration.
This lethality, but not migration defects, is
rescued by maternal effect, since most of
homozygote animals from heterozygote parents
grow to adults with a complete penetrance of
supernumerary turn of DTCs. It seems mig-6
affects all of the three phases of DTC migration,
but separately.
Both classes of mig-6 are rescued by a genomic
fragment containing a single locus. There are
two alternative transcripts, designated mig-6a
and mig-6b. They have the same exon structure
down to exon 10 and have different 3’
extensions. The proteins are putative secreted
proteins with a signal peptide. The N-terminal
half of the proteins has a cluster of
thrombospondin type I domains and N-linked
carbohydrate attachment sites. The C-terminal
half of the proteins has a repeat of Kunitz-type
serine protease inhibitor (KU) domains. Both
forms of the proteins share the same structural
features until the 6th KU domain. The large one
has 5 extra KU domains and a immunoglobulin
C2 type domain in the end. By designing
dsRNA based on transcript specific 3’ sequence,
each transcript was disrupted independently or
together. Quite surprisingly, it was shown that
RNAi of mig-6b phenocopied the class 1
mutant, and RNAi of mig-6a alone or together
with mig-6b phenocopied class 2 mutant
recessive phenotype. All four class 1 alleles are
nonsense mutations that affect just the mig-6b
transcript. At least one of the class 2 mutations
is a missense codon affecting both transcripts.
mig-6 promoter:: gfp reporter showed mig-6
expressions in various tissues including DTCs,
body wall muscles, the head mesodermal cell,
coelomocytes, and some neuronal cells.
Preliminary results suggest that expression of
the two transcripts is under distinct regulation.
Hence the expression patterns of the two
transcripts, in addition to structural difference
may define the class 1 and class 2 phenotypes.
(1) Hedgecock et al. (1987) Development 100,
365-382
(2) Hedgecock et al. (1990) Neuron 2, 61-85
144
(3) Nash et al. (2000) Genes & Development 14,
2486-2500