1. Regulators and Germline Specific Function of DAF-18/PTEN
Gabriel
Chamberlain,
Jun
Liu,
Michael
Zane4
and
Ian
D.
Chin-­‐Sang
Department
of
Biology,
Queen’s
Universty,
Kingston,
ON
Canada
Introduction
Hypothesis / Aims
When
condiKons
become
unfavorable,
C.
elegans
larvae
enter
into
an
alternaKve
state
of
arrested
development
at
the
second
larval
molt
known
as
dauer.
If
hatched
directly
into
the
absence
of
food,
C.
elegans
arrest
development
early
and
remain
suspended
as
L1
larvae,
this
is
known
as
L1
arrest.
Both
forms
of
arrest
require
the
acKon
of
the
phosphatase
DAF-­‐18
(abnormal
DAuer
FormaKon),
the
worm
ortholog
of
the
human
PTEN
(Phosphatase
and
TENsin)
which
is
a
tumour
suppressor.
The
downstream
roles
of
daf-­‐18
in
the
insulin-­‐signaling
pathway
are
well
known
however
few
upstream
regulators
have
been
idenKfied.
In
addiKon,
the
Kssue
specific
funcKon
of
DAF-­‐18
has
been
studied
with
regards
to
vulval
development,
dauer
formaKon
and
neuron
development,
but
unKl
recently,
not
specifically
to
L1
arrest.
Unpublished
data
from
the
Chin-­‐Sang
lab
suggests
a
germline
specific
funcKon
of
daf-­‐18
with
regards
to
L1
arrest
as
well
as
a
new
model
for
daf-­‐18
negaKve
regulaKon
by
the
insulin
receptor
daf-­‐2
and
the
ubiquiKn
ligase
vhl-­‐1.
Using
daf-­‐18
mutants
(ok480)
carrying
our
genomic
daf-­‐18::gfp
reporter
we
will
look
for
increases
in
fluorescence
aZer
feeding
them
RNAi
that
targets
the
candidate
negaKve
regulators.
In
our
translaKonal
reporter
for
DAF-­‐18
fluorescence
is
only
visible
surrounding
the
developing
vulva
at
the
Christmas
tree
stage
of
L4.
Therefore
any
fluorescence
seen
outside
this
area
at
this
developmental
Kme
point
can
be
a]ributed
to
the
effects
of
RNAi
Rescuing
daf-­‐18(ok480)
mutants
with
daf-­‐18
wildtype
genomic
from
an
extra-­‐
chromosomal
array
(quEx518)
only
parKally
reverses
the
defecKve
L1
arrest
phenotype.
As
the
expression
of
extra-­‐chromosomal
arrays
is
silenced
in
the
germline
we
believe
daf-­‐18
must
be
required
in
this
Kssue
for
proper
L1
arrest.
Germline
Specific
Knockdown
of
daf-­‐18
Tissue
specific
RNAi
of
daf-­‐18
in
the
germline,
but
not
the
soma,
results
in
defecKve
L1
arrest.
Germline
Specific
Rescue
of
daf-­‐18
!
Since
germline
silencing
provides
a
hurdle
to
daf-­‐18
rescue
using
extra
chromosomal
arrays
we
will
be
using
MosSci
to
integrate
daf-­‐18
into
the
chromosome
under
a
germline
specific
promoter.
!
MosSci
takes
advantage
of
the
C.
elegans
Mos1
transposon
to
“jump”
transgenes
into
the
chromosome
at
determined
sites
Aim
1
If
DAF-­‐18
is
required
in
the
germline
to
enable
proper
L1
arrest
then:
! Germline
rescue
of
daf-­‐18
will
correct
the
defecKve
L1
arrest
phenotype
in
daf-­‐18
mutants
! Germline
specific
RNAi
of
daf-­‐18
will
result
in
a
defecKve
L1
arrest
phenotype
equivalent
to
a
daf-­‐18
mutant.
Aim
2
If
candidate
genes
are
negaKve
regulators
of
DAF-­‐18
then
DAF-­‐18::GFP
fluorescence
will
increase
with
RNAi
knockdown
of
these
genes.
RNAi to find DAF-18 Regulators Germline Specific Function of DAF-18
Acknowledgments
Dr.
Ian
Chin-­‐Sang
Dr.
Jun
Liu
MSc.C.
Michael
Zane4
MSc.C.
Samantha
Lo
Antonion
Papanicolau
Jeffery
Boudreau
-­‐20
0
20
40
60
80
100
120
0
5
10
15
20
Percent
Alive
(%)
Day
N2
daf-­‐18(ok480)
daf-­‐18(ok480);Ex
(daf-­‐18
genomic)
Using
MosSCI
we
achieved
successful
germline
expression
of
GFP
under
the
promoter
Pie-­‐1
Making
Sense
of
the
Results
!
Integrated
daf-­‐18
cDNA
under
the
pie-­‐1
promoter
was
not
sufficient
to
extend
L1
survival
in
daf-­‐18
mutants
(only
one
line
tested)
!
daf-­‐18
cDNA
expressed
in
an
extrachromosomal
array
causes
dauer
consKtuKve
phenotype
confirming
our
construct
forms
funcKonal
DAF-­‐18
!
Preliminary
results
show
that
the
defecKve
L1
arrest
phenotype
of
daf-­‐18
mutants
can
be
maternally
rescued
from
a
heterozygous
parent
supporKng
an
important
germline
role
PI3K
independent
roles
(ex:
axon
guidance)
Canonical
Insulin
Signaling
pathway
Our
lab
demonstrated
that
DAF-­‐2
binds
and
phophorylates
DAF-­‐18.
We
suggest
that
this
phosphorylaKon
event
results
in
a
conformaKonal
change
of
DAF-­‐18
making
it
vulnerable
to
polyubiquiKnaKon
for
proteosomal
degradaKon.
With
that
in
mind
we
assayed
several
proteosomal
genes
and
ubiquiKn
ligase
genes
using
RNAi
for
negaKve
regulaKon
of
DAF-­‐18
Preliminary
Results
!
From
our
first
RNAi
assay
of
six
candidate
negaKve
regulator
genes;
pbs-­‐4,
pas-­‐3,
rpt-­‐5,
pbs-­‐6,
rpt-­‐6
and
cul-­‐2,
two
candidates,
pas-­‐3
and
pbs-­‐4
caused
increases
in
DAF-­‐18::GFP
levels
during
early
embryonic
development.
Both
candidates
are
genes
encoding
for
proteosomal
subunits
DAF-­‐18::GFP
DAF-­‐18::GFP
DAF-­‐18::GFP
???
Worm
Insulin
Insulin
Receptor
DAF-­‐18
/PTEN
DAF-­‐18/PTEN
degraded
???
Proteosome
DAF-­‐28
Insulin
DAF-­‐2
IR
AGE-­‐1
PI3K
DAF-­‐18
PTEN
PIP2
PIP3
Canonical
Insulin
signaling
pathway
DAF-­‐18/PTEN
PI3K
Independent
roles
Pas-­‐3
RNAi
Pbs-­‐4
RNAi
Z2
Z3
DAF-­‐18::GFP
wildtype
levels
DAF-­‐18::GFP
on
RNAi
for
a
negaKve
regulator