Shining light on Drosophila oogenesis: Live imaging of egg development

Article (PDF Available)inCurrent opinion in genetics & development 21(5):612-9 · September 2011with183 Reads
DOI: 10.1016/j.gde.2011.08.011 · Source: PubMed
  • 1st Li He
    24.72 · Harvard Medical School
  • 29.11 · Paul Sabatier University - Toulouse III
  • 38.51 · University of California, Santa Barbara
Abstract
Drosophila oogenesis is a powerful model for the study of numerous questions in cell and developmental biology. In addition to its longstanding value as a genetically tractable model of organogenesis, recently it has emerged as an excellent system in which to combine genetics and live imaging. Rapidly improving ex vivo culture conditions, new fluorescent biosensors and photo-manipulation tools, and advances in microscopy have allowed direct observation in real time of processes such as stem cell self-renewal, collective cell migration, and polarized mRNA and protein transport. In addition, entirely new phenomena have been discovered, including revolution of the follicle within the basement membrane and oscillating assembly and disassembly of myosin on a polarized actin network, both of which contribute to elongating this tissue. This review focuses on recent advances in live-cell imaging techniques and the biological insights gleaned from live imaging of egg chamber development.
A
vailable online at www.sciencedirect.com
Shining
light
on
Drosophila
oogenesis:
live
imaging
of
egg
development
Li
He,
Xiaobo
Wang
and
Denise
J
Montell
Drosophila
oogenesis
is
a
powerful
model
for
the
study
of
numerous
questions
in
cell
and
developmental
biology.
In
addition
to
its
longstanding
value
as
a
genetically
tractable
model
of
organogenesis,
recently
it
has
emerged
as
an
excellent
system
in
which
to
combine
genetics
and
live
imaging.
Rapidly
improving
ex
vivo
culture
conditions,
new
fluorescent
biosensors
and
photo-manipulation
tools,
and
advances
in
microscopy
have
allowed
direct
observation
in
real
time
of
processes
such
as
stem
cell
self-renewal,
collective
cell
migration,
and
polarized
mRNA
and
protein
transport.
In
addition,
entirely
new
phenomena
have
been
discovered,
including
revolution
of
the
follicle
within
the
basement
membrane
and
oscillating
assembly
and
disassembly
of
myosin
on
a
polarized
actin
network,
both
of
which
contribute
to
elongating
this
tissue.
This
review
focuses
on
recent
advances
in
live-cell
imaging
techniques
and
the
biological
insights
gleaned
from
live
imaging
of
egg
chamber
development.
Address
Department
of
Biological
Chemistry,
Center
for
Cell
Dynamics,
Johns
Hopkins
School
of
Medicine,
855
North
Wolfe
Street,
Baltimore,
MD
21205,
USA
Corresponding
author:
Montell,
Denise
J
(dmontell@jhmi.edu)
Current
Opinion
in
Genetics
&
Development
2011,
21:612–619
This
review
comes
from
a
themed
issue
on
Developmental
mechanisms,
patterning
and
evolution
Edited
by
Sean
Megason,
Shankar
Srinivas,
Mary
Dickinson
and
Anna-Katerina
Hadjantonakis
Available
online
17th
September
2011
0959-437X/$
see
front
matter
#
2011
Elsevier
Ltd.
All
rights
reserved.
DOI
10.1016/j.gde.2011.08.011
Introduction
The
development
of
organs
and
organisms
is
a
dynamic
process,
a
complete
understanding
of
which
requires
study-
ing
living
tissue
with
the
highest
possible
spatial
and
temporal
resolution.
The
combination
of
improved
culture
systems,
light-sensitive
proteins,
and
imaging
techniques
has
revolutionized
developmental
studies
over
the
past
decade.
Analysis
of
mutant
phenotypes
need
no
longer
be
limited
to
end-point
evaluation
of
developmental
failure;
now
investigators
can
observe
how
the
end
result
comes
about,
by
monitoring
the
dynamic
behavior
of
cells
and
molecules.
The
ever-expanding
arsenal
of
genetically
encoded
biosensors
and
caged
proteins
further
provides
opportunities
to
both
monitor
and
manipulate
biological
processes
in
real
time.
Besides
being
a
renowned
genetic
model
for
development
and
disease,
Drosophila
melanoga-
ster
is
becoming
more
and
more
amenable
to
live
imaging,
as
culture
conditions
are
defined
that
support
ex
vivo
de-
velopment
of
larval
and
adult
tissues,
most
notably
the
ovary.
A
few
examples
of
developmental
processes
studied
by
live-cell
imaging
are
shown
in
Figure
1
[18].
Here
we
will
review
the
novel
subcellular,
cellular,
and
multicellular
dynamics
that
have
been
discovered
by
live
imaging
stu-
dies
of
egg
chamber
development
in
the
Drosophila
ovary.
The
generation
and
development
of
egg
chambers
pro-
vides
a
good
model
for
the
study
of
a
great
spectrum
of
biological
processes
required
for
organogenesis
in
general,
including
self-renewal
of
adult
stem
cells,
cell
differen-
tiation,
pattern
formation,
axis
specification,
cell
shape
change
and
migration,
tissue
elongation,
cytoskeleton
dynamics,
RNA
biogenesis,
transport,
localization
and
function,
and
even
tumorigenesis.
Mosaic
analysis
and
RNAi-mediated
gene
knockdown
are
highly
effective
in
this
tissue
that,
unlike
the
embryo,
lacks
significant
maternally
provided
RNA
or
protein.
The
ovary
is
also
readily
permeable
to
drugs
and
even
molecules
as
large
as
antibodies,
which
can
diffuse
between
cellcell
junctions
even
in
living
organs.
Direct
injection
of
material
into
the
germline
cytosol
before
live
imaging
has
also
been
suc-
cessful.
Therefore,
ovarian
development
is
not
only
genetically
tractable,
but
also
accessible
to
various
treat-
ments
that
are
more
commonly
used
in
cell
culture.
Yet
by
maintaining
the
tissue
intact,
processes
that
depend
upon
ensembles
of
cells
and
interactions
of
multiple
cell
types,
which
are
impossible
to
study
in
simple
cell
cul-
ture,
can
be
observed
and
manipulated.
A
brief
introduction
to
the
anatomy
of
the
Drosophila
ovary
Female
flies
possess
a
pair
of
ovaries,
each
of
which
is
composed
of
roughly
15
ovarioles.
Each
ovariole
contains
a
linear
sequence
of
egg
chambers
of
increasing
devel-
opmental
stages.
Germline
and
somatic
stem
cells
reside
near
the
tip
of
the
ovariole
in
a
region
called
the
germar-
ium.
Progeny
of
the
germline
and
somatic
stem
cells
assemble
into
egg
chambers,
which
then
bud
off
from
the
germarium
and
are
linked
to
adjacent
chambers
by
stalk
cells,
like
beads
on
a
string.
Each
egg
chamber
produces
a
single
egg
and
is
composed
of
16
germline
cells
(15
nurse
cells
and
one
oocyte),
surrounded
by
a
monolayer
of
roughly
600
epithelial
follicle
cells.
The
Current
Opinion
in
Genetics
&
Development
2011,
21:612619
www.sciencedirect.com
follicle
cells
serve
several
important
functions
including
patterning
the
oocyte,
synthesizing
and
transporting
yolk
polypeptides
to
the
oocyte,
and
secreting
the
protective
layers
of
the
egg
shell
[9].
The
nurse
cells
produce
and
transport
cytoplasm
into
the
oocyte,
which
is
mostly
transcriptionally
quiescent.
Drosophila
ovarian
develop-
ment
has
been
recently
reviewed
by
Horne-Badovinac
and
Bilder
[10],
and
Bastock
and
St
Johnston
[11].
An
illustrated
developmental
timeline
of
Drosophila
oogen-
esis
is
shown
in
Figure
2.
Ex
vivo
culture
of
fly
ovaries
for
live-cell
imaging
Ex
vivo
culture
and
observation
of
egg
chambers
at
stage
10B
and
later
has
been
possible
since
the
founding
work
of
Petri
et
al.
[12],
and
Gutzeit
and
Koppa
[13
!
].
It
was
also
known
that
egg
chambers
could
develop
normally
follow-
ing
removal
of
the
muscular
sheath
that
normally
encases
each
ovariole,
followed
by
injection
into
a
host
female
[13
!
,14].
However
long-term
ex
vivo
culture
of
earlier
stage
egg
chambers
took
another
16
years
to
achieve
[15
!!
].
To
Shining
light
on
Drosophila
oogenesis
He,
Wang
and
Montell
613
Figure
1
Embryo Larva
Pupa
Adult
cellularization,
gastrulation,
germband extension,
dorsal closure,
myoblast fusion,
hemocyte migration,
PGCs migration &
gonad formation
CNS, PNS, NMJs,
imaginal discs,
salivary gland,
imaginal discs,
SOP, retina,
histoblast,
eye, brain,
heart, testis,
ovary,
Anterior
Posterior
ECM
FAs
Myo
NC
PC
BC
GSC
EC
CB
FS
FSC
Nos
Grk
Bicoid
MT
ovariole
ovary
oocyte
follicle cells
stalk cells
(a)
(b)
stage 10
stage 14
stage 9
stage 8
germarium
Germline stem cell
in their native
niche
Tissue revolution
to achieve global
PCP
Collective
migration of border
cell cluster
Periodic basal
actomyosin
contraction
Ooplasmic
streaming for
polarized
localization of
mRNA
stage 3
-5
Current Opinion in Genetics & Development
(a)
Major
stages
of
the
Drosophila
life
cycle
with
the
tissues
and
developmental
events
studied
by
live-cell
imaging
listed
below.
(b)
Anatomy
of
fruit
fly
ovary
and
expanded
view
of
egg
chambers
in
a
single
ovariole.
Germline
stem
cell
self-renewal,
follicle
rotation,
border
cell
migration,
periodic
actomyosin
contraction,
and
polarized
mRNA
localization
are
further
illustrated
below.
Arrows
indicate
the
direction
of
movement
(PGC:
primordial
germ
cell;
CNS:
central
nervous
system;
PNS:
peripheral
nervous
system;
NMJs:
neuromuscular
junctions;
SOP:
sensory
organ
precursor;
GSC:
germline
stem
cell;
CB:
cystoblast;
EC:
escort
cell;
FSC:
follicle
stem
cell;
FS:
fusome;
ECM:
extracellular
matrix;
PC:
polar
cell;
BC:
border
cell;
NC:
nurse
cell;
Myo:
myosin;
FAs:
focal
adhesion;
MT:
microtubule;
Nos:
nanos;
Grk:
gurken;
and
PCP:
planar
cell
polarity).
www.sciencedirect.com
Current
Opinion
in
Genetics
&
Development
2011,
21:612619
approximate
normal
development,
it
is
crucial
to
maintain
the
tissue
under
physiological
conditions,
which
requires
precise
control
of
environmental
temperature,
nutrients,
oxygen,
pH,
hormones,
and
growth
factors.
Techniques
to
isolate
egg
chambers
for
live
imaging
have
been
described
in
detail
in
several
articles
[15
!!
,1618,19
!!
].
The
key
breakthrough
was
the
discovery
that
pH
of
at
least
6.9
and
insulin
supplementation
are
crucial
for
egg
chamber
growth.
With
these
modifications
and
addition
of
fetal
calf
serum,
stages
69
egg
chambers
can
be
cultured
in
Schnei-
der’s
or
Grace’s
medium
for
up
to
6
h
of
continuous
observation
of
tissue
growth
and
cell
movements
[15
!!
].
More
recently,
proliferation
of
germline
stem
cells
and
production
of
cysts
in
the
germarium
have
also
been
observed
in
living
cultures
for
up
to
14
h
[20
!!
].
While
tremendous
progress
has
been
made,
further
improve-
ments
are
still
needed
since
the
egg
chambers
cultured
under
current
best
conditions
cannot
as
yet
progress
from
stage
9
to
stage
10.
To
achieve
full
development
of
egg
chambers
ex
vivo
may
require
specific
combinations
or
pulses
of
juvenile
hormone,
20-hydroxyecdysone,
insulin
or
perhaps
unknown
factors.
Live
imaging
of
germarium
development
Imaging
the
dynamics
of
stem
cells
within
their
native
niches
has
the
potential
to
reveal
information
inaccessible
in
fixed
samples.
Short-term
imaging
("30
min)
of
the
Drosophila
germarium
during
stem
cell
division
was
used
to
study
asymmetric
distribution
of
Wicked,
a
component
of
the
U3
snoRNP
complex
that
is
important
for
main-
tenance
of
stem
cell
fate
[21].
Long-term
imaging
("14
h)
of
living
germaria
has
recently
been
achieved
by
Morris
and
Spradling
[20
!!
].
In
their
study,
full
cycles
of
division
and
differentiation
of
germline
stem
cells
were
recorded.
Interestingly,
these
live
imaging
studies
revealed
that
the
escort
cells,
which
were
thought
possibly
to
migrate
along
with
the
germline
stem
cell
daughters
and
be
replenished
by
escort
stem
cells,
in
fact
remain
largely
stationary
and
are
mitotically
quiescent.
This
finding
emphasizes
the
necessity
of
observing
developmental
events
as
they
actually
occur
and
the
hazards
of
inferring
dynamic
beha-
vior
from
fixed
samples.
Another
live
imaging
study
in
the
germarium
focused
on
the
fusome,
a
complex
structure
of
endomembranes,
membrane-associated
cytoskeleton,
and
microtubules,
which
ramifies
into
the
interconnected
cells
of
each
germline
cyst
[22].
For
a
long
time,
it
was
unclear
whether
the
fusome
lumen
was
shared
between
all
the
cells
of
the
cyst.
Snapp
et
al.
found
that
photobleaching
one
portion
of
the
fusome
present
in
one
cyst
cell
caused
a
rapid
depletion
of
fluorescence
in
the
whole
structure,
suggesting
that
the
fusome
endomembranes
are
part
of
a
single
continuous
endoplasmic
reticulum
(ER)
[23
!
].
614
Developmental
mechanisms,
patterning
and
evolution
Figure
2
(a)
Timeline of Drosophila
oogenesis
day 0 ~day 5 (stage 2) day 6 (stage6) day 6.5 (stage9) day 7 (stage11) day 7.3 (stage 14)
mature egg
nurse cell dumping
ooplasmic streaming
Grk RNA posterior
localization
follicle cell periodic
basal contraction
border cell migration
tissue rotation
follicle cell proliferation
proliferate of CB into 16-cell cyst
GSC proliferation
differentiation
germarium
stage 5
stage 7
stage 9
stage 10
stage 14
accumulation of Osk (posterior)
and Bicoid (anterior) RNAs
(b)
Current Opinion in Genetics & Development
(a)
Timeline
of
Drosophila
oogenesis
with
major
developmental
events
labeled
below.
The
beginning
of
each
developmental
stage
is
indicated
by
a
mark
on
the
line.
The
interval
between
stages
was
drawn
in
proportion
to
estimated
development
time.
(b)
Micrographs
of
major
developmental
stages
of
Drosophila
oogenesis.
Top
panels
show
three-dimensional
projections
of
z-stacks
of
confocal
images
with
nuclei
labeled
in
blue,
E-cadherin
labeled
in
green,
and
myosin
labeled
in
red.
Bottom
panels
show
single-plane
confocal
images
through
the
middle
of
the
tissue
with
nuclei
labeled
in
white,
E-
cadherin
in
green,
and
myosin
in
red.
Scale
bar
is
50
mm.
Current
Opinion
in
Genetics
&
Development
2011,
21:612619
www.sciencedirect.com
Live
imaging
of
egg
chamber
rotation
What
controls
the
overall
shape
of
an
organ?
In
principle,
it
could
be
achieved
simply
by
the
sum
of
the
shapes
of
its
component
cells.
Alternatively,
the
overall
shape
of
an
organ
might
emerge
dynamically
from
mechanical
inter-
actions
between
different
cells
and
extracellular
com-
ponents.
The
latter
turns
out
to
be
the
case
in
Drosophila
ovary.
Early
stage
egg
chambers
are
spherical
but
they
gradually
elongate
as
they
grow,
ultimately
producing
eggs
that
are
2.5-fold
longer
than
they
are
wide.
This
change
of
tissue
shape
coincides
with
de-
velopment
of
dramatically
polarized
arrays
of
basal
F-
actin
bundles
oriented
perpendicular
to
the
long
axis
of
the
egg
chamber
[24].
At
the
same
time,
the
ECM
proteins
within
the
basement
membrane
surrounding
the
egg
chamber
become
aligned
in
the
same
direction
[25].
The
polarized
basal
F-actin
and
ECM
fibers
have
been
proposed
to
function
as
a
‘molecular
corset’
to
constrain
radial
growth
of
egg
chamber.
However,
images
of
fixed
tissue
did
not
reveal
how
the
F-actin
and
ECM
polarization
was
achieved.
Using
live
imaging
techniques,
Haigo
and
Bilder
made
the
astonishing
observation
that
follicles
rotate
within
the
basement
membrane
and
that
this
rotation
provides
a
novel
mechanism
to
achieve
global
alignment
and
orientation
of
the
F-actin
and
base-
ment
membrane
fibers
required
for
elongation
of
the
tissue
[26
!!
].
During
stages
58,
egg
chambers
rotate
clockwise
or
counterclockwise
around
their
long
axis
at
a
speed
of
"0.5
mm/min
and
thereby
produce
circumfer-
entially
polarized
tracks
of
collagen
IV.
This
directional
rotation
depends
on
integrin-mediated
interactions
of
follicle
cells
with
the
ECM,
and
disruption
of
collagen
IV
or
integrin
expression
prevents
follicle
rotation
and
results
in
round
eggs.
Interestingly,
this
rotation
is
similar
to
follicle
rotation
found
in
gall
midges
by
Went
in
1977,
suggesting
that
it
may
be
a
general
phenomenon
[27].
Many
fascinating
questions
arise
from
the
observation
of
egg
chamber
rotation
including
for
example
how
this
directional
movement
is
achieved.
One
suggestion
is
that
the
follicle
cells
crawl
upon
the
ECM
in
a
coordinated
manner.
Since
the
direction
of
rotation
is
random
from
one
follicle
to
the
next,
it
is
probably
selected
through
a
stochastic
mechanism.
The
nature
of
the
mechanism
is
unclear,
as
is
the
mechanism
by
which
all
the
cells
within
a
single
egg
chamber
choose
the
same
direction.
What
initiates
the
movement
is
another
mystery.
How
the
correct
rotation
axis
is
selected
is
also
an
open
question.
There
are
multiple
possible
explanations
for
how
direc-
tional
rotation
might
lead
to
elongation
of
the
tissue.
One
model
is
that
alignment
of
the
ECM
and
actin
fibers
creates
a
corset
that
constrains
increases
in
egg
chamber
volume
toward
the
poles.
Another
possibility,
which
is
not
mutually
exclusive,
is
that
the
rotation
itself
creates
an
anisotropic
force.
Finally,
the
polarized
actin
cytoskeleton
and
integrin-mediated
adhesion
to
the
ECM
that
occurs
as
a
consequence
of
the
follicle
rotation
serve
as
the
substrate
for
periodic
myosin
contractions,
which
also
contribute
to
egg
chamber
elongation,
as
described
in
the
next
section.
Live
imaging
of
follicle
cell
basal
contractions
Immediately
after
egg
chambers
stop
rotating,
they
start
to
grow
dramatically
and
continue
to
elongate.
Within
10
12
h,
the
egg
chamber
increases
its
volume
by
10-fold
and
elongates
1.6-fold
in
the
absence
of
cell
division.
Achiev-
ing
this
elongation
in
the
presence
of
such
dramatic
tissue
expansion
requires
anisotropic
mechanical
forces
to
con-
strain
the
radial
volume
increase.
One
of
the
major
gen-
erators
of
forces
in
tissues
is
actomyosin
contractility.
Using
time-lapse
imaging
with
fluorescently
tagged
myo-
sin,
we
found
that
contractile
myosin
began
to
accumulate
at
the
basal
surfaces
of
a
subset
of
follicle
cells
beginning
in
early
stage
9.
We
also
observed
that
the
basal
surfaces
of
follicle
cells
actively
contract
and
relax,
shrinking
specifically
along
the
short
axis.
The
contractions
were
strikingly
cyclical
with
an
average
period
of
6.5
min
[28
!!
].
These
myosin-mediated
basal
contractions
require
the
RhoROCK
pathway,
cytosolic
calcium,
integrin-
mediated
cellECM
interactions,
and
E-cadherin-
mediated
cellcell
adhesion.
Both
pharmacological
and
genetic
approaches
show
that
interfering
with
the
con-
tractions
results
in
rounder
eggs
whereas
enhancing
the
contractions
leads
to
longer
eggs.
Since
these
contractions
do
not
occur
in
all
follicle
cells
but
rather
are
mostly
confined
to
follicle
cells
near
the
center
of
the
egg
chamber,
we
postulate
that
the
effect
of
the
contractions
is
to
constrain
the
tremendous
increase
in
tissue
volume
to
the
two
ends.
We
envision
that
this
is
somewhat
similar
to
squeezing
a
ball
of
dough
to
make
it
longer,
although
obviously
the
elastic
properties
of
cells
differ
from
those
of
dough.
Nevertheless,
exertion
of
an
anisotropic
force
near
the
middle
of
the
tissue
as
it
expands
in
volume
over
the
course
of
10
h
does
affect
its
shape,
although
it
is
not
clear
precisely
which
cellular
and
extracellular
elements
respond
to
the
force
and
create
the
lasting
change
in
shape.
Live
imaging
reveals
that
elongation
of
this
tissue
involves
much
more
dynamic
molecular
and
cellular
behaviors
compared
to
the
static
view
of
a
corset
that
was
developed
from
analysis
of
fixed
tissue.
It
will
be
important
in
the
future
to
decipher
the
mechanisms
that
initiate,
sustain,
and
pattern
the
oscillations.
Oscillations
in
myosin
accumulation
and
cellular
contractility
have
been
observed
in
other
epithelia
undergoing
morphogen-
etic
changes
[29,30].
Interestingly
the
oscillation
periods
and
subcellular
locations
of
myosin
accumulation
differ
in
the
different
tissues,
suggesting
that
the
oscillation
mech-
anism
can
be
regulated
tissue-specifically
to
achieve
diverse
morphogenetic
outcomes.
Live
imaging
of
collective
border
cell
migration
At
late
stage
8
a
small
group
of
anterior
follicle
cells
adjacent
to
the
polar
cells,
referred
to
as
border
cells,
round
up
in
response
to
the
cytokine
Unpaired
(Upd),
Shining
light
on
Drosophila
oogenesis
He,
Wang
and
Montell
615
www.sciencedirect.com
Current
Opinion
in
Genetics
&
Development
2011,
21:612619
which
the
polar
cells
secrete.
Upd
activates
the
JAK/
STAT
pathway
in
the
border
cells,
causing
them
to
extend
protrusions,
delaminate
from
the
epithelium
and
migrate
in
between
the
nurse
cells.
These
47
cells
surround
and
carry
the
two
non-motile
polar
cells
from
the
anterior
tip
of
the
egg
chamber,
reaching
the
oocyte
by
stage
10
[3133].
Genetic
screens
and
analysis
of
border
cell
migration
in
fixed
tissue
revealed
multiple
signaling
pathways
that
control
distinct
features
of
the
movement.
Whereas
JAK/STAT
signaling
determines
which
of
the
follicle
cells
acquire
the
ability
to
move,
receptor
tyrosine
kinases,
PVR
and
EGFR,
determine
the
direction
of
movement
in
response
to
ligands
secreted
by
the
oocyte
[3436].
The
steroid
hormone
ecdysone
by
contrast
con-
trols
the
timing
of
border
cell
migration
[32].
Analysis
of
border
cell
migration
using
live
imaging
has
begun
to
reveal
the
dynamic
features
of
their
movement.
For
example,
it
was
surprising
to
find
that
inhibition
of
both
EGFR
and
PVR
function
in
border
cells
did
not
suppress
cell
protrusion
in
the
forward
direction.
On
the
contrary,
multiple
cells
extend
long
and
random
protru-
sions
in
all
directions,
suggesting
that
the
guidance
signals
may
not
only
promote
cell
protrusion
at
the
front
but
also
inhibit
protrusions
in
the
wrong
directions
[15
!!
].
A
major
molecular
driver
of
protrusion
in
cells
is
the
small
GTPase
Rac
and
Rac
has
long
been
known
to
be
required
for
border
cell
migration
[35,37,38].
However,
both
domi-
nant-negative
(DN)
and
constitutively
active
(CA)
forms
of
Rac
cause
strong
migration
defects,
indicating
that
Rac
activity
must
be
spatially
and/or
temporally
regulated.
Recently
it
has
become
possible
to
control
Rac
activity
in
vivo
using
genetically
encoded
and
caged
forms
of
Rac,
created
by
Wu
et
al.
[39].
These
caged
Rac
proteins
provide
a
novel
approach
to
activate
or
inhibit
Rac
activity
with
high
spatial
and
temporal
resolution
in
response
to
flashes
of
blue
laser
light.
Using
this
tool,
we
found
that
activating
or
inhibiting
Rac
activity
in
one
migrating
border
cell
causes
dramatic
responses
of
the
other
cells
in
the
cluster
[40
!!
].
Activating
Rac
in
any
cell,
caused
the
cluster
to
migrate
in
that
direction.
Inhibition
of
Rac
in
the
leading
cell
caused
all
cells
to
lose
their
sense
of
direction
and
thus
to
protrude
outward
in
all
directions.
In
this
study,
the
endogenous
pattern
of
Rac
activity
was
also
monitored
using
a
Rac
FRET
biosensor
that
was
origin-
ally
generated
in
Matsuda’s
lab
and
modified
by
Kardash
and
co-workers
[41,42].
Rac
activity
is
normally
higher
at
the
front
and
lower
at
the
back
of
border
cell
clusters,
and
is
higher
in
the
front
portion
of
the
leading
cell
than
in
the
back
of
the
leading
cell.
Polarity
of
Rac
activity
is
lost
when
guidance
receptor
activity
is
inhibited,
although
some
uniform
Rac
activity
persists.
These
findings
suggest
that
a
low
level
of
uniform
Rac
activity
promotes
protrusion
in
all
directions
in
the
absence
of
guidance
receptor
activity,
and
that
in
response
to
asymmetric
guidance
receptor
activation,
Rac
activity
increases
at
the
front,
enhancing
forward
directed
protrusion.
In
addition,
via
an
unknown
mechanism
enhanced
Rac
activity
at
the
front
inhibits
protrusion
in
other
directions
of
all
the
cells
in
the
cluster.
A
key
open
question
is
by
what
mechanism
the
cells
sense
relative
levels
of
Rac
activity
in
adjacent
cells.
Live
imaging
of
transport
and
polarization
of
RNA
and
protein
in
germline
cells
Initially
discovered
in
developing
oocytes,
the
polarized
localization
of
mRNAs
turns
out
to
be
a
widely
adapted
mechanism
to
establish
cell
polarity
in
germ
cells,
neurons,
and
cells
undergoing
asymmetric
division
[43].
Localization
of
numerous
mRNAs
to
distinct
regions
of
the
fly
oocyte
is
crucial
for
normal
patterning
of
the
embryo
following
fertilization.
One
advantage
of
using
egg
chambers
to
study
the
subcellular
localization
of
molecules
is
the
large
size
of
the
germline
cells.
During
development,
the
oocyte
grows
from
a
"20
mm
diameter
at
stage
3
to
more
than
100
mm
at
stage
10.
Live
imaging
of
RNA
movement
in
the
fly
ovary
was
first
achieved
by
microinjection
of
fluorescently
labeled
molecules
into
late
stage
egg
chambers.
An
active,
long-range,
micro-
tubule-dependent
cytoplasmic
flow
termed
ooplasmic
streaming
occurs
between
late
stage
10
and
stage
13
[44,45].
Streaming
is
inhibited
earlier
in
egg
chamber
development
by
actin
polymerization,
kinesin
and
dynein
motor
activities
and
by
the
two
proteins
Capucccino
(Capu)
and
Spire
[46,47].
Forrest
and
Gavis
pioneered
the
use
of
a
less
invasive
labeling
technique
to
track
in
vivo
movement
of
nanos
RNA
and
found
that
it
translocated
along
microtubules
and
then
became
anchored
at
the
posterior
region
by
an
actin-dependent
mechanism
[48
!
].
This
RNA
labeling
system
takes
advantage
of
the
binding
between
bacterio-
phage
MS2
coat
protein
(MCP)
and
a
specific
RNA
sequence
that
forms
a
stem-loop
structure,
and
was
first
used
by
Bertrand
et
al.
to
study
mRNA
localization
in
living
yeast
[49].
When
MCP
is
tagged
with
fluorescent
protein,
and
co-expressed
with
an
RNA
tagged
with
the
MS2-binding
sequence,
the
position
of
the
RNA
is
revealed
by
the
fluorescent
signal
from
the
MCP
bound
to
it
[50].
This
mRNA
labeling
method
was
later
used
to
localize
many
other
important
mRNAs
in
the
oocyte
including
Gurken,
Bicoid
(bcd),
and
Oskar
[5153].
A
more
detailed
review
of
mRNA
localization
in
the
Drosophila
oocyte
can
be
found
in
Becalska
and
Gavis
[54].
Localized
mRNAs
are
typically
found
in
ribonucleopro-
tein
(RNP)
complexes,
so
in
addition
to
mRNAs,
many
mRNA-binding
proteins,
including
Exuperentia
(Exu),
Staufen
(Stau),
Vasa,
Aubergine
and
Yps,
also
exhibit
specific
localizations
within
the
oocyte.
Live
imaging
has
also
been
used
to
study
the
mechanisms
responsible
for
their
transport
and
localization.
For
example,
fluores-
cently
labeled
Exu,
an
RNA
binding
protein
required
for
616
Developmental
mechanisms,
patterning
and
evolution
Current
Opinion
in
Genetics
&
Development
2011,
21:612619
www.sciencedirect.com
proper
localization
of
bcd
mRNA,
exhibits
a
dynein-de-
pendent
directional
movement
on
polarized
microtubules
(MTs)
and
travels
through
the
ring
canals
that
connect
the
nurse
cells
to
the
oocyte
[55].
Imaging
of
Staufen
(Stau),
a
protein
associated
with
RNPs
containing
oskar
mRNA,
revealed
that
oskar
mRNA
is
randomly
trans-
ported
on
MTs
in
all
directions
with
a
weak
posterior
bias
[53].
Interestingly,
Shimada
et
al.
recently
discovered
that
the
mRNA-binding
protein
Ypsilon
Schachtel
(Yps)
is
transported
via
both
MT-dependent
and
MT-indepen-
dent
mechanisms
and
this
transport
is
regulated
by
nutrient
availability
and
insulin
signaling,
possibly
as
part
of
a
mechanism
to
preserve
oocytes
during
periods
of
nutrient
deprivation
and
allowing
for
rapid
resumption
of
reproduction
when
conditions
improve
[56
!
].
Live
imaging
of
epithelium
morphogenesis
during
dorsal
appendage
formation
Complex
epithelial
movements
also
occur
during
late
stages
of
oogenesis
(from
stage
10B
to
stage
14).
Live
culture
of
late
stage
egg
chambers
was
first
reported
by
William
H.
Petri
in
1979
[12].
The
culture
of
late
stage
egg
chambers
may
be
less
demanding
because
the
egg
chamber
does
not
grow
much
and
starts
to
form
a
vitelline
membrane
and
thus
to
separate
itself
from
the
environ-
ment.
The
formation
of
the
dorsal
respiratory
appendages
during
these
stages
had
been
analyzed
live
by
Dorman
et
al.
[57].
Three
phases
of
morphogenesis
were
revealed,
and
two
cell
types
that
form
the
roof
and
floor
of
the
structure
exhibit
different
morphological
behaviors.
Future
prospects
In
the
past
few
years,
live
imaging
of
earlier
stages
of
oogenesis
has
revealed
patterning
mechanisms
that
were
unimaginable
based
on
analysis
of
fixed
tissue,
such
as
the
rotation
of
follicles
within
the
basement
membrane
and
oscillating
myosin
co ntractions.
The
rapid
develop-
ment
of
new
genetically
encoded
biosensors,
caged
proteins
and
microscopy
technology
provides
unprece-
dented
opportunities
to
address
biological
questions
using
live
imaging.
Biosensors
have
been
enginee r ed
to
reveal
changes
in
pH,
ion
concentrations,
protein
activities,
and
even
the
distribution
of
mechanical
forces
[5860].
The
possibilities
for
manipulating
protein
activities
with
high
spatial
and
temporal
resolution
are
also
likely
to
expand
tremendously
in
the
near
future.
Recently
many
photoactivatable
proteins
have
been
engineered
using
different
approaches
to
cage
a
broad
spectrum
of
signaling
molecules,
including
a
cation
channel
[61],
adenylyl
cyclase
[62],
G
protein-coupled
receptors
[63],
transcription
factors
[64 66],
and
multiple
protein
kinases
[67].
Combining
these
tools
with
inno-
vations
in
whole
organ
cultures
will
allow
investigators
to
both
manipulate
these
pathways
and
monitor
the
immediate
effects,
not
only
on
individual
cells
or
cell
types
but
also
on
the
complex
interactions
between
different
cell
types
and
ECM.
Although
phototoxicity
is
a
factor
that
limits
long-term
3D
imaging
in
many
tissues
including
the
ovary,
advances
in
microscopy
are
likely
to
continue
to
improve
our
ability
to
see
deeper,
with
higher
resolution,
and
over
longer
periods
of
time.
For
example,
light
sheet
microscopy
or
selective
plane
illumination
microscopy
(SPIM)
provides
a
solution
to
image
large
and
deep
samples
with
greatly
reduced
light
exposure
[68].
For
this
technique
the
sample
has
to
be
suspended
in
a
tube
of
transparent
gel
to
allow
imaging
from
multiple
directions,
which
may
require
special
protocol
modifications.
In
addition,
the
quantity
of
data
collected
using
SPIM
challenges
current
software
and
hardware
available
in
most
labs.
Perfusion
systems
may
enhance
survival
of
tissues
that
requires
constant
nutrient
supply
[69].
In
addition,
super-
resolution
techniques
such
as
structured
illumination
microscopy
(SIM),
stimulated
emission
depletion
micro-
scopy
(STED),
4Pi,
and
photo-activated
localization
microscopy/stochastic
optical
reconstruction
microscopy
(PALM/STORM)
have
the
potential
to
reveal
protein
dynamics
at
the
single
molecule
level
[70,71].
Tradeoffs
for
the
increased
resolution
offered
by
these
approaches
include
limitations
in
imaging
speed,
depth,
and
require-
ments
for
specific
fluorescent
probes,
such
as
photoacti-
vatable
or
photoconvertible
fluorescent
proteins.
The
depth
limitation
can
be
overcome
by
combining
PALM/STORM
with
TIRF
for
some
applications,
such
as
imaging
the
basal
myosin
oscillations.
It
is
very
likely
that
with
the
continued
improvements
in
culture
con-
ditions,
biosensor
development,
and
microscopy
tech-
niques,
live
imaging
will
greatly
advance
our
understanding
of
the
dynamic
molecular,
cellular
and
supracellular
mechanisms
that
control
Drosophila
oogen-
esis.
References
and
recommended
reading
Papers
of
particular
interest,
published
within
the
period
of
review,
have
been
highlighted
as:
!
of
special
interest
!!
of
outstanding
interest
1.
Mavrakis
M,
Rikhy
R,
Lilly
M,
Lippincott-Schwartz
J:
Fluorescence
imaging
techniques
for
studying
Drosophila
embryo
development.
Curr
Protoc
Cell
Biol
2008,
Chapter
4:Unit
4.18.
2.
Parton
RM,
Valles
AM,
Dobbie
IM,
Davis
I:
Live
cell
imaging
in
Drosophila
melanogaster.
Cold
Spring
Harb
Protoc
2010,
2010:
pdb.top75.
3.
Aldaz
S,
Escudero
LM,
Freeman
M:
Live
imaging
of
Drosophila
imaginal
disc
development.
Proc
Natl
Acad
Sci
USA
2010,
107:14217-14222.
4.
Cheng
J,
Hunt
AJ:
Time-lapse
live
imaging
of
stem
cells
in
Drosophila
testis.
Curr
Protoc
Stem
Cell
Biol
2009,
Chapter
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JR:
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He,
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619
www.sciencedirect.com
Current
Opinion
in
Genetics
&
Development
2011,
21:612619
    • "Hence, these processes are not essential for egg chamber elongation. Alignment of actin filaments and collagen IV fibers (ECM) during later stages cannot be uncoupled from egg chamber elongation in fat2 mutants expressing Fat2 DICR -GFP, indicating that these structures are important for egg chamber elongation (Gutzeit et al., 1991; He et al., 2011). See also Movie S1. expressing Fat2 DICR -GFP. "
    [Show abstract] [Hide abstract] ABSTRACT: Global tissue rotation was proposed as a morphogenetic mechanism controlling tissue elongation. In Drosophila ovaries, global tissue rotation of egg chambers coincides with egg chamber elongation. Egg chamber rotation was put forward to result in circumferential alignment of extracellular fibers. These fibers serve as molecular corsets to restrain growth of egg chambers perpendicular to the anteroposterior axis, thereby leading to the preferential egg chamber elongation along this axis. The atypical cadherin Fat2 is required for egg chamber elongation, rotation, and the circumferential alignment of extracellular fibers. Here, we have generated a truncated form of Fat2 that lacks the entire intracellular region. fat2 mutant egg chambers expressing this truncated protein fail to rotate yet display normal extracellular fiber alignment and properly elongate. Our data suggest that global tissue rotation, even though coinciding with tissue elongation, is not a necessary prerequisite for elongation.
    Full-text · Article · Mar 2016
    • "Endogenous Abi as well as an EGFP-tagged Abi transgene expressed in abi mutant follicle cells are highly enriched at dynamic protrusions emerging from tricellular junctions and along the membrane (Fig. 2, B and C; and Video 5 A). At later stages (9 and10), follicle cells initiate oscillating acto-myosin contractions driving further egg chamber elongation (He et al., 2011). At this advanced stage of egg chamber maturation, when follicle cell migration ceases, the polarized localization of Abi changes into a more even distribution at basal cell–cell junctions (Fig. 2 D). "
    [Show abstract] [Hide abstract] ABSTRACT: Directional cell movements during morphogenesis require the coordinated interplay between membrane receptors and the actin cytoskeleton. The WAVE regulatory complex (WRC) is a conserved actin regulator. Here, we found that the atypical cadherin Fat2 recruits the WRC to basal membranes of tricellular contacts where a new type of planar-polarized whip-like actin protrusion is formed. Loss of either Fat2 function or its interaction with the WRC disrupts tricellular protrusions and results in the formation of nonpolarized filopodia. We provide further evidence for a molecular network in which the receptor tyrosine phosphatase Dlar interacts with the WRC to couple the extracellular matrix, the membrane, and the actin cytoskeleton during egg elongation. Our data uncover a mechanism by which polarity information can be transduced from a membrane receptor to a key actin regulator to control collective follicle cell migration during egg elongation. 4D-live imaging of rotating MCF10A mammary acini further suggests an evolutionary conserved mechanism driving rotational motions in epithelial morphogenesis.
    Full-text · Article · Feb 2016
    • "To reveal further effects of D-EndoB overexpression in the ovary, the development of egg chambers was examined. The newly eclosed females were collected and fed with yeast powder at 25°C for 36 hours (He et al., 2011), and then the composition of each ovariole with different stages of developing egg chambers was analyzed. Ovarioles from OreR females were classified in the following two groups: one group contained at least one middle stage egg chamber (vitellogenic stage 8-10) and one late stage egg chamber (Fig. 8D, red bar, 56.3%), and the other group contained only one middle stage egg chamber (Fig. 8D, blue bar, 42.3%). "
    [Show abstract] [Hide abstract] ABSTRACT: The nutritional environment is crucial for Drosophila oogenesis in terms of controlling hormonal conditions that regulate yolk production and the progress of vitellogenesis. Here, we discovered that Drosophila Endophilin B (D-EndoB), a member of the endophilin family, is required for yolk endocytosis as it regulates membrane dynamics in developing egg chambers. Loss of D-EndoB leads to yolk content reduction, similar to that seen in yolkless mutants, and also causes poor fecundity. In addition, mutant egg chambers exhibit an arrest at the previtellogenic stage. D-EndoB displayed a crescent localization at the oocyte posterior pole in an Oskar-dependent manner; however, it did not contribute to pole plasm assembly. D-EndoB was found to partially colocalize with Long Oskar and Yolkless at the endocytic membranes in ultrastructure analysis. Using an FM4-64 dye incorporation assay, D-EndoB was also found to promote endocytosis in the oocyte. When expressing the full-length D-endoB(FL) or D-endoBÆ(SH3) mutant transgenes in oocytes, the blockage of vitellogenesis and the defect in fecundity in D-endoB mutants was restored. By contrast, a truncated N-BAR domain of the D-EndoB only partially rescued these defects. Taken together, these results allow us to conclude that D-EndoB contributes to the endocytic activity downstream of Oskar by facilitating membrane dynamics through its N-BAR domain in the yolk uptake process, thereby leading to normal progression of vitellogenesis.
    Full-text · Article · Jan 2014
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