Institut de Génétique et Biologie Moléculaire et Cellulaire, IGBMC/CNRS/ULP/INSERM BP 10142, ILLKIRCH, C. U. de Strasbourg, 67404, France
Author for correspondence (e-mail:
angela{at}titus.u-strasbg.fr)
Accepted 12 August 2004
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SUMMARY |
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Key words: Glia, Migration, Proliferation, Drosophila, Time-lapse
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Introduction |
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Migration and proliferation have been extensively characterised in fixed
tissues or by time-lapse in cell cultures (e.g.
Etienne-Manneville and Hall,
2003; Friedl and Wolf,
2003
; Fulga and Rorth,
2002
; Gerlich et al.,
2003
; Lauffenburger and
Horwitz, 1996
; Meili and
Firtel, 2003
; Welch et al.,
1997
). Ideally, however, one would like to analyse such dynamic
events by time-lapse in the whole animal and to follow individual cells. The
advent of green fluorescent protein (GFP) as a marker of living cells has
opened new perspectives in the field (e.g.
Bellaiche et al., 2001
;
Gilmour et al., 2002
;
Kakita, 2001
;
Kaltschmidt et al., 2000
;
Nadarajah and Parnavelas,
2002
; Ribeiro et al.,
2002
; Wood et al.,
2002
).
In the peripheral nervous system (PNS), glia represent a typical example of
migratory cells. The fact that PNS glia follow axons [fly wing glia
(Giangrande, 1994), fly embryo
(Sepp et al., 2000
), zebrafish
lateral line (Gilmour et al.,
2002
), chicken Schwann cells
(Carpenter and Hollyday, 1992
)]
and migrate as chains of cells strongly suggests that cell-cell interactions
play a role in migration and/or proliferation. The bilayered organisation of
the fly wing epithelium and the presence of purely sensory nerves constitute
advantageous features to study cell migration and proliferation in vivo.
Moreover, the origin of wing glial cells has been characterised
(Giangrande et al., 1993
;
Giangrande, 1994
;
Van De Bor et al., 2000
). We
have therefore established a confocal non-invasive approach that makes it
possible to follow wing glial cells by time-lapse in the whole animal.
We show that extensive and dynamic cell shape remodelling allows glia to polarise along the underlying axon during migration and proliferation. We also identify two populations of migratory cells, pioneer glia, which explore the environment by extending long filopodia, and follower glia, which are less active. Furthermore, we have determined a confocal-assisted ablation protocol that can be used for single cells, which represents a novel and useful tool over the conventional bright-field optic setups. The combined use of time-lapse, cell ablation and genetic manipulation has enabled us to identify the contribution of different cell-cell interactions to distinct aspects of glial cell development: (1) glia-glia interactions control the extent of glia migration; (2) neurone-glia interactions are not necessary for glia motility but do affect the direction of glia migration; and (3) autonomous cues control the final number of glial cells.
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Materials and methods |
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Immunohistochemistry
Fixation, dissection and antibody incubation were performed as previously
described (Giangrande et al.,
1993), using the following antibodies: mouse anti-Repo [1:1000;
Developmental Studies Hybridoma Bank (DSHB)], rabbit anti-Repo (1:4000; A.
Travers), mouse anti-22c10 (Fujita et al.,
1982
) (1:100; DSHB), rat anti-Elav (1:500)
(Robinow and White, 1991
)
(DSHB), rabbit anti-GFP (1:1000; Molecular Probes). TUNEL staining was
performed by using the In Situ Cell Death Detection Kit (Roche) according to
manufacturer's recommendations. Secondary antibodies coupled with FITC, CY3 or
CY5 (Jackson) were used at 1:400. Wings were mounted in Vectashield medium
(Vector Laboratories).
Time-lapse
Living pupae were taped to facilitate dissection. The puparium case was
removed and the exposed wing was covered with 10S oil (Voltalef). Animals were
subsequently transferred, with the wing facing down, to a glass dish
(Willco-dish). Glial cells were imaged in 4D using a TCS SP2 inverted confocal
microscope (Leica) equipped with a heating stage to maintain a constant
temperature (25±2°C).
Confocal laser ablation
The nucleus to be targeted was selected upon GFP labelling and scanned in
the z-axis to identify a focal plane located at the centre of the
nucleus in the three x, y and z axes. A region within the
nucleus was chosen using the Leica bleachpoint function and submitted to UV
laser irradiation (350 nm, 20 seconds pulse at medium intensity). Cell death
was revealed by a strong decrease in GFP fluorescence and was confirmed by
immunolabelling after fixation.
Image processing
Z-stack projections, colour coding
(Bernardoni et al., 1999),
rotations, figure mounting and time-lapse movies were obtained using the
inhouse developed TIMT software. Images were annotated using Adobe Photoshop
and Illustrator, movies were converted to QuickTime format using Adobe
Premiere.
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Results |
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The observation that glia migrate and proliferate to variable extents
(Giangrande, 1994;
Van De Bor et al., 2000
)
suggests a role for extrinsic signals and makes it difficult to follow the
development of glial lineages in fixed tissues. We therefore devised an in
vivo glial marker by crossing the repo-Gal4 driver
(Sepp et al., 2001
) with a
UAS-GFP reporter (repo-GFP).
4D gliogenesis: migration and cytoskeleton remodelling
For time-lapse analyses on repo-GFP pupae, we removed the puparium
case over the wing, leaving the appendage in place
(Fig. 2A). Due to high levels
of autofluorescence, wings were analysed by confocal microscopy (Z stacks of
20-100 optical sections taken at intervals ranging from 90 seconds to 15
minutes). In vivo GFP labelling is comparable to that observed after
immunohistochemistry of fixed preparations (data not shown). Moreover,
processing and laser excitation do not have a major impact on development, as
pupae that have been analysed by time-lapse do reach adulthood and display no
abnormal phenotype. Finally, wings subjected to glial and neuronal labelling
after the time-lapse do not reveal major differences when compared with
unprocessed wings (data not shown). Thus, this in vivo approach is
non-invasive, highly sensitive and allows us to faithfully follow glial
differentiation.
|
|
On L1, high glial density makes it more difficult to follow individual
behaviours. Nevertheless, time-lapse analyses identify the proximal front as a
landmark of directional migration
(Fig. 2C, and Movie 1 in
supplementary material). Cells at the front of migration or pioneer
cells display very long and dynamic filopodia
(Fig. 3A,D, and see Movie 3 in
supplementary material) that have disappeared by the time the pioneers reach
more proximal glia (data not shown). Although the precise number and position
of the pioneer cells is not yet known, their presence suggests that
specialised glia populate different regions of the nerve. To label individual
L1 glial cells, we performed a mosaic analysis with a repressible cell marker
(MARCM) (Lee and Luo, 2001)
(Fig. 3B,C), using the
repo-GFP line. By mitotic recombination, GFP expression is induced in
patches of glial cells and, occasionally, isolated labelled cells are
generated. This approach allowed us to formally demonstrate for the first time
that, in addition to the pioneer, dynamic, glia, another migratory population
showing less filopodial activity is located at more distal positions. All
distal glia (25 wings analysed) show a less elaborate morphology than the
pioneer cells (Fig. 3B-D),
suggesting that they represent the majority of the L1 glia.
|
Glia-glia interactions inhibit the extent of migration
Movies on L3 glia show that cells belonging to one lineage move until they
reach more proximal glial cells. Although both groups of cells continue to
migrate upon establishment of contact, they tend not to intermingle, as
distally located glia do not bypass proximal glia (see Movie 4 in
supplementary material and Fig.
8). Moreover, glial precursor divisions give rise to cells that
always move apart one from the other shortly after being produced (Movie 1).
To test for the role of glia-glia interactions in cell migration, we devised a
confocal-assisted ablation protocol using the repo-GFP line. We
targeted the UV laser to a confined region of a GFP-positive nucleus, using
the Leica bleachpoint function (see Materials and methods), and verified that
cell death is induced in several ways. First, fading of GFP labelling in the
targeted cell is induced within five minutes following UV irradiation
(Fig. 4A). This was observed in
all the cells that were subjected to irradiation (n=38). Labelling
never resumed, even after hours. Second, cell irradiation results in a lack of
Repo labelling. Fig. 4B shows
an example of cell ablation of all L3 glia, verified by lack of both GFP and
Repo labelling throughout the L3 vein. Third, bleaching glial cells using the
488 nm ray at very high intensity does not lead to death. Indeed, bleached
cells resume expressing GFP and go on dividing (data not shown).
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Altogether, these data indicate that glia can move in both directions, although they are less apt to migrate distally than proximally, and that glia-glia interactions inhibit the extent of movement.
Neurone-glia interactions control directional migration but not glial motility
In order to determine the role of neurone-glia interactions, we analysed
migration in wings that lack axons. When the Notch (Nts1) receptor
is absent throughout wing sensory organ development, all cells within the
lineage are transformed into glia. These glia, which initially form a cluster
(Fig. 6A)
(Van De Bor and Giangrande,
2001), eventually organise themselves in a continuous chain,
indicating that they are able to migrate despite the absence of neurones
(Fig. 6D,E). Interestingly,
glial cells reach more distal positions than in wild-type wings
(Fig. 6E,F), suggesting that,
although the axonal substrate does not control glial motility, it affects the
direction of glial migration.
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Control of glial cell number
In the nervous system, the control of cell number is achieved through cell
division and/or death (Hidalgo,
2002; Raff et al.,
1993
; Sepp and Auld,
2003
). For example, glial precursors in the rat optic nerve divide
extensively during development, some progeny subsequently undergoing apoptosis
(Barres et al., 1992
). By using
several approaches, we investigated how the number of wing glia is controlled.
First, we followed an individual glial lineage from 23 hours APF for almost
ten hours, a time frame that includes all divisions (see Movie 6 in
supplementary material). A short interval (thirteen minutes) and the use of a
nuclear GFP enabled us to score for any proliferative and/or apoptotic event.
At the beginning of the movie, the GPI has already divided once, and within an
hour the two GPIIs divide again. This example of a glial lineage of four cells
shows no sign of cell death throughout the time-lapse. Seven L3.3 lineages
were analysed in this way and in no instance did we retrieve dying cells, not
even in lineages containing more than four cells (data not shown). Similar
results were obtained in the L3.1 lineage (n=7 wings analysed by
time-lapse; data not shown).
As a second approach, we analysed the apoptotic profile of wing glia at six developmental stages (see Fig. S1 in supplementary material). Owing to the transient nature of the TUNEL labelling, more than six wings were analysed at each stage (n=7 at 17 hours, n=40 at 20 hours, n=8 at 22 hours, n=25 at 26 hours, n=6 at 30 hours and n=10 at 36 hours APF). Although TUNEL did label sparse cells throughout the wing, in no cases were L1 and L3 glial cells positive for TUNEL labelling. Thus, in the fly wing, the control of cell number is primarily achieved through cell division.
As glia-glia interactions control migration, we investigated whether they
also affect proliferation. Homeostatic control of proliferation, a regulatory
process that involves compensatory divisions, does take place in the neural
crest upon cell ablation (Couly et al.,
1996; McKee and Ferguson,
1984
). This process, however, does not seem to occur in wing glia,
as cells from different lineages keep proliferating even when they are in
touch with each other (n=13) as shown in
Fig. 8. Indeed, the four L3.3
GPIIs all undergo one round of division after the L3.3 lineage establishes
contact with the L3.1 glial cells. Furthermore, L1 glia keep proliferating
(Table 1), even though they are
in contact with each other from very early stages
(Fig. 2C). The formal
demonstration that glia-glia contacts do not control cell division comes from
the observation that, when a GPI is ablated, glial cells from the remaining
lineages do not overproliferate (Figs
5,
9).
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Time-lapse data are consistent with the mode of division proposed in
Fig. 1A. GPIIs are of
comparable size and shape, and both undergo mitosis, which is suggestive of
symmetric rather than asymmetric stem-cell like divisions
(Fig. 11D). Cells of the glial
lineage divide rather synchronously, in contrast with previous results
obtained on fixed material (Van De Bor et
al., 2000). For example, GPIIs of the same lineage divide within
30 minutes of one another (Fig.
11D). Cells from different lineages divide with, at the most, a
one-hour time difference (see Movies 1, 4 in supplementary material). The
discrepancy between fixed and living materials is probably due to the fact
that anti-PH3 antibody recognises dividing cells in a transient manner
(Hendzel et al., 1997
).
In conclusion, glial precursors divide synchronously and symmetrically along the axis of migration.
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Discussion |
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The combined use of cell ablation, genetic and time-lapse approaches allows us to show that: (1) glial cells undergo extensive cytoskeleton and mitotic apparatus rearrangements during movement and division; (2) pioneer glia actively explore the environment during migration; (3) neurone-glia affect directional migration but do not control glial motility; (4) glia-glia interactions control the extent of migration; and (5) the control of glial cell number is achieved via cell proliferation, which is autonomously determined.
Fly wing glia, a model system for cell movement and proliferation
The ability to move is shared by many cells in normal and pathological
conditions (Gammill and Bronner-Fraser,
2003; Keller,
2002
; Perego et al.,
2002
; Small et al.,
2002
; Traver and Zon,
2002
). The fly wing gives us the opportunity to analyze
proliferating cells moving in a chain (L1 glia) and in isolation (early L3
glia).
When we compare L1 glia with fly border and tracheal cells, both
differences and similarities become apparent. First, border cells rely on an
asymmetric structure, the actin based-LCE (long cellular extension), which
triggers movement through a grapple and pull process
(Fulga and Rorth, 2002;
Geisbrecht and Montell, 2002
).
By contrast, wing pioneer glial cells send out numerous filopodia that
dynamically assay the environment in all directions, as is also seen in
tracheal cells (Ribeiro et al.,
2002
; Sato and Kornberg,
2002
). This may reflect the fact that border cells form a cluster
and move simultaneously, whereas, in the chain of glia, distal cells do not
contact pioneer cells and follow the movement of adjacent, more proximal,
cells. Based on time-lapse and ablation data, we propose the following model.
Prior to migration, pioneer cells are free to explore the proximal axonal
substrate whereas follower cells are submitted to bilateral repulsive
glia-glia contacts. As pioneer cells move proximally, they free space at more
distal positions. Follower cells rapidly occupy such space, thus freeing even
more distal regions. This domino-type of migration proceeds until homogenous
axon enwrapping is reached.
Most exploratory activity is spatially and temporally restricted as it
specifically characterises cells that move on naked axons. Whether the
presence of pioneer cells at the front of migration is lineage dependent or
reliant on extracellular signals remains to be determined. In the future,
performing ablations throughout the L1 will help to address the issue of
plasticity. The presence of LCE versus pioneer filopodia may reflect different
modalities of directional migration. It is known that border cells respond to
chemoattractants (Fulga and Rorth,
2002; Montell,
2003
), whereas glial directional migration may be driven by
underlying axons (see below). Interestingly, L3 migrating and proliferating
cells all show filopodia, suggesting yet a different mode of migration
compared with that of cell chains (L1 glia) and clusters (border cells).
Finally, glia, but not tracheal or border cells, divide as they move,
suggesting that the formation of a continuous chain along the axon bundle
requires both migration and proliferation. In the future, it will be crucial
to determine how the two events are coordinated.
The different features shown by border cells, trachea and glia suggest that
cell specification controls motility strategies. The role of cell
specification cues is further demonstrated by the fact that, even within glial
cells, different lineages display distinct features. While wing GPs divide
several times (Van De Bor et al.,
2000) (this study), glia arising from dorsal bipolar dendritic
embryonic lineages (Umesono et al.,
2002
) and microchaete glia
(Gho et al., 1999
;
Reddy and Rodrigues, 1999
) do
not divide, the latter dying soon after birth
(Fichelson and Gho, 2003
).
Understanding the molecular pathways specifying migratory and proliferative
profiles represents one of the future challenges for developmental
biologists.
Like oligodendrocyte precursors in the rat optic nerve that are controlled
by an internal clock (reviewed by Durand
and Raff, 2000), GPIs divide a limited number of times. Different
sets of data speak in favour of an internal clock that limits the absolute
number of divisions to three. First, the average number of glia derived by
different sensory organs is constant (Van
De Bor et al., 2000
) (this study), irrespective of the number of
underlying axons (L3.3 and L3.1 glia line one and four axons, respectively).
Thus, the number of axons does not control proliferation. Second, time-lapse
data show that divisions within and between lineages are rather synchronous.
Third, mitotic clones lacking the Glide/Gcm (Glial cell deficient/Glial cell
missing) glial promoting factor in one gliogenic sensory organ result in fewer
wing glia, indicating that the remaining cells do not compensate for the
missing ones (Van De Bor et al.,
2000
). Finally, our ablation data demonstrate a lack of
compensatory divisions within and amongst glial lineages. Whether vertebrate
and invertebrate clocks rely on the same signals will be a matter of future
studies. In addition to the internal clock, extracellular signals may be at
work and may control the fine-tuning of proliferation (four to eight cells per
lineage). A role of cell-cell interactions in the control of glial
proliferation has also been observed in the fly embryo
(Griffiths and Hidalgo,
2004
).
Specific cell-cell interactions control different aspects of migration
One of the most peculiar features of glia is that they tend to form a chain
of cells. This might suggest that glial cells display affinity for axons as
well as for other glial cells, the equilibrium between these affinities
dictating the extent of migration and triggering the formation of a continuous
glial sheath. However, even in the absence of axons (Nts1
data), glial cells form a continuous chain, rather than staying as a cluster
or moving apart from one another. Thus, axons do not trigger glial cell
alignment, clearly showing that glia are endowed with an intrinsic migratory
potential. The chain of glia present in Nts1 wings is
unbranched, as if the surrounding vein were providing a physical channel or
instructive cues for migration. Although we cannot formally exclude a
participation of veins in axonal navigation and/or glia migration, veinless
wings still contain properly organised axons and glia (data not shown).
Moreover, ectopic axons present in the intervein space of Hw wings
carry properly lined glial cells (data not shown), thus indicating that veins
are not instructive for glial migration.
One way to reconcile all data is that different types of interactions take place. On the one side, glia tend to fully occupy and enwrap naked axons, probably in response to neuronal signals. On the other side, counteracting interactions are at work between glia. Thus, while affinity between glial cells induces them to stay together, repulsive contacts prevent them from forming a cluster and trigger the formation of a chain. The observation that the cytoplasmic processes of adjacent glia largely overlap (data not shown) is in agreement with this hypothesis, and leads us to propose that glial cells tend to reduce the extent of contact by sliding over each other. The equilibrium between all these forces allows the formation of a continuous chain and controls the extent of movement, compatibly with the substrate available for migration.
Glial cells move in a stereotyped direction and, as shown by the ablation
data, do not require the presence of guide-post glia to find their way.
Instead, neurone-glia interactions affect directional migration. Indeed, both
in fly wings and in the zebrafish lateral line, misrouted axons result in
redirected glia (Giangrande,
1995; Gilmour et al.,
2002
) (this study). Furthermore, in both systems, glial arrest is
observed upon axonal arrest (Giangrande et
al., 1993
; Giangrande,
1994
; Gilmour et al.,
2002
). The fact that glia use axons as a substrate suggests an
unpredicted axonal feature. Indeed, although the polarised nature of the axon
is well characterised with respect to microtubule growth, how does the axon
convey directional information to the enwrapping glia?
Our data clearly show that glial migration relies on complex and dynamic glia-glia and neurone-glia interactions. Establishing time-lapse protocols that simultaneously monitor neurones and glia, or aiming at simultaneously identifying the whole glial population and a subset of glia, will be crucial to gaining a better understanding of the precise nature and role of such homo- and heterotypic interactions.
Glial cell polarity and division
Most migratory cells undergo an epithelial to mesenchymal transition that
implies changes in cell polarity (Hay,
1995). Similarly, glial cells originate through the apico-basal
division of the IIb precursor (Gho et al.,
1999
; Reddy and Rodrigues,
1999
; Van De Bor et al.,
2000
). The newly formed GPIs wait almost ten hours before
dividing, whereas GPIIs divide and migrate rapidly soon after birth
(Van De Bor et al., 2000
)
(this study). By the end of this latency period, the GPI acquires a very
polarised shape, and divides along the proximodistal axis. Altogether, these
results indicate that a change in cell polarity occurs in the GPI, the cell
that starts migrating. Thus, latency probably serves to build up the GPI
competence for proliferation and migration.
The fact that glial cells divide and migrate along the same axis suggests that the signalling pathways controlling cell polarity, division and motility are coordinated. The analysis of mutations affecting these processes will be fundamental for understanding the molecular bases of their integration.
Conclusions
The development of transgenic animals carrying GFP and cell-specific
promoters makes it now possible to analyse dynamic behaviours such as
migration and proliferation at the level of individual cells in wild-type and
mutant backgrounds. Furthermore, following the consequences of cell ablation
allows analysis of the role played by cell-cell interactions. In future,
combining genetics with cell biology in living animals will give novel
insights into the cellular and molecular network controlling cell movement and
division in physiological and pathological conditions.
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ACKNOWLEDGMENTS |
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Footnotes |
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Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/131/20/5127/DC1
* Present address: University of Edinburgh, Wellcome Trust Centre for Cell
Biology, King's Buildings, Mayfield Road, Edinburgh, EH9 3JR, UK
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