UMR 7622, CNRS-Université Paris VI, 9, Quai St. Bernard, 75252 Paris Cedex 05, France
* Author for correspondence (e-mail: michel.gho{at}snv.jussieu.fr)
Accepted 4 October 2002
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SUMMARY |
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Key words: Apoptosis, Axogenesis, gcm, Microchaete, Phagocytosis, p35, reaper, hid, grim, Asymmetric cell divisions
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INTRODUCTION |
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Mechanosensory organs on the thorax of Drosophila, also called
microchaetes, have become a model system to study how cell division and cell
determination intermingle (Jan and Jan,
1998). Microchaetes are formed by two outer support cells (the
socket and the shaft cell) and two inner cells (the neurone and the sheath
cell). These cells arise from a primary precursor cell, called pI, after four
asymmetric cell divisions that occur during pupal stages of development. The
division of pI gives rise to two secondary precursor cells, pIIa and pIIb.
During the next cycle of divisions, pIIb divides prior to pIIa to generate a
glial cell and a tertiary precursor cell, pIIIb. The division of pIIa produces
a socket and a shaft cells. Finally, pIIIb divides, giving rise to a neurone
and a sheath cell (Gho et al.,
1999
).
The asymmetry of these divisions is controlled by the differential
segregation of cell determinants during mitosis
(Rhyu et al., 1994;
Manning and Doe, 1999
). For
example, during pIIb division, Numb and Prospero are inherited exclusively by
the glial cell (Gho et al.,
1999
). Numb is a negative regulator of the Notch receptor
(Rhyu et al., 1994
;
Guo et al., 1996
), therefore
among the two pIIb daughter cells, this pathway is only activated in pIIIb,
the glia sibling cell. Prospero, a transcription factor, regulates the
expression of several genes. In embryonic cells, Prospero represses neuronal
specific genes such as deadpan and activates the expression of glial
specific genes such as glial cell missing (gcm/glide)
(Vaessin et al., 1991
;
Freeman and Doe, 2001
).
It is generally admitted that glial cells are involved in axonal guidance
and neuronal survival (Jones,
2001). In Drosophila, glial cells are characterised by
their expression of gcm, which encodes a DNA-binding protein
promoting expression of glial cell determinants such as the homeobox
transcription factor Repo (Jones,
2001
). In the microchaete lineage, we have previously reported
that the glial cell migrates away from the sensory cluster along the axon
(Gho et al., 1999
). Similar
observations were obtained in sensory campaniform in the wing
(Van De Bor et al., 2000
). In
this case, glial cells show precursor properties and divide to generate
clonally related glial cells which migrate towards the proximal region of the
wing blade.
In this study, we have examined the fate of glial cells in the thoracic microchaete lineage by time-lapse confocal microscopy in living pupae and by immunodetection in dissected nota. This analysis has revealed that glial cells do not actually migrate away but rather undergo apoptosis. Furthermore, glial cells die even after transformation of their fate, suggesting that these cells are committed to programmed cell death independently of the acquired identity. These observations have led us to ask what the role of glial cells is in the final configuration of microchaete organs, particularly during axogenesis.
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MATERIALS AND METHODS |
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Clonal analysis
Somatic clones were obtained using the FLP/FRT recombination system
(Xu and Rubin, 1993). The w;
FRT40A gcme1 line (gift of V. Rodriguez) were crossed to
the flp, FRT40A ubq-nls::GFP (gift of Y. Bellaiche) line to generate
gcm null somatic clones. The FRT2A H99 (gift of B. Bello) line was
crossed to the flp, FRT2A ubq-nls::GFP (gift of J.-R. Huynh) to generate H99
deficient somatic clones. The FRT82B prospero17
(pros17) (Reddy and
Rodrigues, 1999
) line was crossed to the flp, FRT82B ubq-nls::GFP
(gift of J-R. Huynh) to generate pros-null somatic clones. To induce
mitotic recombination, second instar larvae from these crosses were heat
shocked twice at 38°C for 30 minutes at 1 hour interval and kept at
25°C for recovery.
Time-lapse confocal microscopy
In vivo imaging was carried out as described previously
(Gho et al., 1999). Confocal
images were acquired every 3 minutes on a Leica TCS confocal microscope at
24°C. Time-lapse movies were assembled using NIH image software.
Macrophages labelling
Indian Ink (Pébéo, Gemenos, France) was injected in early
third instar larvae. Development was then allowed to proceed at 18°C until
pupation. After pupation, samples were transferred at 25°C until
dissection and antibody staining.
Immunohistology
Dissected nota from pupae at 22-30 hours after pupal formation (APF) were
processed as described previously (Gho et
al., 1996). The following primary antibodies were used: rabbit
anti-ß-galactosidase (Cappel, 1:1000), mouse anti-GFP (Roche, 1:500),
rabbit anti-GFP (Santa-Cruz, 1:500), mouse anti-Cut (DSHB, 1:500), rabbit
anti-Repo (gift of A. Travers, 1:500), rat anti-Elav (DSHB, 1:10), mouse
anti-Futsch 22C10 (DHSB, 1:100) (Hummel et
al., 2000
), rabbit anti-Croquemort (1:1000)
(Franc et al., 1999
),
guinea-pig anti-Senseless (1:1000) (Nolo
et al., 2000
). Alexa 488- and 568-conjugated secondary antibodies
anti-mouse, anti-rat and anti-rabbit were purchased from Molecular Probes and
used at 1:1000. DNA fragmentation was assayed by TdT-mediated dUTP nick end
labelling (TUNEL kit, Roche Molecular Biochemicals). Images were obtained on
an Olympus BX41 fluorescence microscope (x63 immersion oil objective)
equipped with a CoolSnap camera driven by Metaview software (Universal
Imaging). Images were processes with NIH Image and Photoshop software.
Only sensory clusters located in row 1 (close to the midline) were considered for statistical analysis.
Physiological assay
Cleaning reflex was tested on decapitated adult as described elsewhere
(Corfas and Dudai, 1989).
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RESULTS |
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At about 23 hours APF (on average 1.5 hours after pIIb division), the nucleus of the glial cell condensed (observed as a change in the nucleus shape and in the level of fluorescence) and then rapidly fragmented (Fig. 1A). Fragments dispersed quickly in about 10 minutes, generally towards a posterior direction (arrowheads in Fig. 1B), and disappeared. Before fragmentation, we frequently observed that the glial cell lost contact with the other cells in the cluster (see film clip 1 available at http://ifr-bi.snv.jussieu.fr/FichesPerso/Gho.html). The fragmentation of the glial cell nucleus was observed in every cluster studied. In 90% of the cases (n=18), fragmentation occurred after pIIIb division, when the neurone and the sheath cell were already present. In the other cases, fragmentation was observed before the division of pIIIb (Fig. 1B). Thus, the elimination of the glial cell was independent of the division of its sister cell pIIIb.
Cell fragmentation and glial cell elimination were also observed when UAS-H2B::YFP or UAS-nls-GFP constructions were driven by other GAL4 lines such as SOP-GAL4 (which expresses GAL4 in secondary precursor cells and their descendants, not shown) and scabrous-GAL4 (which expresses GAL4 in pI and its descendants) (Fig. 1C). This indicates that fragmentation of the glial cell is not an artefact because of the overexpression of the UAS-H2B::YFP construction in the lineage cells nor a positional effect of the transgenic insertions. Similar nuclei fragments were observed in the A101 strain, which express ß-Gal under the control of the neuralized promoter (not shown). Thus, in all genetic contexts studied, cell fragmentation and glial cell disappearance were observed.
In w1118 pupae, nuclear staining with DAPI revealed that the glial cell (identified with the sensory cell specific anti-Cut antibodies) had a very condensed nucleus, which was not seen in other cell types suggesting that this cell was in the process of DNA condensation and further fragmentation (not shown). To detect DNA fragmentation in w1118, we performed a TUNEL assay. At 23 hours APF, TUNEL staining in wild-type pupae revealed labelled fragments in the proximity of many sensory clusters, suggesting that a cell undergoes apoptosis nearby. To analyse whether the fragments belong to the lineage, we took advantage of Senseless antibodies which preferentially recognised the neurone, the sheath and the glial cells. This double-staining revealed that most Senseless-labelled fragments were also TUNEL-positive (arrow in Fig. 1D). These observations confirm that the TUNEL-positive fragments pertain to the sensory lineage. Furthermore, our experiments showed that sensory clusters are formed either by four cells and a TUNEL-positive fragment or by four cells and a Repo-positive cell (arrowhead in Fig. 1D). Clusters with a cell co-labelled with TUNEL and Repo were rarely observed. This strongly suggests that, in general, the glial cell lost Repo immunoreactivity prior to fragmentation.
All together, our observations reveal that glial cell fragmentation and further elimination is a bona fide phenomenon in the thoracic bristle lineage. Those events are reminiscent of programmed cell death by apoptosis. Therefore, we analysed whether inhibition of apoptosis would result in the loss of glial cell fragmentation.
The glial cell undergoes apoptosis
Genes within the H99 region are involved in the death of the glial
cell
To highlight a potential role of the pro-apoptotic genes rpr, hid
and grim in the death of the glial cell, we generated somatic clones
deficient for the H99 region that covers these three genes.
At 24 hours APF, Repo staining revealed that the glial cell was present in 95% of the clusters within H99 deficient clones (n=83, Fig. 2A), whereas clusters within the twin wild-type clones showed a glial cell in only 8% of the cases (n=59). This striking effect of the H99 deletion on the survival of the glial cell reveals an involvement of the pro-apoptotic genes of the H99 region in the death of the glial cell.
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Surprisingly, in the H99 heterozygous parts of the nota, glial cells were visible in 73% of the clusters (n=156, not shown) at 24 hours APF. This increased presence of glial cells in H99 heterozygous clusters compared with wild-type clusters reveals a dose-dependent action of the pro-apoptotic genes of the H99 region.
Finally, at 30 hours APF, fewer glial cells were observed compared with at 24 hours APF. Glial cells were detected in 63% of the clusters within H99-deficient clones (n=89, Fig. 2B), in 45% of the clusters in heterozygous regions (n=144, not shown) and in none of the clusters within homozygous twin spots (n=35, not shown).
p35 overexpression prevents fragmentation of the glial cell
To investigate whether the viral caspase inhibitor p35 could prevent
fragmentation of the glial cell, we monitored the presence of Repo-positive
cells in neuP72 UAS-H2B::YFP UAS-p35 flies.
At 24 hours APF in neuP72 UAS-H2B::YFP animals, only 6% of the clusters showed a Repo-positive cells (n=65). At the same time in neuP72 UAS-H2B::YFP UAS-p35 pupae, glial cells were present in 86% of the clusters (n=60, Fig. 2C) and no fragments could ever be seen. At later stages (30 hours APF), the proportion of sensory organs presenting Repo-positive glial cell in p35 overexpressing flies decreased to 15% of the clusters (n=40, Fig. 2D).
Taken together, these observations show that glial cells are eliminated from the epithelium by a H99-dependent and p35-sensitive apoptosis. In addition at late stages, we observed that both p35 overexpressing pupae and H99 homozygous clones showed a decrease in glial cell number. Therefore, we monitored the fate of these cells in neuP72 UAS-H2B::YFP UAS-p35 living pupae.
Surviving glial cells do not remain in the epithelium
In neuP72 UAS-H2B::YFP UAS-p35 living pupae, the glial
cell remained associated to the corresponding sensory cluster in an
unfragmented form between 21 and 24 hours APF
(Fig. 3A), confirming the
observations obtained in fixed material. This is about 2 hours longer than in
control pupae (see Fig. 1A).
After 24 hours APF, the fluorescence of the glial cell gradually weakened as
the cell moved away from the cluster (Fig.
3B; see film clip 2 at
http://ifr-bi.snv.jussieu.fr/FichesPerso/Gho.html).
Moreover, the glial cell progressively fell down into the internal cavity
(Fig. 3B') where it could
no longer be observed (Fig.
3C'). It is important to note that fragmentation was never
observed. Thus, from these experiments we conclude that, although glial cell
fragmentation is inhibited by the viral caspase inhibitor p35, these cells
nevertheless leave the epithelium.
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Ectopically surviving glial cells do not affect axon morphology and
connectivity
Given the important role of glial cells in axonal guidance and neuronal
survival, it was surprising that in wild-type specimens, most glial cells had
already undergone apoptosis at 25 hours APF when axonal growth cones became
visible (data not shown). In addition, as shown in
Fig. 1B, glial fragmentation
sometimes occurred before the division of pIIIb, as such before neurone
formation, rendering any interaction between the glial cell and the neurone
very unlikely in wild-type flies. Under experimental conditions in which glial
cell death was inhibited, we asked whether surviving glial cells would affect
the morphology and/or the development of microchaetes.
We first analysed possible ectopic interactions between glial cells and axonal processes. In p35-expressing pupae at 24 hours APF, glial cells were tightly associated with 22C10-positive axonal processes in 72% of the sensory organs studied (n=69). In 55% of these cases, glial cells were located at the growth cone (Fig. 4A, another example of such a location, on the second row of microchaetes, is shown in Fig. 4D), while they were associated to the axon in the remaining 17.5% of the cases (Fig. 4B). In the rest of the sensory organs analysed (27.5%), glial cells remained within the proximity of the cluster (Fig. 4C). The fact that in 72% of the clusters, a close association was observed between the axon and the ectopically surviving glial cell shows that the ectopic cell retains `glial' characteristics.
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Second, we studied the consequence of surviving glial cells on the morphology of the axonal arborisation. At 27 hours APF, the axonal network and its orientation, showed no major difference between wild-type and neuP72 UAS-H2B::YFP UAS-p35 pupae (Fig. 5A,B, 27 hours). Therefore, it seems unlikely that glial cells provide any guidance clue to the neurones. In addition, we performed physiological studies testing the cleaning reflex in p35-expressing and control adult flies. In both cases, we observed that, after stimulation of the thoracic bristle, the cleaning reflex was elicited normally in p35-expressing adults (not shown). Taken together, these observations suggest that ectopic survival of glial cells has no effect on either the final axonal connectivity or the physiology of the sensory bristle.
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The presence of the glial cell promotes axonal outgrowth
Although, glial cell survival does not seem to affect axonal projections,
we wondered whether ectopic presence of glial cells could influence the time
course of axogenesis. Therefore, we analysed axonal growth at different times
of development in neuP72 UAS-H2B::YFP UAS-p35 pupae. While
wild-type specimens exhibited no processes at 24 hours APF
(Fig. 5A, 24:00 h), axonal
growth cones were already observed 30 minutes before, in p35 overexpressing
flies (Fig. 5B, 23:30 h). At 25
hours APF, the first growth cones appeared in neuP72
UAS-H2B::YFP pupae, whereas axons originating from the first row of pIs were
already reaching the second line in neuP72 UAS-H2B::YFP
UAS-p35 pupae (Fig. 5A,B, 25:00
hours).
Similar results were obtained when glial cells were forced to survive in H99-deficient clones. Fig. 5C shows a H99 clone in a pupa at 25 hours APF, sensory neurones inside the clones extend their axons towards the second row of microchaetes (Fig. 5C, arrowheads), whereas axons from sensory organs in the contralateral H99 heterozygous heminotum are still absent or just appearing. The fact that, in H99 heterozygous tissue, axogenesis occurs at the same time as in control tissue was a surprise because, under these conditions, glial cells survive longer than in wild type (see above). This suggests that H99 heterozygous glial cells, even if they survive longer, are unable to affect neuronal development (see Discussion).
Early axogenesis was not due to an advanced rate of neuronal development because, first of all, the time of the pIIIb division was not altered and, second, the determination of neurones, as measured by Elav immunoreactivity, was similar in wild-type and in p35-overexpressing pupae (data not shown). Furthermore, the time course of axonal appearance was not affected after overexpression of p35 using the neuronal-specific driver elav-GAL4 (not shown).
Therefore, we conclude from these observations that the presence of the glial cell promotes axonal differentiation and outgrowth.
Glial cell death is independent of glial identity
In order to test the role of cell identity acquisition in triggering
apoptosis of glial cells, we analysed the pattern of apoptosis in gcm
mutant somatic clones. No Repo-immunostained cell was revealed in clusters
inside gcm clones compared with, at most, one cell for clusters
outside the clone, confirming that pIIIb sibling cell did not acquire glial
cell identity in gcm mutant clones (data not shown). Thus, at 24
hours APF, two types of sensory organs were observed in gcm clones
(Fig. 6A), those composed of
four cells (82%, n=66) and those composed of five cells (18%,
n=66). Interestingly, a similar situation was observed outside
gcm clones. There, clusters were composed of four (84%,
n=62) or five cells (16%, n=62). In addition, two
Elav-positive cells were detected in five-cell clusters inside gcm
clones (arrowhead in Fig. 6A),
this situation was never observed in clusters outside gcm clones. In
four-cell clusters within gcm clones, only one cell was Elav
positive, verifying that the entire lineage had been produced at this pupal
age. The absence of Repo-positive cells in gcm clones and the extra
neurone observed in five-cells clusters confirmed that in absence of
gcm, glial cells adopted a neuronal fate. Moreover, the number of
cells by cluster (four or five) and the number of neurones in each cluster
(one or two, respectively) observed in gcm clones suggests that one
extra neurone was eliminated from the five-cell clusters. This was confirmed
by the observation of Elav-positive fragments in gcm clones
(arrowhead Fig. 6B), suggesting
that the extra Elav-positive cell underwent programmed cell death. These
results indicate that the glial cell undergoes apoptosis independently of
gcm expression. As this cell dies when it acquires a glial or a
neuronal identity, this could indicate that apoptosis is triggered in the
future glial cell before this cell is determined.
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Glial cell death is independent of prospero expression
Previous studies have shown that the glial cell strongly expresses the
transcription factor Prospero (Gho et al.,
1999). Therefore, we investigated a potential involvement of this
gene in glial cell apoptosis using mosaic flies for the null allele
pros17. No Prospero-immunostained cell was observed in
clusters inside pros17 mutant clones, confirming the
amorph quality of this allele. Surprisingly, one Elav-positive neurone and one
Su(H)-immunoreactive socket cell were observed in every sensory clusters
inside the clones (data not shown). This strongly suggests that the bristle
lineage occurred normally in absence of Prospero, allowing the study of the
glial cell fate under these conditions.
Sensory organs in pros17 clones at 24 h APF were composed by five (29%, n=69) or four (71%, n=69) Cut-positive cells. In addition, one Repo-immunoreactive cell was observed in every five-cell cluster within the clone, showing that Repo-positive glial cells are formed in the absence of Pros. Furthermore, the lack of Repo-positive cell in four-cells clusters suggests that the Repo-positive glial cell was eliminated from the cluster as in control tissue (see above). This was confirmed by the observation of Repo-positive (arrows in Fig. 6C) as well TUNEL-positive cell fragments (arrows in Fig. 6D) co-labelled with senseless nearby sensory clusters within pros17 clones. These results show that the glial cell undergoes apoptosis independently of pros expression.
Fragments are phagocytosed
Macrophages are known to be involved in the removal of apoptotic cells.
Therefore, we analysed the behaviour of macrophages during the period of glial
cell fragmentation. In embryos, macrophages can be visualised by
immunostaining against the membrane protein Croquemort (Crq)
(Franc et al., 1999). However,
we failed to detect Crq-immunoreactive cells in the notum. As an alternative
procedure, we labelled macrophages by injecting Indian Ink into larvae.
Macrophages are known to accumulate ink following its injection into the
general cavity (see Lanot et al.,
2001
).
Indian Ink was injected in neuP72UAS-H2B::YFP early third instar larvae. Fifty percent of the injected larvae (n=250) survived until pupation and only 20% of the pupae were still alive when dissected at 24 hours APF. Dissected nota from these pupae frequently exhibited black-labelled multicellular structures located at posterior lateral positions. In addition, spread throughout the nota, we observed subepithelial groups of `black-dots', which we interpreted as macrophages containing ink conglomerates. We next examined the glial cell fragments, identified as highly YFP-positive particles. This analysis showed YFP-positive nuclear fragments surrounded by `black-dots' suggesting that fragments were engulfed by ink marked macrophages (Fig. 7). This evidence together with the observation that fragments move away (see arrowhead in Fig. 1B) from the clusters indicates that they are phagocytosed by mobile macrophages.
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DISCUSSION |
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Inhibition of apoptosis does not prevent loss of glial cells from the
epithelium
Overexpression of the caspase inhibitor (p35) or removal of pro-apoptotic
genes (H99 deletion) blocks apoptosis of the glial cell, as evidenced by the
absence of nuclear fragmentation. Nevertheless, in both cases, the glial cell
disappears later on from the epithelium. We propose three possibilities to
explain this disappearance: loss of specific markers, cell migration or cell
death.
Time-lapse study of neuP72 UAS-H2B::YFP UAS-p35 specimen showed that YFP was always present in this cell until it went too deep in the specimen to be observed. We believe that this movement towards the inside of the pupa is the reason for the loss of Repo-positive cells observed between 24 and 30 hours APF. Therefore the hypothesis that the glial cell disappearance is due a loss of markers seems unlikely.
The possibility that surviving glial cells migrate away could be in
agreement with the observation that those cells tend to be associated to the
subepithelial axonal processes. Nevertheless, the drop of these cells,
perpendicularly to the plane of the neuronal processes, suggests that they
lost their close associations with the axons. This drop appears reminiscent of
a process know as `cell competition' in which cells lose their epithelial
position and fall down into the internal cavity
(Simpson, 1979).
We favour the hypothesis that surviving glial cells in H99-deficient clones
as well as in p35 overexpressing pupae still present characteristics that
either trigger a redundant programmed cell death or are recognised by
executioner cellular agents. Accordingly, phagocytosis of exceeding cells
after apoptosis inhibition was recently observed in embryos
(Mergliano et al., 2002).
Glial cell survival promotes axonal outgrowth
The ability of glial cells to induce ectopic expression of neuronal
specific markers such as Futsch protein has already been observed
(Klambt et al., 2001).
Consistently, our data show that ectopically surviving glial cells induce a
premature outgrowth of 22C10-positive axonal processes. Nevertheless, this
observation is difficult to reconcile with the fact that axonal outgrowth was
similar between wild-type and H99 heterozygous pupae where glial cells showed
prolonged survival. However, as we observed that these cells undergo nucleus
fragmentation (data not shown), we think that the H99 heterozygous glial cells
present apoptotic characteristics that prevent them from promoting axonal
outgrowth.
We believe that the premature axogenesis is indeed due to the action of the ectopic glial cells for the following reasons. First, similar observations were obtained when apoptosis was blocked by two different procedures, overexpression of p35 and H99 deletion. This ruled out the possibility that premature axogenesis could be a consequence of the genetic background on the sensory cells. Second, overexpression of p35 in the neurone using elav-GAL4 had no effect on the timing of axogenesis. This excludes the possibility of cell-autonomous effects of p35 on neurones. Finally, the time of neuronal determination was unaffected after apoptosis blockade. These observations strongly suggest that premature axogenesis is promoted by the presence of the glial cell that has been forced to survive.
The close association observed between p35 overexpressing glial cells and axons could reveal a common clue recognised by both cells and used for pathfinding. Alternatively, the glial cell could migrate towards the axon or vice versa. From our study, we cannot distinguish between these hypotheses. Nevertheless, the similarity of the axonal arborisation in control and p35-expressing flies strongly suggests that ectopic glial cells do not have major influence on axonal pathfinding. If some subtle effect occurs, the normal functionality of bristles observed in p35 expressing flies would suggest that the precise path of the axon does not matter for making a functional connection.
Our results also revealed that, under normal conditions, axonal outgrowth
occurred in the absence of glial cells. Thus, microchaete glial cells are
dispensable for axonal guidance, connectivity and sensory function. Instead,
the principal clue to guide microchaete axons of the thorax seems to be a
scaffold of persistent larval multidendrite neurones
(Usui-Ishihara et al., 2000).
Thus, we suggest that the acquisition of glial cell identity by one of the
cell in the microchaete lineage is an evolutionary relic.
Mechanisms responsible for the apoptosis of the glial cell
Diverse studies of developing vertebrate and invertebrate embryos have
demonstrated the essential role of extrinsic factors in promoting or
preventing cell survival (see Rusconi et
al., 2000; Durand and Raff,
2000
). Two types of extrinsic signals can be distinguished: the
endocrinal signal, which has a global action on the organism; and the
paracrine signals, which act locally. During the bristle lineage, it is
unlikely that hormonal signals or other endocrine factors are involved in
glial cell death, as apoptosis occurred in the absence of any synchrony. A
synchrony would be expected for a large-scale signal unless cells exhibit
different responsiveness at different times. Our study revealed that ecdysone,
an essential hormone controlling metamorphosis, did not trigger glial cell
death. More precisely, glial cell death occurred normally in
ultraspiracle mutant clones that affect nuclear ecdysone receptor
complex (data not shown) (Thummel,
1995
). Among the paracrine signals analysed, we focused our
attention on the EGF and JNK pathways, both of which are involved in
triggering apoptosis in Drosophila
(Rusconi et al., 2000
;
Adachi-Yamada et al., 1999
).
However, in our system, neither upregulation of the EGF pathway (by
overexpression of the activators Secreted Spitz, Rhomboid and an activated
form of the EGF receptor) (Klambt,
2000
) nor blockade of the JNK pathways (in basket mutant
clones) (Noselli, 1998
) had an
effect on glial cell apoptosis (data not shown).
As a consequence, we favour the idea that an intrinsic mechanism is
involved in the apoptosis of the glial cell. We have first ruled out a
potential involvement of gcm in this process. This result was
unexpected, knowing the key role of gcm in determining glial cell
identity, as revealed by the absence of Repo- and gain of Elav-expression in
sensory organs within gcm clones (our data)
(Van De Bor and Giangrande,
2002). Other intrinsic factors potentially involved in glial
apoptosis are Numb and Prospero, which segregate during pIIb division into the
glial cell (Gho et al., 1999
).
According to this possibility, those intrinsic factors, when inherited by one
pIIb daughter cell, would trigger apoptosis in the glial cell or would prevent
it in its sibling pIIIb cell. Recent studies have shown that Numb prevents
apoptosis mediated by the Notch pathway in an embryonic sensory lineage
(Orgogozo et al., 2002
). This
observation is difficult to reconcile in our system, as glial cells receive
Numb during the asymmetric cell division of its progenitor, pIIb. In the
bristle lineage, Numb plays an essential role in cell determination and any
modification of the segregation of this cell determinant has consequences for
the cell identity (Rhyu et al.,
1994
; Wang et al.,
1997
; Reddy and Rodrigues,
1999
). This precludes any well-defined experiment designed to test
a possible role of this factor on the glial apoptosis.
Previous studies have shown that Prospero promotes pIIb cell fate. Thus,
pros-overexpression transforms the pIIa cell into its sibling pIIb
(Reddy and Rodrigues, 1999;
Manning and Doe, 1999
). In
pros null clones, we observed at least two of the pIIb cell progeny,
the neurone and the glial cell. This shows that removal of pros gene
does not strongly affect the pIIb identity. In addition, we also observed
glial cell apoptosis. Two conclusions can be drawn from these results.
Firstly, Prospero seems sufficient but not necessary to induce pIIb identity
and secondly, this gene is not involved in triggering glial cell
apoptosis.
A common pattern of cell divisions at the basis of sensory organ
lineages
In the Oncopeltus larvae, nuclear breakdown in one of the five
cells that makes up the mechanosensory organs has been described
(Lawrence, 1966). Moreover, it
has been shown that, for a period, these organs comprise five cells but only
four survive throughout the life of this bristle, confirming that one cell of
the sensory cluster is eliminated. Although the origin of the cell that
underwent nuclear breakdown was not conclusively determined in these early
observations, we believe that this cell is the neurilemma cell, which is
homologous to the Drosophila glial cell of microchaete organs. Thus,
these observations strongly suggest that the elimination of the glial cell in
mechanosensory organs has been conserved throughout evolution.
Several cell lineages that give rise to various Drosophila sensory
organs share the same common pattern of cell divisions
(Fig. 8)
(Orgogozo et al., 2001;
Orgogozo et al., 2002
;
Gho et al., 1999
;
Brewster and Bodmer, 1995
;
Van De Bor et al., 2000
). This
core sensory lineage is composed by a primary precursor cell which divides
giving rise to two secondary precursor cells. One of these cells produces
three inner cells after two rounds of divisions and the other gives rise two
outer cells after one division. We propose that diversity amongst the
different lineages arises from adding different processes to this core
(Fig. 8). Two types of
processes may be distinguished: first, processes in which cell identity is
changed without altering the configuration of the common core pattern of cell
divisions: and, second, processes in which new complexity is added to this
core without profound modifications in cell identity (red and blue arrows in
Fig. 8, respectively).
Chordotonal organs and es-md sensory organs in the embryo are two examples of
the former type of process (Brewster and
Bodmer, 1995
; Orgogozo et al.,
2001
). Both of them originate from precursor cells after a cell
lineage identical to the core sensory lineage discussed before. However, the
identities of the cells are different. Chordotonal cell lineage gives rise to
an internal sensory organ while es-md lineage produces a mono-innervated organ
and a multidendritic neurone (Fig.
8). Situations that involve processes of the second type, which
add complexity without extensive changes in cell identity, are found in
microchaete (this work), in embryonic md-lineage
(Orgogozo et al., 2002
) and in
wing campaniform sensilla (Van De Bor et
al., 2000
). Specifically, the cell lineages at the origin of
microchaete and embryonic md-neurones are similar to the core of sensory
lineages. However, programmed cell death occurs in one inner cell (the glial
cell) in microchaetes and in the outer secondary precursor cell and the
tertiary precursor cell in the md-lineage
(Orgogozo et al., 2002
).
Complexity can also increase by adding cell proliferation. Thus, in
campaniform sensilla of the wing, the same cell, which dies in the thoracic
microchaete lineage, shows proliferative characteristics and after symmetric
division produces several mature glial cells
(Van De Bor et al., 2000
). In
the same way, it seems very likely that such extra proliferative features are
involved in the formation of chemosensorial bristles
(Nottebohm et al., 1994
).
|
In conclusion, Drosophila sensory organs appear to be homologous
structures that originate from an ancestral cell lineage and possess a common
core of cell divisions. In agreement with Bellaiche and Schweisguth
(Bellaiche and Schweisguth,
2001), we propose that sensory organ diversity arises by adding
new features to this core, which is mainly achieved through cell
proliferation, cell determination and programmed cell death. It is interesting
to note that these three processes are also identified as a source of
morphological and functional variation during the formation of complex systems
such as the central nervous system of vertebrates.
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ACKNOWLEDGMENTS |
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Footnotes |
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