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First published online December 12, 2006
doi: 10.1242/10.1242/dev.02707
Institute of Genetics, University of Mainz, Saarstrasse 21, D-55122 Mainz, Germany.
* Author for correspondence (e-mail: technau{at}uni-mainz.de)
Accepted 18 October 2006
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: CNS, Programmed cell death, Segmental patterning, Neuroblasts, Lineages, H99, Drosophila
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). Selective cell death also provides an elegant mechanism for
spatial patterning of developing tissues and organs (reviewed by
Rusconi et al., 2000), and is
employed in refining the nervous system in both vertebrates and invertebrates
(reviewed by Oppenheim, 1991;
Truman et al., 1992), in digit
formation in higher vertebrates, in removing larval tissues during insect
morphogenesis and in a number of other developmental processes. In these
processes, tight spatio-temporal regulation of PCD is required to ensure
precise elimination of redundant cells. How this regulation is achieved, and
how PCD is integrated with cell determination and differentiation during
development, are fundamental questions in developmental biology that remain
largely unanswered. The fruitfly Drosophila melanogaster renders itself useful to investigations of PCD, as the genes required for the initiation and execution of PCD have been cloned and there are numerous genetic tools available to allow manipulation of genes and developmental processes. Our goal has been to investigate mechanisms involved in the regulation of PCD in the developing CNS of the Drosophila embryo, particularly with respect to its role in segmental patterning. We provide a basis for these investigations by pursuing a more detailed analysis of cell death in the embryonic CNS, and by establishing single-cell models for further examination of mechanisms regulating developmental cell death.
In Drosophila, significant amounts of apoptotic cells have been
observed in the embryonic CNS from the early stages of CNS formation to the
end of embryogenesis (Abrams et al.,
1993). Several studies over the last decade have identified
different kinds of apoptotic cells in the CNS
(White et al., 1994;
Sonnenfeld and Jacobs, 1995;
Hidalgo et al., 2001;
Peterson et al., 2002;
Lundell et al., 2003;
Miguel-Aliaga and Thor, 2004;
Karcavich and Doe, 2005). In
most cases, the developmental signals responsible for inducing PCD in these
cells are unclear. Embryonic neuroblast (NB) PCD has been shown to require the
proapoptotic gene reaper (rpr)
(Peterson et al., 2002), but
it is not known how rpr is activated to induce PCD in these NBs. In
the third-instar larva, a pulse of the Hox protein Abdominal-A induces PCD in
the dividing abdominal postembryonic NBs through activation of one or more of
the three proapoptotic genes Hid (Wrinkled - Flybase),
rpr and grim, and thus limits the production of neural cells
in the abdominal CNS (Bello et al.,
2003). Whether a similar signal is involved in the death of the
embryonic NBs remains to be investigated. Other groups have reported PCD
occurring in postmitotic differentiated neural cells. Sonnenfeld and Jacobs
(Sonnenfeld and Jacobs, 1995)
were the first to report degeneration of differentiated midline glial cells
upon completing their function in the morphogenesis of commissural axon tracts
in early embryogenesis. Hidalgo and colleagues
(Hidalgo et al., 2001) showed
that survival of longitudinal glia (LG) depends on the Neuregulin trophic
factor homolog Vein. Miguel-Aliaga and Thor
(Miguel-Aliaga and Thor, 2004)
found that the pioneer neurons dMP2 and MP1 undergo segment-specific PCD at
the end of embryogenesis, and that the Hox gene Abdominal-B is
required for the survival of these cells in posterior segments of the ventral
nerve cord (VNC). Several studies (Novotny
et al., 2002; Lundell et al.,
2003; Karcavich and Doe,
2005) have reported apoptosis among the progeny of the neuroblast
NB7-3. One to two of the six postmitotic cells produced in this lineage
undergo apoptosis and these are the first reported examples of the death of
clearly identifiable, undifferentiated cells in the Drosophila
embryonic CNS. Furthermore, Notch was identified as the apoptotic signal in
the NB7-3 lineage (Lundell et al.,
2003), but exactly how it activates the apoptotic pathway is
unknown.
Despite the obvious importance of PCD in Drosophila development,
only a very general overview of the occurrence of PCD in the developing
embryonic CNS has been provided to date
(Abrams et al., 1993). A
systematic analysis of the number, segmental pattern and identity of dying
cells has not been made. Such a detailed analysis would provide an important
foundation for further research on mechanisms regulating developmental cell
death. We present here the results of three approaches taken to gain insight
into the occurrence and role of PCD in the embryonic CNS of Drosophila
melanogaster: (1) tracing the spatio-temporal pattern of apoptotic cells
in the developing wild-type CNS, as well as comparing the total cell numbers
in thoracic and abdominal neuromeres of wild-type and PCD-deficient embryos;
(2) examining the clonal origin, development and axonal projection patterns of
additional cells in PCD-deficient embryos by DiI labeling of NB lineages; and
(3) analysis of specific cell subpopulations in PCD-deficient embryos using
various cell markers, and determination of the timing of PCD and the identity
of some of these cells in the wild type, in order to establish models for
studying mechanisms of PCD regulation.
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MATERIALS AND METHODS |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Immunohistochemistry
Following dechorionization in 7.5% bleach, embryos from overnight
collections were devitellinized and fixed in heptane with 4% formaldehyde in
PEMS buffer (0.1M Pipes, 1mM MgSO4, 1mM EGTA, 1.2 M Sorbitol, all
Sigma) for 25 minutes. The fixed embryos were dehydrated by a 10 minute wash
in methanol. For staining with diaminobenzidine (DAB, Sigma), embryos were
incubated in 3% H2O2 solution in ethanol for 15 minutes.
Primary antibodies used were mouse BP102 (1:20), mouse anti-FasII (1:10),
mouse anti-Engrailed/Invected (1:2) and mouse anti-Even-skipped (1:2), all
from Developmental Studies Hybridoma Bank; mouse anti-BrdU (1:3.5,
Becton-Dickinson), rabbit anti-human activated caspase-3 (1:50, Cell
Signalling Technology), rat anti-Gooseberry distal (1:2, R. Holmgren,
Northwestern University, Evanston, IL, USA), rat anti-Gooseberry proximal
(1:2, R. Holmgren), guinea pig anti-Hb9 (1:1000, J. Skeath, Washington
University School of Medicine, St. Louis, MO, USA), rabbit anti-Repo (1:500)
(Halter eta al., 1995), mouse
anti-Ladybird early (1:2, K. Jagla, Institut National de la Santé et de
la Recherche Médicale, Clermont-Ferrand, France), rabbit anti-Eagle
(1:500) (Dittrich et al.,
1997), mouse anti-Eagle (1:10, C. Doe, University of Oregon,
Eugene, OR, USA), rabbit anti-Even-skipped (1:1000, M. Frasch, Mount Sinai
School of Medicine, New York, NY, USA), rabbit anti-ß-gal (1:2000,
Cappel). The secondary antibodies used were anti-mouse-biotin,
anti-rat-biotin, anti-guinea pig-biotin, anti-rabbit-biotin, anti-mouse-FITC,
anti-rat-FITC, anti-rabbit-FITC, anti-guinea pig-Cy5, anti-rat-Cy5,
anti-rabbit-Cy5, anti-mouse-Cy3, anti-guinea pig-Cy3, anti-rabbit-Cy3 (1:250,
all from donkey, all Jackson Immunoresearch Laboratories), anti-mouse-Cy5 from
goat (1:250, Jackson Immunoresearch Laboratories) and donkey
anti-mouse-Alexa488 (1:250, Molecular Probes). For DAB stainings, the ABC Kit
from Vectastain was used. Color images were produced using a Zeiss Axioplan 2
microscope. The Leica TCS SPII confocal microscope was used for fluorescent
imaging, and the images were processed using Leica Confocal software and Adobe
Photoshop.
Cell counts
Embryos were fixed as described above, then incubated for 40 minutes in a 2
µg/ml RNase solution. Following washes in PBT and PBS, embryos were
embedded in 70% glycerol. Fillet preparations were made and stacks recorded
with Nomarski optics. Sections were taken every 0.98 µm, using a Zeiss
Axioskop 2 microscope equipped with a motorized stage. Cells were counted in
one hemineuromere of segments T2 and T3, and from A3 to A5. To this purpose,
cells in each section of the stack were marked using Adobe Photoshop. To avoid
marking cells that had been marked in a previous section of the stack,
subsequent sections were projected on top of each other and compared. The
marked cells counted in each section were added to give the sum of all cells
in one stack.
BrdU labeling
BrdU (Sigma) was injected as previously described
(Prokop and Technau, 1991).
Injected embryos were allowed to develop until stage 17 at which point fillet
preparations of the CNS were made and fixed in 18% formaldehyde for 2 minutes.
After washing, the preparations were treated for 4 minutes with 2N HCl and
blocked in 10% goat serum for 15 minutes. Antibody staining was performed as
described above.
DiI labeling
Dil labeling was performed as previously described
(Bossing et al., 1996). Embryos
from the Df(3L)H99/TM6, ubi-GFP fly stock were labeled. Heterozygous
embryos were used as controls, as their CNS lineages did not differ from the
published description of the wild type (see
Bossing et al., 1996;
Schmidt et al., 1997). The
Df(3L)H99 homozygous embryos were distinguished on the basis of head
involution and thicker midline phenotypes. Clones were imaged using the Zeiss
Axioskop 2 microscope and the images processed as described above. For
illustrations, a Zeiss Axioplan microscope with a Camera lucida was used and
the drawings produced using Adobe Illustrator software.
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RESULTS |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). One
typical feature of H99 embryos is a variably penetrant defect in head
involution, visible from stage 15 to the end of embryogenesis (Abbot and
Lengyel, 1991). In other tissues, these embryos show a range of phenotypes
from problems with germ-band retraction (the earliest phenotype we were able
to observe) and defects in gut development and CNS condensation, to those with
hardly any macroscopically visible phenotype. In order to maximize our chances
of observing differences between the wt and H99 CNS, we analyzed
embryos at late developmental stages (late 16 or early 17), and we considered
cases both with and without defects in CNS condensation, as this did not seem
to affect our observations.
Comparison of morphology and cell number in the CNS of wt and H99 embryos
As a consequence of the lack of PCD, the CNS in late H99 embryos
is wider than in wt, but it has a fairly normal appearance. The midline is
widened and disrupted due to the survival of several midline glia
(Sonnenfeld and Jacobs, 1995;
Zhou et al., 1995). As has
been reported previously (Zhou et al.,
1995; Dong and Jacobs,
1997), the commissures and the longitudinal connectives,
visualized by BP102 staining, are broadened and the junctions between them
thickened due to additional axons, but their pattern is not changed
(Fig. 1A,B). This indicates
that at least some of the supernumerary neurons differentiate and extend
axonal projections within the normal commissures and longitudinal connectives.
In general, the axons seem to find and follow their normal pathways in
H99 embryos, as there was no obvious phenotype in the FasII pattern
(Fig. 1C,D). The three
longitudinal fascicles formed and, apart from a variably `bumpy' appearance,
looked similar to wt. The peripheral transverse, segmental and intersegmental
nerves appeared normal, as well as the four nerve branches (SNa-d) (data not
shown). The nerves all appeared to be of normal thickness and it was difficult
to tell whether they contained supernumerary axons. The glia pattern, apart
from a moderate misplacement of some cells, was also surprisingly normal, both
in the VNC (Fig. 1E,F) and in
the periphery (data not shown). Also, as explained in more detail below, the
number of Repo-positive glial cells was unaltered in H99 embryos. We
conclude that the CNS structure in late H99 embryos is not
drastically affected at the macroscopic level, although there must be a large
number of additional cells present.
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). In H99 embryos injected with BrdU at
early stage 16 and fixed at late stage 17, there were only a few more labeled
cells than in wt embryos injected at the same stage (data not shown). When
BrdU was injected at early stage 17, we found two classes of H99
embryos at late stage 17: those that differed little from wt
(Fig. 3, compare A,B), and
those with a greater number of labeled cells than in the wt
(Fig. 3, compare A,C). These
results show that some of the supernumerary cells in H99 embryos are
cells that are never born in the wt CNS. Also, the variability in the BrdU
uptake within the H99 mutant strain appears to be a consequence of
surviving NBs or GMCs either not dividing at all, or going through a variable
number of divisions. We also observed a difference in the amounts of
BrdU-labeled cells between the thorax and abdomen of H99 embryos
injected at early stage 17 (Fig.
3B,C). In the thoracic region, the majority of BrdU-labeled cells
were lateral, whereas in the abdomen the labeled cells were distributed more
equally between the lateral and medial regions of each segment. There were
fewer cells stained in the thoracic region in general, which is in agreement
with the previously published observation that the majority of abdominal NBs
undergo PCD, whereas the thoracic ones mostly enter quiescence at the end of
embryogenesis (Truman and Bate,
1988).
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), and it does not show any staining in homozygous
H99 embryos (data not shown). In the CNS of wt embryos, PCD first
appeared around the beginning of stage 11. PCD then continued to occur until
the end of embryonic development (Fig.
4) (see also Abrams et al.,
1993). The number of dying cells per abdominal hemisegment
increased steadily from stage 11 to reach a peak in mid-development (stage
14), at about 20 cells/hs (n=33). After this point, the number of
dying cells stayed more or less constant until the end of embryonic
development. As has been reported previously
(Abrams et al., 1993), the
spatial distribution of activated Caspase-3-positive cells at any stage shows
both a regular, segmentally repetitive distribution, as well as a random one
(data not shown).
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in homozygous H99 embryos (preferentially in the abdomen). We
obtained clones for almost all 30 NBs, analyzed their cell number and axonal
projections and compared them with the published descriptions of wild-type
clones (Bossing et al., 1996;
Schmidt et al., 1997;
Schmid et al., 1999). The
results are summarized in Table
1 (for selected images see Figs
5,
6, and Figs S1, S2 in the
supplementary material). The clones were sorted into four groups on the basis
of their appearance: (1) clones showing no difference in cell number or
morphology as compared with wt; (2) clones with additional cells and
wild-type-like axonal projections; (3) clones with additional cells and axonal
projections different from those in wt counterparts; and (4) clones showing no
tagma-specific phenotype in wt, but differing between abdomen and thorax in
H99. The groups are described and the most interesting examples of
clones are shown below.
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). In only
one out of five cases in H99 did we observe an axon which branched
out of the typical axon bundle (data not shown); however, the clone contained
a wild-type number of cells (11) and we therefore assume that the axon had
been misrouted. We observed two other lineages (thoracic NB1-3t and abdominal
NB6-4a) in which the H99 embryos did not seem to differ from their wt
counterparts (Table 1 and data
not shown); however, due to a low sample number (n=1), we consider
these observations inconclusive.
2. Clones with additional cells and wild-type-like axonal projections
For 14 NB lineages we obtained clones in H99 embryos which clearly
and repeatedly contained more cells than their wt counterparts but showed a
wild-type-like projection pattern. These were NB2-1, NB2-2a, NB2-4a, NB2-5,
NB3-1a, NB3-2, NB3-5, NB4-4, NB5-1, NB5-4a, NB5-5, NB6-1, NB7-1 and NB7-3 (for
details see Table 1). All of
these clones were easily identifiable on the basis of their axonal projections
(for selected clones, see Figs S1, S2 in the supplementary material). Due to
the lack of information on axon numbers per fascicle in wt clones, and because
of tight packaging of axons in the bundles, we were generally not able to
determine whether the projections in H99 contained additional axons
or not. The only exception was NB7-3, which contains only four cells in the
wt: three contralaterally projecting interneurons (EW1-3) and one
ipsilaterally projecting motoneuron (GW)
(Fig. 5D)
(Higashijima et al., 1996;
Bossing et al., 1996;
Dittrich et al., 1997;
Schmid et al., 1999;
Novotny et al., 2002). We
obtained five examples of this clone in H99 embryos, comprising 9-10
cells (Fig. 5E,F). Although
their projections followed the wt pattern, we were able to identify an
additional motoprojection in all five cases. Regarding the interneuronal
projections, it was not possible to determine the number of axons they
contained, as these are bound together too tightly.
We found four further NBs to have larger clones in H99 than in wt (NB2-2t, NB4-1, NB5-6a and NB6-4t) (Table 1 and data not shown) but as we only obtained one clone for each of these, we can draw no solid conclusion about PCD in these lineages.
3. Clones with additional cells and atypical axonal projections
We obtained clones of four NB lineages (NB4-2, NB5-3, NB7-2 and NB7-4) in
H99 embryos which showed additional cells and axonal projections that
have not been observed in their wt counterparts.
NB4-2 contains 10-16 cells (7-13 interneurons, 3 motoneurons) in wt
(Bossing et al., 1996)
(Fig. 6A). We obtained three
clones of this lineage in H99, one thoracic containing 16 cells, and
two abdominal with 17 and 25 cells (Table
1, Fig. 6B,C). One
of the abdominal clones exhibited a wild-type-like projection pattern, whereas
the other abdominal and the thoracic clone contained two additional
motoneurons each, whose axons project ipsilaterally in the anterior direction.
This lineage can also be placed in group 4, and as such is mentioned again
below.
NB5-3 is another example of a lineage with additional cells and projections
in H99 (Table 1,
Fig. 6D-F). In wt this lineage
contains 9-15 cells. These are mostly interneurons, except for one motoneuron
in the thoracic and first abdominal segments
(Fig. 6D)
(Schmid et al., 1999). The
cells are arranged in two clusters, one lying medially and projecting across
the anterior commissure, and the other lying laterally and projecting through
the posterior commissure (Bossing et al.,
1996; Schmidt et al.,
1997; Schmid et al.,
1999). We obtained seven abdominal clones in H99 embryos,
containing 19-27 neurons. In two of these clones, we found at least one
additional ipsilateral axon projecting anteriorly
(Fig. 6E,F), and in four
further clones we identified structures that resembled the beginnings of axons
growing out in the same direction. In addition, all seven clones contained a
motoneuron. As the labeled clones were found in various abdominal segments
(A1, A2, A3, A6 and A7), we conclude that in the wt the motoneuron is born in
all segments and undergoes PCD (most likely before growing an axon) in A2 to
A8, thus representing an example of segment-specific cell death.
The NB7-2 lineage normally consists of 8-14 interneurons (mostly 12), whose
projections form two fascicles. One traverses contralaterally across the
posterior commissure (7-2Ic) and the other extends ipsilaterally to the
posterior (7-2Ii) (Fig. 6G)
(Bossing et al., 1996). In
H99, we obtained two clones, with 21 and 28 neurons, that project an
additional axon contralaterally through the posterior commissure, alongside
the wild-type-like fascicle (Table
1, Fig. 6H,I). We
believe this to be a separate axon, and not a case of loose fasciculation,
because the position of the axon was exactly the same in both clone examples,
i.e. it comes from the cells lying laterally within the clone. In a loose
fascicle, one would expect the axons to be positioned more variably and fairly
close together.
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). In
H99, this lineage contained 18-24 neurons and 4-7 glia. Additional
axons projected contralaterally through the anterior commissure of the next
segment in all clones obtained (six abdominal and one thoracic,
Fig. 6K,L). NB5-4t also exhibited atypical projections in H99 (see Fig. S2 in the supplementary material); however, as we obtained only one clone we cannot draw firm conclusions about this lineage.
4. Clones whose phenotypes differ between abdomen and thorax
In six cases we obtained NB lineages which seemed differently affected in
the abdomen and thorax of H99 embryos. These were NB4-3, NB3-3,
NB4-2, NB5-2, NB6-2 and NB1-2. In wt, NB4-3 consists in both tagma of 8-13
motoneurons whose projections all leave the CNS through the segmental nerve
(Schmidt et al., 1997). This
lineage often comprises an epidermal and a sensory subclone, which we have
also observed in two out of five cases in H99 (data not shown). The
cell numbers in these epidermal subclones did not differ from wt. However, the
abdominal CNS clones (n=3) all showed a higher cell number than in wt
(15, 15 and 22), whereas the thoracic clones (n=2) did not (12-13 and
8) (Table 1). As the axonal
projections of NB4-3 in H99 did not differ from those in wt, we
conclude that the additional cells either do not differentiate and extend
axons, or they project through the wt fascicles. NB3-3, NB5-2
(Table 1 and data not shown)
and NB4-2 (Fig. 6A-C), showed
the same kind of phenotype, namely a normal cell number in the thoracic, and
more cells in abdominal clones. However, more thoracic clones would need to be
labeled (n=1 in each case) in order to obtain conclusive data.
The opposite phenotype was observed for NB6-2
(Table 1). This NB normally
makes 8-16 interneurons, and also shows no tagma-specific differences
(Bossing et al., 1996). We
obtained four abdominal NB6-2 clones which did not differ from wt (13, 13-14,
13-14 and 14 cells), whereas the two thoracic clones showed an increase in
cell number (18 and 19 neurons). NB1-2 also appeared similar to wt in the
abdomen, and contained more cells in the thorax
(Table 1). However, no solid
conclusion could be drawn about NB1-2 based on only one thoracic clone.
Identification of dying cells
We next attempted to identify the dying cells in the CNS more closely. To
do this, we selected a number of molecular markers that are known to be
expressed in smaller or larger groups of cells in the VNC [e.g. Repo, dHb9
(Exex - Flybase), Eve], and compared the extent of their expression in late
developmental stages of wt and homozygous H99 embryos. We reasoned
that any cell which is determined to express one of these markers, but
undergoes cell death at some point in development, would be likely to continue
to express this marker if PCD is prevented. In fact, this has been shown for
apoptotic midline glia (Sonnenfeld and
Jacobs, 1995; Zhou et al.,
1995) and other apoptotic cells in the CNS
(Novotny et al., 2002;
Miguel-Aliaga and Thor, 2004).
In H99 embryos, we therefore expected to see all the additional cells
which continued to express a particular marker. In parallel, we examined the
overlap between the activation of Caspase-3 and individual marker expression
in various developmental stages of wt embryos, in order to determine the time
of death for some of these cells. We chose embryos in mid-development (stages
13 and 14) as our analysis of cell death distribution indicated that it is
most frequent in these stages. We also examined embryos in a late
developmental stage (late stage 16) to identify cells that are removed towards
the end of embryogenesis.
The glial marker Repo did not show any obvious difference in the extent of expression between wt and H99 embryos (see Fig. 1E,F). We therefore performed precise cell counts for Repo-expressing cells at late stage 16, including glia in the CNS and the peripheral glia that are born in the CNS and then migrate out along the nerves. In wt embryos, a total of 34.17±0.65 cells/hs were counted (n=30), and in H99 we found 34.77±0.73 cells/hs (n=30). We conclude that the number of glial cells is not significantly changed in H99 embryos, and that the great majority of dying cells in the embryonic CNS are neurons or undifferentiated cells.
As anticipated, markers expressed in large groups of cells, such as dHb9,
Gooseberry and Engrailed (Fig.
7A,B and data not shown), stained more cells in H99 than
in wt. Most of these markers also showed, at least in some of the
developmental stages examined, overlap with activated Caspase-3 staining in a
few cells in wt embryos (Fig.
7C and data not shown). In most cases, we were not able to
identify these cells due to the extent of marker expression. Closer
identification was possible only for cells expressing dHb9, a homeodomain
protein expressed in a specific subset of neurons
(Broihier and Skeath, 2002). At
stage 14, for example, we found cells that most likely correspond to one of
the RP motoneurons (RP 1, 3, 4 or 5), co-labeled with activated Caspase-3.
This cell death is specific to segments A7 and A8, and activated Caspase-3
staining of this cell was detected at this stage in 27.3% of hemisegments
analyzed (n=22; Fig.
7C).
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;
Novotny et al., 2002),
interesting pattern changes in H99 embryos
(Fig. 8). Ladybird early (Lbe)
is a homeobox transcription factor involved in several developmental processes
(Jagla et al., 1994;
Jagla et al., 1997;
Jagla et al., 1998). In the
embryonic CNS it is required for the correct development of glial cells
derived from NB5-6 and of neurons derived from NB5-3
(De Graeve et al., 2004). De
Graeve et al. also showed that late H99 embryos have additional
Lbe-positive neuronal cells in the VNC. We made the same observation, and
determined that these Lbe-positive cells die in stages 13 and 14
(Fig. 8A-C). As for the dHb9
marker, this suggests that these cells die prior to or at an early stage of
differentiation. The experiments we are currently undertaking should provide
us with the possibility to precisely identify each of these cells based on the
combination of markers it expresses. Another marker we used, the zinc-finger
protein Eagle (Eg), has been described as being required for proper
differentiation of cells in four NB lineages, NB2-4, NB3-3, NB6-4 and NB7-3
(Higashijima et al., 1996;
Dittrich et al., 1997). We
focused our analysis on the NB7-3 lineage, which consists of only four
neurons: three interneurons and one motoneuron. Previously published data show
that PCD is involved in the formation of this lineage, as late H99
embryos have additional cells in the NB7-3 lineage
(Novotny et al., 2002). It has
been shown that these cells are not fully differentiated, and that the death
of some of these cells depends on Notch activity
(Lundell et al., 2003). Our
results are in agreement with the published data
(Fig. 8D,E), although we also
observed apoptotic Eg-positive cells at stage 13 (not shown) and at late stage
16 (Fig. 8F). We identified the
cell undergoing PCD at late stage 16 as the GW motoneuron, and determined that
it is removed specifically in segments T3 to A8 in 38.9% of cases
(n=108). We thus showed that differentiated NB7-3 progeny can also
undergo PCD. In T1 and T2 this cell survives into larval stages. Even-skipped
(Eve), another marker we used, is a homeodomain transcription factor expressed
in a very restricted and well-described pattern in the embryonic CNS
(Frasch et al., 1987;
Doe et al., 1988). Comparing
the Eve expression pattern in anterior abdominal segments at late stage 16, we
did not see any difference between wt and H99 embryos. However, some
or all of the U neurons in segments A6 to A8 of wt embryos were missing,
whereas they were still present in homozygous H99 embryos
(Fig. 8G,H). These motoneurons,
belonging to the NB7-1 lineage, underwent PCD in stages 14 and 15 as seen by
Caspase-3 activation (Fig. 8I).
Further experiments are underway to investigate possible factors that regulate
PCD of these cells.
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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The CNS of PCD-deficient embryos is not drastically affected at the macroscopic level
In our analysis of PCD distribution we found that, macroscopically, the CNS
of wt and PCD-deficient (H99) embryos do not show large differences.
Our observations indicate that the supernumerary cells do not disturb
developmental events in the CNS of H99 embryos, such as cell
migration and axonal pathfinding. The glial cells mostly find their
appropriate positions accurately. The DiI-labeled NB lineages were, in the
majority of cases, easily identifiable based on their shape, position and
axonal pattern, despite the supernumerary cells. The FasII pattern showed that
the axonal projections form and extend along their usual paths. In fact, the
supernumerary cells themselves are capable of differentiating i.e. expressing
marker genes and extending axons, as shown by clones of several NBs and by
cell marker expression analysis in H99 (e.g. NB7-3).
Pattern and degree of cell death in the ventral nerve cord
It has been shown that a large number of CNS cells undergo PCD during
embryonic development (Abrams et al.,
1993). The distribution of activated Caspase-3-positive cells in
wt embryos suggests that the death of some cells is under tight spatial and
temporal control, as revealed by their regular, segmentally repeated
occurrence. Other dying cells were rather randomly distributed, suggesting a
certain amount of developmental plasticity. The overall counts of
Caspase-3-positive cells give an estimate of the numbers of dying cells at a
given time. They indicate that PCD becomes evident in the CNS at stage 11 and
is most abundant in the late embryo (from stage 14). It is however difficult
to estimate the total number of apoptotic cells throughout CNS development by
anti-Caspase-3 labeling, because the cell corpses are removed fairly quickly.
We therefore counted the total number of cells per thoracic and abdominal
hemineuromere in the late embryo. Comparison between stage 16 and stage 17 wt
embryos indicates that 25-30 % of all cells are removed in both tagmata after
stage 16, which in turn suggests that the total percentage of removed cells
must be high, as PCD occurs at high levels already from stage 14 on. In
comparison to the developing nervous system of C. elegans, where PCD
removes about 10% of cells, and of mammals, where this number can be as high
as 50-90%, PCD in the fly CNS appears to show an intermediate prevalence. This
lends support to the hypothesis of an increasing contribution of PCD in
shaping more advanced nervous systems during evolution.
Comparisons between wt and H99 reveal, as expected, a greater
number of cells in both tagmata of H99 embryos (151% increase in the
thorax and 162% in the abdomen at stage 17). These additional cells in
H99 may reflect the total number of cells normally undergoing cell
death until stage 17. However, there is a large variability in the total
number of cells, especially within the H99 strain. In wt embryos, it
seems to be more pronounced in the thorax and at stage 17, which might be a
consequence of variable amounts of PCD occurring until this stage. The even
higher variability within the H99 strain (both in thorax and abdomen)
is likely to reflect variable numbers of additional cell divisions. The great
majority of abdominal NBs are normally removed by PCD after they have
generated their embryonic progeny (Bray et
al., 1989; White et al.,
1994; Peterson et al.,
2002), whereas in the thoracic neuromeres most of the NBs enter
quiescence at the end of embryogenesis and continue dividing as postembryonic
NBs in larval stages (Truman and Bate,
1988). Thus, there are few mitoses occurring in the wt CNS from
stage 16 onwards (Prokop and Technau,
1991). Our BrdU labeling experiments revealed a high number of
BrdU-positive cells in some H99 embryos injected at early stage 17.
We assume that these are progeny of mitotic NBs and/or GMCs that survive and
continue dividing, generating cells that do not exist in wt. Clones obtained
by DiI labeling in H99 confirm this conclusion (see below). Our
finding that surviving cells divide already in the embryo complement the
results of Peterson et al. (Peterson et
al., 2002), who found that, in reaper mutants, NBs in the
abdominal neuromeres survive and generate progeny in larval stages.
Supernumerary cells can be specified as neurons but not as glia
Among the DiI-labeled clones in H99 embryos, we obtained very few
NB lineages which did not differ from their wt counterparts. The majority
contained, as expected, supernumerary cells. In some cases we could identify
axons projected by these cells, which shows they are specified as neurons. In
fact, in three cases (NB4-2, NB5-3 and NB7-3), we found these additional cells
to be specified as motoneurons. As additional axons within a fascicle were
generally difficult to identify, it is possible that these are not the only
lineages which make additional motoneurons in H99. Whether these
cells are normally born and apoptose, or originate from additional divisions
of surviving NBs or GMCs, cannot be determined from these experiments, but
similar observations have been made for both cases. Lundell et al.
(Lundell et al., 2003) have
shown that the normally apoptotic progeny of NB7-3 can express the neuronal
differentiation markers Ddc and Corazonin when cell death is prevented. Also,
the additional progeny of the surviving NBs in the reaper mutant
larvae express the neuronal marker Elav, showing that cells which are never
born in the wt are capable of becoming neurons
(Peterson et al., 2002). It is
interesting that none of these cells, regardless of their origin, are
specified as glia. We did not observe any additional glia in the NB clones in
H99 embryos, and we also found equal numbers of Repo-expressing glial
cells in wt and H99. We conclude that PCD occurs almost exclusively
in neurons and/or undifferentiated cells, and that lateral glia are not
produced in excess numbers in the embryo. Furthermore, because it is likely
that NBs, which normally die, stay in a late temporal window in H99,
one could speculate that NBs in this window normally do not give rise to glia.
Our results are not in agreement with the notion that LG are overproduced, and
their numbers adjusted through axon contact
(Hidalgo et al., 2001).
Hidalgo et al. observe occasional apoptotic LG and it is possible that our
method of counting does not allow a resolution fine enough to account for an
occasional additional Repo-positive cell in H99 embryos. However, if
LG were consistently overproduced, we would expect to observe a higher number
of glia in H99 embryos. We assume that LG cell death may reflect a
small variability in the number of cells needed, and not a general mechanism
for adjusting glial cell numbers.
As already mentioned, we generally found no difference between Repo-expressing glia numbers in wt and H99. However, a small difference does become apparent when one separates the total cell counts into those in the CNS and those in the periphery: 25.67±0.45 cells/hs and 28.42±0.64 cells/hs for wt and H99, respectively, were counted in the CNS, whereas 8.50±0.28 cells/hs and 6.35±0.82 cells/hs for wt and H99, respectively, were found in the periphery. The reasons for this difference might be the greater width of the CNS in H99 embryos, and that the cues required for proper migration of the peripheral glia are disturbed by additional cells. Alternatively, the difference might be due to differentiation defects in these cells.
Atypical axonal projections in DiI-labeled H99 clones
In addition to NB clones with too many cells and wild-type-like axon
projections in H99, we also obtained some lineages whose clones
exhibited atypical projection patterns. We found these projections to belong
both to motoneurons (e.g. in NB4-2) and interneurons (e.g. NB5-3, NB7-2 and
NB-7-4). NB4-2 normally produces two motoneurons (RP2 and 4-2Mar) and 8-14
interneurons (Bossing et al.,
1996). In two out of three NB4-2 clones in H99 we found
two additional motoneurons that project anteriorly, similar to RP2. One of the
two clones was found in the thorax and had a normal cell number (16), whereas
the other was abdominal and had too many cells (25). Thus, the two additional
motoneurons are likely to be the progeny of divisions occurring in the wt, and
not of an additional NB or GMC mitosis. The fact that the third NB4-2 clone
(found in the abdomen and comprising 17 cells) did not show the same
motoneuronal projections could be due to these cells not being differentiated
at the time of fixation (we have occasionally observed clones of different
ages in the same embryo), or they may not have differentiated at all. It would
be interesting to determine the target(s) of these additional motoneurons and
thereby perhaps gain insight into physiological reasons for their death.
However, such an experiment has to await tools that allow us to specifically
label the NB4-2 lineage, or these motoneurons, in the H99 mutant
background.
The other three lineages (NB5-3, NB7-2 and NB7-4) all have atypical
interneuronal projections. The cells which these atypical axons belong to may
represent evolutionary remnants that are not needed in the Drosophila
CNS. Alternatively, they might have a function earlier in development and be
removed when this function is fulfilled. Such a role has been shown for the
dMP2 and MP1 neurons, which are born in all segments and pioneer the
longitudinal axon tracts. At the end of embryogenesis these neurons undergo
PCD in all segments except A6 to A8, where their axons innervate the hindgut
(Miguel-Aliaga and Thor,
2004). It is known that some cells of the NB5-3 lineage express
the transcription factor Lbe, and that H99 mutants show about three
additional Lbe-positive neurons per hemisegment, which mostly likely belong to
NB5-3 (DeGraeve et al., 2003).
Our DiI-labeling results complement this finding in that we also find four or
more additional neurons in H99 clones. The supernumerary Lbe-positive
neurons in H99 could possibly be the ones producing the atypical
axonal projections.
Tagma-specific differences in H99 embryos
In the wt embryo, only eight NB lineages show obvious tagma-specific
differences in cell number and composition
(Bossing et al., 1996;
Schmidt et al., 1997).
Tagma-specific differences among serially homologous CNS lineages have
previously been shown to be controlled by homeotic genes (e.g. Prokop et al.,
1994a; Berger et al., 2005).
Therefore, these lineages provide useful models for studying homeotic gene
function on segment-specific PCD. In H99 embryos, we observed further
lineages that were differently affected in the thorax and abdomen. How these
tagma-specific differences arise in a PCD-deficient background is an
interesting question. For example, NB4-3 shows a wild-type cell number in the
thorax (8 and 12-13), but has too many cells in the abdomen (15, 15 and 22).
There are a couple of plausible scenarios to explain this observation. First,
the development of the NB4-3 lineage, including the involvement of PCD, could
actually differ in the thorax and abdomen of wt embryos, with the final cell
number being similar by chance. The DiI-labeled clones allow determination of
the final cell number, but do not reveal how this number is achieved. The
difference would become obvious in an H99 mutant background, at least
regarding the involvement of PCD. Second, and this possibility does not
exclude the first one, the thoracic NB4-3 could become a postembryonic NB
(pNB) and the abdominal NB4-3 might undergo PCD after generating the embryonic
lineage. In H99, the abdominal NB would be capable of undergoing a
variable number of additional divisions to generate a variable number of
progeny. This would easily explain larger discrepancies in cell number between
individual clones in H99 (e.g. the abdominal NB4-3 clone with 22
cells), and is in agreement with our occasional observations of H99
embryos with a very high CNS cell number per segment, and with the two
observed classes of H99 embryos with high and low numbers of
BrdU-positive cells.
NB6-2 is another lineage whose clones differ in the two tagmata of
H99 embryos. In this case, the abdominal clones showed no difference
to their wt counterparts, whereas the thoracic clones did (18 and 19 cells).
Although no difference in cell number between thoracic and abdominal clones
was reported for this lineage, a rather large count range (8-16 cells) was
given (Bossing et al., 1996),
which would allow for a thorax-specific PCD of two to three postmitotic
progeny. Alternatively, the thoracic NB6-2 might undergo cell death upon
generating its progeny, which would make it the first identified apoptotic NB
in the thorax. When PCD is prevented, this NB may undergo a few additional
rounds of division. The data obtained in our experiments do not counter this
notion, but the number of clones obtained in the thorax was not sufficient to
draw a definite conclusion. As the abdominal NB6-2 lineage in H99 did
not differ from the one in wt, its NB may be one of the few abdominal
postembryonic NBs (see below).
Identities of neuroblasts dying in the late embryo and of surviving neuroblasts resuming proliferation in the larva
As mentioned above, a specific set of NBs undergoes PCD in the late embryo,
whereas surviving NBs resume proliferation in the larva as pNBs, after a
period of mitotic quiescence (Bray et al.,
1989; White et al.,
1994; Peterson et al.,
2002; Truman and Bate,
1988; Prokop and Technau,
1991; Prokop and Technau,
1994b). The identities of the individual NBs undergoing PCD versus
those surviving as pNBs are still unknown. The sizes of NB lineages obtained
in H99 embryos may provide hints for identifying candidate pNBs in
the abdomen [12 NBs/hs in A1, four in A2 and three in A3 to A7 according to
Truman and Bate (Truman and Bate,
1988)], and NBs that undergo PCD in the thorax at the end of
embryogenesis [seven NBs/hs in T1 to T3
(Truman and Bate, 1988)]. In
the abdomen, NB1-1a and NB6-2 are obvious candidates for pNBs, as they
remained consistently unchanged in H99 embryos
(Table 1). Two other NBs, NB1-2
and NB3-2, are also potential abdominal pNBs as they mostly did not differ
from their wt counterparts, and only occasionally contained one additional
cell. On the other hand, clones which showed more than twice the cell number
in H99 (NB2-1, NB5-4a and NB7-3, see
Table 1) than in wt, strongly
suggest that these NBs normally undergo PCD in the abdomen (but perform
additional divisions in H99), because, even if one daughter cell of
each GMC undergoes PCD, they still cannot account for all cells found in
H99 clones.
Regarding thoracic NBs, we can only speculate on account of low sample numbers. NBs which seem to become pNBs in the thorax, as they showed no difference between wt and H99 clones, are NB3-2 (n=2 clones in H99), NB4-3 (n=2) and NB4-4 (n=3) (Table 1). Potential candidates for NBs which do not become pNBs, but undergo PCD in the thorax, are expected to consistently have a significant increase in cell number in H99. These are NB5-1 (n=2 clones in H99) and NB5-5 (n=5) (Table 1). In addition, lineages for which we obtained only one clone in H99 but which also showed many more cells in the thorax than normal are NB2-2t, NB5-4t and NB7-3 (Table 1, and see Fig. S2 in the supplementary material).
Established models for studying the mechanisms of developmental PCD in the CNS
In order to investigate the developmental signals and mechanisms involved
in the regulation of PCD in the embryonic CNS, we identified some of the
apoptotic cells which will be used as single-cell PCD models. These are the
dHb9-positive RP neuron from NB3-1, Lbe-positive neurons from NB5-3, the
Eg-positive GW neuron from NB7-3 and the Eve-positive U neurons from NB7-1. As
not much is known about the dying RP motoneuron or the Lbe-positive neurons,
our first goal is to characterize each of these cells more closely, based on
the combination of expressed molecular markers.
Some of the dying NB7-3 cells are already known to be undifferentiated
daughter cells of the second and third GMC, which undergo PCD shortly after
birth. Notch has been identified as the signal initiating PCD. The surviving
daughters receive the asymmetrically distributed protein Numb, which
counteracts the PCD-inducing Notch signal
(Lundell et al., 2003). The
same had been shown in a sensory organ lineage of the embryonic peripheral
nervous system, where cells produced in two subsequent divisions undergo
Notch-dependent PCD (Orgogozo et al.,
2002). Both the PCD in the NB7-3 lineage and in the sensory organ
lineage require the Hid, rpr and grim genes
(Novotny et al., 2002;
Lundell et al., 2003;
Karcavich and Doe, 2005;
Orgogozo et al., 2002). It
will be interesting to see whether the Notch-Numb interaction also plays a
role in the segment-specific PCD of the differentiated GW motoneuron, or if
another signal is used for the removal of this, and possibly other,
differentiated cells.
The U motoneurons also show a segment-specific cell death pattern (they
apoptose in A6 to A8), thus somewhat resembling the MP1 and dMP2 neurons
(Miguel-Aliaga and Thor,
2004). However, in contrast to MP1 and dMP2, the U neurons survive
in the anterior segments and undergo PCD in the posterior ones. Whether
homeotic genes play any role in the survival or death of these cells remains
to be investigated.
In summary, we present here descriptions of PCD in the developing CNS of the wt Drosophila embryo, and of the CNS of PCD-deficient embryos. We find the pattern of Caspase-dependent PCD to be partly very orderly, suggesting tight spatio-temporal control of cell death, and partly random, which suggests a certain amount of plasticity already in the embryo. The CNS of PCD-deficient embryos is nevertheless well organized, despite the presence of too many cells. We find these superfluous cells to come from both a block in PCD and from additional divisions that surviving NBs go through. We were able to link the occurence of cell death to identified NB lineages by clonal analysis in PCD-deficient embryos, to uncover segment-specific differences, and to establish single-cell PCD models that will be used in further studies to investigate mechanisms responsible for controlling PCD in the embryonic CNS.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/1/02707/DC1
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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