Programmed cell death (PCD) is a prominent feature of
development in all metazoan organisms, functioning in the control
of cell numbers, in the removal of redundant structures and in the
elimination of misspecified or harmful cells (reviewed by Jacobson
et al., 1997). 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), rprand 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
Programmed cell death in the embryonic central nervous
system of Drosophila melanogaster
Ana Rogulja-Ortmann, Karin Lüer, Janina Seibert, Christof Rickert and Gerhard M. Technau*
Although programmed cell death (PCD) plays a crucial role throughout Drosophila CNS development, its pattern and incidence
remain largely uninvestigated. We provide here a detailed analysis of the occurrence of PCD in the embryonic ventral nerve cord
(VNC). We traced the spatio-temporal pattern of PCD and compared the appearance of, and total cell numbers in, thoracic and
abdominal neuromeres of wild-type and PCD-deficient H99 mutant embryos. Furthermore, we have examined the clonal origin and
fate of superfluous cells in H99 mutants by DiI labeling almost all neuroblasts, with special attention to segment-specific differences
within the individually identified neuroblast lineages. Our data reveal that although PCD-deficient mutants appear morphologically
well-structured, there is significant hyperplasia in the VNC. The majority of neuroblast lineages comprise superfluous cells, and a
specific set of these lineages shows segment-specific characteristics. The superfluous cells can be specified as neurons with extended
wild-type-like or abnormal axonal projections, but not as glia. The lineage data also provide indications towards the identities of
neuroblasts that normally die in the late embryo and of those that become postembryonic and resume proliferation in the larva.
Using cell-specific markers we were able to precisely identify some of the progeny cells, including the GW neuron, the U
motoneurons and one of the RP motoneurons, all of which undergo segment-specific cell death. The data obtained in this analysis
form the basis for further investigations into the mechanisms involved in the regulation of PCD and its role in segmental patterning
in the embryonic CNS.
KEY WORDS: CNS, Programmed cell death, Segmental patterning, Neuroblasts, Lineages, H99, Drosophila
Development 134, 105-116 (2007) doi:10.1242/dev.02707
Institute of Genetics, University of Mainz, Saarstrasse 21, D-55122 Mainz, Germany.
*Author for correspondence (e-mail: email@example.com)
Accepted 18 October 2006
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.
MATERIALS AND METHODS
OregonR was used as the wild-type strain. Df(3L)H99/TM3, Sb flies were
obtained from the Bloomington Drosophila Stock Center and rebalanced
over TM6b, abdA-lacZ and TM6, ubi-GFP balancer chromosomes.
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% H2O2solution 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-?-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.
Embryos were fixed as described above, then incubated for 40 minutes in a
2 ?g/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 ?m, 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 (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.
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.
We began our investigations by comparing the CNS morphology and
total CNS cell number in wild-type (wt) embryos with PCD-
deficient embryos homozygous for the deficiency Df(3L)H99(H99)
(White et al., 1994). 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
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 H99embryos
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
Development 134 (1)
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.
We then determined the number of additional cells in these
embryos. We counted all cells within specific segments of the VNC
of stage 16 and 17 wt and H99 embryos (for practical reasons,
counting was restricted to a small number of embryos; see Materials
and methods). Two thoracic (T2 and T3) and three abdominal (A3
to A5) hemisegments were counted per embryo and the results
compared (Fig. 2). In wt embryos, the cell number in the thorax
dropped by about 30% from mid-stage 16 (503±31, n=8) to stage 17
(354±58, n=8; P<0.001, two-tailed t-test), suggesting that there is a
high occurrence of cell death near the end of embryogenesis. The
same was true for the abdominal hemisegments (about 26%
reduction): 386±12 (n=12) at mid-stage 16 and 286±34 (n=12) at
stage 17 (P<0.001). In H99 embryos at mid-stage 16, average cell
numbers per thoracic hemisegment (hs) (524±34, n=10) and per
abdominal hs (451±36, n=12) were slightly higher than in wt.
Despite the greater variability in the H99 strain, the difference
between the wt and H99 abdominal cell counts is statistically
significant (P<0.001). In contrast to wt, cell numbers in H99did not
significantly change from stage 16 (see above) to 17: 534±83 (n=10)
in thoracic neuromeres and 462±80 (n=18) in abdominal
neuromeres. At stage 17, we observed considerable differences
between wt and H99 embryos in both thoracic [354±58 (n=8) and
534±83 (n=10), respectively] and abdominal segments [286±34
(n=12) and 462±80 (n=18), respectively] (Fig. 2). The difference is
statistically significant in both cases (P<0.001).
The supernumerary cells in the CNS of H99 embryos are most
likely to be cells whose fate in wt would be PCD. It is, however, also
conceivable that at least some of these cells derive from the
continuing division of NBs which would normally undergo PCD
after they have generated their progeny, or from the division of
ganglion mother cells (GMCs) that are born, but normally undergo
apotosis without dividing. To test this, we performed BrdU labeling
experiments. It has been shown that in the wt VNC, there are few
divisions taking place after stage 16 (Prokop and Technau, 1991). 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 H99embryos
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 H99embryos 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
Cell death in the embryonic CNS of Drosophila
Fig. 1. The CNS of homozygous H99 embryos is not grossly
deformed. (A,B). Axon tracts are visualized using the BP102 antibody
in wt (A) and H99 (B) embryos. Their pattern is similar, although
thickened junctions between the longitudinal connectives and the
commissures (arrow), and a widened midline (bracket) are visible in
H99. Note that the CNS of H99 embryos is generally wider than that of
wt due to additional cells. (C,D) FasII staining of axons reveals a variably
altered pattern. The H99 embryo shown in D has a more extreme
phenotype in that the fascicles are somewhat disordered and fuzzy.
(E,F) Glial cells, visualized with the anti-Repo antibody. A stack of scans
was made throughout the CNS and the scans then projected together
to show all Repo-positive cells. The positioning of glia is only slightly
affected in H99. All images show four abdominal segments (A3 to A6)
of late stage 16 embryos; anterior is up.
wt th.H99 th.
St. m16 St. 17
Fig. 2. CNS cell count comparison of mid-stage 16 and stage 17
wt and H99 embryos. White and black bars represent cell counts
from wt and H99 embryos, respectively. For exact numbers see text.
Asterisks mark statistically significant differences between wt and H99
(P<0.001, two-tailed t-test). The thoracic CNS shows a difference
between wt and H99 only at stage 17. The abdominal part of the CNS
of H99 embryos contains more cells already at mid-stage 16, and the
difference increases further at stage 17. th, thorax; abd, abdomen.
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).
Temporal profile of cell death throughout
embryonic CNS development
In order to examine the occurrence of PCD in the developing
nervous system of the Drosophila embryo, we made use of a
polyclonal antibody raised against the activated form of the human
caspase-3 protein. This antibody has previously been shown to
recognize apoptotic cells in Drosophila tissues (Brennecke et al.,
2003), 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
DiI labeling of neuroblast lineages in H99
We next investigated the development of individual NB lineages in
embryos that lack PCD, with a view to determining how many
additional cells, if any, each NB lineage would make, how the
additional cells develop (e.g. whether they differentiate, migrate,
extend axons, etc.), and how their potential axons project. We
performed labeling experiments with the cell lineage tracer DiI
(Bossing and Technau, 1994) 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.
1. Clones with no additional cells
Abdominal NB1-1a was the only NB lineage which we repeatedly
(n=5) found to be unchanged in H99embryos (two clones of 10 cells
and three clones of 11 cells) (Fig. 5A-C, Table 1). The wt consists of
9-11 cells (three subperineural glial cells, the aCC motoneuron, the
pCC interneuron, and a cluster of 4-6 interneurons) (Udolph et al.,
1993). 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.
Development 134 (1)
Fig. 3. BrdU staining in late stage 17 wt and H99 embryos. All
embryos were injected at early stage 17. (A) Wt VNC. Sporadic staining
can be seen in the abdominal segments. In the thoracic region, BrdU-
labeled cells are found only in the very lateral region of each
hemisegment. (B,C) VNCs of two H99 embryos, representing the two
classes of staining we found in H99 mutants. One shows a similar
amount of staining as in wt (B), whereas the other has many more
BrdU-positive cells (C). In both cases, there is a concentration of
dividing cells in the lateral region of each abdominal hemisegment. The
thoracic segments in B and C show reduced staining in the medial
regions. Anterior is to the left in all images.
Fig. 4. Profile of Caspase-dependent PCD in the embryonic CNS.
Activated-Caspase-3-positive cells in the wt embryonic CNS were
counted in abdominal hemisegments over the course of development.
PCD in the CNS begins at stage 11 and increases until stage 14. The
levels remain high until the end of embryonic development. Bars
represent s.d.; n=36-51 (for all stages). Developmental stages are
indicated above the bars.
2. Clones with additional cells and wild-type-like axonal
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
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.
Cell death in the embryonic CNS of Drosophila
Table 1. Comparison of DiI-labeled clone sizes and projections
between wt and H99 embryos
projections NB Group‡
*Candidate abdominal pNBs.
†Candidate thoracic NBs that undergo apoptosis.
‡The group that each NB belongs to is indicated as follows: 1, Clones with no
additional cells; 2, Clones with additional cells and wild-type-like axonal projections;
3, Clones with additional cells and atypical axonal projections; 4, Clones whose
phenotypes differ between abdomen and thorax.
a, abdominal clones; t, thoracic clones.
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.
The NB7-4 lineage contains 8-12 interneurons and 5-7 glial cells
in the wt. The interneurons project contralaterally across the posterior
commissure (Fig. 6J) (Schmidt et al., 1997). 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
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 H99did 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 H99we 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.
Development 134 (1)
Fig. 5. DiI-labeled clones of NB1-1a and
NB7-3 in H99 embryos. Each plate shows a
semischematic illustration of a wt and an H99
clone, and the corresponding image of the H99
clone. Glia are depicted in green, motoneurons
in red. Dashed lines indicate the CNS midline.
(A-C) No difference can be seen between NB1-
1a in wt (A) and H99 (B,C). Shown is an H99
clone containing 11 cells (including three glial
cells). Both the cell numbers and the axonal
projection pattern are unchanged in H99.
(D-F) NB7-3 consists of more cells in H99 (E,F)
than in wt (D), including an additional
motoneuron that projects its axon outside the
CNS (arrow). The image shows an abdominal
H99 clone with 9 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
Cell death in the embryonic CNS of Drosophila
Fig. 6. DiI-labeled clones of NB4-2, NB5-3,
NB7-2 and NB7-4. Each plate shows an
illustration of a wt and an H99 clone, and the
corresponding image of the H99 clone. Glia are
depicted in green, motoneurons in red. Dashed
lines indicate the CNS midline. (A-C) The NB4-2
lineage has two motoneuronal projections in wt
(A). In H99, there is a third motoneuronal axon
(arrow in C) projecting ipsilaterally through the
posterior root of the intersegmental nerve. The
image shows an abdominal clone comprising 25
cells. (D-F) The wt NB5-3 shows an ipsilateral
motoneuronal and two contralateral
interneuronal projections (D). In H99 (E,F), there
is an additional ipsilateral interneuronal
projection extending anteriorly (arrow in F).
Shown is an image of an abdominal clone
containing 25 cells. (G-I) In the wt, NB7-2
interneurons form two fascicles, an ipsilateral
one extending posteriorly, and a contralateral
one projecting through the posterior commissure
(G). The H99 clones (H,I) show another
contralateral projection, extending alongside the
wt one (arrow in I). Shown is an abdominal H99
clone with 21 cells. (J-L) In the wt, NB7-4
interneurons form one fascicle that traverses
contralaterally through the posterior commissure
(J). In H99 (K,L), NB7-4 clones exhibit an atypical
projection that runs through the anterior
commissure of the next segment (arrow in L).
Shown is an abdominal clone with 29 cells.
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).
Some molecular markers which are expressed in small subsets
of cells in the VNC showed, as has already been reported (De
Graeve et al., 2004; 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
Programmed cell death is an integral part of animal development,
and as such is also involved in spatial patterning of tissues and
organs. In the Drosophila embryonic CNS, factors regulating this
developmental cell death have just begun to be identified. In order
to establish a basis for further investigations into these mechanisms,
we set out to analyze the distribution of PCD throughout CNS
development, and to identify apoptotic cells in order to use them as
models for these investigations.
Development 134 (1)
Fig. 7. Caspase-dependent PCD of dHb9-expressing cells. (A,B) At
late stage 16, H99 mutants (B) have more dHb9-positive cells than wt
(A). (C) Activated-Caspase-3 staining reveals that some of these cells die
at stage 14 (arrow). In this case, the dying cell lies in a position
corresponding to the RP (1, 3, 4 or 5) neurons. dHb9 staining is in
green, activated Caspase-3 in red. Anterior is up in all images.
Fig. 8. Caspase-dependent PCD of Lbe-, Eg- and Eve-expressing
cells. (A-C) Lbe-positive cells, most likely from the NB5-3 lineage, are
present in higher numbers in H99 mutants (B) than in wt (A). Apoptotic
Lbe-positive cells can be seen at stage 14 (arrow in C). (D-F) The Eg-
expressing NB7-3 lineage also shows too many cells in H99 (E, compare
with wt in D). The GW motoneuron undergoes apoptosis in segments
T3 to A8 at late stage 16 (arrows in F). (G-I) Eve-positive U
motoneurons are not present in segments A7 and A8 in wt embryos
(G). In H99, these cells survive (H). U neurons in A7 and A8 die at stage
14 (arrows in I). Lbe staining in C, Eg in F and Eve in I are in green,
activated Caspase-3 in red. A,B,D-H show late stage 16 embryos; C,I
show stage 14 embryos. Lines in G-I demarcate segments A7 and A8
for clarity. Anterior is up in all images.
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
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 ofC. 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
reapermutant 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.
Cell death in the embryonic CNS of Drosophila
Atypical axonal projections in DiI-labeled H99
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
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 H99mutant 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 H99embryos 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-
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
Development 134 (1)
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
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
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
We thank R. Holmgren, J. Skeath, K. Jagla, C. Doe and M. Frasch for reagents;
J. Urban and R. Cantera for critical reading of the manuscript; and C. Berger
for helpful discussions. This work was supported by a grant from the Deutsche
Forschungsgemeinschaft to G.M.T.
Supplementary material for this article is available at
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