5.) NEURONAL CELL DEATH AS A MEAN OF DIVERSIFICATION
As I have outlined in Chapter 2, there are three principal ways in which
elements of a diversifying network could behave: New elesments could
differentiate; elements could be preserved without significant change; or
elements could be removed from the network. The reduction of elements could be
achieved in two ways. Either cells forming certain elements would not
differentiate at all; or they do differentiate to a certain stage and then get
eliminated at a later stage in development.
As far as the muscle pattern is concerned, I have outlined in Chapter 2 that the
absence of muscles in the dorsal domain of A8 is a very distinctive feature.
This applies in particular to dorsal longitudinal muscles. Apart from the minute
and barely characterised MO1, there is only the large muscle DO1 in A8 in a
region that contains eight muscles in the reference segment. Taking the muscle
pattern as the most accessible part of the neuromuscular machinery, one can ask
whether the observed reduction of elements is echoed in the CNS on the level of
the respective motorneurons.
The removal of neurons leading to a diversification of the neuromuscular
architecture is a phenomenon well known in vertebrates. Between 50% and 80% of
all embryonic motorneurons undergo apoptosis after extending their axons as a
result of a failure in finding their appropriate target (OPPENHEIM, 1991).
Experiments investigating similar mechanisms in the grasshopper Schistocerca
gregaria could not show a target-dependence in motorneuron survival: If muscles
are surgically removed from grasshopper embryos, their respective motorneurons
still extend their axons into the domain of their target and continue growing
even though they fail to find it (BALL et al., 1985; WHITINGTON et al., 1982).
Previously, segmental differences in the number of neurons and glia have been
linked to changes in the lineage of specific neuroblasts (TRUMAN & BALL, 1998;
PROKOP & TECHNAU, 1994; SCHMID et al., 1999; UDOLPH et al., 1993; PROKOP et al.,
1998; BERGER et al., 2004).
These changes include both alterations in proliferation and apoptosis. One study
showed that apoptosis occurs in postembryonic neuroblasts, so that the number of
neurons in the adult is locally reduced. A pulse of expression in the Hox-gene
abdominal-A specifies the time at which cell death occurred. Thereby, the
authors showed a mechanism that linked abdominal-A with the determination of the
final number of the progeny that a single neuroblast generates (BELLO et al.,
Recently, apoptosis has been shown to occur segment-specifically in post-mitotic
neurons of Drosophila: In some segments, the pioneer neurons dMP2 and MP1
undergo cell-death at stage 17, well after their axons have been extended and
innervate the hindgut. This cell-death occurs specifically in segments anterior
of A6. It was linked to the differential expression of the Hox-gene Abdominal-B,
which prevents death in these cells in A6, A7 and A8 by repressing the apoptotic
genes reaper and grim (MIGUEL-ALIAGA & THOR, 2004; see also Chapter 6).
Based on my muscle map and the studies described above, I asked two questions:
Is the absence of muscles in the periphery correlated with the loss of their
respective motorneurons? If so, what mechanism is responsible for this loss – do
the relevant neurons fail to differentiate or are they apoptotically “ablated”
at post-mitotic stages?
5.2 No Indication of a Differentiating DA1 Provides a Model System
Muscles DA1 and DA2 receive their innervation from the motorneurons aCC and RP2
respectively. As I have described in detail in Chapter 3, these muscles appear
to be absent from segment A8. Their motorneurons are contained in the RN2-gal4
The aCC and pCC are generated in one segment in early stage 11 and then migrate
just beyond the segment border into the next anterior metamere by late stage 11
(BROADUS et al., 1995). However, aCC still innervates muscles DA1
intersegmentally in the segment it originated from. RP2 remains in the segment
of its origin and innervates DA2 in this segment, along with other dorsal
longitudinal muscles (LANDGRAF et al., 1997; LANDGRAF et al., 2003; Matthias
Landgraf, personal communication). With the absence of DA1 and DA2 in A8, aCC in
A7 and RP2 in A8 would thus be supernumerary neurons. The RN2 line therefore
provided me with a model system with which to address my questions (see the
diagram of figure 5.1). Beforehand, however, I looked for further arguments in
support of my claim that the dorsal acute muscles are not present in A8.
As described in Chapter 4, the expression pattern of the transcription factor
evenskipped includes dorsally projecting motorneurons, some interneurons,
pericardial cells and a ring of cells outlining the anal pad in the late embryo.
It is also expressed in DA1 in all segments from T2 to A7 and in muscle GS4 in
A9 (see also Chapter 4). Using evenskipped as a marker for the differentiating
DA1, I traced the development of this muscle back to approximately stage 12,
when individual muscle founder cells (MFCs) appear darkly stained. One can
clearly see one evenskipped positive MFC differentiate per hemisegment from A1
to A7 in addition to one or two pericardial cells (see figure 5.2).
In this figure one can also see evenskipped-positive cells differentiating in
the mesoderm of A9, although they are labelled less strongly. They later migrate
ventrally and become difficult to discriminate from the cells outlining the anal
pad. It is likely that they will seed the formation of muscle GS4. In A8, there
is no indication of a differentiating DA1 muscle founder cell. This supports my
claim that DA1 is missing in this segment. With this in mind one can consider
the motorneuron aCC in A7 to be a supernumerary neuron.
5.3 Cell-Death of Differentiated Motorneurons: aCC in A7 and RP2 in A8
In a 13 hrs AEL embryo of the RN2 line, the pattern of aCC, pCC and RP2 is
similar in all abdominal segments from A1 to A8 (see figure 5.3). In A9, only
RP2 is present along with two to three neurons posterior of RP2 that extend
axons and innervate the hindgut through the hindgut nerve (see also Chapter 4).
Note that this pattern is consistent with the anterior migration of aCC and pCC
as well as the position of A9 as the most posterior segment.
Cells in all segments extend axons that grow to the dorsal domain of the muscle
field. This includes aCC in A7 and RP2 in A8, highlighting the ISNa in A8 up to
the level of DO1. There they defasciculate and clearly show growth cones (see
fig. 3.8, Chapter 3). At around 14 hrs AEL, the cells show first signs of
disintegration: the axons start to fragment and the cell bodies shrink until
they show a clearly pyknotic appearance or have disappeared at around 15 hrs AEL
(see figure 5.4).
This is around the time when aCCs and RP2s in other segments have established
contact with their target muscles. Preparations of first instar larvae show aCC,
pCC and RP2 with their elaborate dendritic projections in all segments. Only aCC
in A7 and RP2 in A8 are missing from the pattern. I concluded from these
experiments that these supernumerary neurons are ablated from the neuromuscular
network after they have differentiated and went on to show that this ablation
was achieved through apoptosis.
5.4 Cas-3 Signal as direct Evidence for Apoptosis
Caspases are a group of cysteine proteases that are required for apoptosis
(HENGARTNER, 2000). Caspase-3 (Cas-3) protein is a reliable marker for apoptotic
cells. Antibody staining against Cas-3 clearly co-localises with the pyknotic
cell bodies of aCC in A7 and RP2 in A8 at 15.5 hrs AEL (see fig. 5.5).
This provides direct evidence that the ablation of these cells is achieved
apoptotically. I then asked whether aCC in A7 and RP2 in A8 would survive in an
apoptosis mutant. This was of interest not only because of the neuronal
apoptosis, but also to investigate whether the missing muscles DA1 and DA2 would
differentiate in such a mutant.
5.5 Survival of aCC in A7 and RP2 in A8 in a deficiency that removes grim,
reaper and hid
In Drosophila, apoptosis depends on the RHG-motif genes reaper (rpr) (WHITE et
al., 1994), grim (CHEN et al., 1996) and head involution defective (hid)
(GRETHER et al., 1995) (reviewed in BERGMANN et al., 2003). Embryos that are
homozygous for the chromosomal deletion Df(3L)H99 lack these three genes; they
are considered to be deficient for apoptosis and have not been found to show any
sign of programmed cell death (WHITE et al., 1994). Using once again antibody
staining against Evenskipped, I could highlight not only the neuronal pattern of
dorsally projecting motorneurons (including aCC and RP2) together with some
interneurons, but also the forming muscle DA1 in the dorsal musclefield.
The formation of DA1 is not disrupted in Df(3L)H99 and identical to the wildtype
pattern (see fig. 5.6). Muscle DA1 is clearly visible in all abdominal segments
from A1 to A7. There is no sign of an evenskipped signal in the dorsal domain of
A8 other than in a single pericardial cell. This finding shows that apoptosis is
not required for the absence of DA1 in A8. It seems therefore that this muscle
fails to differentiate in this segment.
On the other hand, the evenskipped pattern in the ventral nerve cord is clearly
different from the one seen in the wildtype: At 14.5 hours, the wildtype aCC in
A7 and RP2 in A8 have either clearly started to disintegrate or disappeared –
together with U neurons and lateral cells of the eve pattern in A7, A8 and A9.
In homozygous Df(3L)H99 embryos, however, these neurons do not undergo cell
death (see figures 5.7 and 5.8). This finding provides further evidence that the
ablation of supernumerary neurons is achieved through apoptosis.
The involvement of apoptosis that I have described in the diversification of
this neuromuscular network provides a potentially rewarding system to which one
could address a variety of questions which I outline below. It is the first
known case of apoptosis in post-mitotic neurons in the Drosophila CNS that can
be linked to a specific function – the innervation of their respective target
As Miguel-Aliaga and Thor have pointed out before (MIGUEL-ALIAGA & THOR, 2004),
apoptosis in invertebrate embryos in contrast to vertebrate embryos typically
occurs in cells that have just arisen (referring to LUNDELL et al., 2003;
SULSTON & HORVITZ, 1977; SULSTON et al., 1983). They propose the midline
precursor cells dMP2 and MP1 as a model system to study the developmental
regulation of apoptosis and the mechanisms that control programmed cell death
and associated events. These include in particular the role of macrophages and
glia in engulfing and removing apoptotic neurons from the CNS (SONNENFELD &
JACOBS, 1995; SEARS et al., 2003; BAEHRECKE, 2002), as well as the molecular
mechanisms that are involved in initiation and execution of programmed cell
death. The three apoptotic genes have been demonstrated to work in an additive
manner (ZHOU et al., 1997), but the cues that underlie their activation remain
to be resolved.
There are several reasons why the death of aCC and RP2 might provide a rewarding
system to study these aspects of developmental apoptosis in Drosophila. The
cells of the RN2 pattern are very dorsally located, easily accessible and
extremely well characterised. Cell death occurs relatively late and not only in
posterior segments, but also in the suboesophageal ganglion S3 and in RN2 cells
anterior of this segment (Li Feng, personal communication). Furthermore, the
sibling of aCC, the interneuron pCC, does not undergo apoptosis in any segment.
Its axon runs along the intermediate FasII fascicle, but nothing is known about
its function. This provides us with a system of two sibling cells that face very
different fates – further studies could identify cues that contribute to these
cells’ specification and in the case of aCC in A7, the programmed cell death.
There are a variety of evenskipped-related GAL4 lines available that allow
precise manipulations of the cells that are contained in the RN2 pattern.
For my own project, the episode of cell death provided me with a clear example
for an elimination of network elements in which the reduction of muscles in the
periphery and of their partners in the CNS goes hand in hand. It also showed
that the mechanism that leads to the loss of DA1 in A8 must be independent of
apoptosis. This is consistent with the fact that no instance of apoptosis has
been found in the developing Drosophila mesoderm (BATE, 1993). I base my claim
that DA1 does not differentiate primarily on the fact that I could not detect
Evenskipped in any muscle or MFC of A8. Therefore, one can hypothesise that the
muscle progenitor that is equivalent by lineage to the muscle progenitor of the
MFC for DA1 in the reference segment, differentiates differently in A8. It could
either give rise to an adult muscle precursor rather than an MFC, or to an MFC
with a different identity that does not express eve, or it could be that the
progenitor is simply not formed in this segment of the mesoderm.
To investigate this issue, the most obvious experiment to do would be to trace
the lineage of muscle progenitors and MFCs in the dorsal domain of A8. Labelling
against twist, l’sc and eve at different developmental stages could help to
resolve the question of the fate in A8 of the lineal equivalent to DA1 of the
reference segment. Investigating the cues that underwrite this difference might
be the next step.
The hypothesis that the cell-death of aCC and RP2 could be mediated by their
failure to find target muscles is tempting, especially if one looks at the
prominent growth cones that aCC and RP2 develop to explore the dorsal domain of
A7. However, the muscle ablation experiments in Schistocerca (BALL et al.,
1985;wrong reference see above) and other studies of cell-autonomous cell death
in the developing Drosophila (MIGUEL-ALIAGA & THOR, 2004; LOHMANN et al., 2003)
point in another direction. A genetic ablation of the mesoderm showed that
motorneurons differentiate and form dendrites independently of the absence of
target muscles (LANDGRAF et al., 2003).
Another interesting aspect of my findings is the stage at which the observed
apoptosis occurs. Why do aCC and RP2 differentiate normally, extend an axon, and
then die? One could think that the underlying cues that encode positional
information in these cells and that tell them to die could equally be used for
changes in the neuroblast lineages. There is no obvious advantage in
differentiating a whole pattern of cells from which certain elements are then
ablated. However, it could indicate that aCC and RP2 have essential roles aside
from the obvious one of motorneurons controlling the contractions of specific
muscles. In the case of aCC, the survival of its sibling pCC gives an example
for this model: Both cells arise from the lineage of neuroblast 1-1 (BROADUS et
al., 1995). Interneuron pCC has never been observed to undergo apoptosis in any
of the abdominal or even more anterior segments; one can assume that it is
required for whatever its function is in all segments. This prevents the system
from ablating or altering the lineage to an extent that would lead to the loss
of both aCC and pCC.
Of course this does not explain why aCC extends an axon before it dies. However,
it indicates that “developmental constraints” (developmental pathways leading to
its formation that cannot be overridden) or a “burden”, (the sum of all
subsequent events or structures that rely on a feature) (LAUBICHLER, 2000),
demand its conservation until later stages. One can see this differentiation of
seemingly supernumerary, useless motorneurons as a consequence of developmental
constraints or burdens in a diversifying system. Further studies investigating
this aspect will have to characterise other features in the CNS that depend on
the differentiation of aCC and RP2.
Most importantly for my own studies, the death of aCC and RP2 gave me a very
obvious feature in the process of neuronal diversification. I used it as a
handle to move from my first question (what mechanisms contribute to the
diversification of the network) to my second one: What are the underlying cues
that tell cells in certain segments how they have to behave? For example, what
signal triggers the activation of a developmental pathway leading to cell death
in the aCC of A7, whilst its equivalent in A6 differentiates and innervates a
muscle? The drastic and relatively accessible event of cell death in two
superficial and well-characterised neurons opened a way to investigating the
role of positional cues in this process of diversification.
Figure 5.1: The RN2 pattern as a model for element reduction
RN2-gal4:CD8GFP expression highlights the motorneurons RP2 and aCC, as well as
aCCs sibling, the interneuron pCC. RP2 innervates muscle DA2 segmentally (for
instance, RP2 in A7 innervates DA2 in A7), whereas aCC innervates muscle DA1
intersegmentally from the next anterior segment of the VNC (for instance, aCC in
A6 innervates DA1 in A7).
Since both muscles DA1 and DA2 are not present in A8, aCC in A7 and RP2 in A8
appear to be redundant. Therefore, the RN2 pattern provides a system to
investigate the fate of supernumerary motorneurons.
Figure 5.2: Differentiation of a muscle founder cell for DA1
Antibody staining for evenskipped reveals the differentiation of a muscle
founder cell and pericardial cells in all thoracic and abdominal segments except
A8. This suggests that DA1 does not have an equivalent muscle in segment A8 (n >
Figure 5. 3: The RN2 pattern in an embryo and a larva
The expression pattern of RN2 in a 13.5 hrs AEL embryo (A) and a first instar
larva (B). RP2, aCC and pCC differentiate normally in A7 and A8 in the early
The same pattern in the larva, however, reveals the absence of aCC in A7 and RP2
in A8. The single cell shown in this picture in A7 is most likely a pCC
Figure 5.4: Death of supernumerary motorneurons
Disintegration of supernumerary yet differentiated motorneurons: The RN2-gal2
driven expression of CD8GFP reveals the disintegration of nerve A8 (arrow) and
the cell bodies of aCC in A7 as well as RP2 in A8 at this 14.5 hrs AEL embryo.
Figure 5.5: The enzyme caspase-3 as a marker for apoptosis
In this preparation of a 14.5 hrs AEL embryo with CD8-GFP expression in the RN2
cells, the localisation of caspase-3 highlights aCC in A7 and RP2 in A8. Thereby,
it provides direct evidence for the apoptosis of these neurons (n > 8).
The intensity of the signal increases towards the posterior and is particularly
strong in A8 and even more so in A9. In this respect, it looks very similar to a
staining against AbdB. This shows that cell death occurs most frequently at the
Figure 5.6: evenskipped expression in the dorsal muscle field
evenskipped is expressed in the forming DA1 muscle in wildtype and the H99
apoptosis deficiency mutant: Shown here in a dorsal view of 16 hrs AEL embryo.
There is no apparent difference between the formation of DA1 muscles in wildtype
and H99 embryos, showing that apoptosis is not involved with the absence of DA1
in A8 (n > 8).
Figure 5.7: Overview of eve expression in the ventral nerve cord
evenskipped expression in the ventral view: The images are overviews, see the
red outline boxes in high magnification in figure 5.8 (n > 8).
Figure 5.8: evenskipped expression in the ventral nerve cord
In this overlay of pictures, you can see the pattern of RP2, aCC and RP2 along
with some interneurons in a wildtype and an H99 apoptosis deficiency embryo.
U-neurons and other interneurons that are known to express evenskipped are not
in focus. The embryos were fixed at 15 hrs AEL (n > 8).
Note that in the wildtype animal, aCC in A7 and RP2 in A8 show a fading of the
evenskipped signal as well as the disintegration of one aCC and the lack of any
signal for one RP2. In the H99 apoptosis deficiency mutant, there is no sign of
cell death in any of the neurons, providing evidence for the role of apoptosis
shaping segmental differences in the RN2 pattern.
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