4.) RELATING MUSCLES & NEURONS OF A8/9 TO THOSE OF A2-7
The characterisation of muscles and their respective motorneurons in the
previous chapters led me directly to the task of relating these pairs of cells
from A8/9 to pairs in the reference segment. This is ideally done through the
identification of muscles and neurons in the reference segment that are
equivalent to those in A8/9. Once this is achieved, one can ask whether the
identity of muscles and neurons can be altered, how and to what extent; and
whether the match between a muscle and its neuron is fixed in both segments or
is open to re-arrangements.
In order to avoid engaging in a discussion about “homologues” with its many
functional, phylogenetic and developmental implications (WAGNER, 1989), I am
using the word “equivalent” to describe the relationship between muscles and
neurons among the compared segments. I will base my claim for equivalence
between two muscles or neurons on shared features, which I will describe in
every individual case.
In muscles, these features include position, size, orientation, insertion sites
in the epidermis, patterns of gene expression and innervation. In neurons, these
features include position, dendritic and axonal projections, patterns of gene
expression and targets. These features can be subject to dynamic changes in a
developmental context and therefore, ought to be compared only at equivalent
developmental stages. As in any comparative study, determination of equivalence
gains weight the more features are shared among the compared segments. Of
similar interest are features they do not share – if equivalence can be derived
from other properties of the cells, such instances can point towards means of
4.2 Relating Muscles: Marker Expression Profiles
In muscles, I regard morphological information (position, size, orientation and
insertion sites into the epidermis) as relatively unreliable indications of
equivalence; the large changes in A8/9 morphology could easily account for
passive changes in any of these features and thereby, disguise equivalence. I
have discussed such a case in the apparent shift of dorsal muscles to the
ventral side of the body in A9 (see Chapter 2). However, there are four muscles
in A8 with almost completely or completely conserved anatomical features: VL1 to
VL4 in A8 are very similar to those in the reference segment. The conserved
morphology of their respective neurons (see Chapter 3) adds further support for
claiming equivalence: Their dendritic projections look very similar to those of
equivalent neurons in more anterior segments. This notion also implies that
“posteriorness” alone does not appear to be sufficient for changes in the
dendritic morphology. This supports the view that the grossly enlarged dendritic
projections of aCC in A8 and RP2 in A9 (see Chapter 3) are indeed related to a
change in the function of these neurons and their respective target muscles.
For all other muscles, more evidence is required. The identity of muscles can be
reliably determined by their patterns of gene expression (reviewed in BAYLIES et
al., 1998). This includes in particular the transcription factors evenskipped (FRASCH
et al., 1987), Krüppel (GAUL et al., 1987; RUIZ-GOMEZ et al., 1997), ladybird (JAGLA
et al., 1998), vestigial (WILLIAMS et al., 1991) and S59 (DOHRMANN et al., 1990;
CARMENA et al., 1995); and the cell adhesion molecule connectin (NOSE et al.,
1992). As an ongoing endeavour during my research, I progressively mapped the
expression of these markers in the differentiated muscles of A8 and A9 at around
14 hrs AEL.
The findings of these experiments are summarised in the maps of figure 4.1 and
in table 4.1. Images of the original specimens for the most relevant findings
(profiles of LT1, DT1 and DO1 in A8; and TO1, TO2 and GS4 in A9) are given in
figures 4.2 to 4.6. In the following, I will discuss the implications of these
profiles concerning a possible equivalence.
Firstly, it is worth noting that the expression of vestigial in the VLs of A8
fits with their role as equivalents of other VLs in the reference segment.
Furthermore, I could not detect the expression of any other markers, which again
matches with more anterior VLs. The only exception for this is Krüppel, which I
was unable to detect in any of the VLs; however, Krüppel expression in the
muscles is dynamic and the signal in VA1 of A8 might have obscured the signal in
the overlapping VLs.
Secondly, it has been reported (JAGLA et al., 1998) that there is no expression
of ladybird posterior of the SBM between A7 and A8. This is interesting, since
the position and orientation of muscle TT1 suggests that it could be the
equivalent to the SBM. It runs transversally along the segment border between A8
and A9; however, there is no indication of ladybird expression in TT1, as one
can see in the images published by (JAGLA et al., 1998).
Thirdly, and much more interestingly, evenskipped is expressed in muscle GS4.
Originally, I had failed to detect signal in this muscle, which is easily missed
due to the strong eve expression in cells surrounding the anal pad. The only eve
expressing muscle in the reference segment is DA1, which receives its
innervation intersegmentally from the motorneuron aCC (see Chapter 3). After I
had found no sign of a DA1 in A8 and subsequently discovered the death of aCC in
A7 (see Chapter 5), I noticed that aCC in A8 survives. Therefore, I asked
whether there might be an evenskipped expressing muscle in A9. Staining in flat
preparations revealed that muscle GS4 actually does express evenskipped. It is
expressed in the most dorsal muscle from T1 to A7, suggesting that it might be a
feature of the dorsal-most muscle. I take this finding as further evidence that
the very ventral muscles of GS4, TO1 and TO2 are actually in the dorsal domain
of A9 (as discussed in Chapter 2 with respect to the arrangement of nerve A9 and
the targets of ventrally versus dorsally projecting motorneurons). Later I
tested if the surviving aCC in A8 innervates muscle GS4 (see chapter 4.5).
There are other features of GS4 that it does not share with DA1: I could not
find vestigial signal, whereas there is strong vestigial expression in TO1 and
TO2. Nor could I detect any Krüppel signal. Dissections of RN2-Gal4:mCD8GFP and
CQ-Gal4:mCD8GFP embryos showed that GS4 received innervation from at least one
motorneuron of the RN2 pattern and at least one U-neuron (see figure 3.8 and
figure 3.7a, Chapter 3). It is worth noting that both RN2 and CQ neurons express
evenskipped. I regard GS4 as a dorsal muscle with no direct equivalent, which is
innervated by at least two motorneurons, of which at least the one within the
RN2 pattern can be easily compared with its equivalent from the reference
segment. This made the identification of GS4’s neurons a priority (see 4.5
Relating Neurons: aCC and RP2).
Finally, the expression profiles of LT1, DT1 and DO1 in A8 allow some
interesting conclusions. I could not detect any signal of Krüppel in DO1,
whereas LT1 and DT1 show strong staining (see figure 4.5b). DO1 in the reference
segment expresses Krüppel, but not vestigial. DO1 in A8 expresses vestigial,
only. Generally, the marker expression profiles of DO1 are more like those of
the smaller DA muscles of the reference segment (such as DA2), rather than the
morphologically more similar DO muscles. From dissections of embryos of
CQ-Gal4:CD8-GFP (LANDGRAF et al, 2003), I know that DO1 of A8 is innervated by a
U neuron (see NMJs in figure 3.7, Chapter 3). I did not find this neuron through
flipout experiments. Backfill experiments in combination with staining for
evenskipped could resolve which of the U neurons innervates this muscle. In
summary, I did not find a conclusive basis for determining equivalence between
DO1 in A8 and any particular muscle of the reference segment.
The expression profiles of LT1 and DT1 in A8 are identical: they consist of
connectin, Krüppel and S59. This matches with DT1 from the reference segment but
contradicts my original hypothesis that LT1 might be an equivalent to an LT
muscle from the reference segment. Initially, this hypothesis had been supported
by the similarities in the basic branching pattern of the motorneuron for LT1 in
A8 with the one of LT3 in the reference segment (see figure 4.7): The cell
bodies of both neurons are located very laterally in the VNC and both neurons
have large dendrites that are divided into three branches. However, the LT3
neuron is located in the posterior domain of the segment, whereas the cell body
of the LT1 neuron lies more anterior. Therefore, I consider these two neurons
not to be equivalent.
I try to avoid irrelevant details, but an evaluation of how cells relate to each
other among different segments requires a lot of attention to a variety of
features. The matching expression profiles between LT1 and DT1 in A8 and DT1 in
the reference segment brought a new hypothesis into favour: That the two muscles
in A8 are a “duplicated” variation of what is DT1 in the anterior segments.
One can speculate that a functional muscle could be quite easily generated by a
single mutation that affected the segregation lineage in the mesoderm.
Progenitor cell divisions that give rise to muscle founder cells are
characteristically asymmetric. Each of these divisions gives rise to two
different classes of cells with each sibling either maintaining or losing the
expression of genes that were originally expressed by the parent progenitor
(RUIZ-GOMEZ & BATE, 1997). This outcome of the progenitor division depends on an
asymmetric distribution of the cytoplasmic protein Numb between the two daughter
cells (RUIZ-GOMEZ & BATE, 1997; UEMURA et al., 1989; SPANA et al., 1995; RHYU et
al., 1994). The cell receiving Numb maintains progenitor gene expression; its
sibling that lacks Numb loses this expression.
Previous experiments (RUIZ-GOMEZ & BATE, 1997; CARMENA et al., 1998)
demonstrated that during wildtype development, two ventral S59 expressing muscle
progenitors divide sequentially to give rise to four cells, the founders for
muscles VA1, VA2 and VA3; as well as the ventral adult precursor VAP. Numb is
distributed asymmetrically in both progenitors. The generation of the four
individually distinct cells requires the segregation of Numb to one cell in each
pair of siblings. Therefore, after the division of the most dorsal progenitor,
Numb is segregated to the VA2 founder and excluded from VA1. Similarly the
division of the second progenitor generates a VA3 founder containing Numb and
the VAP that lacks Numb.
In numb mutant embryos, both siblings of a division adopt the fate of the cell
that does not receive Numb in the wild type. Therefore, two duplicated VA1s and
two VAPs are generated. If numb is expressed ectopically in the mesoderm, the
opposite fates are acquired by the two sibling cells of each pair: Two VA2s and
two VA3s differentiate.
If a mutation seeded the formation of two DT1s in A8, the independent phenomena
of muscle attachment and innervation could contribute to the maintenance of both
of these muscles in the course of evolution. If this had been the case with LT1
and DT1, this would provide an example for a novel mechanism of muscular
Alternatively, one could argue that the matching expression profiles have
functional reasons; this is supported by the notion that the two muscles are
perfectly aligned and roughly in the same area (latero-dorsal). Therefore, it is
likely that they function as a unit. In any case, I decided to focus on the
“novel” muscle LT1 with its unusual motorneuron. Noting that this muscle
expresses S59, I decided to use this marker to trace the differentiation of LT1
over the course of its development.
4.3 Relating Muscles: Development of S59 Positive Muscles
The development of the S59 expressing cells in the somatic mesoderm of the
reference segment has been characterised in great detail (DOHRMANN et al., 1990;
CARMENA et al., 1995). S59 is first expressed in a pattern of mesodermal cells
towards the end of stage 11. These are muscle progenitors that divide and give
rise to muscle founder cells or adult muscle precursors. The muscle founder
cells form three clusters: I (2 cells) in the ventral, anterior region of the
segment; II (4 cells) in the ventral, posterior region; and III (2 cells), in
the dorso-lateral region of the segment (see figures 4.9 to 4.11).
By stage 12, the dorsal muscle progenitor of cluster III divides and gives rise
to two muscle founder cells. One of these seeds the formation of muscle DT1, the
other one probably seeds muscle DO3 (CARMENA et al., 1995). By stage 13, the
muscle precursor of DT1 strongly expresses S59. The two progenitors of the
ventral, anterior cluster II give rise to four cells: Muscle founder cells for
VA1, VA2 and VA3; and one adult ventral muscle precursor. Of these cells, only
the precursor for VA2 maintains expression of S59 beyond stage 13. In all other
muscles, S59 expression ceases.
The single progenitor of ventral posterior cluster I gives rise to the muscle
founder cells of LO1 and VT1. The founder of LO1 expresses S59 very strongly and
migrates dorsally and to the anterior, crosses the segment border and seeds
precursor formation. However, by stage 13, the expression of S59 in the LO1
precursor is very faint. The muscle founder cell for VT1 migrates slightly more
dorsally and seeds the formation of a precursor. It is crucial to understand
that VT1 forms in the ventral, anterior domain of the segment, in which its
founder cell was born, whereas its sibling migrates to one segment further
In A8, the situation is different. By late stage 11, a cluster of four
S59-expressing cells can be seen in the posterior, ventral part of the segment
and a cluster of two cells in the anterior, ventral part. These cells are most
likely the equivalent of cluster II and cluster I respectively. However, the
pattern of migration and the differentiation into muscle precursors proceeds
differently compared to the one shown for cluster II of the reference segment.
It is likely that muscle VA1 in A8 is derived from this cluster; it maintains
the expression of S59 beyond stage 13. There is also a cluster of two cells in
the dorsal domain of A8, approximate equivalent to cluster III in the reference
segment. A cell from this cluster seeds the formation of DT1 in A8. Two more
muscles express S59, one in A8, one in A9, but their identity is unclear and
they are labelled with question marks in the figures. From its position and
orientation, the muscle in A8 might be muscle LL1 or VL1. In A9, I found S59
expression exclusively in the neuroectoderm.
Looking at the anterior cluster I in A8, one can see that the progenitor
divides, as previously described at late stage 11. This is followed by the
migration of a very darkly stained muscle founder cell across the segment
boundary and into the lateral domain of A7, where it seeds the formation of LO1.
Its sibling, which seeds the formation of VT1 in the reference segment, remains
in A8 and migrates dorsally. There it seeds the formation of the original
objective for these observations: muscle LT1.
One can now ask whether VT1 in the reference segment and LT1 in A8 are therefore
equivalents. In this instance it is obvious why I refrain from using the term
“homologue” to describe the relationship between these muscles: some features
are shared, others are not. Both VT1 and LT1 are derived from equivalent
lineages and muscle founder cells; both muscles are transverse. They are
different in their position (ventral versus lateral); size (approximately 5
myoblasts contribute to VT1 as against approximately 10 that contribute to LT1;
n>10); and to some extent gene marker expression (LT1 expresses Krüppel and
connectin, whereas VT1 does not appear to).
This covers lineage, orientation, position, size and patterns of gene
expression. From a developmental perspective, I regard VT1 and LT1 clearly as
equivalents at least up to the stage of myoblast recruitment. It is not fruitful
to argue about homologies, yet most interesting to explore how two elements of a
network starting as identical units diverge over the course of their
specification to finally serve two different functions. One of the features that
I have outlined in the introduction to this chapter has so far been neglected:
the innervation. In terms of different functions, the next logical step was to
investigate the neurons that control these two muscles.
4.4 Relating Neurons: VT1, LT1 and the Role of the Transverse Nerve
The transverse nerve (TN) contains the axons of only two motorneurons per
hemisegment: A posterior one that branches together with its equivalent in the
other hemisegment; and an anterior one. The posterior TN neuron innervates the
alary muscle dorsal of the SBM and is located at the same level as pCC. The
anterior TN neuron innervates muscle VT1 and is on the same level as RP2 (see
figure 4.17 A; MACLEOD et al., 2003). These motorneurons project their axons to
the midline, exit the VNC dorsally and grow along the posterior segment border
(GORCZYCA et al., 1994; CHIANG et al., 1994; MACLEOD et al., 2003; LANDGRAF et
al., 1997). The external VT1 is the only somatic muscle innervated from the next
anterior segment through the transverse nerve (TN); however, the nerve extends
beyond this muscle to the lateral domain of the segment (see figure 3.15,
Chapter 3; figure 4.18). On the posterior edge of the SBM, two sensory neurons
join the transverse nerve: the lateral bipolar dendrite neuron (LBD) and the
tracheal dendrite neuron (TDN) (GORCZYCA et al., 1994; see figure 4.18).
Muscle VT1 is specific to abdominal segments A2 to A7 (see figure 2.4 in Chapter
2). Its equivalent muscle LT1 in A8 has a prominent neuromuscular junction and
receives its innervation from a motorneuron with very elaborate dendritic
branches (see Chapter 3), which does not extend its axon through the TN. Through
flip-out experiments, I never found a cell resembling a VT1 neuron in A7.
However, as I have shown in Chapter 2, there is clearly a transverse nerve in
exiting A7 and running along the A7/8 border. Therefore, I wondered whether,
despite my findings with the flip-out approach, an anterior VT1 neuron existed
in A7, for example a cell, which, like the A7 aCC, was eliminated by apoptosis.
In 13.5 hrs AEL embryos, one can see a transverse nerve exiting the VNC from T1
to A7; at around 14 hrs AEL, the TNs of T1 to T3 progressively disintegrate and
finally do not project into the muscle field at around 15 hrs AEL (data not
shown). As the VNC contracts progressively during this time, the mesodermal
cells on the dorsal surface that are part of the TN delaminate and the nerves
get “pulled” to the posterior.
To investigate whether there is a VT1-like neuron in A7 and in the thoracic
segments, I performed retrograde labelling experiments by directly applying a
DiI crystal to the transverse nerve of A8, of the thoracic segments and of the
reference segment (see Chapter 8 for Material and Methods). Such a treatment
often leads to the labelling of the posterior TN neuron in both hemisegments,
because the posterior neuron is branched and therefore, both cells have axons
exiting on each side.
To avoid labelling a cell that was assigned to die, I first carried out these
experiments as late as I was technically able (around 15 to 15.5 hrs AEL). This
is well after aCC in A7 and RP2 in A8 have started to disintegrate. In most
specimens, they have completely disappeared by this stage. By using embryos of
the RN2-Gal4 line crossed to UAS-GFP, I had reliable landmarks to discriminate
between the anterior TN neuron (aligned with RP2) and the posterior TN neuron
(aligned with pCC).
In A7 (from where the TN of A8 is derived), I labelled one motorneuron with a
cell body at the same anterior-posterior level as pCC and dendritic projections
at the level of the anterior commissure (see figures 4.15 and 4.17; n > 10).
This identified it as the “posterior TN neuron” innervating the alary muscle as
previously described (MACLEOD et al., 2003).
In the reference segment (anterior of A7), I labelled two motorneurons (see
figures 4.15 and 4.17; n > 10). One motorneuron with a cell body at the same
anterior-posterior level as pCC and dendritic projections to the anterior. This
identified it as the “posterior TN neuron” innervating the alary muscle as
previously described (LANDGRAF et al., 1997; LANDGRAF et al., 2003; MACLEOD et
al., 2003). The other motorneuron had a cell body at the same level as RP2. This
identified it as the “anterior TN neuron” innervating muscle VT1 as previously
described (MACLEOD et al., 2003).
In the thoracic segments, I applied the DiI crystal to the already disintegrated
transverse nerve. By this stage, the TN did not project into the muscle field,
although the nerve endings were still in place on the dorsal surface of the VNC.
The dye revealed a motorneuron with a cell body at the same level as RP2 and
with dendritic projections at the same level as the anterior commissure. Based
on its position, I regard this neuron as being equivalent to the anterior TN
neuron of the reference segment, where it innervates muscle VT1 (n = 3). I did
not label any posterior TN neurons, but this could be due to the small number of
specimens. The absence of alary muscles in the thoracic segment also implies
that the respective neuron might not differentiate in the first place.
My interest now focused on the apparent “absence” of the anterior TN neuron in
A7. In order to see whether it differentiated and extended an axon earlier in
development, I repeated the experiment using younger embryos (13.5 hrs AEL or
younger; preceding the death of aCC in A7). In most specimens, I succeeded in
labelling only the posterior TN motorneuron (n = 8). Thus, I can conclude that
there is probably no motorneuron extending its axon through the TN into A8
besides the posterior one that innervates the alary muscle (see discussion
I then decided to investigate the neuromuscular junctions formed by axons of the
TN in the reference segment and in A8 at 15.5 hrs AEL using DiI. In the
reference segment, the motorneuron axons project into the muscle field along the
segment border. At this stage, the most distal part of the nerve extends to the
level of VL1 and the SBM (see figure 4.17; n > 20).
In A8, the nerve projects into the muscle field along the segment border, too.
The most distal part extends to the level of VL3, which is approximately where
one would expect a muscle VT1. In most specimens examined (7 of 9), the nerve
bifurcated. Since I was not able to find a NMJ in any segment at this stage, I
decided to investigate specimens later in development.
I repeated my observations with glue preparations of first instar larvae (for
methods see Chapter 8.9). I was not able to preserve the cuticle of segment A8
or the TN for this segment. However, labelling the TN in the reference segment
revealed the VT1 neuron (n = 4). I could trace the axon to the periphery and
identify a neuromuscular junction on VT1. In two specimens, the dye also
revealed the posterior TN motorneuron (alary muscle) in the same hemisegment
with its cell body posterior of the VT1 neuron.
In order to investigate the patterns of innervation of the two TN motorneurons
in greater detail and at later stages, I examined the projections of
motorneurons in third instar larvae of the OK371-Gal4 line. The arrangement of
muscles with respect to features of the cuticle such as denticles is fixed.
Therefore, denticles can serve as landmarks to determine the position of
neuromuscular junctions from the OK371 pattern. Using the G203 2C12144 line with
GFP:titin (expresses GFP in the muscles), I determined the position of VT1 with
respect to the denticles (see fig. 4.18a). Then I localised this position in a
OK371:CD8GFP third instar larva (expresses GFP in motorneurons and allows one to
trace axons) and found NMJs on VT1 derived from the TN (see figures 4.18b and
4.18c). I could not find any NMJ ventral of the SBM derived from the TN in A8
(see figures 3.15 and 3.16 in Chapter 3).
In the reference segment, the TN nerve continues to grow beyond the region of
VT1. It extends to the region of the LBD neuron without forming a detectable
NMJ. Interestingly, the TN in A8 projects slightly more dorsally: it ends dorsal
of the NMJ of the LT1 neuron (see figure 3.15, Chapter 3). From these
observations, I draw the following conclusions for larval TNs: There are two
motorneurons per hemisegment in the TN of A1 to A7, of which the anterior one
innervates VT1, the posterior one the alary muscle (as described in MACLEOD et
al., 2003). There is one motorneuron per hemisegment in the TN of A8, which is
the posterior one that innervates alary muscle. The very dorsal extension of the
TN of A8 suggests that the alary muscle of this segment is probably located more
dorsally than in the reference segment.
To compare the motorneuron innervating VT1 in the reference segment and the LT1
neuron in A8 genetically labelled with mCD8:GFP, I repeated the flipout
experiments described in Chapter 3. This time, I specifically looked for the
motorneuron of VT1. I found a total of three specimens in which I could identify
this cell based on two features: Its axon exits the VNC through the TN; and its
cell body is anterior of its dendrite. Figure 4.16B shows a fourth specimen
provided by Annemarie North. It looks identical to the neurons I found; however,
the quality of the specimen is significantly better in terms of its general
preservation and the lack of background from labelled cells in nearby segments.
In comparison with the previously described LT1 neuron, neither cell body
position nor dendrite morphology or the axonal projection bears similarities. An
equivalence of the two neurons is therefore extremely unlikely.
The fate of the anterior TN neuron of A8 is unclear. There are several
possibilities: The neuron could never differentiate in this segment; it could
fail to extend an axon through the TN; it could die even before the TN
differentiates; or it could differentiate into a completely different
motorneuron that extends its axon through a different nerve. With the aid of NB
markers or labelling techniques, it seems conceivable that the tracing of the
lineage could finally reveal the answer. For my work, however, I reject the
hypothesis that there is a skeletal muscle in A8 that receives its innervation
from the TN, based on the (negative) evidence presented above.
I showed in Chapter 3 that LT1 in A8 is innervated by a neuron that does not
project through the TN. Tracing the nerves of OK371:CD8GFP larvae showed that
VT1 in the reference segment and LT1 in A8 are most likely innervated by only
one excitatory motorneuron each (see figures 3.15 and 3.16 in Chapter 3). In the
light of the developmental equivalence of these two muscles determined earlier
in this chapter, these findings have interesting implications for the
understanding of neuron/muscle relationships (see 4.6 Discussion).
4.5 Relating Neurons: aCC and RP2
I have described the pattern of the RN2-Gal4 line in detail in Chapter 3. In
brief, the motorneuron aCC is located with its sibling interneuron pCC, just
anterior to the segment border of the metamere it originated in, into which it
projects intersegmentally and innervates muscle DA1. For example, motorneuron
aCC in A6 innervates muscle DA1 in A7. The second motorneuron in the pattern,
RP2, segmentally innervates - among others - muscle DA2. For example,
motorneuron RP2 in A6 innervates muscle DA2 in A6 (see figure 5.1 in Chapter 5).
Muscles DA1 and DA2 do not have obvious equivalents in A8 and the
“supernumerary” neurons aCC in A7 and RP2 in A8 differentiate and then die (see
Posterior of the dying RP2 in A8 at approximately 14 hrs AEL, there is a pair of
cells that I take to be aCC/pCC in the same segment; there is also an RP2 in A9
and two to three unidentified cells per hemisegment at the posterior-most margin
of A9 (aCC, pCC and RP2 were identified in the RN2 pattern, see figure 3.3,
Chapter 3). I had found that muscle GS4 (which expresses evenskipped, a marker
for the most dorsal muscle) alongside with TO2 (which expresses vestigial, a
marker for longitudinal muscles) receives innervation from a motorneuron of the
RN2 pattern (see figure 3.8, Chapter 3). There is also a contribution of the RN2
pattern to the hindgut nerve, which I have resolved later in this chapter.
TO2 and GS4 have obviously different features from DA1 and DA2 in the reference
segment, which are innervated by aCC and RP2 motorneurons. Note that GS4 is also
innervated by a U-neuron of A8 (see Chapter 3, figure 3.8). Since aCC, RP2 and
U1 motorneurons can be reliably identified as equivalents at 14 hrs AEL among
different segments, the RN2 and CQ line provided me with the ideal model to
explore morphological changes of motorneurons accompanying alterations in the
I used flies of the RN2-flp, tub-frt-stop-frt-Gal4, UAS-mCD8GFP line (see
PIGNONI & ZIPURSKI, 2001 for tubulin-CD2 stock; Annemarie North, unpublished) to
label single RN2 neurons in third instar larvae. In this line, the gene for the
flipase is placed under the control of a weak evenskipped promoter. I exposed
embryos at a variety of stages to 29ºC for 24 hours and raised the larvae at
25ºC to third instar (see Chapter 8, Material and Methods). Flip-out events
occurred only in a subset of cells in the RN2 pattern, leading to Gal4
expression under the control of the Tub-promoter. I dissected larvae in which
single cells expressed GFP and then stained the CNS for evenskipped as a
reference marker, as well as GFP.
In this way I identified aCC in A8 (n = 7; see figure 4.19) and RP2 in A9 (n =
6; see figure 4.20). The aCC in A8 has two symmetrical dendritic branches, one
ipsilateral, one contralateral and both originating from the cell body. They
extend far to the anterior, covering much of the neuropile in A8 and probably
reaching into A7. The RP2 in A9 has a dendrite that projects to the anterior and
then bifurcates into an ipsilateral and a contralateral branch. Just as in the
aCC, these two branches are symmetrical and cover much of the neuropile in A8.
In both these neurons, the dendritic projections are strikingly different from
those of their equivalents in the reference segment (see figure 4.21 for direct
comparison; and the discussion of this chapter for implications).
The two or three neurons posterior of RP2 in A9 extend axons to the hindgut (see
figure 3.8, Chapter 3) but have no detectable dendrites (data not shown).
Looking at my specimens of aCC in A8, I realised that a neuron I had identified
through the flipout experiments of Chapter 3 and matched with TO2 was actually
an aCC with strongly altered dendrites (see figure 3.12g in Chapter 3).
Since phalloidin injections failed to reveal the most posterior muscles of A9, I
could not confidently match aCC in A8 and RP2 in A9 individually with their
target muscles GS4 and TO2. To identify the targets confidently, I filled these
neurons with the intracellular dye Lucifer Yellow in 14.5 hrs AEL embryos (see
Chapter 8, Material and Methods). I traced the axonal projection of aCC in A8 to
muscle TO2 (n = 7; see figure 4.22a) and RP2 in A9 to muscle GS4 (n = 5; see
I began this chapter by outlining the idea that elements of the neuromuscular
network can share particular features at different stages of their development.
Depending on the degree of resemblance, one can consider certain iterative
elements to be equivalent and investigate their role in the locomotor network,
their partners in this network, and their developmental fate.
By tracing the lineage of the S59 positive muscle progenitors and their progeny,
I could determine the equivalence of the muscle founder cells of VT1 in the
reference segments and LT1 in A8. These two muscle founder cells fuse with
specific numbers of naïve myoblasts to form two distinct types of muscle
precursors. I investigated the cues underlying this diversification in Chapter
6. More detailed information about how the recruitment of certain numbers of
myoblasts is regulated in a segment-specific manner is of great interest. It
could help to understand the interaction of genes that are involved with
myoblast recruitment and the underlying spatial cues on a molecular level.
I showed in this and the previous chapter that VT1 in the reference segment and
LT1 in A8 are innervated by two different motorneurons. Not only do they have
very distinct dendritic arbours, they also project to their target muscles
through different nerves. This demonstrates that the match between a neuron and
its target muscle is not fixed among all segments and raises the question of
what features have to change in order to accomplish a switch in the innervation.
The current understanding how the match between a motorneuron axon and its
specific target muscle is achieved is based on two models that are not mutually
exclusive: The “relative balance” (WINBERG et al., 1998) and the “lock-and-key”
model (HOANG & CHIBA, 1999) both predict the existence of molecular interactions
between motorneurons and muscles, as well as repulsive and attractive cues that
modulate those. Several studies have demonstrated that many genes affecting
motor axon pathfinding are widely expressed by muscles and motorneurons:
Alterations in the levels of cell surface proteins such as Fasciclin II in
muscles can lead to a biased innervation of specific muscle fibres (DAVIS et al.
1997). This supports the “relative balance“ model, in which the final step in
target recognition would be controlled by the overall balance of attractive and
repulsive cues mediated by a combination of cell adhesion molecules (CAMs) and
other guidance cues.
The “lock-and-key” model, on the other hand, proposes a complementary code of
molecular cues expressed by the motorneuron and its target muscle that ensures a
precise match during the process of target recognition. Support for this model
comes from NetrinB, which is expressed in only three muscles: VL3, VL4 and DA2.
Motorneuron RP3 innervates specifically muscles VL3 and VL4, but fails to do so
in 35% of the cases in NetrinB mutants (MITCHELL et al., 1996).
In this context it is worth noting that LT1 in A8 expresses connectin, which has
also been shown to play a role in target finding (NOSE et al., 1994), whereas
VT1 in the reference segment does not. Similarly, Krüppel is expressed in LT1,
but not in VT1. If an evolutionary change in the expression profile of muscle
marker proteins had occurred at the level of muscle founder cells, this would
have determined a different identity, and could have been accompanied by a new
neuron finding this novel target. This could be a neuron that is supernumerary
in anterior segments where it dies at later stages in development.
If the lineage of the LT1 motorneuron in A8 was known, one could investigate
whether such supernumerary neurons differentiate in the reference segment. A
network that arises not only from a range of muscles that one particular
motorneuron has to choose from, but also from a range of motorneurons that can
be attracted by a particular muscle would allow dynamic re-matching through
mutations leading to altered identities of either motorneurons or muscles.
With respect to the VT1 innervation, one can ask why the VT1 neuron in the
thoracic segments survives until about 15.5 hrs AEL and maybe even well into
larval life. The labelling was done on disintegrated nerves that did not reach
into the muscle field anymore and at a time when aCC in A7 and RP2 in A8 have
already disintegrated. Yet there is no obvious difference in this neuron’s
dendrite or cell body and its equivalents in abdominal segments, where it forms
a functional NMJ. In the thorax, this neuron could act as a pioneer and die at
much later stages.
With respect to alterations in motor neuron identity, I approached the
neuromuscular network from the opposite angle. Instead of looking for the
neurons innervating muscles that are derived from equivalent founder cells, I
investigated the development of motorneurons aCC and RP2 that innervate
different muscles in different segments. Until approximately 14 hrs AEL, the aCC
and RP2 motorneurons of all abdominal segments look identical. Within the next
hour, however, the supernumerary aCC in A7 and RP2 in A8 disintegrate and die
(see Chapter 5). The neurons that started as aCC in A8 and RP2 in A9, however,
differentiate into two very distinct neurons that make contact with target
muscles in A9. However, these muscles are different from DA1 and DA2, which are
normally innervated by aCC and RP2. In larval stages, aCC in A8 and RP2 in A9
have bilateral dendrites that extend far to the anterior.
These distinct features of aCC and RP2 dendrites could be explained through
passive alterations due to the over-all changes in this segment’s morphology.
Alternatively, one could hypothesise that the death of aCC in A7 and RP2 in A8
leaves a “gap” in the neuropile into which neurons of more posterior segments
can project dendrites. This would mean that the presence of a cell in one
segment would create a repulsive signal at least for its equivalents from other
segments. Previous experiments showed that neighbouring neurons do not extend
axons into the domain of a dead neuron of a different class (Matthias Landgraf,
personal communication). However, this applies to neurons of a different class
and not to equivalent neurons from other segments that are more likely to
respond to the same attractive and repulsive cues in the neuropile. In this
context, it is worth noting that branches of dendrites do occasionally expand
beyond the segment in which they normally branch. Thereby they span up to three
segments even with their equivalents developing normally and occupying this
domain in the neuropile (Matthias Landgraf, personal communication).
To test this hypothesis, either the flipout system or laser ablation could be
used to selectively eliminate neurons from the RN2 pattern early in development
and investigate whether other (equivalent) cells take over their dendritic
domains. An alternative explanation for the altered dendrite morphology of aCC
in A8 and RP2 in A9 is their “different” function with respect to equivalents
from more anterior segments. Because they innervate two muscles with a different
position, size and orientation than their equivalents in the reference segment,
one might expect that these pairs of neurons and muscles play a different role
in locomotion. This is likely to require specific synaptic input and an
appropriate dendritic morphology to accommodate this.
A different issue that applies to both muscles and neurons is the one of
identity. Are muscles VT1 in the reference segment and LT1 in A8 homologues? Is
aCC in A8 still an aCC? These examples show how important it is to “study things
in the process of development from the beginning” (ARISTOTELES, 360 BC). The
cells described start as identical entities that differentiate into
morphologically and functionally very distinct elements of the neuromuscular
network depending on their environment (A8/9 versus the reference segment). The
question as to whether the resulting structures are still homologues or not is a
rather semantic issue and will not be discussed in further detail. It is more
important to reveal the molecular and cellular basis of the developmental
pathways that lead to the diversification of originally identical elements of
the networks. This leads directly to the basis of segmental identity, which I
investigated in Chapter 6.
For such investigations, the RN2-Gal4 line has provided an exceptionally
rewarding system. Within the three segments of A7, A8 and A9, I could describe
three different muscle patterns. This is accompanied on the level of central
neurons by all three principal ways of diversification, as I have defined them
in Chapter 2: The RN2 motorneurons for A7 differentiate the same way as they do
in more anterior segments (conservation of elements); the RN2 motorneurons for
A8 die postmitotically (removal of elements); and the RN2 motorneurons for A9
develop new features (differentiation of novel elements). The small number of
cells contained in the RN2 line, their accessibility for mechanical manipulation
and the detailed knowledge about the specification of dorsally projecting
motorneurons makes this system particularly attractive to study the mechanisms
Investigating the development of U1 neurons in A6, A7 and A8 could also lead to
interesting findings. U1 in A6 innervates muscle DO1 in A7; U1 in A7 most likely
the much bigger muscle DO1 in A8; and U1 in A8 probably muscle GS4. This system,
in which one finds a conserved, a slightly different and a drastically different
target could also provide insights into the process of neuromuscular
Figure 4.1: Expression of muscle markers
The expression profile of marker proteins, such as transcription factors or cell
surface proteins, gives a unique identity to groups of muscles or even
individual muscles in the reference segment. In A8/9, they can provide valuable
cues for evaluating the relationships among muscles of different segments.
This figure gives a diagrammatic summary of the expression profiles of connectin
(cell surface protein); evenskipped, Krüppel, ladybird, S59 and vestigial (transcription
factors) in approximately 15 hrs AEL embryos. These results are derived from a
large number of specimens, each set of experiments: n > 10. This applies also to
the figures that show staining in the actual cells (fig. 4.2 to 4.6).
Table 4.1 Summary of the expression profiles of muscle markers
Summary of the expression profiles of connectin (cell surface protein);
evenskipped, Krüppel, ladybird, S59 and vestigial (transcription factors) in
approximately 15 hrs AEL embryos. In addition to the diagram in figure 4.1, this
table contains references to positive, but not identified muscles.
Figure 4.2: Expression pattern of connectin
Expression of connectin in a flatprep (~15 hrs AEL). The yellow arrow points to
DT1, the blue one to LT1. Slide provided by Matthias Landgraf.
Figure 4.3: Expression of vestigial in A8/9
Expression of vestigial in a flatprep (~15 hrs AEL). PS marks the posterior
spiracles. Note that the TO2 muscles (white arrows) are torn as they usually do
in flat preparations.
Figure 4.4: Expression of vestigial in A8/9
Expression of vestigial in a whole mount embryo (~15 hrs AEL). PS marks the
posterior spiracles, the black arrow indicates the midline. The nuclei of DO1
are clearly visible (red dotted circles).
Figure 4.5: Expression of S59 and Krüppel
Expression of S59 (A) and Krüppel (B) in whole mount embryos (~16 hrs AEL). The
yellow arrow points to DT1, the blue one to LT1. Note that the S59 specimen is
shown in a dorso-lateral view, the Krüppel one in a dorsal view. PS = posterior
Figure 4.6: Expression of evenskipped
Expression of evenskipped in a flatprep (~15 hrs AEL). Note the expression in
DA1 of A6 and A7 and strong expression on the margin of the anal pad in A8.
Arrows indicate eve expression in two parallel GS4 muscles of the two
Figure 4.7: Similarities in neuronal morphology
LT3 neuron for A7 (A) in a phalloidin background (red) and the LT1 neuron for A8
(B) in a background for evenskipped (red). The cell bodies do not lie in the
same antero-posterior region of the neuropile, however, the dendritic
projections of the two neurons share some similarity. Both are divided into
three major branches (not visible in the two dimensional z-projections) that
project over two or even three segments (LT1 for A8).
Figure 4.8: Lineage of S59 positive cells in the mesoderm
This diagram summarises the formation of S59 positive muscles in the segments
A6, A7 and A9.
(A) S59 positive muscle progenitors give rise to muscle founder cells (MFCs) in
three clusters (I, II and II). At stage 12, these clusters are the same in all
abdominal segments from A1 to A7. In A8, a cluster of four cells similar in
their position to cluster II expresses S59.
(B) The two muscle founder cells of cluster I migrate; MFC Ib migrates into the
lateral domain of the segment it originates from, whereas MFC Ia migrates to the
anterior and crosses the segment border.
(C) MFC Ia seeds the formation of muscle LO1 in the reference segment. MFC Ib
seeds the formation of muscle VT1 in the reference segment, and LT1 in A8. Other
muscles in the reference segment that are derived from S59 expressing founder
cells include DT1 and VA2.
Muscle VA1 in A8 is likely to be derived from the cluster ~II of A8 and is
probably an equivalent to muscle VA2 from the reference segment. Muscle DT1 in
A8 strongly expresses S59 from around 13 hrs AEL. The observations that led to
this diagram were made in a set of experiments with n > 10. This applies to the
following fig. 4.9 to 4.14.
Figure 4.9: Differentiation of S59 positive cells
Over the course of germband retraction, S59 is detectable in three clusters of
cells: A ventral anterior cluster I, a ventral posterior cluster II, and a
dorsal cluster III. In A8, only clusters I and II can be detected at this stage.
In A9, only neuroectodermal cells are S59 positive. The black arrow indicates
Figure 4.10: Migration of S59 positive Muscle Founder Cells
Towards the end of germband retraction, individual muscle founder cells within
the clusters differentiate and start to migrate. For example, cluster I is
clearly discernible due to its size and intense S59 staining (see orange arrow).
Figure 4.11: Migration of S59 positive MFCs
In this specimen, one can see an overview of the final position in which the
MFCs of the three clusters of S59 positive cells will start to recruit myoblasts
for fusion in the reference segment: the anterior, ventral cluster Ib will give
rise to VT1 (note that in some anterior segments, myoblasts are visible); the
darkly stained MFC of Ia that will give rise to LO1 is in the lateral domain;
the ventral, posterior cluster II consists of four cells; and the dorsal cluster
III that will give rise to DT1.
In A8, MFC Ib has migrated to the anterior, lateral domain of the segment where
it will seed the formation of muscle LT1. In the ventral, posterior cluster II,
four MFCs are clearly labelled.
Figure 4.12: Ia has migrated to the next anterior segment
Just before muscle founder cells start to recruit myoblasts for fusion, MFC Ia
(orange arrow) that will give rise to muscle LO1 has crossed the segment border
and is now in the next anterior segment. The sibling MFC Ib that will give rise
to LT1 in A8 has migrated to the lateral domain (compare with the more ventrally
located Ib of A7).
In this specimen, there is one cell associated with Ib in A8 with an uncertain
identity (black arrow). It is likely to be a myoblast fusing with the aligned Ib
Figure 4.13: S59 signal in differentiating muscles
S59 positive syncytia outline the forming muscles of DT1, LO1, VT1 and VA2 in
the reference segment and LT1 as well as an unidentified muscle ventral of LT1
in A8. Note that at this stage, there is no indication of S59 signal in DT1 of
A8; the signal can be detected later than in the DT1 of the reference segment.
Figure 4.14: S59 in fully differentiated muscles
Fully differentiated muscles with S59 signal reveal DT1, LO1 faintly), VT1 and
VA2 in the reference segment. In A8, muscles DT1 and LT1 are stained as well as
an unidentified muscle ventral of LT1 and one unidentified muscle in A9.
Figure 4.15: Fills of the embryonic Transverse Nerve (TN)
DiI (red) fills of the transverse nerve (TN) in an approximately 15 hrs AEL
embryo (post cell death of aCC in A7 and RP2 in A8). RN2 background provides
(A) Overview of the specimen of which figures C and D were taken (see yellow
(B) DiI fill of the transverse nerve in the reference segment. Nerve endings
reach out to the muscle field. The neuron is aligned with RP2, which identifies
it as the anterior TN neuron that innervates VT1.
(C) DiI fill of the transverse nerve in the thoracic segment T3 (initially
innervates A1). By this stage, the nerve has already disintegrated and does not
enter the muscle field anymore. Here, too, the neuron is aligned with RP2, which
identifies it as the anterior TN neuron that innervates VT1.
(D) DiI fill of the transverse nerve in A7 (innervates A8). Two neurons were
labelled in this specimen, as is often the case with the TN. Note that aCC in A7
and RP2 in A8 have already disappeared. The cell body is approximately aligned
with pCC, therefore, it is more posterior than the labelled neurons in figures B
and C. This identifies it as the posterior TN neuron, which innervates the alary
Figure 4.16: Fills of the larval Transverse Nerve (TN)
DiI (red) fills of the transverse nerve (TN) in first instar larva (glue
preparations). RN2 background provides landmarks (green). Note the strong GFP
expression in aCC of A8 (white arrow in the top image).
Note also that the segment boundaries in the larva do not follow straight lines
– the guidelines given as label for the segments are based on the position of
aCC and RP2.
(A) DiI fills of the transverse nerve in the reference segment. In the larva,
two neurons per hemisegment can be discerned: one posterior (innervating the
alary muscle, yellow arrow) and one anterior (innervating muscle VT1 in the next
posterior segment, blue arrow). Note that in this specimen, two TNs (A5 and A6)
(B) Flip-out clone of the VT1 neuron in a 3rd instar larva. The dashed line
indicates the midline. Specimen provided by Annemarie North.
(C) Neuromuscular junction of the anterior motorneuron of the transverse nerve
on muscle VT1 – the approximate outline of this muscle is indicated by a dotted
line. The muscle is difficult to see in a glue preparation, since it is covered
by VL and VO muscles. NMJs are indicated by the arrows.
Figure 4.17: Projections of the embryonic Transverse Nerve (TN)
DiI (red) fills of the transverse nerve (TN) in an approximately 13.5 hrs AEL
embryo (4.17 A) and a 15 hrs AEL embryo (4.17 B-D). RN2 background provides
(A) Reference Segment: A fill of a young (13.5 hrs AEL) embryo in the TN of A7 (cell
bodies in A6) reveals one anterior TN neuron (blue arrow) that innervates muscle
VT1; as well as the posterior TN neurons that innervate the alary muscles (one
in each hemisegment; yellow arrow).
(B) A8/9: The TN of A8 (cell bodies in A7) grows along the segment border. The
specimen shows only the posterior TN neurons – this applies to both young (approximately
13.5 hrs AEL embryos; n=8) and older (post aCC death, 14.5 hrs AEL and older;
(C) The TN of A7, growing along the segment border A7/8 in a different specimen.
Note the bifurcation.
(D) The TN of A4, growing along the segment border A4/5 as an example for the TN
in the reference segment. The nerve does not bifurcate; no obvious sign of a
neuromuscular junction can be seen.
Figure 4.18: Innervation of VT1 through the TN
(A) Muscle VT1 (outlined in blue) is highlighted under UV light in a third
instar larva of a line that expresses GFP associated with the muscle protein
titin. Its position with respect to the denticle bands is fixed.
(B) and (C) The same region in a third instar larva of a line that expresses
mCD8-GFP in all motorneurons (OK371), thereby, highlighting their axons and
neuromuscular junctions in the periphery. Brightfield in (B), UV light in (C).
The transverse nerve is clearly visible and forms a neuromuscular junction (C;
yellow arrows). It continues growing dorsally, as expected since there are at
least two neurons contributing to the TN in the larva (blue arrow).
The position of the neuromuscular junctions corresponds with the position of
VT1, as determined through the denticle bands as landmarks. The VA and VO
muscles are also visible in this image, shining through from underneath the
Figure 4.19: Dendritic morphology of aCC in A8
In order to visualise individual cells of the RN2 pattern, virgins of a RN2-flp,
tub-frt-stop-frt-Gal4, UAS-mCD8GFP were used. In this line, the gene for the
flipase is placed under the control of a weak evenskipped promoter. If embryos
are raised at 29ºC, flip-out events occur only in a subset of cells in the RN2
pattern, leading to Gal4 expression under the control of the Tub-promoter.
Motorneuron aCC can often be recognised even in the full pattern, due to the
“gap” that the death of aCC in A7 and RP2 in A8 leaves. In the specimen shown,
evenskipped staining (red) provided additional landmarks to identify aCC in A8.
The dashed line indicates the midline. The specimen shown was a third instar
Figure 4.20: Dendritic morphology of RP2 in A9
Protocol as above. In the figures shown above, evenskipped staining (red)
provided information on landmarks to identify RP2 in A9. The dashed line
indicates the midline. The specimen shown was a third instar larva.
Figure 4.21: Direct comparison of aCCs and RP2s
A direct comparison between RP2 in the reference segment (A) with RP2 in A9 (B);
and between aCC in the reference segment (C) and aCC in A8 (D).
The dashed line indicates the midline. All specimens are third instar larvae.
Figure 4.22: Fills of aCC in A8 and RP2 in A9
Intracellular dye fills with Lucifer Yellow (LY) allow one to trace the axonal
projections of individual neurons from the CNS to the target muscle (green). A
background of RN2 (red) provides positional information as a landmark and proves
the identity of the filled cell. The specimens are flat preparations of 14 to
14.5 hrs AEL embryos.
(A) Fill of aCC in A8: The axon projects through nerve A9 to muscle TO2 (n = 7).
(B) Fill of RP2 in A9: The axon projects through nerve A9 to muscle GS4 (n = 5).
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