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6.) CUES UNDERLYING THE DIVERSIFICATION
6.1 Introduction
The mechanisms that I have described in the previous chapters that lead to the
diversification of features over the course of development occur within temporal
and spatial boundaries. Cues that provide individual cells with positional
information are the substrate from which the diversification arises. In this
chapter, I will finally investigate these cues.
The mechanisms by which the body plans of metazoan animals are patterned are
highly conserved (FERRIER & HOLLAND, 2001). A group of homeodomain transcription
factors, the Hox proteins, are expressed along the anterior-posterior (AP) axis
(LEWIS, 1978). Hox proteins regulate downstream target genes in the individual
cells where they are expressed. These target genes and their respective targets
participate in developmental programmes that shape increasingly distinct
features in specific domains of the body by governing mitotic rates, cell
adhesion, cell shape, cell migration and apoptosis. Therefore, Hox genes
determine alternative developmental pathways (GARCIA-BELLIDO, 1975).
In Drosophila, Hox genes are expressed from early embryogenesis onwards
throughout development (reviewed in AKAM, 1987). The patterning of the
blastoderm into segments is achieved by the expression of gap and pair rule
genes (MCGINNIS, 1992). They act in a combinatorial way that involves activator
genes such as fushi-tarazu, evenskipped or tailless, and repressors such as
hunchback. These genes also establish the domains of Hox expression with spatial
and temporal limits. Generally, Hox genes do not autoregulate; later in
development, genes of the Polycomb and the trithorax groups maintain the
patterns originally established by the segmentation genes (reviewed in
CASTELLI-GAIR, 1998).
These patterns of Hox gene expression correspond approximately with the initial
parasegmental boundaries. Hox genes that are included in this pattern belong to
two complexes: The Antennapedia complex, comprising empty spiracles (ems),
labial (lab), proboscipedia (pb), Deformed (Dfd), Sex combs reduced (Scr) and
Antennapedia (Antp), is primarily expressed in the gnathal and thoracic segments.
The Bithorax complex, comprising Ultrabithorax (Ubx), abdominal-A (abd-A) and
Abdominal-B (Abd-B), is primarily expressed in the abdominal segments.
A disruption of the expression patterns of homeotic genes often elicits an
alteration of segmental identities. The consequences for the phenotype depend on
where and when this disruption occurs. For example, mutations in the bithorax
cluster of genes can transform the third thoracic segment of Drosophila into
something morphologically resembling the second thoracic segment, giving the
adults two sets of wings (LEWIS, 1978). Nevertheless, what appear to be
“segmental identity shifts” led to a simplistic model: Hox genes are expressed
from an early developmental stage within initial parasegmental boundaries, and
act to establish the segmental identity through a combinatorial expression in an
anterior-posterior gradient. The Hox proteins then act as transcription factors
and through the regulation of target genes, determine developmental pathways
that lead to the differentiation of segment specific features.
Such a static, domain-based model fails to predict the actual expression
profiles of Hox genes and their effect on the determination of developmental
pathways. Its weaknesses were concisely summarised by Castelli-Gair as such (CASTELLI-GAIR,
1998): “First, in all cases the Hox genes are expressed in fewer segments at
early than at late stages of development. Second, at a single time point, a
segment is a mosaic of cells expressing and not expressing a given Hox gene.
Third, although the expression of Hox genes is frequently parasegmental, there
are many cases in which this is not true.” (referring to MARTINEZ-ARIAS et al.,
1985; DELORENZI & BIENZ, 1990; IRVINE et al., 1991).
Therefore, it is necessary to think of the expression of Hox genes and their
effect on the cells in which they are expressed as highly dynamic. Cells express
different Hox genes at different stages in development. In addition, Hox genes
can also activate different mechanisms in cells depending on when, where and how
strongly they are expressed (CASTELLI-GAIR & AKAM, 1995; MICHELSON, 1994).
The understanding of the spatial and temporal dynamics of Hox expression is well
advanced in Drosophila. Several recent studies demonstrate ways in which Hox
genes specify cell fate by direct regulations of target genes.
In a stage 12 embryo, Deformed (Dfd) was shown to directly activate the
apoptotic gene reaper and thereby lead to the death of specific cells on the
boundary between the maxillary and mandibular head lobes (LOHMANN et al., 2002).
The same study demonstrates that Abdominal-B is sufficient to regionally induce
cell-death through the activation of reaper transcription, thereby regulating
the segment boundaries between A6/A7 and A7/A8. Therefore, these two Hox genes
regionally sculpt the body into morphologies specific for a particular segment.
Mechanisms akin to this operate in the morphogenesis of the vertebrate limbs
(SAUNDERS & FALLON, 1966; HURLE et al., 1996).
Another study investigated the role of Hox genes on segment-specific alterations
of neuroblast lineages through the apoptotic elimination of certain neuroblasts
in the larval abdomen (BELLO et al., 2003). A burst of abdominal-A expression
activates the cell death program, while the neuroblast is still engaged in the
cell cycle. The death of the neuroblast puts an end to its divisions and
thereby, the final number of neurons and glia of this lineage is constrained.
Mutants that lacked either abd-A, or the H99 apoptotic genes showed a threefold
excess progeny with respect to the wildtype.
Another study used segment-specific differences in the lineages of equivalent
neuroblasts in the abdomen and the thorax to investigate the way in which Hox
genes influence the cell cycle (BERGER et al., 2004). It shows that the thoracic
lineage represents a ground state which is altered in the abdomen through the
action of abd-A and Abd-B. The two Hox proteins specify the lineage by
down-regulating CycE, the cyclin of the G1 phase. In turn, CycE specifies a
sublineage. In mutants that lack this protein, thoracic lineages are show
abdominal phenotypes. Ectopic expression leads to abdominal lineages showing
thoracic phenotypes.
Segment-specific regulations of neuroblast lineages were also observed in other
studies (PETERSON et al., 2002; PROKOP et al., 1998; ISSHIKI et al., 2001;
SCHMID et al., 1999; UDOLPH et al., 1993; PROKOP & TECHNAU, 1994).
Most recently, the segment specific apoptosis of post-mitotic dMP2 and MP1
pioneer neurons at stage 17 was shown to be determined by the differential
expression of Abdominal-B (MIGUEL-ALIAGA & THOR, 2004). In A6, A7 and A8, Abd-B
represses reaper and grim cell-autonomously and thereby, prevents apoptosis in
dMP2 and MP1 in these segments. Together, these studies show that the activity
of Hox genes can determine the segmental specification of neuronal architecture
by the direct activation of apoptotic programs.
Segmental variations of the muscle pattern have been demonstrated to be governed
by Hox gene expression, too (MICHELSON, 1994). In the abdomen, Ubx and abd-A
underlie the iterative formation of particular muscle precursors in the more
anterior segments, whereas Abd-B suppresses this formation in A8/A9. Ectopic
expression of Ubx and abd-A at high levels in the same mesodermal cells as Abd-B
can “override” its inhibitory activity, resulting in the induction of anterior
muscle precursor fates that are normally suppressed in the wildtype A8.
Interestingly, this study also demonstrates a case in which ectodermal and
mesodermal cells are matched in response to the expression of Hox genes:
Ectodermal tendon cells and matching mesodermal muscle precursors are both
subjects to the over-all diversification of the pattern (MICHELSON, 1994).
A more recent study provides could show that the muscle-identity gene apterous
is under direct control of Antennapedia, which is expressed in the muscles of
the thoracic segments T2 and T3 (CAPOVILLA et al., 2001). The authors show in
the LT1-4 muscles that Antennapedia positively regulates apterous, thereby
probably determining the identity of these particular muscles. It is worth
noting that LT1-4 muscles contain more nuclei in the thorax than in the abdomen.
This finding suggests that Hox proteins regulate the number of myoblasts
recruited for fusion similarly to my own findings with VT1.
To investigate the role of Hox genes on the diversification of the neuromuscular
machinery, I had to choose features that are strikingly different in A8/9
compared to the reference segment. I consider the cell-death of aCC in A7 and
RP2 in A8 as very distinctive segment-specific features in the CNS. In the
periphery, the same applies to the loss of muscle DA1 in A8 and the change in
the differentiation of the VT1 muscle founder cell in the same segment that
leads to the formation of muscle LT1. In this chapter, I investigate the role of
Hox proteins on these segment-specific alterations in the neuromuscular network.
6.2 Motorneurons aCC in A7 and RP2 in A8 survive in Abd-B Mutants
It is a very straightforward assumption to think of Abd-B as being responsible
for the specification of features in A8/9: It is the most posteriorly expressed
Hox gene (see figure 6.1) and is known to be responsible for a variety of
A8-specific features such as the formation of posterior spiracles (JUERGENS et
al., 1984; CASTELLI-GAIR, 1998).
To investigate whether cell death occurs in aCC in A7 and RP2 in A8 of an Abd-B
null-mutant, I stained embryos of the AbdBM1 line (Abd-B null mutation, see
Chapter 8, Materials and Methods) with an antibody against Evenskipped. In
embryos of this line, no Abd-B can be detected and they are considered to be
deficient for Abd-B expression (SANCHEZ-HERRERO et al., 1985). Embryos at stage
17 were selected and mutants identified on the basis of two features: the
decreased size of the anal pad (it appears to be confined to A9 in Abd-B
mutants; see the overview in figure 6.2) and – less reliably – the lack of
posterior spiracles.
In the wildtype, the disintegration of aCC in A7 and RP2 in A8 is clearly
visible at 15 hrs AEL. At this stage, the cell bodies are either pyknotic or
have completely disappeared from the pattern of evenskipped expressing cells
(see figure 6.3, top). In Abd-B mutants, however, neither aCC in A7 nor RP2 in
A8 shows any sign of disintegration or cell death at 15 hrs AEL. They appear
identical to their equivalents in more anterior segments (see figure 6.3,
bottom). In addition, there are several cells posterior to RP2 in A8 that are
eve-positive that are not present in wildtype embryos. These cells make it
difficult to reliably identify aCC and pCC in A8 or RP2 in A9.
This experiment shows that Abd-B function is required for the death of aCC in A7
and RP2 in A8.
6.3 Abd-B Null Mutation affects the Formation of DA1
I then used the same specimens to look at the dorsal muscle field, where
evenskipped is expressed in pericardial cells and muscle DA1 (see figure 6.4).
As I have shown in Chapter 5, the wildtype DA1 is clearly visible from A1 to A7
at 15 hrs AEL, but there is no indication for an eve-positive muscle in A8. In
Abd-B mutants, however, there is a muscle forming dorsally in all abdominal
segments including A8. This eve-positive muscle in A8 comprises of seven or
eight nuclei.
This experiment shows that Abd-B is required for the mechanisms that prevent DA1
from forming in A8.
6.4 Abd-B Null Mutation disrupts the Muscle Pattern
To investigate the extent to which the muscle pattern in Abd-B null-mutants is
altered in A8, I dissected AbdBM1 mutant embryos at 15 hrs AEL. Mutants were
selected based on the absence of spiracles and the tracheae being attached
directly to the cuticle on the segment border of A7 and A8 (HU & CASTELLI-GAIR,
1999). The muscle pattern was revealed with Alexa 568 fluorophor-conjugated
phalloidin, which binds to actin (see Chapter 8, Materials and Methods).
The muscle pattern in A8 looked different from what the wildtype; there was a
significant increase in dorsal longitudinal muscles. I could detect significant
changes in the ventral domain. Overall, the alterations looked inconsistent
among different specimens and I could not quantify the presence or absence of
specific muscles. The disrupted muscle morphology could be a secondary effect
due to the loss of posterior spiracles, which grossly alters the anatomy of A8
in Abd-B mutants. It might also be an artefact caused by the dissection of the
mutant embryos. To avoid such artefacts, I stained whole embryos of the AbdBM1
line with an antibody against myosin.
Specimens treated in such a way showed a clear increase in the number of muscles
in A8 (see figures 6.7 and 6.8) with respect to the wildtype A8. In the ventral
muscles, specific differences are difficult to determine due to the spherical
shape of the posterior end. Myosin stains all muscles and the density of muscles
in this region makes the determination of individual muscle identities
unreliable. In the lateral and dorsal musclefield, the comparison can be done
with more precision. In several mutants, A8 had at least two muscles that shared
size, shape, orientation and position with LT muscles from more anterior
segments (see figure 6.8). All in all, I can say that the muscle pattern of A8
in Abd-B mutants is transformed, but I was not able to find a consistent
alteration in particular muscles. To follow this line of experiments, one would
need to break up the muscle pattern by staining against markers for specific
muscles, such as S59 or connectin. I investigated the effect of an Abd-B null
mutation on the S59 positive muscles in 6.7.
I conclude from this experiment that Abd-B function is crucial for the wildtype
formation of the A8 muscle pattern. It is very likely that Abd-B expression
suppresses the differentiation of other specific muscles in addition to DA1.
6.5 Ectopic Expression of Abd-B leads to Cell Death in specific Neurons
I then asked whether the ectopic expression of Abd-B in more anterior segments
would be sufficient to ablate aCC and RP2 motorneurons. I used the RN2-Gal4 line
to drive UAS-Abd-B and examined embryos at 15 hrs AEL. Cells of the RN2 pattern
in all segments frequently showed the pyknotic appearance typical for apoptotic
cells (see figure 6.5). Notably, in all my experiments pCC (which in wild type
embryos does not undergo apoptosis in A7 or A8) has never shown any signs of
cell death.
To provide further evidence that the disintegration of aCCs and RP2s was due to
apoptosis, I repeated the experiments and stained embryos against caspase-3. I
could show a co-localisation of pyknotic cell bodies of aCCs and RP2s with the
cas-3 signal (see figure 6.6).
This experiment shows that the ectopic expression of Abd-B is sufficient to
induce the mechanism leading to the death of aCC and RP2 motorneurons in all
abdominal and thoracic segments.
I also repeated this experiment in a Hb9-Gal4 line to test if ventrally
projecting motorneurons can also be ablated. I found clear Cas-3 signal in many
motorneurons, however, the expression pattern at 14.5 hrs AEL contains many more
cells than in the RN2 line and the identity of the dying cells was unclear.
6.6 Ectopic Expression of Abd-B Does Not Prevent the Formation of DA1
I then asked whether the ectopic expression of Abd-B could suppress the
formation of DA1 in the reference segments. I used the Gal-4 line 24B (BRAND &
PERRIMON, 1993) that is expressed throughout the mesoderm of early stage 11
embryos and continues, so that by stage 14, expression can be found in the
somatic muscle precursors (MICHELSON, 1994). I crossed this line with UAS-Abd-B
and stained embryos against evenskipped to visualise muscle DA1.
I examined 10 embryos at stage 17, when cell death in the VNC had already
occurred. With only one exception, embryos showed the wildtype pattern in the
muscle field, with one Eve-positive muscle from T1 to A7. In the only exception,
the specimen lacked DA1 in one hemisegment of A7 (data not shown).
I then examined 12 embryos at stage 11 or 12, when muscle founder cells can be
individually identified in the evenskipped pattern (see Chapter 5). In all 12
specimens, the eve signal matched with the one known from wildtype embryos.
There is one muscle founder cell in each thoracic and abdominal segment from A1
to A7 and another one in A9, but none in A8.
In order to express Abd-B earlier than 24B, I repeated the experiment using the
twi-Gal4 line (GREIG & AKAM, 1993). twist is expressed early in embryogenesis,
at high levels in progenitors of somatic muscles and at low levels in other
mesodermal derivatives (see Chapter 1; THISSE et al., 1987; LEPTIN, 1991;
BAYLIES & BATE, 1996). I crossed this line with UAS-Abd-B and stained embryos
against evenskipped to visualise muscle DA1. I examined embryos at various
stages of their development as described above. All embryos showed the wildtype
pattern of evenskipped expression (see Chapter 5; data not shown).
I conclude from these experiments that the ectopic expression of Abd-B alone
using the means described is not sufficient to suppress the formation of DA1 in
segments anterior of A8. As the absence of Abd-B results in the formation of an
eve expressing muscle in A8, I assume that the failure of DA1 to form in A8 in
the wild type embryo must depend on a combination of different factors, of which
Abd-B is only one.
Furthermore, in addition to the data on the S59-expressing muscle progenitors
and muscle founder cells in A8/A9, this experiment suggests that there is no
segmentally repeated ground plan of mesodermal stem cells analogous to the array
of ectodermal neuroblasts. It is conceivable to assume that segmental
differences in the mesoderm of A8/A9 are - on contrast to the ectoderm - already
present on the level of stem cells and a “sculpting” of a network through
apoptosis is therefore redundant.
6.7 Ectopic Expression of Abd-B changes the Morphology of VT1
I then asked whether the ectopic expression of Abd-B could change the identity
of VT1 in anterior segments into its A8-equivalent LT1 (see Chapter 4). There
are two obvious features that make LT1 distinct from VT1: Its dorso-lateral and
medial position as opposed to the ventral and anterior position of VT1. More
reliably, LT1 is larger in size: It contains approximately 9 nuclei (9.3, n =
9), whereas VT1 in anterior segments contains only approximately 5 (4.75, n =
12). For further details on the statistical analysis of this experiment, please
see the end of this chapter.
I used embryos from the cross of 24B-Gal4 with UAS-Abd-B and stained them with
anti-S59 to reveal - among others - muscles VT1 in the reference segments and
LT1 in A8 at 15 hrs AEL. The position and orientation of VT1 was normal with
respect to VA1 and other landmarks such as the denticle bands: Ventral and at
the anterior margin of the segment, oriented transversally and approximately
aligned with VA1. The size of the muscle, however, was significantly increased
now comprising of approximately 9 nuclei (8.56, n = 9) (see figure 6.9).
This experiment shows that ectopic expression of Abd-B in the mesoderm is
sufficient to change segment-specific features of muscles. However, Abd-B is not
sufficient to change the position of muscle insertion sites. This suggests that
the mechanisms leading to the segment-specific differentiation of muscle
insertion sites might depend on cell-autonomous expression of Abd-B in the
ectoderm, as proposed elsewhere before (MICHELSON, 1994).
6.8 Mapping of Hox-proteins in the CNS at 14 hrs AEL
From the experiments described above, one could derive a very simple model: The
presence of Abd-B in particular cells determines the activation of certain
developmental programs such as apoptosis, the recruitment of a certain number of
myoblasts for fusion and possibly the suppression of muscle formation.
This resembles the simplistic model of the “static” expression of Hox genes to
specify anterior-posterior identities in whole segments. As I have emphasised in
the introduction to this chapter, the function of Hox genes cannot be explained
by such a model, since it neglects temporal patterns, spatial patterns
(CASTELLI-GAIR, 1998), combinatorial aspects and expression levels of Hox genes.
To investigate the role of Hox genes in the specification of cells in
consideration of these four aspects, I decided to map Hox proteins with higher
resolution. I chose the distinctive fate of apoptosis of aCC in A7 and RP2 in A8
as a model system and studied the expression of Hox genes known to be present in
the abdomen: Antennapedia, Ultrabithorax, abdominal-A and Abdominal-B. For these
experiments, RN2 served as a marker line. I did this with the aim of identifying
the Hox proteins that were present in specific cells of the RN2 pattern at
around 14 hrs AEL, the time just before cell death becomes detectable.
The results of these experiments are listed in table 6.1 (see also figures 6.10
to 6.25). The value of positive (clear expression) or negative (no expression or
too little to be clearly not background) was assigned to aCC, pCC and RP2 for
A6, A7 and A8. The values given for A6 also count for the more anterior
abdominal segments, with the exception of Abd-B, which is expressed in aCC and
pCC or A6, but negative in all abdominal RN2 cells anterior of this segment.
Most notably, the model “expression of Abd-B determines A8-fate” does not hold
up: on a single-cell level it becomes clear that several cells (for example, aCC
in A6) express Abd-B, but do not die. Looking at the expression profiles, one
can also see that a combinatorial code of Hox genes is insufficient to explain
the determination of the “apoptosis path”, too. Neither “absence of any Hox
protein” nor a combination of Hox proteins gives a unique profile to the dying
neurons aCC in A7 and RP2 in A8.
Since I have also considered the specification of the earliest born U-neuron
(U1), I started to map Hox proteins in the CQ line in the same way as I did for
the RN2 line. However, aCC in A8 and RP2 in A9 also have “altered” muscle
targets with respect to their homologues in the reference segment; since the RN2
pattern is more accessible for flip-out experiments (see Chapter 4), it provided
a more attractive system than the CQ line and I did not perform flip-out
experiments with this line. Nevertheless, the map of Hox proteins in the CQ
cells is given in table 6.3 (see also figures 6.10 to 6.25) and might be a
useful piece of information for further experiments on motorneuron
specification. In contrast to the RN2 neurons, the Hox expression pattern of the
CQ line does assign individual identities to the “altered” U1 neurons in A7 and
A8 simply through the expression of Abd-B.
The expression pattern of Hox genes in the RN2 line could not resolve the
question how Abd-B assigns a certain identity to aCC in A7 and RP2 in A8.
Therefore, I decided to look for proteins that could potentially act as
co-factors for Abd-B in the process of specifying cells of the RN2 pattern.
6.9 Mapping Co-Factors of Hox Proteins in the CNS at 14 hrs AEL
Since the DNA binding properties of Hox proteins are not very specific, it is
unclear how the high specificity in regulating appropriate target genes is
achieved (CARR & BIGGIN, 1999). There are several known co-factors of Hox
proteins that can account for increased specificity. This might include a
network of zinc finger proteins that are expressed within certain domains where
they enable specific Hox genes to function (ROBERTSON et al., 2004; reviewed in
MAHAFFEY, 2005) or segmentation genes with the potential to act as co-factors
(GEBELEIN et al., 2004). However, there is no comprehensive model that would
give an explanation on how Hox proteins choose target genes to determine
developmental pathways, and the role of potential co-factors is far from being
understood.
Investigating the expression of co-factors as a possible cue to further specify
a cell’s identity was therefore a risky endeavour. I restrained my experiments
to a set of genes that have previously been discussed as possible co-factors:
teashirt (tsh; DE ZULUETA et al., 1994; ALEXANDRE et al., 1996), extradenticle
(exd; CHAN et al., 1994), disconnected (disco; MAHAFFEY et al., 2001), engrailed
(en; GEBELEIN et al., 2004; MANN, 1994) and Krüppel (Kr; WHITE & LEHMANN, 1986).
The expression profiles in the RN2 background are given in table 6.2 (see also
figures 6.10 to 6.25), arranged in the same fashion as for the Hox proteins.
None of the mapped proteins appeared to be expressed with segmental specificity
that would make individual cells distinct. Curiously, however, Krüppel is
selectively expressed in pCC of A5 and A6 – as the only cells of the RN2 pattern
in the VNC. Disco is selectively expressed in a set of four lateral neurons per
abdominal hemisegment, but in no RN2 cell of the abdominal VNC.
6.10 Discussion
The experiments of this chapter show that Abd-B is responsible for the
determination of developmental paths that lead to the specification of the
neuromuscular network in A8/9 and make it distinct from the reference segment.
These paths include the recruitment of a specific number of myoblasts; selective
cell death in certain neurons; or the formation of entire muscles.
Ectopic expression of Abd-B is sufficient to induce the differentiation of
A8-features in more anterior segments. However, examining the expression pattern
of Abd-B at single cell resolution does not permit a simple hypothesis according
to which Hox expression alone would assign a segmental identity to individual
neurons in the RN2 pattern and thereby determine their fate. The investigation
of known co-factors did not help to resolve this issue.
Future experiments could study the temporal patterns of Hox expression in
greater detail. One could ask whether the specification of a neuron’s identity
was already achieved through an earlier wave of Hox expression than the one I
have been looking at (14.5 hrs AEL). This could explain why the ectopic
expression of Abd-B is sufficient to ablate RN2 cells in anterior segments,
whereas my experiments show that the expression of Abd-B in individual cells at
14 hrs AEL does not allow predicting how they will be specified. This would also
fit with the observation that Abd-B is expressed in A9 first, and later its
expression increases in more anterior segments (for example, compare fig. 6.14
and 6.18). In this context, it is worth noting that aCC in A8 and RP2 in A9 do
survive and become fully functional neurons in control of target muscles (see
Chapter 4), although they do express Abd-B just like their dying siblings in A7
(aCC) and A8 (RP2).
Another fruitful line of enquiry would be the one of other known co-factors.
Future experiments could focus on genes like disco-related (MAHAFFEY et al.,
2001), lines (CASTELLI-GAIR, 1998) or buttonhead (SCHOCK et al., 2000; ALEXANDRE
et al., 1996), which have been shown to be crucial for specification of features
in the head region. Similarly, segmentation and gap genes known to underlie the
expression of Hox genes could provide further insights (GEBELEIN et al., 2004;
WHITE & LEHMANN, 1986) as well as the genes of the “terminal class” (nanos,
pumilio, torso, trunk, torsolike, polehole and Nasrat; NÜSSLEIN-VOLHARD et al.,
1987). If these experiments also failed to provide insights on the role of AbdB
and its co-factors, a genome-wide deficiency screen would finally and reliably
identify all proteins involved in the specification of A8/A9.
6.11 t – Test for the increased size of VT1 in AbdBM1 embryos
To test whether the increased number of nuclei in muscle VT1 of embryos of the
AbdBM1 line with respect to the same muscle in the wildtype was significant, I
performed a t-Test analysis.
Hypotheses: H0: nuclei WT = nuclei mutant
H1: nuclei WT < nuclei mutant
Nuclei counts:
WT = 4,75 (n=12, variance = 0.36, standard deviation = 0.60).
Mutant = 8,56 (n=9, variance = 0.62, standard deviation = 0.78).
This led to a calculated t-value of 12.29.
The chosen threshold for the analysis is 5% (a=0.05), the degree of freedom is
19 (df=19). For a=0.05, t(19) = 1.729.
Since 1.729 < 12.29, one can abandon H0 and conclude that the number of nuclei
in VT1 muscles of the AbdBM1 line is indeed significantly increased.
Figures
Figure 6.1: Abd-B expression in a RN2 background
Expression of AbdB (red) in an RN2 (green) background: aCC in A7 and RP2 in A8
are disintegrating in this late 15 hrs AEL specimen. The function of
Hox-proteins in patterning the anterior-posterior body axis suggests a possible
role for AbdB in the underlying segmental diversification in the most posterior
segments – such as the observed cell death.
Figure 6.2: Neuronal eve expression in Abd-B mutants
Overview of the CNS of wildtype and Abd-B null mutant embryos, labelled for
evenskipped (n > 8). For details in high magnification (red outline), see figure
6.3.
Figure 6.3: Eve expression in motorneurons of Abd-B mutants
The neuronal evenskipped pattern in high magnification (overlay, n > 8), ~14.5
hrs AEL: The wildtype embryo shows the disintegration of aCC in A7 and RP2 in
A8. Note that one RP2 has already completely disappeared.
In the Abd-B mutant embryo, neither aCC in A7 nor pCC in A8 show any signs of
disintegration. Beyond that, there are several eve-positive cells in A9 and
possibly in A8 that do not appear in the wildtype pattern (note the area
labelled with “unclear”).
Figure 6.4: Eve expression in the dorsal muscles of Abd-B mutants
Dorsal view of the same specimens as above: The muscle DA1 expressed evenskipped
from T2 to A7 in the wildtype embryo. Note that in T1, there is a smaller muscle
expressing eve. There is no indication of an eve positive muscle in A8 (arrow; n
> 8).
In the Abd-B mutant embryo, however, there are eve-positive cells that form a
muscle in A8 (arrow). Note also that in the mutant, the eve-positive cells that
surround the anal pad demonstrate a dramatic decrease of the size of it with
respect to the wildtype.
Figure 6.5: Ectopic expression of Abd-B in the RN2-Gal4
Using the RN2-Gal4 line to drive Abd-B ectopically, one can initiate cell death
in some aCC and RP2 neurons (arrows for examples) in segments anterior of A7 (n
> 8).
Note that I never found cell death in pCC. To provide evidence that this
ablation of cells is caused by apoptosis, I repeated the experiment with an
antibody against caspase-3 (see figure 6.6).
Figure 6.6: Cas-3 staining in RN2-Gal4xUAS-Abd-B embryos
RN2 (green) anti cas-3 (red) shows a co-localization in RP2 of A6 (arrow A6). In
A7, one aCC is still clearly outlined and expresses cas-3 (arrow A7). RP2 in A8
cannot be detected in the left hemisegment, on the right one, however, it does
co-localize with cas-3 (arrow A8).
Note also the gradient of cas-3 signal towards A8 and A9, indicating an
increased number of apoptotic cells in these segments. The bar indicates the
posterior margin of the VNC.
Figure 6.7: Myosin staining in Abd-B mutant embryos
This overview of a wildtype and an Abd-B null mutant embryo shows the area given
in high magnification in figure 6.8 (orange outlines).
Figure 6.8: Myosin staining in Abd-B mutant embryos
High magnification images of the same specimens as in figure 6.7, a wildtype and
an Abd-B null mutant embryo. Note the absence of posterior spiracles in the
Abd-B mutant.
In the lateral region of A8, mutant embryos show at least two muscles that look
like LTs. There is also an increase in the number of muscles in the dorsal
region of this segment.
Figure 6.9: Misexpression of Abd-B in the mesoderm
If mesoderm-specific line 24B-Gal4 drives Abd-B ectopically, the size of muscle
VT1 almost doubles: It consists of 5 nuclei in the wildtype, whereas in the
misexpression embryos VT1 consists of 9 nuclei. This matches with the size of
LT1 in A8, the equivalent of VT1. The specimens shown are approximately 14 hrs
AEL old. Observations were drawn from a larger sample of specimens (n > 8).
Note that the position of VT1 remains the same in the mutant, suggesting that
the Abd-B expression in the mesoderm only is not sufficient to change the
insertion sites in the (ectodermal) bodywall.
Green is antibody signal for S59 (at this stage specific for VT1, VA2 and DT1),
red is antibody signal for engrailed as a marker for segment borders. This
allows the identification of VT1 as the muscle in the anterior domain of the
segment and VA2 in posterior. The arrows indicate other VT1 muscles.
Green arrows indicate trachaeal branches, which fluoresce in the specimen shown
in the figure at the bottom.
Figures 6.10 to 6.25: Expression of Hox genes and co-factors
Images of the original specimens used for the mapping of Hox proteins and
co-factors in the RN2 and CQ background.
Green is the GFP expression in the reference neurons (RN2 or CQ), Red the
antibody signal.
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