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.


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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