3.) CHARACTERISATION OF MOTORNEURONS FOR MUSCLES IN A8/A9

3.1 Introduction: Motorneurons of the Reference Segment

I have described the development of the central nervous system as it is known from the reference segment in Chapter 1. In the following, I will concentrate only on a characterisation of the pattern of post-mitotic, differentiated motorneurons in the reference segment as they appear from approximately stage 16 throughout larval development.

In the reference segment, there are 36 motorneurons per hemisegment, connecting to 30 muscles in the same segment (segmental innervation) or next posterior segment (intersegmental innervation). Due to the intersegmental innervation, segment A6 of the ventral nerve cord (VNC) is the most posterior “reference segment” in the sense of a stereotypical abdominal metamere. Segment A7 holds neurons that contact muscles in A8 intersegmentally. As mentioned before, the target muscles for most of these motorneurons have been identified and characterised in great detail (LANDGRAF et al., 1997; SINK & WHITINGTON, 1991).

The motorneurons can be divided into three subgroups: Unique motorneurons that are specific for only one muscle; common excitatory neurons that contact several muscles; and common neuromodulatory motorneurons (VUM neurons with axons extending to both hemisegments) (reviewed in LANDGRAF & THOR, 2006). In the reference segment, there are seven dorsally projecting motorneurons that express evenskipped (aCC, RP2 and U1 to U5) and approximately 29 ventrally projecting ones per hemisegment that express Nkx6 and Hb9.

3.2 Motorneurons of A8/9

It is important to realise that a characterisation of motorneurons in segments A8 and A9 will not include all motorneurons that innervate muscles in the periphery of these segments. This is because some of the muscles in A8 are innervated intersegmentally by motorneurons located in A7. However, an overall description of the motorneurons should answer questions such as whether or nor the much smaller number of muscles in A8/9 with respect to the reference segment is met with a similarly decreased number of motorneurons in the VNC.

The line OK371-Gal4 drives specifically in glutamatergic neurons (K.G. Moffat, J.B Connolly, J. Keane, S.T. Sweeney, and C.J. O'Kane, unpublished; see also MAHR & ABERLE, 2005) and allowed me to visualise the complete pattern or motorneurons (see figure 3.1; for neuromuscular junctions, see figures 3.14 to 3.16). I used the position of the interneuron pCC as a landmark to determine the segment border and counted a maximum of 15 cell bodies in A8 and 3 in A9 per hemisegment. As described in Chapter 2, there are up to 15 muscles in A8 that receive innervation through nerve A8; and there are up to 9 muscles in A8 and A9 that receive innervation through nerve A9. Since these numbers provided me with little more than a confirmation that a reduction of muscles is accompanied by a reduction of neurons, I decided to break the pattern of motorneurons down by concentrating on the dorsally projecting ones.

3.3 Dorsally Projecting Motorneurons: The RN2 and CQ Pattern

With the notion in mind that alterations of muscles between the reference segment and A8/9 were most obvious in the dorsal muscle field, the seven dorsally projecting motorneurons (dMN) per hemisegment were of particular interest to me. These neurons provided a favourable system for practical reasons too: Gal-4 lines specific for U-neurons (CQ-Gal4; LANDGRAF et al., 2003) and aCC and RP2 (RN2-Gal4; LANDGRAF et al., 2003) allow the analysis of even smaller subsets of neurons and make them amenable to precise genetic manipulations.

These two lines would also provide a system in which I could study equivalent neurons in all abdominal segments and ask whether they have different functions in specific metameres. This made these lines a valuable model in which I could readily relate pairs of neurons and muscles in A8/9 with their equivalents from the reference segment (see Chapter 4).

The pattern of RN2-Gal4: In the reference segment at 14 hrs AEL, the RN2-Gal4 expression pattern consists of 4 neurons per hemisegment: the motorneurons RP2 and aCC, the interneuron pCC (the sibling of aCC) and one unidentified interneuron between and ventral of RP2 and aCC (see figures 3.2 to 3.4; for neuromuscular junctions, see figures 3.8). This interneuron is likely to be the sibling of RP2 (Matthias Landgraf, personal communication). The neurons aCC and pCC develop in early stage 11 in one segment and then migrate just beyond the segment border into the next anterior metamere by late stage 11 (BROADUS et al., 1995). From there, aCC innervates muscle DA1 intersegmentally, whereas RP2 remains in its segment of birth and innervates – among others – muscles DA1 and DA2 segmentally (LANDGRAF et al., 1997; LANDGRAF et al., 2003).

In A7 and A8, the pattern is identical to the one described for the reference segment until around 14 hrs AEL (see figure 5.3, Chapter 5). At this stage, aCC in A7 and RP2 in A8 start to disintegrate and die and are missing from the pattern in late embryos by about 15 hrs AEL. This cell death is addressed in detail in Chapter 5.

In A9, there is only RP2, which fits with the notion that aCC and pCC migrate to the anterior. The cell bodies of RP2 in A9 are closer to the midline than in other segments. Posterior of RP2, there are one to three cells per hemisegment that maintain GFP expression throughout larval life. At least some of these cells extend axons into the hindgut nerve (see figure 3.8). The development of aCC in A8 and RP2 in A9 (both neurons that innervate muscles in A9) is described in Chapter 4.

The pattern of CQ-Gal4: In the reference segment at 14 hrs AEL, the CQ-Gal4 pattern consists of five U motorneurons (U1 to U5) per hemisegment. These innervate the muscles DO1 (U1), DO2, DA1, DA2 and LL1 (LANDGRAF et al., 1997; LANDGRAF et al., 2003). In addition to these, there are several interneurons of unknown function contained in the pattern. U1 and U2 are the earliest to express Gal-4 and can be easily identified (see figures 3.5 and 3.6; for neuromuscular junctions, see figure 3.7a).

In A7, there are three U neurons per hemisegment. I showed with evenskipped-staining that the line ST27-Gal4 (Stefan Thor, unpublished) drives exclusively in U1 (data not shown) and used this line to identify a cell that I therefore consider to be U1 in A7 and A8. Based on its position, the second U-neuron in A7 is likely to be U2. In A8, there is the mentioned U1 and one other U-neuron per hemisegment. In A9, there are no U motorneurons, only small clusters of approximately four interneurons in each hemisegment. This fits with the finding in other segments that U-neurons extend their axons intersegmentally (i.e. U neurons from the VNC segment A8 innervate muscles in the periphery of A9).

I decided to concentrate on analysing the RN2 pattern, as I found different kinds of segmental specifications within this line (see Chapters 4 and 5). However, the U neurons provide a valuable system to supplement this analysis. Some data on the U-neurons are contained in Chapter 4 and Chapter 6. The neuromuscular junctions of the ventrally projecting Hb9 positive motorneurons are shown in figure 3.7b.

3.4 Characterisation of Motorneurons for A8/9 and their Targets

I was particularly interested in the neuron innervating muscle LT1, which is an example of a “novel” or drastically altered element in the network. Furthermore, I wanted to find neurons innervating the VL muscles, which are examples of “unchanged” elements in the network (see Chapter 1). Since Gal-4 lines such as CQ, RN2 or OK371 are expressed in neurons with overlapping dendrites, whose axons exit the CNS in bundles, the full patterns are unsuitable for a single-cell analysis and do not allow the pairing of neurons with their specific target muscles.

Therefore, I chose a different approach in which I used the OK371-Gal4 line crossed with flies carrying the heatshock-Flipase and UAS-FRT-STOP-FRT-CD8-GFP constructs (heatshock flipase; UAS-Stop-CD8-GFP/Cyo; Tm6/MKRS; for details, see Chapter 8, Materials and Methods) (WONG et al., 2002). This allowed me to label single motorneurons in which flipout events had occurred. I raised heatshocked embryos to third instar larvae and looked for animals with only one or very few, non-overlapping neurons in the VNC. Axons in such animals can be traced to their target muscle, where they form a clear neuromuscular junction (NMJ). I injected Alexa-568 fluorophore-conjugated phalloidin (for details, see Chapter 8, Materials and Methods) into animals with NMJs in A8 or A9 to visualise the muscle pattern. In this way, I could identify individual motorneurons and their respective target muscles (see figures 3.9 and 3.10).

The identification of the neuron controlling muscle LT1 was the original aim of these experiments. Therefore, I dissected and processed the CNS that contained this particular cell with antibody staining against evenskipped. The expression pattern of eve highlights reliable landmarks for segment boundaries. These include segmentally repeated interneurons. Through the position of the interneuron pCC just posterior of the segment boundary I could also determine the position of the cell body of the labelled motorneuron and the size of its dendritic field (i.e., the number of segments it spans). The dendrite of the LT1 neuron is strikingly large, extending over two segments: Whilst the cell body itself is located at the anterior margin of A8, the dendrite expands in three branches to the anterior and covers parts of the neuropile in A8, A7 and A6 (see figure 3.11). The size and shape of the dendrite did not suggest a homology to any motorneuron known from the reference segment.

I limited my experiments on other motorneurons to a simple characterisation of cell body position and dendrite morphology. Movies in two channels taken under UV light and brightfield illumination recorded the individual motorneurons and their position with respect to the midline and exiting nerves. To present the data in print, I generated z-projections of the UV channel. This resulted in a loss of detail in some specimens; however, the overall shape and position of the neurons and their dendritic trees remained recognisable and made this form of presentation the most suitable one. The images are given in figures 3.12A to 3.12H with the respective target muscles.

3.5 Characterisation of 3rd Instar Motorneurons in the Reference Segment

Like the LT1 neuron, several other motorneurons for A8/9 muscles showed very extensive dendritic fields. This raised the concern whether the characterised morphology of embryonic motorneurons (LANDGRAF et al., 1997) was suitable for a direct comparison with flipout motorneurons in third instar larvae. I did another series of heatshock experiments to identify motorneurons of the reference segment and investigate to what extent motorneurons change their morphology over the course of larval development. I was also curious to learn whether motorneurons for muscles in A8/9 had bigger dendritic fields than motorneurons of the reference segment.

Results of these experiments are shown from figure 3.13A to figure 3.13E. In comparison to embryonic motorneurons, the dendrites of third instar larvae are bigger and much denser. However, the overall morphology remains the same and the motorneurons are clearly recognisable. In comparison to motorneurons from A8/9, neurons from the reference segment tend to have less elaborate dendritic trees. Implications of these findings are discussed below.

3.6 Discussion

In analogy to the map of the muscle pattern, one can see the principle means of diversification on the level of motorneurons: Reduction; conservation; or the differentiation of novel elements.

Muscles of A8 that are identical to their equivalents in the reference segments are innervated by neurons that look unchanged. Examples for such a case are motorneurons for muscles VL1 or VL3; they receive innervation from neurons that look like the embryonic V neuron and RP3 as they were characterised previously in the embryo (LANDGRAF et. al, 1997), respectively. The same applies for muscle VL4 and its respective motorneuron. The reduction of elements can be seen by simply counting the number of motorneurons in A8 and A9; much more precise, however, is the investigation of the death of aCC in A7 and RP2 in A8, which I will deal with in Chapter 5; and the reduced number of U neurons in A7, A8 and A9. The differentiation of a novel element can be followed in the case of LT1 in A8; this muscle without an obvious equivalent in the reference segment receives innervation from a neuron with elaborate dendritic projections unknown from any anterior motorneuron.

One can speculate that change of a muscle’s morphology is linked with a change of its function. Similarly, as the neuron receives input through its dendrite, the change in the dendritic morphology is likely linked to an altered function of the neuron-muscle pair. Alternatively, one could argue that the posterior position of the A8/9 motorneurons demands dendritic projections that span further to the anterior than in the reference segment if there was not enough space in the posterior region. These two interpretations are not mutually exclusive. However, there are motorneurons for muscles in A8/9 whose dendrites are constrained to one or two segments; this applies for instance to the mentioned neurons for VL1 and VL3.

The characterisation of motorneuron morphologies did not enable me to match equivalent motorneurons among segments right away. However, in combination with the comparison of other features it provided me with a crucial basis to relate pairs of muscles and neurons from the reference segment to equivalent ones in A8/9. This was key to my questions regarding the means and underlying cues of diversification. From here, I explored the relationship between neurons and muscles in different segments using segmentally repeated motorneurons (RN2 and CQ; the neuron of the transverse nerve), the well-characterised LT1 neuron and more sophisticated means of determining muscle homologies. The findings of these investigations are the core of this thesis and are given in Chapters 4 and 5.

Figures

Figure 3.1: Characterisation of the OK371 pattern

An embryonic ventral nerve cord of the OK371 line (specific for glutamatergic neurons) at approximately 14.5 hrs AEL (post cell death of aCC in A7 and RP2 in A8). GFP in green, staining anti evenskipped in red, n > 8.

Figure 3.2: Characterisation of the RN2 pattern

An embryonic ventral nerve cord of the RN2 line (includes most prominently motorneurons aCC and RP2 and the interneuron pCC) at approximately 14.5 hrs AEL (note that aCC in A7 and RP2 in A8 display pyknotic cell bodies as indicated by white arrows). GFP in green, staining anti engrailed in red, n > 8.
 

Figure 3.3 and 3.4: The abdominal pattern of the RN2 line

Figure 3.3: Motorneurons RP2, aCC and interneuron pCC. The diagram and the specimen to the left (14.5 hrs AEL) show the segmental pattern of these three cells. This pattern occurs from A1 to A8.

Segment A9 contains only RP2; note also that the cell bodies of these RP2s are much closer at the midline than in other segments. At the most posterior margin of the VNC, there are one to three interneurons.

Figure 3.4: In addition to these cells, the RN2 pattern includes a pair of ventral, segmentally repeated interneurons between RP2 and aCC (white arrows; digitally enhanced).
 

Figures 3.5 and 3.6: The abdominal pattern of the CQ line

The motorneurons U1, U2, U3, U4 and U5 differentiate in segments A1 to A6. In A7, there are three motorneurons per hemisegment; U1 and U2 are very central and express GFP very early. Therefore, they can be readily identified – for these figures by using anti-GFP staining.

In A8, there are two neurons per hemisegment, of which I consider the central one U1. In A9, there are no motorneurons, but a small cluster of two to four interneurons per hemisegment. In anterior segments there are several interneurons lateral of the U-neurons.
 

Figure 3.7: The innervation of CQ neurons in A8/9

Specimens at approximately 14.5 hrs AEL. The figure on top shows the innervation of muscle GS4 through at least one U-neuron.

The figure below shows the innervation of muscle DO1 in A8. Fluorescence in the VNC was cut out digitally to remove very strong signal.

In the reference segments, the innervation of U-neurons includes muscles DO1, DO2, DA3 and LL1.

The embryos shown in these figures are of the CQ Gal4 line driving UAS-CD8GFP, which reveals the CQ motorneuron cell bodies in the ventral nerve cord (not shown) and their axons in the periphery. Observations from specimens n > 10.
 

Figure 3.7b: The innervation of CQ neurons in A9

Specimen at approximately 14.5 hrs AEL. The figure shows the innervation of muscle TO2 in A9 through at least one U-neuron. The NMJs on GS4 in A9 can not be seen in this specimen.

Fluorescence in the VNC was cut out digitally to remove very strong signal.

The embryo shown in this figure is of the CQ Gal4 line driving UAS-CD8GFP, which reveals the CQ motorneuron cell bodies in the ventral nerve cord (not shown) and their axons in the periphery. Observations from specimens n > 10.
 

Figure 3.8: The innervation of RN2 neurons in A8/9

Specimens at approximately 14.5 hrs AEL (n > 10). The figure on top shows the innervation of TO2, GS4 and the hindgut by at least one RN2 neuron each. Fluorescence in the VNC was cut out digitally due to very strong signal (even stronger than in the VNC of the figure at the bottom). Green is GFP, red is phalloidin staining in the muscles.

The figure below shows the extension of an axon into the muscle field of A8. Note that it has already started to disintegrate (compared to nerve A7 or A9), but the growth cone is still clearly visible and reaches out towards the most dorsal muscles. This is the axon of aCC of A7 and / or RP2 of A8 extending into the dorsal domain of A8, where it does not have a target muscle.

The specimens shown here are embryos of the RN2 Gal4 line, recombined with CD8GFP (green). To highlight the muscles in the periphery, they were exposed to fluorophor conjugated phalloidin (red; see “Materials and Methods” for details).
 

Figures 3.9 and 3.10: Identification of specific motorneurons

OK371Gal-4 flies are crossed with flies carrying the heatshock-flipase and UAS-FRT-STOP-FRT-CD8-GFP. Heatshocked embryos were raised to 3rd instar larvae. These were screened for specimens with only a single neuron expressing GFP in A7, A8 or A9.

Such larvae were injected with a solution of phalloidin, methanol and fixative to visualise the muscle pattern (see figure 3.9A).

Thereby, individual motorneurons could be identified and matched with their respective target muscles, in this example muscle LT1 in A8 (see figures 3.10; and the overlay in figure 3.9B). The black arrow in 3.10A indicates the fluorescence on the outer margin of the anal pad.
 

Figure 3.11: Processing of the LT1 motorneuron

Figure 3.11A: Following the identification of the innervated target muscle, the VNC was dissected out, if appropriate fixed and documented.

Figure 3.11B: As a specimen of particular interest, the VNC containing the neuron of muscle LT1 in A8 was then stained for GFP (green) and evenskipped (red). Note that the outline of the segment borders in this specimen are based on the position of pCC and the exit points of nerves; therefore, they are not very precise and meant to be a guideline.
 

Figure 3.12: Neurons for muscles in A8/9

Z-projections of motorneurons with their respective target muscles. The red, dashed line indicates the position of the midline in the VNC.

On the bottom of each image the nerve through which the neuron projects is given (A8 or A9) and the subbranch (ISN or SN).

Motorneurons that project to the dorsal muscle field first, then lateral and ventral. Finally, there are at least two VUM neurons projecting into A8/9: One projects to the latero-dorsal domain of A8, the other one to the field just lateral of the anal slit.

Note: Figure 3.12A seems to show two dendrites – however, this is only one neuron with two very elaborate branch domains. n-numbers range between 2 and 8.
 

Figure 3.13: Neurons for muscles in the reference segment

Z-projections of motorneurons with their respective target muscles. The red, dashed line indicates the position of the midline in the VNC.

On the bottom of each image the nerve through which the neuron projects is given and the subbranch (ISN or SN). Motorneurons that project to the dorsal muscle field first, then lateral and ventral. N-numbers range between 2 and 8.
 

Figure 3.14: Dorsal neuromuscular junctions of the OK371 line

Dorsal view of the neuromuscular junctions (NMJs) of A7 and A8 as revealed in the OK371 pattern. The dashed line indicates the dorsal midline. The most likely target muscles of the NMJs are given. Note the much higher density of cuticular hairs on A8 compared to A7 (Observations derived from several specimens, n > 10).
 

Figure 3.15: Lateral neuromuscular junctions of the OK371 line

Lateral view of the neuromuscular junctions (NMJs) of A7 and A8 as revealed in the OK371 pattern. The most likely target muscles of the NMJs are given. TN indicates the transverse nerve, which will be of special interest in Chapter 4.

Figure 3.16: Ventral neuromuscular junctions of the OK371 line

Ventral view of the neuromuscular junctions (NMJs) of A7, A8 and A9 as revealed in the OK371 pattern. The most likely target muscles of the NMJs are given.
 

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