In this study, we have focused on the growth of axons from the dorsal and lateral sensory neuron clusters in abdominal segments A1 to A7. During normal development, sensory axons contact other sensory neurons or their axons shortly after emerging from the neuron cell body. The dorsal cluster axon fascicle initially grows internally to the transverse connective branch of the trachea, turns ventrally, joins the intersegmental nerve (ISN) in the dorsal body wall region and continues growing ventrally towards the central nervous system (CNS) 1114. It has previously been reported that the dorsal branch of the ISN is pioneered by the dorsal hair es neurons desA2 1116. However, we find that when dbd and other dorsal cluster neurons are labeled with DiI in different hemisegments of the same embryo, dbd's axon has always advanced further along the ISN than other axons (Steinel and Whitington, unpublished observations). Lateral cluster axons fasciculate as they initially grow anteriorly, following the pioneering axon for the lateral cluster, lch5-1. They then turn internally along the spiracular branch of the trachea, fasciculate with efferent motor axons of the ISN and advance ventrally towards the CNS parallel to, but separated from, the dorsal cluster sensory fascicle 12. While v'ch1 lies in the lateral cluster, its axon follows a different path to the rest of these neurons: it initially grows ventrally to contact cell bodies of sensory neurons in the v' (ventral') cluster and follows their axon fascicle towards the CNS within the segmental nerve (SN) 15. SN axons converge with ISN axons at the edge of the ventral nerve cord. The sensory axonal pathways are summarized in schematic form in Figure 1a. After entering the CNS, sensory axons advance into the neuropile. The lateral cluster lch5 axons pause at this stage (early stage 16) and acquire a flattened growth cone morphology with multiple filopodia, before turning antero-posteriorly along the longitudinal connectives (LC). Sensory neurons of different modalities and from different positions in the body wall diverge and form specific central arborization patterns within the neuropile. These specific sensory projections have been established by stages 16–17, depending on the neuron type 17. Figure 1b–d shows the mature embryonic projections of the lateral cluster neuron lch5-5, the v'ch1 neuron and the dorsal cluster neuron dbd, as revealed by single neuron, juxtacellular staining with DiI.

We visualized sensory axon morphology in mid-stage 16 to stage 17 (gastric caecae length 50–200 μm) nrg LOF mutant embryos by DiI labeling. While there is some normal variability in the timing of axon growth, all of the identified sensory neurons we examined have entered the neuropile and begun to form terminal arborizations by mid-stage 16 in 100% of wild-type embryos (n = 19 for lch5-1, 27 for lch5-5, 19 for v'ch1, and 8 for dbd). Sensory neurons showed a highly penetrant axon stalling defect in nrg mutant embryos. Axons were judged to have stalled if they had not entered the neuropile. Stalled axons consistently showed a simple morphology, terminating in the nerve with a simple, club-shaped growth cone with, at most, one or two projecting filopodia (Figure 2b–g). Cell body and dendrite morphology of sensory neurons were relatively normal, although, as previously reported 7, the regular alignment of lch5 cell bodies was often disrupted in nrg mutant embryos (Figure 2f,g).

Axon stalling occurred at a variety of positions along the normal axon trajectory. Axons seldom stalled before entry into the ISN: Figure 2e shows an example of such an early lch5-3 stall. Most lateral cluster axon stalls occurred in the periphery some distance along the peripheral nerve (Figure 2a–c), but in a number of cases axons stalled just before they entered the CNS or shortly after entry (Figure 2a,d). Dorsal cluster axon stalls occurred most frequently just before entry to the CNS. The location of axon stalls did not obviously correlate with the position of nerve branches or other structural features. The inherent variability in the stalling point is highlighted by the observation that adjacent neurons of the same lch5 cluster can stall at very different points along the nerve (Figure 2f,g).

We injected a range of identified lateral and dorsal cluster sensory neurons, including the five lateral chordotonal neurons lch5-1 to lch5-5, v'ch1, lesB, ldaA, ldaB, dbd as well as other cells in the lateral and dorsal clusters that could not be unambiguously identified at the individual neuron level. Axon stalling was seen for all of the neurons examined, except for dbd. The overall stalling frequency for lch5-5 and for all types of neurons combined, excluding dbd, was the same for the nrg¹⁴ and nrg¹⁷ LOF alleles (Table 1). We collected sufficient data for two neurons, lch5-1 and lch5-5, to enable us to compare their stalling rates: lch5-1 stalled less frequently than lch5-5 (P < 0.01) in nrg¹⁴/Y embryos (Table 1).

The dorsal cluster neuron dbd appears to be unaffected by loss of nrg function. In all 14 dbd neurons filled with DiI, the axon showed the bifurcating projection in the CNS, typical of a wild-type neuron at this stage (compare Figures 1d and 2h; Table 1).

Sensory axons were never seen to project along inappropriate pathways in nrg mutants. This was established by injecting single sensory neurons with DiI, then immuno-staining the embryo with mAb1D4 or mAb22C10, which reveals the morphology of all motor axons and sensory axons, respectively. Stalled sensory axons in the mutant showed the same close association with ISN motor axons and separation from dorsal sensory axons (0.5–1.0 μm) as seen in wild-type embryos (Figure 2b,d,i). The overall distribution of sensory nerves was normal in nrg mutants (Figure 2j). This result provides further support for our conclusion that sensory axons fasciculate normally in nrg mutants. Sensory axon stalling defects were not evident in embryos stained with mAb22C10 alone (as has been noted previously 7), presumably because individual sensory axons stall at different, variable locations rather than consistently at particular sites along the nerve. The relative timing of axon outgrowth from lateral cluster neurons, as assayed by mAb22C10 immuno-staining, is also unaffected by loss of nrg function: as in wild-type embryos, lch5-1 is the pioneer for the lateral cluster fascicle in nrg¹⁴ mutants (in 24/24 hemisegments, Figure 2k).

During embryogenesis, Nrg shows a widespread pattern of expression. Of particular relevance to its role in the regulation of sensory axon growth is the expression of Nrg¹⁸⁰ on sensory neuron cell bodies and sensory and motor axons as well as the expression of Nrg¹⁶⁷ on glial cells and trachea 618. Nrg could, therefore, mediate sensory axon extension through its expression in sensory axons or one or more of these various cell types, all of which have been implicated as possible growth substrates in sensory axon guidance.

To determine whether Nrg expression in sensory neurons is sufficient to promote sensory axon extension, we drove expression of the wild-type form of Nrg¹⁸⁰ using the P0163-GAL4 driver in the nrg¹⁴ null mutant background. We focused on the two neurons for which we had multiple fills in nrg¹⁴ mutant embryos – lch5-1 and lch5-5. To confirm that expression of Nrg¹⁸⁰ had been specifically restored in sensory neurons, we carried out BP104 immunohistochemistry (which recognizes an epitope specific to Nrg¹⁸⁰) on nrg¹⁴/Y;P0163-GAL4/UAS-nrg¹⁸⁰ embryos. Positive staining was seen in sensory neurons, including the lch5s and their axons, but not in central neurons (compare Figure 3a and 3b).

Embryos of this genotype were selected and lch5-1 or lch5-5 neurons were injected with DiI. The rate of lch5-5 axon stalling was significantly lower frequency (P < 0.001) than in the nrg¹⁴ mutant (Table 1). None of the axons that stalled did so in the PNS, whereas the majority (57%) of the stalls in the nrg¹⁴ mutant occurred in the PNS.

We injected 22 lch5-1 neurons in nrg¹⁴/Y;P0163-GAL4/UAS-nrg¹⁸⁰ embryos. None of these axons had stalled: all had either turned rostro-caudally or had an expanded growth cone located in a normal ventro-medial position in the LC (Table 1).

We conclude that Nrg expression in sensory neurons is sufficient to mediate its effects on both lch5-1 and lch5-5 axon extension.

To test whether the extracellular region alone can mediate the effects of Nrg on sensory axon growth, we attempted to rescue the nrg mutant phenotype by driving expression of a GPI-anchored form of the Nrg extracellular domain 19 in sensory neurons using the P0163-GAL4 driver.

We injected a total of 25 lch5-5 neurons with DiI in nrg¹⁴/Y;UAS-nrg^GPI;P0163-GAL4 embryos. The axon stalling rate in these embryos was significantly lower (P < 0.01) than for the nrg¹⁴ mutant (Table 1). Furthermore, only 2 of the 12 axons that did stall did so in the PNS. These results show that the extracellular region of Nrg can at least partially rescue the mutant phenotype.

The UAS-nrg^GPI stalling rate is significantly higher than the UAS-nrg¹⁸⁰ rate (albeit only at a low significance level, P = 0.05). This may suggest that the nrg^GPI construct is not as effective in promoting axon advance as the full length nrg construct, although it is also possible that differences in the level of expression of the two constructs could account for this apparent difference in rescuing ability.

To determine whether, as in the adult Drosophila wing 10, overexpression of Nrg results in sensory axon defects, we over-expressed Nrg¹⁸⁰ in the embryonic PNS using either the P0163-GAL4 driver or the pan-neural elav-GAL4 driver. Confocal microscope observations on wild-type and P0163-GAL4 X UAS-nrg¹⁸⁰ embryos stained with BP104 under the same conditions confirmed that Nrg¹⁸⁰ was expressed at higher levels in the transgenic embryos than in wild-type embryos (Figure 3c,d).

Neither form of overexpression resulted in sensory axon stalling as determined by DiI injections (n = 31 lch5-5 neurons in P0163-GAL4 X UAS-nrg¹⁸⁰ embryos, n = 30 lch5-5 neurons in elav-GAL4 X UAS-nrg¹⁸⁰ embryos; Table 1). In one P0163-GAL4 X UAS-nrg¹⁸⁰ embryo, the lch5-5 axon bifurcated in the PNS, but both branches grew into and arborized within the CNS. Furthermore, no axonal misprojections were evident in mAb22C10 stained embryos (n = 98 hemisegments in P0163-GAL4 X UAS-nrg¹⁸⁰ embryos, n = 91 hemisegments in elav-GAL4/UAS-nrg¹⁸⁰ embryos). mAb49c4 immunohistochemistry, which labels chordotonal neurons specifically, did reveal an aberrant dorsal misprojection of lch5 axons but only in 1/85 hemisegments of elav-GAL4 X UAS-nrg¹⁸⁰ embryos.

We also overexpressed the GPI-anchored form of Nrg using the P0163-GAL4 or elav-GAL4 drivers. Of 27 lch5-5 neurons labeled with DiI in P0163-GAL4 X UAS-nrg^GPI embryos, 2 showed axon stalling in the PNS, while the other 25 had a normal axon projection in the CNS (Table 1). Only one lch5-5 axon stall was seen in 16 elav-GAL4 X UAS-nrg^GPI embryos (Table 1). Thus, unlike adult wing sensory neurons 10, expression of Nrg^GPI in embryonic sensory neurons has only a weak effect on their axon growth.

Misexpression of Nrg¹⁸⁰ in the oenocytes (with the sal-GAL4 driver) or the trachea (with the btl-GAL4 driver) – cell types that lie in the vicinity of the lch5 neurons and their axons 1215 – has no apparent effect on sensory axon growth (Table 1).

All stocks were raised on standard cornmeal and sugar medium. Two nrg mutant alleles were used, nrg¹⁴ and nrg¹⁷ (previously called nrg¹ and nrg², respectively 7). nrg¹⁴ is a protein null and nrg¹⁷ is a hypomorph that shows a markedly reduced expression of Nrg protein in embryonic tissues. Immuno-staining of nrg¹⁴/Y embryos with BP104 confirmed the absence of Nrg¹⁸⁰ protein in these embryos during the period of sensory axon outgrowth (Figure 3e,f). Two UAS-nrg lines were used in nrg over-expression and mutant rescue experiments: full-length neuronal Nrg¹⁸⁰ (UAS-nrg¹⁸⁰) and an artificial Nrg isoform (UAS-nrg^GPI), in which the extracellular region of Nrg is membrane-tethered with a GPI-moiety 26. The following GAL4 lines were used to drive expression of these UAS lines: P0163-GAL4, which drives expression in all sensory neurons and their support cells 27; the pan-neural elav-GAL4 line 28; sal-GAL4, which drives expression in oenocyte precursors 29; and btl-GAL4, which drives expression in trachea 30. All mutant or transgenic stocks were maintained over green fluorescent protein- or lacZ-marked balancer chromosomes to enable genotyping of embryos by fluorescence microscopy or anti-β-galactosidase immunohistochemistry. Crosses were conducted at 25°C with the exception of UAS-GAL4 crosses, which were carried out at either 25°C or 29°C.

Eggs were collected for 6 hours at 25°C then placed at 18°C for an additional 16 hours, or collected overnight at 25°C on apple juice-agar plates with yeast paste. Embryos were staged on the basis of gut morphology 31 and length of gastric caecae (see Figure 9 in 32). Embryos were stained using standard immunohistochemical methods 33, with the exception that DiI-labeled embryos were treated individually on-slide. Primary antibodies used were mAb22C10 (mouse IgG, developed by S Benzer and obtained from the Developmental Studies Hybridoma Bank (DSHB) developed under the auspices of the NICHD and maintained by the University of Iowa, diluted 1:10 in phosphate-buffered saline with 0.1% Tween-20 (PBT), anti-Nrg¹⁸⁰, BP104 6 (mouse IgG, developed by C Goodman and obtained from DSHB, used at 1:4), 49C4 34 (mouse IgM, gift from Y-N Jan, used at 1:10), 1D4 35 (mouse IgG, obtained from DSHB, used at 1:5), anti-sex-lethal (mouse IgG, developed by P Schedl and obtained from DSHB, used at 1:20) and anti-β-galactosidase (mouse IgG, Promega, Madison, Wisconsin, USA used at 1:500). HRP or Alexa488-conjugated anti-mouse IgG, IgM or anti-rabbit IgG secondary antibodies were used at dilutions of 1:250 or 1:500 (obtained from Jackson Immunologicals, West Grove, Pennsylvania, USA).

Sensory neurons were individually stained in intact embryos by juxtacellular injection of DiI, as previously described 1217. Following dye injection, preparations were photo-converted in the presence of 0.2% diaminobenzidine to give a permanent dark reaction product, and in some cases immuno-stained with mAb1D4 to reveal the relationship between the injected neuron and motor axons. Stained embryos were mounted in 70% glycerol in phosphate-buffered saline and viewed with a Zeiss Axioskop. Digital images were captured with a Dage-MTI DC330 video camera and a Scion CG-7 frame grabber. Projections of in-focus images in multiple focal planes were made manually using Adobe Photoshop 7.0/8.0 software. DiI fills of the dorsal cluster neuron dbd and embryos stained with Alexa488-conjugated secondary antibodies were imaged with a Zeiss LSM 5 Pascal microscope. Z-projections of multiple focal planes were made using Zeiss LSM Image Browser software.