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A Targeted Expression of Tetanus Toxin Light Chain Resulting in an Eclosion Phenotype

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Sean T. Sweeney*, Simon H.B. Maddrell† and Cahir O'Kane*

Department of Genetics (*) and Department of Zoology (†), University of Cambridge, Downing Street, Cambridge, CB2 3EH. E.mail (STS): ss2@mole.bio.cam.ac.uk

Summary

During a behavioural screen employing crosses between 400 P[GAL4] lines and UAS-tetanus toxin light chain (TeTxLC, which abolishes synaptic vesicle release), a cross between one P[GAL4] line and UAS-TeTxLC produced a fly which eclosed but subsequently failed to mature further. These flies were unable to inflate their wings, excrete their meconium, sclerotise their cuticle or straighten their metathoracic legs. Many of these processes are thought to be mediated by neuropeptide hormones such as cardioacceloratory peptide (CAP; implicated in control of wing inflation and meconium excretion) and bursicon (implicated in cuticle sclerotisation). These neuropeptide hormones may in turn be under the control of eclosion hormone which triggers the performance of ecdysis behaviours. We postulated that inhibition of secretion of a neuropeptide hormone or hormones may be the cause of the observed phenotype of these 'juvenile' flies. We undertook a characterisation of the TeTxLC expression pattern in the CNS of these flies to assess the extent of expression. The expression pattern of P[GAL4]76 was primarily limited to a subset of the mushroom body fibres, two cell bodies in the cephalic ganglion (between the optic lobes and the central brain, at the posterior surface) and four large cells which lie in the posterior ventral region of the abdominal ganglion. We then screened through a panel of anti-neuropeptide antisera to ascertain whether any of the expression patterns of candidate neuropeptide hormones co-localised with the TeTxLC expression pattern.

Here, we present a description of these 'hormonally challenged' flies, a characterisation of the TeTxLC expression pattern and a co-localisation of a neuropeptide hormone expression pattern coincident with the TeTxLC expression pattern.

Introduction

We have previously demonstrated that the ectopic expression of Tetanus toxin light chain (TeTxLC) in Drosophila neuronal tissue leads to the loss of neuronal synaptobrevin (n-syb) and evoked synaptic transmission (Sweeney et al., 1995). Synaptobrevin has previously been implicated in small synaptic vesicle (SSV) mediated neurotransmitter release and large dense core vesicle (LDCV) mediated neuropeptide release (Martin et al., 1994). Many eclosion and post-eclosion behaviours in Drosophila are thought to be mediated by neuropeptide hormones which are likely to be secreted via LDCV mediated exocytosis. We have previously used the P[GAL4] targeted expression system to express TeTxLC as the basis for a behavioural screen (Sweeney et al., 1995). During this screen, the outcome of a particular cross came to our attention. This cross produced flies which eclosed but subsequently failed to mature further. These flies were unable to inflate their wings, excrete their meconium (waste products from metamorphosis which is normally excreted upon eclosion as a bright green liquid), sclerotise their cuticle or straighten their hindmost legs. The head sac or ptilinum is also retained. The ptilinum is an inflatable air sac which physically aids exit from the pupal case and is normally lost within 1-2h of eclosion. Many of these processes are thought to be mediated by neuropeptide hormones such as cardioacceloratory peptide (CAP; implicated in control of wing inflation and meconium excretion; Tublitz and Truman, 1985) and bursicon (implicated in cuticle tanning; Reynolds 1983; 1985). These neuropeptide hormones may in turn be under the control of eclosion hormone which is thought to trigger eclosion behaviour (Truman, 1985). We postulated that inhibition of secretion of a neuropeptide hormone or hormones may be the cause of the observed phenotype of these 'juvenile' flies. I undertook a comparison of the TeTxLC expression pattern in the CNS of these flies with the expression patterns of known neuropeptides to assess whether the toxin could be causing the eclosion phenotype by preventing release of one or more specific peptides. Furthermore, if neuropeptides were present within the TeTxLC expression pattern, it would be likely that such peptides would be secreted via large dense core vesicles (LDCVs). If the phenotype was caused by the TeTxLC induced inhibition of neuropeptide release, this would indicate that n-syb is involved in LDCV mediated exocytosis in Drosophila since neuropeptides are normally secreted via LDCVs as opposed to SSVs (Martin, 1994).

Here, we present a description of these 'hormonally challenged' flies, a characterisation of the TeTxLC expression pattern and a co-localisation of a neuropeptide hormone expression pattern coincident with part of the TeTxLC expression pattern.

Figure 1a / Figure 1b. Phenotype of flies produced by the cross 76xTNT-E. Panel A shows 5 day old female wild type flies which have been raised at 18oC. Panel B shows three 5 day old flies produced from the cross 76xTNT-E raised at 18oC. Note the crooked meta-thoracic legs (arrow), the difference in colour of the cuticle in comparison to Panel A, the retained meconium (asterisk, the green colouration under the cuticle) and the retained ptelinum (arrowhead).

Figure 2. Expression pattern of TeTxLC in the cross 76xTNT-E. Top left and right panels are reconstructions of two wholemount preparations of the VNC showing ß-PDH-immunoreactive neurons in the ventral midline of the abdominal ganglia (Abg). Top left panel is a ventral view and top left panel is a lateral view. Four large (arrowheads) ß-PDH immunoreactive cell bodies can be seen. Fibres project from these cells via the median abdominal nerve trunk. Thg, thoracic ganglia; DN2-3, dorsal nerves; VN1-3, ventral nerves innervating the legs. Scale bars = 100µm. Reproduced from Helfrich-Förster and Homberg (1993) Lower panels, Ventral nerve chords (VNCs) from offspring of the cross 76xTNT-E were dissected out and stained with an antibody to TeTxLC at a dilution of 1/10,000. Lower left panel shows a ventral view of such a VNC. Four large cells are clearly visible at the posterior end of the terminal fused abdomino-thoracic ganglion. No other cell bodies appear to stain positively for TeTxLC. Lower right panel shows a lateral view of a TeTxLC stained VNC. Two large cell bodies stain positively for TeTxLC expression. These cells appear to send extensions posteriorly through the median abdominal nerve. There also appear to be TeTxLC positive fibres dorsal to these cells which are similar to projections from similarly positioned ß-PDH immunoreactive cells in Phormia (Nässel et al., 1993). See Figure 3. for the expression pattern of TeTxLC in the cephalic ganglia of 76xTNT-E flies.

Figure 3 TeTxLC expression does not coincide with ß-pigment dispersing hormone expression in the cephalic ganglion of 76xTNT-E flies. The cephalic ganglia were dissected from 76xTNT-E flies within an hour of eclosion and immunocytochemically probed for the expression of ß-PDH and TeTxLC simultaneously. Rabbit anti-ß-PDH was used at a dilution of 1/500 and mouse monoclonal anti-TeTxLC was used at 1/10,000 dilution. Secondary antibodies employed were goat anti-mouse-FITC conjugate and goat anti-rabbit-Texas Red conjugate. TeTxLC expression is depicted in green and ß-PDH expression is depicted in red. ß-PDH expression is restricted to the previously identified lateral neurons (Helfrich-Förster and Homberg, 1993) whilst TeTxLC expression is found in a subset of the mushroom bodies and one cell on each side of the mushroom bodies dorsal to the lateral neurons.

Figure 4a / Figure 4b. ß-pigment dispersing hormone expression overlaps with TeTxLC expression in the ventral nerve chord of 76xTNT-E flies. The ventral nerve chords were dissected from 76xTNT-E flies within an hour of eclosion and immunocytochemically probed for the expression of ß-PDH and TeTxLC simultaneously. Rabbit anti-ß-PDH was used at a dilution of 1/500 and mouse monoclonal anti-TeTxLC was used at 1/10,000 dilution. Secondary antibodies employed were goat anti-mouse-FITC conjugate and goat anti-rabbit-Texas Red conjugate. TeTxLC expression is depicted in green and ß-PDH expression is depicted in red. Panel A shows the TeTxLC expression whilst Panel B depicts the ß-PDH expression. The two panels were not overlapped because detection of ß-PDH expression is much weaker than that of TeTxLC. Panel A is one of a series of optical sections of 10µM thickness. Panel B is an accumulated series of optical sections within which lies the section from which the picture from Panel A was taken at a different excitation and emission appropriate for the fluorescent label. This was performed to maximise the weaker staining observed for ß-PDH.

Figure 5. Schematic dissected view of the nervous system, intestinal tract and aorta of a dipteran. The cross hatched areas are putative release sites of neuropeptides. In the majority of cases these areas probably represent neuroheamal release sites. Release sites can be found in the anterior aorta (a. aorta), corpora cardiaca (triangular cross hatched structure below anterior aorta), dorsal sheath of thoracic-abdominal ganglion (T1-3, A1-8), pericardial septum at posterior aorta (abd aorta) and hindgut. Cell bodies (filled circles; not accurate numbers) of neurons are shown in one hemisphere only. Systems displayed are (1) protocerebral neurosecretory cells with axons to corpora cardiaca, anterior aorta and crop duct (CD); (2) subesophageal system (serotonergic) with axons to thoracic-abdominal dorsal neural sheath and several other targets not shown here; (3) thoracic system with terminals in dorsal neural sheath; (4) Lateral abdominal system with axons to pericardial septum of abdominal aorta; (5) median abdominal system with axons to hindgut and sometimes rectal pouch (RP) and its papillae. It is in the median abdominal system that the identified cell bodies containing ß-PDH potentially have a role. MT=Malpighian tubules. SEG=subesophageal ganglion. Reproduced from Nässel et al (1994).

Discussion

We have found a P[GAL4] line, which, when crossed to a UAS-TNT line produces a fly which ecloses but fails to mature further. We think it likely that expression of TeTxLC under control of the GAL4 insertion that is expressed in a relatively small number of neurons causes an eclosion behaviour phenotype. We cannot say at this stage which cells are responsible for the phenotype, but the most obvious candidates are four large neurosecretory cells in the abdominal ganglion where TeTxLC expression is driven. TeTxLC expression is also seen in a subset of fibres of the mushroom body and two cells which lie either side of the mushroom body, near the lateral neurons identified by Helfrich-Förster and Homberg (1993). It has previously been shown that ablation of the mushroom bodies has no consequences for the development of the fly (deBelle and Heisenberg, 1994). The two cells which lie either side of the mushroom bodies and which also express TeTxLC, send axons to the antennal lobe (data not shown), and therefore we think that the TeTxLC induced block in exocytosis in these cells is unlikely to be the cause of the phenotype we observe. The four large cells in the abdominal ganglion express ß-pigment dispersing hormone (ß-PDH), an octadecapeptide first identified in crabs (Rao et al., 1985) which has since been shown to be widely present as a neuropeptide in crustaceans and in orthopteroid and dipteran insects (Rao et al., 1987; Rao et al., 1991; Rao and Reihm, 1989; Nässel et al., 1993).

This data would suggest that ß-PDH could be a candidate neuropeptide hormone for mediating aspects of post-eclosion maturation behaviour. Large dense core vesicles (LDCVs), which commonly are thought to release neuropeptides (Nicholls, 1994), often contain two or three different neuropeptides (O'Brien and Taghert, 1994) and thus ß-PDH may only be part of a cocktail of neuropeptides mediating post-eclosion maturation. Clearly more anti-neuropeptide antisera require to be screened in order to identify other neuropeptide hormones which are secreted by the four abdominothoracic neurosecretory cells identified here. Furthermore, this data would also imply that n-syb or another TeTxLC target may be involved in LDCV release.

In the blowfly, Phormia terraenovae, the ß-PDH immunoreactive fibres which project through the median abdominal nerve produce synapses on the posterior part of the midgut, hindgut and rectal pouch (in the second part of the rectum. (Nässel et al., 1993)). The neurosecretory cells which produce these axonal projections appear to be homologous to the four ß-PDH producing cells in the abdominal neuromere of Drosophila, termed the VA neurons by Nässel et al., (1993, see Figures 2. and 5). It is therefore possible that the pattern of the ß-PDH positive axons projecting through the median abdominal nerve in Drosophila share similar synaptic targets to those in Phormia. Whether the ß-PDH positive synaptic arbors on the hindgut are neurohaemal in nature or synapse directly on to the individual regions of the hindgut has yet to be determined. We cannot at this point determine whether ß-PDH is acting directly as an effector of hindgut function or as a neurohormone. To distinguish between these possibilities will require standard physiological and behavioural analysis of the kind that can be applied to conventional mutants with a similar phenotype. It is also likely that ß-PDH is acting in concert with one or a number of other neuropeptides which are known to be expressed in the abdominal ganglion. The search should be continued uncover other neuropeptides which may co-express with ß-PDH in the cells in the abdominal neuromere in which we have in this study expressed TeTxLC. The VA neurosecretory cells also send projections dorsally to targets which have yet to be characterised and hence the role of these projections in a pathway or circuit can only be guessed at present. Similar projections have been observed arising from the ß-PDH immunoreactive cells in Phormia but they have not been observed previously in Drosophila (Nässel et al., 1993).

Much of the characterisation of the phenotype of these flies remains to be carried out in terms of defining the exact nature of the physiological lesion caused by TeTxLC expression. However, the ability to reproduce such phenotype repeatably and reliably will be invaluable to such a study. Clearly, more anti-neuropeptide antibodies need to be tested for co-localisation with the TeTxLC expression pattern. Once we have a clearer picture of the peptides the secretion of which are being inhibited in these flies, we may be able to inject such peptides to attempt to rescue the phenotype.

References:

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Helfrich-Förster, C. and Homberg, U. (1993) J. Comp. Neurol. 337, 177-190.

Martin, T. F. J. (1994) Curr. Opin. Neurobiol. 4, 626-632.

Nässel et al., (1993). J. Comp. Neurol. 331, 183-198.

Nässel., (1994) Zoological Science 11, 15-31.

Nicholls, D. G. (1994). Proteins, Transmitters and Synapses. (Oxford: Blackwell Scientific Publications).

O'Brien, M. A. and Taghert, P. H. (1994) Zoological Science 11, 633-645.

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Rao et al., (1991) In J. J. Menn,T. J. Kelly, & E. P. Masler (Ed.), Insect Neuropeptides: Chemistry Biology and Actions., 453 (pp. 110-122). American Chemical Society, Washington.

Reynolds, S. E. (1983) In Downer, L.G.H. and Laufer, H. (Eds.), Endocrinology of Insects (pp. 235-248). New York: Alan Liss.

Reynolds, S. E. (1985) In Kerkut, G. A. and Gilbert, L.I. (Eds.), Comprehensive Insect Physiology, Biochemistry and Pharmacology (pp. 335-351). New York: Pergamon.

Sweeney, S.T. (1995) Neuron 14, 341-351

Truman, J. W. (1985) In Kerkut, G. A. and. Gilbert, L.I. (Eds.), Comprehensive Insect Physiology, Biochemistry and Pharmacology (pp. 413-440). New York: Pergamon.

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