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): email@example.com
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.
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).
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.
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).
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.
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