1 Department of Biology and Program in Neuroscience, University of Fribourg,
CH-1700 Fribourg, Switzerland
2 Institute of Genetics, University of Mainz, D-55099 Mainz, Germany
* Author for correspondence (e-mail: reinhard.stocker{at}unifr.ch)
Accepted 22 September 2003
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SUMMARY |
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Key words: Pharyngeal sense organs, Sensory neurons, Metamorphosis, FLPout labeling, Embryonic labeling, Mitotic labeling
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Introduction |
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As a seeming exception to the rule of sensory replacement, small subsets of
neurons associated with leg imaginal discs or abdominal segments have been
shown to persist during metamorphosis (Jan
et al., 1985; Shepherd and
Smith, 1996
; Tix et al.,
1989a
; Williams and Shepherd,
1999
). However, experimental evidence suggests that these neurons
might have a specialized function, serving as pioneers for growing adult
afferents (Usui-Ishihara et al.,
2000
; Williams and Shepherd,
2002
). Whether they become truly integrated in the adult nervous
system is not known.
The external gustatory sensilla of the Drosophila larva appear to
follow the general holometabolan fate: they degenerate during metamorphosis
and are replaced by adult-specific sensilla that derive from the labial
imaginal disc (Ray et al.,
1993; Ray and Rodrigues,
1994
; Wildermuth and Hadorn,
1965
). Here, we study whether this rule also applies to the
internal gustatory system that is located along the pharyngeal tube.
Interestingly, the adult pharynx derives essentially from small, densely
packed imaginal cells the clypeolabral bud that are closely
associated with the larval pharyngeal skeleton
(Bryant, 1978
;
Gehring and Seippel, 1967
;
Struhl, 1981
). Does this imply
that adult pharyngeal sensilla are born during metamorphosis, like their
external counterparts, or do the anatomical similarities of certain larval
(Python and Stocker, 2002
;
Singh, 1997
;
Singh and Singh, 1984
) and
adult pharyngeal organs (Nayak and Singh,
1983
; Stocker,
1994
; Stocker and Schorderet,
1981
) rather suggest persistence of sensilla through
metamorphosis?
To study possible links between the two sets of sense organs, we undertook a combined approach involving reporter expression from P[GAL4] driver lines, horseradish peroxidase (HRP) injections into syncytial blastoderm stage embryos, cell labeling through heat shock induced FLPout in the late embryo, bromodeoxyuridine (BrdU) birth dating and staining for programmed cell death. Our data include, neuron by neuron, the entire pharyngeal sensory system. They demonstrate that each of the three adult pharyngeal organs is of embryonic origin and derives from mature larval sense organs. Only a few sensilla are added through metamorphosis and few larval sensilla degenerate. This design is in marked contrast to nearly all other sense organs, which originate entirely from imaginal discs. The overall persistence of the larval sensory system is particularly striking because the pharynx undergoes extensive reorganization during this period.
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Materials and methods |
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Dissection
Larvae were cut at about half of the body length in PBS containing 0.2%
Triton X-100 (PBST). Anterior halves were turned inside out. Gut, fat body and
salivary glands were removed. Tissues were then fixed on ice for 1 hour in 4%
paraformaldehyde (PA) in PBST. In the fragile pupae, spiracles and the
posterior end of the pupal case were removed. An incision was made along the
dorsal midline from posterior to the thoracic region. After fixation for 1
hour in PA on ice, pupae were transferred to PBST and dissected from the pupal
case. Heads were cut, fat bodies were removed by gentle flow from a pipette
and tissues were transferred back to PA for 1-2 hours. In adults, the
proboscis was opened distally with a razor blade and cut into distal and
proximal parts that were fixed in PA for 2-3 hours on ice. At all stages,
tissues were prepared for whole mounts. In adults, cryosections (10-16 µm)
were also made.
Tracing embryonic lineage
HRP was used as a cellular tracer of embryonic lineage, as described
previously (Technau, 1986;
Tix et al., 1989a
). Briefly,
HRP 10% in 0.2 M KCl was injected into stage 4 (syncytial blastoderm) embryos
(wild-type CS or MJ94/UAS-lacZ). The injected embryos were allowed to
develop to adulthood. 1-2-day-old adults were dissected, fixed, cryosectioned
and processed for HRP and/or lacZ histochemistry (see below). A
second method for tracing embryonic origin was to generate
mCD8-GFP-labeled cells by heat-shock-induced FLPout. For this
purpose, the progeny of the cross MJ94 x y w hs-flp;
Sp/CyO; UAS>CD2 y+>mCD8-GFP
(Wong et al., 2002
) was
exposed to 35°C for 1 hour. Owing to egg-laying periods of 6 hours, the
age of these animals varied between 18-24 hours and 12-18 hours after egg
laying (AEL).
BrdU labeling
BrdU labeling (Stocker et al.,
1995; Truman and Bate,
1988
) was performed in the progeny of the cross MJ94
x UAS-lacZ or MJ94 x UAS-mCD8-GFP at 0
hours, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 12 hours and 18
hours APF. BrdU was applied topically
(Winberg et al., 1992
): 2-3
µg of a BrdU solution (100 mg ml1) in a mixture of
dimethylsulfoxide and acetone (1:1) was applied onto the animals' intact
anterior surface. In pupae at more than 12 hours APF, the puparium was
partially removed. Animals were then allowed to undergo metamorphosis and were
sacrificed as 2-3-day-old adults.
Immunocytochemistry and histochemistry
Green fluorescent protein (GFP) and anti-CD8 antibody (Caltag) coupled with
green fluorescence label Alexa 488 (Molecular Probes) were used as fluorescent
markers for the confocal microscope. This was done often in combination with
the neuronal nuclear marker anti-Elav 9F8A9 (Developmental Studies Hybridoma
Bank;
http://www.uiowa.edu/~dshbwww)
coupled with red fluorescence Cy3 (Jackson ImmunoResearch) or Alexa 568
(Molecular Probes). These double labelings were performed in both whole mounts
and cryosections (Python and Stocker,
2002). The embryonic tracer HRP was identified in cryosections
using the diaminobenzidine method. For lacZ labeling or
HRP/lacZ and BrdU/lacZ double labeling, ß-galactosidase
was visualized histochemically using X-Gal (Gibco)
(Brand and Perrimon, 1993
).
BrdU immunocytochemistry with an anti-BrdU antibody (G3G4; Developmental
Studies Hybridoma Bank) was performed as described by Stocker et al.
(Stocker et al., 1995
).
Programmed cell death
Apoptosis was studied in flies MJ94/UAS-mCD8-GFP by applying an
antibody against Drosophila caspase (-active Drice),
generously supplied by B. Hay (California Institute of Technology, Pasadena,
CA). Double labeling was accomplished by applying two secondary antibodies,
tagged with green fluorescence Alexa 488 (Molecular Probes) for CD8 and with
red fluorescence Alexa 568 for caspase, respectively.
Confocal and light microscopy
Multiple series of confocal images were taken at 0.5-1 µm intervals with
a BioRad MRC 1024 microscope (equipped with a Kr/Ar laser). Image analysis was
performed on a Macintosh computer using the public domain NIH Image program
(http://rsb.info.nih.gov/nih-image/).
Color selection of images was done using Adobe PhotoShop. Light microscopic
images were taken with a Zeiss AxioCam digital camera at 1300x1030
resolution and stored and processed with the AxioVision program.
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Results |
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Most of the adult pharyngeal neurons are persisting larval neurons born in the embryo
To study whether some of the adult sensory neurons were indeed born during
embryonic development, we followed two different approaches. First, we
injected the lineage tracer HRP at the syncytial blastoderm stage
(Technau, 1986). Using this
technique, adult cells born during embryogenesis remain labeled, whereas adult
cells born during postembryonic stages undergo an almost complete dilution of
the marker owing to massive proliferation and growth
(Tix et al., 1989a
). Second,
we generated mCD8-GFP-labeled pharyngeal neurons by
heat-shock-induced recombination in the progeny of a cross between
MJ94 and y w hs-flp; Sp/CyO; UAS>CD2
y+>mCD8-GFP (cf. Wong
et al., 2002
). When the heat shock is applied late during
embryogenesis, the presence of labeled single neurons in adult multineuronal
sensilla should prove their embryonic origin. This is because multineuronal
sensilla derive from a common sensory mother cell, the neurons being born last
in the cell lineage (Ray et al.,
1993
).
In the adult, the embryonically injected HRP was observed in many cells, except in tissues that are known to derive from imaginal discs, such as the antennae. Remarkably, many cells in all three pharyngeal sense organs showed the HRP product, mostly in their cytoplasm. In the lso, strong HRP staining was present in sensillum 7 (Fig. 2A), but label was weak or absent in the other sensilla. Whether HRP resided in neurons or in sheath cells was not clear. Intense HRP staining was also present in the vcso and dcso. In the vcso, the label was strongest in what seemed to be dendritic extensions (Fig. 2B). In the dcso, HRP product was clearly present in neurons, as judged by the labeling of dendritic and axonal processes. All six neurons were probably HRP positive (Fig. 2C,C'). No label was found in fishtrap bristle sensilla (Fig. 2D).
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Extra neurons are added during metamorphosis
Interestingly, late embryonic FLPouts always led to pairs of labeled
neurons in sensilla 8 and 9 of the lso
(Fig. 2H,I,
Table 2), suggesting that cell
divisions occurred after the FLPout. To discover whether the terminal cell
divisions of these neurons had taken place during metamorphosis, we used the
mitotic marker BrdU (Truman and Bate,
1988; Stocker et al.,
1995
) to MJ94 and wild-type CS animals, in experiments
ranging from 0 hours to 18 hours APF. Indeed, in adult flies that had been
treated in this way as pupae, subsets of neuronal and nonneuronal cells of the
pharyngeal sense organs were labeled. In the lso, we observed BrdU-positive
neuronal and non-neuronal cells at the distal tip, belonging to sensilla 8 and
9 (Fig. 3A-C). Additional
take-up had occurred in cells situated more proximally in the lso
(Fig. 3A-E), for example in the
neuron associated with sensillum 6 (Fig.
3D). In the vcso and dcso, none of the preparations revealed any
labeled neurons. However, two accessory cells were clearly BrdU positive in
both of these organs (Fig.
3F-I). Also, we observed labeling in the neurons of fishtrap
bristles (Fig. 3H) and massive
take-up in cells of the pharyngeal wall
(Fig. 3G,H). Thus, the BrdU
studies suggest that subsets of cells in the three sense organs undergo
terminal division during metamorphosis. In summary, the adult pharyngeal sense
organs appear to consist of both embryonically and postembryonically derived
neurons and accessory cells.
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In contrast to the pps and vps, the dps underwent complex transformation. Initially, its dendrites appeared to be collected in two clusters, anterior and posterior (a, p1) (Fig. 4G). Two neurons of the dpo (Fig. 1A,I, Fig. 4G) then joined the dps, leading to an additional cluster p2 (Fig. 4H,I). From 14 hours APF onward, this entire complex began to split into two distinct organs (vcso and lso) in parallel with the distal elongation of the pharynx (cf. Fig. 4J,K,M). The posterior organ, vcso, was derived from p1 and p2; p1 gave rise to the distal and middle sensilla of the vcso, p2 to the proximal sensillum (Fig. 4K,L). Five to six additional neurons, probably remnants of the dps and dpo, were found scattered on the associated nerve, outside the vcso proper (Fig. 4J-L). They persisted up to the adult stage (Fig. 1M, Fig. 2K). At 24 hours APF, Elav staining revealed for the first time the two rows of fishtrap bristles (Fig. 4L).
The anterior organ, lso, derived mostly from cluster a. It gave rise to two sensilla: a complex one with eight neurons (sensillum 7) and a mononeuronal sensillum (Fig. 4M), perhaps sensillum 3, judged by its distinct size. Around 20 hours APF, an additional four cells in the distalmost part of the lso, encompassing bineuronal sensilla 8 and 9, began to express Elav and GAL4, and another five neurons appeared more proximally (Fig. 4M-O). The latter formed mononeuronal sensilla 1-6 together with the persisting putative sensillum 3 (see above).
These observations are fully compatible with the experimental data regarding the persistence of most of the larval neurons and the birth of a new, smaller set during metamorphosis. In addition they demonstrate the conservation through metamorphosis of entire sense organs, pps and dps, the existence of complex morphogenetic movements including the split of a larval sense organ in two, and the degeneration of the vps.
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Discussion |
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HRP injected at the syncytial blastoderm stage becomes incorporated into
every cell upon cellularization. During subsequent development, the marker
remains at high levels in cells that divide only a few times but becomes
diluted in cells that undergo repeated divisions
(Technau, 1986). Consequently,
labeling in the adult is expected in many neurons of the central nervous
system known to be persisting larval neurons [e.g. optic lobe pioneers
(Tix et al., 1989b
)] but
should be absent from tissues derived from imaginal discs
(Levine et al., 1995
;
Tissot and Stocker, 2000
;
Truman, 1996
). This
corresponds to what we observe and allows us to postulate an embryonic origin
for the elements containing high HRP levels in adult pharyngeal sense
organs.
This interpretation is supported by the FLPout experiments (cf.
Wong et al., 2002) performed
at late embryonic stages with the neuron-specific MJ94 line. In
adults deriving from this treatment, we detected exclusively single labeled
neurons in sensillum 7 of the lso (containing eight neurons) and in the five
multiply innervated sensilla of the vcso and dcso
(Table 2). Although we did not
study the cell lineage of these sensilla, they are probably homologous to
other multineuronal terminal-pore gustatory sensilla, which derive from a
common sensory mother cell (Ray et al.,
1993
). Indeed, apart from its eight neurons, sensillum 7 of the
lso corresponds to a typical insect gustatory sensillum in terms of fine
structural and cellular organization, containing no more than three accessory
cells (Nayak and Singh, 1983
).
Hence, the single labeled neurons in this sensillum and in all sensilla of the
vcso and dcso must have been postmitotic during FLPout. This agrees with the
observation that formation of head nerves is complete by embryonic stage 15
(Campos-Ortega and Hartenstein,
1997
).
Could these neurons have remained immature during larval life,
differentiating only during metamorphosis, similar to subsets of postmitotic
cells in the larval central nervous system CNS
(Booker and Truman, 1987;
Truman, 1990
)? We believe this
rather unlikely because it would require either the entire sensillum or
subsets of neurons in multineuronal sensilla to remain immature. Moreover,
there is no indication for immature neurons from tracing their development
with the marker line mCD8-GFP. Thus, we suggest that all the neurons
of the dcso and vcso, and sensillum 7 of the lso derive from mature,
functional larval neurons. Also, continuous reporter expression through
metamorphosis suggests that one of the mononeural lso sensilla (perhaps
sensillum 3) might be another persisting larval sensillum.
Cell proliferation and cell death in pharyngeal sense organs through metamorphosis
The persistence of mature larval neurons does not exclude the addition of
cells during larva-adult transition. Indeed, BrdU applied during metamorphosis
labeled subsets of cells in all three sense organs and in the fishtrap
bristles. In agreement with these data, late embryonic FLPouts displayed pairs
of labeled neurons in the sensilla 8 and 9 of the lso. Neuronal identity of
the labeled cells was also established for the lso sensillum 6 as well as for
the fishtrap bristles. Consequently, these neurons must have arisen by a
terminal division after the FLPout and after BrdU application (i.e. during
metamorphosis) (Fig. 5). Hence,
of the total of 32 sensory neurons of the three main adult sense organs, only
the birth dates of four monoinnervated lso sensilla remain unclear. Yet, their
metamorphic origin is suggested by BrdU uptake in sensillum 6 and the gradual
appearance of Elav staining at 24 hours APF in all four sensilla.
Taken together, these data suggest that the lso is composed of both larval and adult-specific sensilla, whereas the vcso and dcso consist exclusively of larval sensilla. Seen from a larval perspective, both the pps and the dps are conserved through metamorphosis, although with considerable modification of the dps (Fig. 5). By contrast, the vps undergoes programmed cell death, and the fishtrap bristles are entirely adult specific, lacking any larval counterpart. In conclusion, metamorphosis of the internal gustatory sensory system is an intricate process involving neuronal persistence, generation of additional neurons and neuronal death.
Larval sense organs persist despite extensive reorganization of the pharynx
The fact that the pps and dps persist through metamorphosis is remarkable
given the origin of the adult labrum and cibarium from imaginal cells of the
clypeolabral bud (Bryant, 1978;
Gehring and Seippel, 1967
;
Struhl, 1981
). In agreement
with these reports, we observe massive labeling of pharyngeal epithelial cells
after early pupal BrdU application. Moreover, the pharyngeal cuticle is shed
and regenerates, a process that includes the cuticular part of the sensilla in
question. Perhaps the birth of additional accessory cells during metamorphosis
(e.g. in the dcso or vcso, containing exclusively persisting neurons) is
related to this modification. Formation of new cuticular structures is also
known from persisting external sensilla during larval molts, but the survival
of pharyngeal sensilla during the extensive remodeling of the pharynx remains
stunning. The morphogenetic movements we observe in the sensory system
certainly reflect these dramatic changes.
Why is the larval pharyngeal sensory apparatus largely conserved through metamorphosis?
Small subsets of neurons associated with leg imaginal discs or with
abdominal segments have previously been shown to persist through metamorphosis
(Jan et al., 1985;
Shepherd and Smith, 1996
;
Tix et al., 1989a
;
Williams and Shepherd, 1999
).
In the fly Phormia, such leg-disc-associated neurons remain immature
(Lakes-Harlan et al., 1991a
;
Lakes-Harlan et al., 1991b
),
implying that they are non-functional. Laser ablation studies suggest that
persisting neurons might help adult afferents to navigate from the imaginal
discs to their central targets
(Usui-Ishihara et al., 2000
;
Williams and Shepherd, 2002
).
Whether they become truly integrated in the adult nervous system or die after
reaching adulthood (having completed their pathway role) remains to be shown.
Recently, tracing the expression pattern of a Kr-driven reporter line
suggested the incorporation of four receptor neurons of the larval eye into
the so-called adult eyelet
(Helfrich-Förster et al.,
2002
; Hofbauer and Buchner,
1989
), but this was not tested experimentally.
Our data demonstrate for the first time experimentally the integration of larval sensory neurons into the adult nervous system of Drosophila. Particularly striking and novel is the fact that entire, fully differentiated larval sense organs become incorporated. Also, this is the first observation of metamorphic survival in the chemosensory system.
Concerning the persisting neurons of the lso, a pathway function for the
newly developing afferents towards and inside the central nervous system is
certainly possible. However, the integration of the surviving pharyngeal
neurons into the adult sensory system invites other interpretations. For
example, these neurons and/or their central projections might be particularly
precious, allowing, for example, the persistence of specific
feeding-associated gustatory tasks through metamorphosis. As an alternative
explanation, survival might be due to reasons of economy, a principle that
governs the metamorphosis of the nervous system
(Tissot and Stocker, 2000).
Although neuronal reorganization is indispensable owing to the changing
demands of larval and adult life, it is kept at a minimum, as shown by the
survival of most larval interneurons and motor neurons
(Truman, 1996
). Sophisticated
adult sense organs, however, might be easier to build de novo than by the
transformation of simple larval organs, explaining the almost complete
replacement of the larval sensory system. Why pharyngeal sense organs do not
follow this general rule might relate to their largely conserved function at
the two stages of life (analyzing the quality of ingested food of similar
composition). The presence of larva-specific and adult-specific sensilla,
however, suggests the existence of stage-specific gustatory tasks.
Using the genetic potential of the fly, it will be intriguing to dissect the functions of the different types of sensilla. Moreover, our analysis invites us to study at single cell level the genetic basis of many essential developmental processes, including cell determination, differentiation and apoptosis.
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ACKNOWLEDGMENTS |
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