Institute for Molecular Biology, University of Zürich, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland
* Author for correspondence (e-mail: noll{at}molbio.unizh.ch)
Accepted 16 September 2002
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SUMMARY |
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Key words: Drosophila, Pox neuro, Enhancer, Courtship behavior, Male fertility, PNS, CNS, Appendages
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INTRODUCTION |
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In an attempt to determine all functions of Poxn, we have prepared
a Poxn null mutant. Surprisingly, this mutant turned out to be male
sterile, a phenotype not previously observed for Poxn mutants
(Awasaki and Kimura, 1997;
Awasaki and Kimura, 2001
).
Although the fertility of males depends on their initiation of courtship,
which is stimulated through chemosensory bristles
(Stocker, 1994
), such a
phenotype was not expected because male courtship is influenced by a number of
additional sensory modalities such as visual, olfactory, mechanosensory and
auditory signals (Hall, 1994
;
Greenspan and Ferveur, 2000
).
These sensory cues allow the male to recognize its own species, discriminate
against males and to find females that have not been mated recently. The male
initiates courtship by following an evading female, taps her abdomen with his
foreleg, extends and vibrates the wing on the side oriented towards her (thus
producing a `love song' that reduces her locomotor activity), extends his
proboscis and licks the genitalia of the female with the labellum, and finally
attempts to copulate (Hall,
1994
). Copulation is established by the male bending and thrusting
his abdomen forward and grabbing the genitalia of the female, anchoring
himself with his claspers and penis, and finally mounting the female to remain
in this copulation position for about 15 minutes to transfer his sperm and
accessory gland fluid.
Males are stimulated to initiate courtship by female pheromones, which are
transferred over short distances to olfactory trichoid sensilla on the third
antennal segment and maxillary palp or through direct contact with
chemosensory bristles and sensilla on the labellum, forelegs and wing
(Robertson, 1983;
Gailey et al., 1986
;
Stocker and Gendre, 1989
;
Stocker, 1994
;
Ferveur et al., 1996
;
Ferveur et al., 1997
;
Yamamoto et al., 1997
). Males
not only follow chemical cues, they also react to visual stimulation by a
moving object of the appropriate size. Although the visual modality seems to
be a facultative input to induce courting behavior in Drosophila
melanogaster (Spieth and Hsu,
1950
; Cook, 1980
),
males with reduced or no eye pigment are at a distinct disadvantage in
competition experiments (Conolly et al., 1969;
Reed and Reed, 1950
).
As Poxn null mutant males are sterile, we began to explore the impact of the absence of Poxn functions on male fertility and courtship behavior. Surprisingly, many different functions of Poxn are involved. To determine the contribution of each of these functions to male courtship and fertility, a complete dissection of all Poxn enhancers was necessary. This analysis revealed an intriguing complexity of the arrangement and substructure of Poxn enhancers. In addition, it demonstrated that male courtship behavior and fertility functions of Poxn include: (1) the regulation of the development of chemosensory bristles on the labellum, legs and wings, which receive and propagate female pheromones; (2) the regulation of the development of defined neurons in the adult brain whose proper connections are required at least for the processing of olfactory signals produced by females pheromones; (3) the regulation of proper copulation behavior, which depends on the expression of Poxn in specific neurons of the embryonic ventral cord; and (4) the regulation of proper development in the male genital disc to give rise to the penis, claspers and posterior lobes.
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MATERIALS AND METHODS |
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Poxn rescue and Poxn-Gal4 driver constructs
To map the enhancers of the Poxn gene, rescue constructs were
designed, using Poxn chromosomal and/or cDNA sequences
(Fig. 1B), and inserted into
the polylinker of the P-element vector pW6, which carries a
mini-white marker gene (Klemenz
et al., 1987). To gain additional information about the spatial
and temporal expression patterns of Poxn, different Poxn
enhancer fragments, the Poxn promoter, leader and N-terminal-coding
sequence were fused at amino acid 1 or 28 of the Poxn paired domain to the
coding region of the yeast transcriptional activator Gal4, joined to the
3'UTR/poly A addition site of Poxn
(Fig. 1B), and cloned into the
polylinker of pW6. For transformation, the constructs were injected into
w1118 embryos, and the resulting lines crossed
into the appropriate w; Poxn
M22-B5 mutant
background.
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Analysis of GFP expression patterns and dissection of adult
brains
Poxn-Gal4 driver lines were used in combination with
P{y+ UAS-GFP} to reveal the Poxn
expression patterns in various tissues and the axonal projection patterns of
the Poxn-expressing neurons in the PNS and CNS. Green fluorescent
protein (GFP) expression patterns were analyzed with a Zeiss Axiophot
microscope under fluorescence and the filter set 450-490/FT510/LP520, or with
a Leica SP1 confocal microscope and standard settings for FITC detection. The
resulting Z-stacks were processed by the use of Leica `LCS Lite' analysis
software.
Adult brains were dissected in 0.1 M PIPES/NaOH, pH 7.5, 2 mM EGTA, 1 mM MgCl2 (PEM-buffer), 4% formaldehyde, fixed for about 15 minutes, and mounted in Ringer's solution on microscope slides bounded on two sides by cover slips to support the cover slip.
Immunohistochemistry
Tissues were fixed with 4% formaldehyde in PEM buffer, blocked with 2%
fetal calf serum and incubated overnight at 4°C with purified, 1:200
diluted rabbit anti-Poxn antiserum (Bopp et
al., 1989). To detect bound antibody, tissues were further
incubated with biotinylated goat anti-rabbit IgG (Vector Labs), followed by
incubation with avidin-peroxidase (ABC-Kit, Vector Labs) and development with
3,3'-Diaminobenzidine/H2O2.
Morphological analysis of the adult cuticle
Flies were anaesthetized and dissected. Body parts were incubated in 10%
NaOH for 30 minutes at 80°C to remove the tissue from the cuticle
structures, rinsed with PBS and mounted in glycerol. Photographs were taken
with a Zeiss Axiophot microscope equipped with a Hamamatsu CCD camera.
Courting tests
Single choice courting tests were performed at room temperature in a
modified 24-well microtiter plate (15x8 mm wells covered with microscope
glass slides), which was placed in a tray humidified with wet filter paper.
All tests were performed at room temperature either under daylight or in a
darkroom 10 cm from a 15 W lamp shielded by a Kindermann 2038 red filter. Test
males were collected within 12 hours of eclosion and kept isolated in a tube
with fly food for 5 days at 18°C and at a regular 12-hour light/dark
cycle. w; PoxnM22-B5/CyO females
were collected as virgins and kept for 5 days in groups of about 10 animals in
tubes under the same conditions as the males. Before courting tests, flies
were adapted to room temperature for about 4 hours. Four to 8 hours after
lights had been turned on, females were aspirated without anaesthesia and
placed into the courting chamber followed by the male. Observation time was
limited to 15 minutes or until copulation started. Latency time for the
beginning of courting (wing extension) and copulation were recorded.
The fertility of males was assessed during long-term single crosses with
w; PoxnM22-B5/CyO virgins in food
tubes at 25°C and 60% humidity for 4 days at a 12-hour light/dark cycle.
Males of low fertility were tested in groups of six with six virgins. In both
tests the number of larvae and the genotype of the offspring were
recorded.
Fly stocks
The following fly stocks were prepared:
Additional stocks used were Df(2R)WMG, Sp Bl lt/In(2LR)Gla
(Bloomington stock 1887), w1118 (Bloomington stock 5905),
w; sliF81/CyO
(Rothberg et al., 1990), y
w; P{y+ UAS-GFP} (P element on third chromosome; from
D. Nellen) and Oregon-R (Munich) (from W. McGinnis).
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RESULTS |
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To obtain Poxn null mutants, the P element in P{Lac-W}M22
was mobilized to produce a set of imprecise excisions
(Salz et al., 1987) that
deleted the neighboring Poxn gene. One of these deficiencies,
Poxn
M22-B5, when tested over Df(2R)WMG,
indeed resulted in the expected phenotype of missing chemosensory bristles.
This deficiency extends over 17 kb, from its proximal breakpoint in exon 2 of
Poxn to its distal breakpoint upstream of the adjacent gene encoding
a sugar transporter homolog CG8249, and includes, in addition to the
Poxn promoter region, all upstream enhancers of Poxn
(Fig. 1A). Because it also
deletes the entire coding region of the neighboring sugar transporter gene and
possibly affects the control region of the next distal transcription unit
CG8253, a transgene, Resdistal, that completely includes both these
genes (Fig. 1A) was crossed
into Poxn
M22-B5 mutants. Such transgenic
Poxn
M22-B5 flies have a phenotype indistinguishable
from that of Poxn
M22-B5 or
Poxn
M22-B5/Df(2R)WMG flies and are viable, but all
males are sterile. As no Poxn protein is detectable at any time in these
mutants, Poxn
M22-B5 is a null allele of
Poxn.
Transformed chemosensory bristles of Poxn null mutants
In homozygous PoxnM22-B5 flies, all taste
bristles on wings, legs and labellum are affected. Thus, all taste bristles on
the anterior wing margin (Fig.
2A) are transformed into mechanosensory bristles, and the
transformed dorsal bristles no longer constitute a second posterior row, but
are interspersed with the anterior row of mechanosensory bristles, with which
they form a single dorsal row (Fig.
2B). The transformed bristles are easily distinguished from
wild-type mechanosensory bristles by their increased length and thickness. On
the legs, taste bristles (Fig.
2C) are transformed into bracted mechanosensory bristles, most of
which are indistinguishable in morphology from wild-type mechanosensory
bristles (Fig. 2D), or
occasionally lost. Only on the tibia, some transformed bristles are longer and
thicker than the surrounding mechanosensory bristles (not shown). Finally, on
the labellum, the number of taste bristles
(Ray et al., 1993
)
(Fig. 3A) appears unchanged,
but their shafts are longer, have pointed tips like those of mechanosensory
bristles and are often bent or kinked (Fig.
3B).
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Late enhancer active in chemosensory neurons of all taste
bristles
To delimit the enhancers regulating the various Poxn functions, a
large number of partial Poxn genes were constructed, in which the
coding region and promoter of Poxn were combined with different
upstream, downstream or intron regions
(Fig. 1B) and which were
subsequently tested as transgenes for rescue of Poxn functions in
homozygous PoxnM22-B5 flies. In addition, several
Poxn-Gal4 transgenes, expressing Gal4 under the control of the
Poxn promoter and different Poxn enhancers
(Fig. 1B), were combined with a
UAS-GFP reporter transgene and tested for GFP expression. The rescue
experiments demonstrated that the Poxn functions required for early
development of recurved taste bristles on legs and wings or on the labellum
are controlled by separate `early' enhancers active during late third larval
instar and early pupal stages. Rescue of the taste bristles on the legs and
anterior wing margins depends on an enhancer of complex structure located in
the upstream region, whereas rescue of the labellar taste bristles depends on
a distinct enhancer located in the downstream region of Poxn
(Fig. 1C). In addition to these
enhancers, expression of Poxn indicates that all chemosensory
bristles share an enhancer required for their late development the functional
significance of which, however, has not yet been demonstrated.
This enhancer for late Poxn expression in chemosensory neurons is
located close to the transcriptional start site. This is evident from the
observation that either one of six transgenes, Poxn-Gal4-14, Poxn-Gal4-13,
Poxn-Gal4-9, Poxn-Gal4-BasiK, Poxn-Gal4-581 or Poxn-Gal4-6,
which share only upstream and leader sequences downstream of the AgeI
site at -0.13 kb (Fig. 1B), is
sufficient to drive this late expression of a UAS-GFP reporter gene.
In contrast to the early expression of Poxn in developing adult chemosensory
organs, initially observed in their SOPs of the corresponding third instar
discs (Dambly-Chaudière et al.,
1992), late Poxn expression in chemosensory neurons, as assayed by
GFP expression, is first detectable at about 36 hours APF (after puparium
formation) and is maintained in adults.
Usually four GFP-expressing neurons innervate each taste bristle on the
legs (Fig. 2E) or wings, while
two to four neurons innervate each open-tipped taste bristle on the labellum
(Fig. 3D). All neurons are of
the chemosensory type and project into the lumen of the individual shafts.
This is obvious in the labellar bristles where GFP is clearly visible in
shafts (Fig. 3D), whereas the
dendritic projections into shafts is not as easily detectable in legs
(Fig. 2E). Two additional
groups of neurons labeled with GFP innervate the labral sense organs (LSO)
(Stocker, 1994;
Mitchell et al., 1999
). This
paired organ of the internal mouth parts is composed of six mechanosensory and
eight chemosensory neurons (Singh,
1997
), about six of which express GFP
(Fig. 3D) driven by the
Poxn-Gal4 constructs (Fig.
1B).
Complex early enhancer for development of taste bristles in legs and
wings
In addition to the late enhancer active in all chemosensory bristles, an
early enhancer located further upstream is necessary for the activation of
Poxn functions in third instar larvae and pupae to restore proper
morphology in taste bristles on legs and wings of Poxn null mutants.
The complex properties of this enhancer are apparent from studies of
Poxn transgenes under the control of incomplete enhancers
(Fig. 1B) that rescue different
parts of the wild-type pattern of taste bristles. Because some of these
transgenes exhibit a slight dose dependence, they were always used as two
homozygous copies to delimit the functions of this enhancer, which extends
over a XbaI-HindIII fragment, located between 3.0 and 8.2 kb
upstream from the transcriptional start site. Driving Poxn expression under
the control of increasing lengths of the upstream region rescues some taste
bristles on legs and wings in homozygous
PoxnM22-B5 flies only when regions
upstream of the PstI site in this fragment are present as in the
BsK, ScK, SaK, BaK and EvK transgenes
(Fig. 4). Nevertheless, the
enhancer crucially depends also on sequences downstream of PstI, as
evident from the complete absence of taste bristles in
Poxn
M22-B5 flies endowed with the
transgenes C1,
KBs,
XBs and
XP (Fig. 4),
all of which lack the 0.9 kb XbaI-PstI fragment
(Fig. 1B). Omission of DNA
regions proximal to the XbaI site, as in the P2 or
KX transgenes, does not affect the rescue of bristles on legs
and wings and thus sets a proximal limit of the early enhancer. Its distal
limit is approximated by a complete rescue obtained by the EvK
transgene that includes 8.2 kb of upstream sequences up to the
HindIII site (Fig.
1B).
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Thus, although the part of the enhancer included in the 0.9 kb proximal to
PstI of Poxn transgenes is not sufficient for the rescue of
any taste bristles, it is necessary for the development of all taste bristles
on legs and wings. The inclusion of more distal regions of the enhancer
rescues increasing numbers of taste bristles, apparently with a general
distal-to-proximal polarity along the leg and wing. All taste bristles on the
tarsal segments 2, 4, 5 and about 40% of those on the anterior wing margin,
mostly on its distal part, are rescued by the BsK transgene, which
extends only by 0.35 kb beyond PstI
(Fig. 1B). By contrast, only
few taste bristles are rescued on the tibia (<25%) and virtually none on
tarsal segments 1 and 3 (not shown). Addition of increasing distal regions of
the enhancer up to the BamHI site in transgenes ScK, SaK (or
SH) and BaK (or
BH) does not
significantly change the number of rescued bristles on the leg
(Fig. 4). Only further
inclusion of the most distal region of the enhancer in EvK restores
the bristle pattern of the tibia and all tarsal segments although an excess of
taste bristles appears at the expense of mechanosensory bristles in the tibia
(Fig. 4). Most notably, the
number of these ectopic taste bristles, which appear to arise from transformed
mechanosensory bristles, are reduced by the additional presence of the first
two introns in the Full transgene, yet completely suppressed only
when also downstream control regions are present as in the SuperA
transgene (Fig. 1B, Fig. 4). Although addition of
the 2.2 kb between BstXI and BamHI in the absence of the
most distal part of the early enhancer does not significantly alter its rescue
efficiency for leg bristles, rescue of taste bristles on the anterior wing
margin is enhanced from 40% to about 90%
(Fig. 4). Hence, the enhancers
for taste bristles on legs and wings overlap, but do not behave identically
and seem to have different requirements.
Early enhancer for development of labellar taste bristles
In contrast to the taste bristles on legs and wings, the labellar bristles
of PoxnM22-B5 mutants are not simply
transformed into mechanosensory sensilla, but the shafts show tips of bizarre
forms (Fig. 3B, arrows).
Moreover, the morphology of the labellar chemosensory bristles cannot be
rescued by the presence of enhancers in the upstream region of Poxn
transgenes. The early enhancer that, in combination with the late enhancer,
restores wild-type morphology of labellar bristles is located in the
downstream region of Poxn. This region is delimited by the
SuperA transgene (Fig.
1B), which is sufficient for rescue of labellar bristle morphology
(Fig. 3C), while the
E77 transgene including only 3.0 kb of the downstream region
(Fig. 1B) is not.
Poxn males do not court receptive females in the absence of
proper visual input
The observation that PoxnM22-B5 males
are sterile prompted us to examine their mating behavior towards receptive
virgin females. Under daylight conditions, two thirds of the
Poxn
M22-B5 males do not initiate courtship
in single choice experiments within 15 minutes under standard conditions (see
Materials and Methods), while the remaining third courts females very weakly,
but proceeds through the entire courting sequence (for a review, see
Greenspan and Ferveur, 2000
).
Although these males attempt to copulate by bending their abdomen, no
copulation is observed. All males have well developed testes with motile
sperm, accessory glands, ejaculatory duct and sperm pump. However, the
cuticular structure of their genitals is aberrant, as will be shown below.
Interestingly, w1118;
Poxn
M22-B5 double mutants do not take note
of females at all under daylight (32 males tested). Similarly,
Poxn
M22-B5 or
w1118;
Poxn
M22-B5 males carrying the
Resdistal construct, whose associated mini-white marker gene restores
wild-type eye pigmentation, do not court females under red light, but only at
daylight (Fig. 4). Clearly, in
the absence of Poxn functions, male courting behavior depends
entirely on proper visual input.
Influence of number of taste bristles on courting behavior of
males
Surprisingly, PoxnM22-B5 males carrying
the BasiK transgene display a partially rescued courting behavior
even though none of the recurved taste bristles on legs, wings or labellum are
rescued. While under daylight they court slightly more often and with a
somewhat shorter latency time before the onset of courting than their mutant
counterparts, a striking difference becomes apparent when their mating
behavior is compared in the absence of proper visual input. In contrast to
Poxn males, which exhibit no courtship behavior under red light, one
quarter of the transgenic Poxn males do court receptive females
(Fig. 4). Although the
BasiK transgene lacks the early Poxn function required for
development of chemosensory bristles, it includes the late function expressing
Poxn in chemosensory neurons. It was thus conceivable that the partial rescue
resulted from the expression of Poxn in `chemosensory' neurons able to develop
even without the preceding early Poxn function. By introducing
Poxn-Gal4-13 (Fig. 1B)
and UAS-GFP transgenes, we therefore investigated whether such
neurons expressing Poxn, as assayed by GFP, are present in
Poxn
M22-B5 adults partially rescued by the
BasiK transgene. Indeed, five to 20 neurons, often in groups of two,
are observed to express GFP in a prothoracic leg of these males
(Fig. 2F), whereas about 180
chemosensory neurons (four neurons per taste bristle) express GFP (and thus
Poxn) in the corresponding leg of a wild-type male
(Fig. 2E). A similarly reduced
fraction of GFP expressing neurons is also present in meso- and metathoracic
legs (not shown) and in the labellum (Fig.
3E), as well as in legs (Fig.
2G) and labellum (not shown) of
Poxn
M22-B5 mutants that have not been
partially rescued by the BasiK transgene. Thus, the potential of
expressing Poxn under control of the late enhancer in the remaining
`chemosensory' neurons associated with some of the transformed bristles of
Poxn mutants does not depend on the presence of the BasiK
transgene. In legs, these neurons are most often associated with transformed
bracted mechanosensory bristles, but rarely innervate structures resembling
short and unpigmented bristles (less than one per leg; not shown). However,
although the strongly reduced number of these neurons expressing Poxn in the
partially rescued Poxn males would be consistent with the observed
partial rescue of courting behavior, this explanation appears improbable in
view of additional evidence discussed below.
If PoxnM22-B5 males are rescued by
Poxn transgenes that include increasing lengths of upstream control
regions (Fig. 1B), their
enhanced courting behavior appears to parallel their increasing number of
rescued taste bristles on legs rather than wings. Thus, while only about one
third to half of all males court under red light if no taste bristles are
present, all males court if taste bristles on tarsal segments 2, 4 and 5 are
rescued by BsK (not shown). Similarly, further increase of the
upstream region in ScK, SaK and BaK transgenes does not
change significantly the latency time before courting under daylight or red
light conditions, nor the number of rescued chemosensory bristles on the legs,
whereas the number of taste bristles on the wings varies by a factor of two
(Fig. 4). Only when all taste
bristles on the legs and wings are rescued by the EvK transgene,
which includes 8.2 kb of the upstream region
(Fig. 1B), the latency time of
the rescued males is significantly reduced to that of Oregon-R males
(Fig. 4).
Despite their normal courting behavior,
PoxnM22-B5; EvK males, which
carry the full set of chemosensory bristles on legs and wings, but none on the
labellum, have not been observed to copulate in courting tests. In fertility
tests, two out of 90 Poxn
M22-B5 males
carrying two copies of the EvK transgene produced a dramatically
reduced number of offspring, while none out of 480 males carrying a single
copy was able to generate any offspring.
Defective genitalia of Poxn males
The observation that PoxnM22-B5 males
whose taste bristles were rescued by EvK exhibit a wild-type
courtship behavior, but are unable to copulate suggests that male fertility
depends on additional functions of Poxn. Examining the expression of
UAS-GFP under the control of Poxn-Gal4-14 in pupae, we found
a sexually dimorphic pattern expressed by about 30 hours APF in males
(Fig. 5A), but absent in
females (Fig. 5B). This
male-specific pattern can be divided into two non-overlapping subpatterns
regulated by separate enhancers: one located in introns 1 and 2; the other in
the upstream region of Poxn (Fig.
5C). The absence of these enhancers can be correlated with missing
Poxn functions resulting in defects of the male genitalia. Thus,
Poxn
M22-B5 males have no penis, although
the penis apodeme and protractor muscle are still present
(Fig. 5D). The enhancer
regulating the Poxn functions required for normal development of the
penis is delimited by the 1.2 kb BstXI-ScaI fragment of the
upstream region (Fig. 1C), as
evident from the rescue of the penis in
Poxn
M22-B5 males with Poxn
transgenes, such as ScK, EvK or
KBs, which include
this fragment, but not with transgenes like BsK or L1, in
which this fragment is missing (Fig.
1B, left side of Fig.
5D). A second, more subtle defect is observed on the outer
genitalia of Poxn
M22-B5 males whose
posterior lobes are degenerate and whose claspers display slightly aberrant
bristle patterns (Fig. 5D). An
essential part of the enhancer(s) regulating these functions is located in the
second intron of Poxn (Fig.
1C). This is evident from an almost complete rescue of claspers
and posterior lobes by the E77 transgene (not shown), which includes
this intron as an upstream enhancer (Fig.
1B), and from the full rescue of these structures by L1
(Fig. 1B), whereas
Poxn transgenes that include no intronic sequences, such as
EvK, do not rescue the morphology of the posterior lobes and claspers
at all (right side of Fig.
5D).
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The copulation rate and fertility of
PoxnM22-B5 males is restored efficiently
only when Poxn functions regulated by the enhancers in the introns as well as
the upstream region of Poxn are supplied, for example by EvK
and L1 (Fig. 4).
Hence, male fertility clearly depends on the integrity of all genital
structures.
Strongly reduced male fertility in the absence of Poxn expression in
the embryonic ventral nerve cord
Transgenic PoxnM22-B5;
SH (Fig. 1B)
males, which are able to sense females despite a reduced set of chemosensory
bristles and whose genitals are perfectly restored, begin courting after about
3 minutes under daylight conditions (Fig.
4). Nevertheless, no copulation was observed within the 15 minutes
of our tests. Long-term courting tests reveal that the males have difficulties
in establishing physical contact with the females' genitalia and, after
mounting, in remaining in the copulation position. In mass crosses and
long-term single crosses, such males exhibit a strongly reduced fertility
(<20% of single crosses have offspring), and
Poxn
M22-B5;
SH flies
cannot be maintained as stable lines. However, the fertility and copulation
behavior of Poxn
M22-B5;
SH
males can be restored by adding a copy of C1
(Fig. 1B,
Fig. 4). Although this
transgene includes an enhancer region required for the rescue of the full
complement of taste bristles on legs and wings, it neither enhances the
incomplete number of taste bristles of
Poxn
M22-B5;
SH males when
provided in trans nor rescues any taste bristles by itself
(Fig. 4). Therefore, the
C1 transgene must include a distal upstream enhancer, whose function
is necessary for the full rescue of male fertility but which differs from the
taste bristle enhancer of this region.
Surprisingly, this enhancer, which is included in a 2.57 kb
HindIII-SalI fragment, drives expression in the ventral
nerve cord of the embryo (Fig.
6A-C), as evident from the expression patterns of Poxn
transgenes and UAS-GFP transgenes under Poxn-Gal4 control
that comprise this upstream region (Fig.
1B). Most of the enhancer is located in the distal 1.76 kb
HindIII-BamHI fragment as BH expresses Poxn
only weakly in the ventral cord, while ventral cord expression of C1,
Full, or
XBs is very strong. By contrast,
SH shows no Poxn expression in the ventral cord. Moreover,
fertility of Poxn
M22-B5 males rescued by
these transgenes and their expression in the ventral cord are in excellent
agreement (Fig. 4). In
accordance with our earlier conclusion, the fertility of these males does not
correlate with the rescue of leg and wing taste bristles, as males rescued by
XBs have no taste bristles but a considerably higher fertility
than those rescued by
SH or
BH, which possess
at least half of all leg and wing taste bristles
(Fig. 4).
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Poxn expression under the control of the ventral cord enhancer is first
detectable during late stage 10 or early stage 11 of embryogenesis
(Bopp et al., 1989) and fades
only at the end of embryonic development. Many Poxn-expressing cells in the
ventral cord are of neuronal morphology, as shown in a stage 17 embryo
expressing GFP under the control of Poxn-Gal4-13
(Fig. 6B,C). No Poxn expression
is detectable in the ventral CNS of third instar larvae
(Fig. 7B). Similarly, no
expression is apparent in the adult ventral CNS (not shown). However, GFP
expressed under control of the late taste bristle enhancer reveals a sexually
dimorphic projection pattern of the chemosensory afferents
(Fig. 6D,E), which has been
observed previously for axons of some taste bristles on the prothoracic legs
(Possidente and Murphey,
1989
). A considerable number of chemosensory neurons from the male
prothoracic legs project contralaterally into the prothoracic leg neuromere,
while no arborizations of this kind are detected in the meso- or metathoracic
leg neuromeres or the wing neuromere (Fig.
6D). In the female ventral CNS, all projections are exclusively
ipsilateral (Fig. 6E).
|
Male courting behavior crucially depends on Poxn expression
in the brain
Male fertility of Poxn mutants is rescued by XBs
to a much larger extent than by
KBs, even though both
transgenes include the ventral cord enhancer and do not rescue any taste
bristles (Fig. 4). The only
difference between these two transgenes is a 1.55 kb
XbaI-KpnI upstream fragment absent from
KBs
(Fig. 1B). This region includes
a considerable region of an enhancer that regulates Poxn expression
in the embryonic, larval and adult brain. Poxn transgenes without
this upstream region, such as
KBs,
KX or
BasiK, exhibit only a very faint Poxn expression in the brain,
whereas all transgenes including this upstream fragment, such as
XBs,
XP or XK, rescue the complete
Poxn expression pattern in the brain of Poxn mutants at all stages.
As C1 exhibits no expression in the brain, it follows that the brain
enhancer is completely included in a 2.38 kb XbaI-EcoRI
fragment, 0.62 kb upstream of the transcriptional start site
(Fig. 1C), though most of it is
located in its distal two thirds.
Poxn protein in the brain is first detected during embryonic stage 12 and
continues to be expressed in the embryonic, larval and adult brain throughout
development. In the embryonic brain lobes, two groups of cells express Poxn
with bilateral symmetry (Fig.
7A), while it is detectable only in a single group in each brain
hemisphere of third instar larvae (Fig.
7B). In the adult brain, again two bilateral-symmetric groups of
cells express Poxn, a ventral cluster of about 100 cells, forming a ventral
arc around the region where the antennal nerve enters the brain, and a cluster
of about 200 cells, located in a dorsolateral region adjacent to the antennal
lobe on the surface of the ventrolateral protocerebrum
(Fig. 7C). Most of these cells
have the morphology of neurons, as evident from the GFP pattern driven by
Poxn-Gal4-13 (Fig.
7D). The majority of the neurons of the ventral cluster project
into the antennal lobe (Fig.
7D,G) (Laissue et al.,
1999), while the major target of the dorsolateral cluster is the
ellipsoid body neuropil (Fig.
7D,F), a part of the central complex neuropil involved in
locomotion (Strauss and Heisenberg,
1993
; Martin et al.,
1999
). The position and architecture of the dorsolateral neurons
resemble those described for large-field R neurons
(Hanesch et al., 1989
;
Renn et al., 1999
). In
addition, projections from the dorsolateral cluster of cells target the
lateral triangle (Hanesch et al.,
1989
; Renn et al.,
1999
), and a domain dorsal of it
(Fig. 7F), while the nerve
emanating from the ventral cluster arborizes on at least two additional
targets in the dorsolateral part of the brain
(Fig. 7G), but does not seem to
connect to the dorsolateral cluster of neurons. The positions of the cell
bodies and the projection pattern are symmetric in the two brain hemispheres
and do not exhibit an obvious sexual dimorphism.
Both groups of cells are also present in the adult brain of a Poxn mutant (Fig. 7E). The cells have a neuronal shape, as evident from GFP expression under control of Poxn-Gal4-13, and their number is comparable with that of the wild type. However, the projection pattern has completely changed. The ellipsoid body and other brain centers are not specifically targeted, and the arborizations are diffuse and asymmetric for the two hemispheres.
To evaluate the contribution to male fertility of Poxn expression
in the brain, the courting behavior of
PoxnM22-B5;
KBs males was
compared with that of Poxn
M22-B5;
XBs males (Fig.
4). Both transgenes do not rescue any chemosensory bristles, but
restore the ventral cord expression and rescue the male genitalia as well as
the late Poxn expression in the `chemosensory' neurons associated with the
transformed bristles. However, the
XBs transgene also restores
Poxn expression in the brain, whereas
KBs does not and
supports it only weakly. This difference in brain expression correlates with a
reduced courting intensity and fertility of the Poxn males rescued by
KBs when compared with those rescued by
XBs
(Fig. 4). Interestingly,
Poxn males whose Poxn expression is rescued in the brain by
XBs exhibit similar courting intensity and fertility as
Poxn males rescued by
KX, which show only a faint
expression in the brain, but whose taste bristles on legs and wings are
largely rescued (Fig. 4). By
contrast, Poxn males rescued by
KBs exhibit a
considerably reduced fertility. Because these males show only faint expression
of Poxn in the brain and have no taste bristles, it follows that male
fertility is enhanced by the rescue of high levels of Poxn expression in the
developing brain by
XBs to a similar extent as through the
rescue of most taste bristles on legs and wings by
KX.
The faint Poxn expression observed in the brain of Poxn males
rescued by BasiK is important for their initiation of courtship under
red light, as evident from the following observations. If late Poxn expression
in `chemosensory' neurons associated with the transformed mechanosensory
bristles is rescued by E77 or C1, Poxn males initiate
courtship at daylight, but not when their vision is compromised under red
light (Fig. 4). With
C1, however, additional rescue of their embryonic ventral cord
expression is observed. It follows (1) that late Poxn expression in these
neurons is not sufficient to rescue any male courtship behavior under red
light, even if Poxn expression in the ventral cord is rescued as well, and (2)
that ventral cord expression does not affect initiation of courtship at
daylight. In addition to the late Poxn expression in the remaining
`chemosensory' neurons, BasiK rescues only the faint expression in
the brain, which is not rescued by C1 or E77. Therefore, the
observed initiation of courtship by
PoxnM22-B5; BasiK males under red
light probably depends on the faint brain expression rather than the late
expression in `chemosensory' neurons, also observed in Poxn males
rescued by C1 or E77, which exhibit no courtship behavior
under red light (Fig. 4).
Expression of Poxn in the brain is also important for the initiation of male
courtship at daylight. This is not only evident from a comparison of courtship
behavior of Poxn males rescued by
KBs or
XBs, but also from the observation that at daylight the
courting activity of Poxn
M22-B5 males is
significantly enhanced by the BasiK or XK transgenes
(Fig. 4).
In summary, male courtship behavior strongly depends on an enhancer that regulates Poxn expression in the adult brain.
A Poxn wing hinge defect impairs flying but not wing
extension and vibration during male courting
Homozygous PoxnM22-B5 flies display
morphological aberrations in the hinge region of the wing, a Poxn
phenotype described previously (Awasaki and
Kimura, 2001
). Although homozygous
Poxn
M22-B5 mutants cannot fly properly,
males are able to extend and vibrate the wing during the initial courting
steps. The ability to fly is controlled by an enhancer located downstream or
in the last intron of Poxn as it is rescued by the SuperA or
E77, but not by the Full transgene
(Fig. 1B). Because the courting
song of Poxn males was not examined, it is not clear if it is
affected by the absence of this Poxn function. However, when
Poxn
M22-B5; Full males are
compared with completely rescued
Poxn
M22-B5; SuperA males, no
significant time difference between their onsets of courting and copulation is
observed (Fig. 4). This finding
suggests that the wing hinge phenotype of Poxn males does not
interfere with courting.
![]() |
DISCUSSION |
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These male courtship and fertility functions of Poxn include
functions required for the development of (1) taste bristles on tarsal
segments and tibia, anterior wing margin and labellum, whose chemosensory
neurons in part respond to female pheromone signals; (2) a ventral and
dorsolateral cluster of neurons in the brain, entrusted with targeting the
antennal lobe, ellipsoid body, lateral triangle and at least three additional
centers in the brain and processing signals, some of which presumably
originate from stimulatory olfactory signals propagated by the antennal nerve;
(3) specific neurons in the larval ventral nerve cord during embryogenesis, on
which the copulation behavior of the male depends; and (4) male genitalia,
including penis, posterior lobes and claspers. The multitude of these
courtship functions emphasizes the redundancy in the exchange of sensory
information between males and females during courtship, an essential feature
common to all communication systems
(Shannon, 1948a;
Shannon, 1948b
), and obviously
crucial for the survival of the species and its evolutionary success
(Greenspan and Ferveur,
2000
).
In addition, we have identified enhancers (W. B. and M. N., unpublished)
for previously reported Poxn functions affecting the segmentation of
legs and antennae and the structure of the wing hinge
(Fig. 1C)
(Awasaki and Kimura, 2001). Our
analysis correlates the activity of enhancers not only with the expression
patterns which they control, but also with the partial and complete rescue of
mutant phenotypes of structural and behavioral nature. This approach thus
reveals not merely the size, but also the complex arrangement and
substructures of enhancers. The amazing complexity of the organization and
substructures of the enhancers reflects their evolutionary history and thus
may provide insights into the origin of their present functions.
Complex arrangement and substructures of Poxn enhancers
The dissection of the entire Poxn control region into different
enhancers regulating its many functions is illustrated in
Fig. 1C. The overall
arrangement of the enhancers reveals an astounding density and complexity. We
have delimited 11 Poxn enhancers in this study and estimate a total
of at least 15, if we include our unpublished results that characterize
enhancers active mainly in the embryo (Fig.
1C). Thus, at least nine enhancers are located in the upstream
region, five in the introns and one in the downstream region of Poxn.
Not all enhancers are separable from each other, but some overlap or
interdigitate. For example, the enhancers for taste bristle development on
legs and wings overlap to a large extent, but are not identical. Moreover, the
region over which they extend includes completely the enhancers for (1)
embryonic ventral cord expression, (2) penis development and (3) larval p-es
organ development (W. B. and M. N., unpublished). Two extreme models for the
arrangement of these five enhancers are conceivable. They truly overlap by
sharing all or in part some of the same transcription factor binding sites, or
they interdigitate without sharing any binding sites. The two models are, of
course, not mutually exclusive, and in the case of Poxn enhancers we
might indeed deal with a mixed model. Thus, the leg and wing taste bristle
enhancers, located in the XbaI-HindIII fragment, might share
binding sites at both ends (Fig.
1C), while the central region might include part of an
interdigitating wing bristle enhancer and not be required for the development
of leg taste bristles (Fig. 4;
W. B. and M. N., unpublished). Interestingly, this region also includes the
enhancers for penis development, which thus might be interdigitating or
overlapping with this part of the wing taste bristle enhancer.
The leg and wing taste bristle enhancers exhibit a complex substructure. They both depend at their proximal end on binding sites in the XbaI-PstI fragment, which cannot activate Poxn transcription sufficiently to support taste bristle development. Only the addition of the adjacent upstream region supports taste bristle development in distal parts of both legs and wings, while further addition of a large central region affects taste bristle development only in the wing. However, we do not know if this central region is required, together with the most distal region of the XbaI-PstI fragment, to support the development of taste bristles in the tibia and in the first and third tarsal segments. Both proximal and distal parts of the leg or wing taste bristle enhancer are active only in cis, but not in trans, with each other or with the central region (Fig. 4; W. B. and M. N., unpublished), which implies that the leg and the wing bristle enhancer, included in the 5.2 kb XbaI-HindIII fragment, are both single enhancers rather than each being composed of several independent enhancers.
An additional complication of the leg taste bristle enhancer is the fact
that, if intron and downstream control regions are absent, it produces in the
tibia a large excess of ectopic taste bristles at the expense of
mechanosensory bristles, an effect that is more pronounced in the male than in
the female (Fig. 4; W. B. and
M. N., unpublished). Thus, the balance between taste and mechanosensory
bristles in the tibia, yet not in other leg segments, clearly depends on the
presence of additional intron and downstream elements. Interestingly, one to
three ectopic taste bristles, located in the proximal region of the wing, are
similarly suppressed by the additional presence of introns
(Fig. 4; W. B. and M. N.,
unpublished). This situation is further complicated by preliminary results
with PoxnM22-B5 flies rescued by a Poxn
transgene that completely lacks the upstream enhancers for wing and leg taste
bristles, but includes all downstream and intron enhancers. As expected, all
labellar taste bristles of these flies are rescued. Surprisingly, however,
some of the leg taste bristles are rescued only in male forelegs, but none in
female legs, while all ventral and about a third of the dorsal wing taste
bristles are rescued in both males and females. It appears, therefore, that
the downstream labellar taste bristle enhancer shows considerable redundancy
with the upstream wing and leg taste bristle enhancers, yet not vice versa
(Fig. 1C). Future detailed
analysis of which binding sites are part of these enhancers is expected to
shed light on their intricate structure and function relationships and to
reveal insights into their evolutionary origin.
Role of Poxn in the development of leg, antennal, wing and
male genital discs
In addition to the enhancers that control male courtship functions
discussed below, we have identified two enhancers
(Fig. 1C) whose function is
required in the male genital disc for the development of the penis, claspers
and posterior lobe (Fig. 5).
Moreover, in leg and antennal discs, Poxn is expressed in, and
required for the development of, two segment primordia (W. B. and M. N.,
unpublished) that give rise to homologous segments
(Fig. 4)
(Postlethwait and Schneiderman,
1971). Their homology is reflected at the molecular level by the
fact that their expression in leg and antennal discs is regulated by the same
enhancer (Fig. 1C). It has been
proposed that the genital disc is a ventral disc which behaves in a manner
similar to the leg and antennal discs (e.g.
Gorfinkiel et al., 1999
).
However, our identification of two enhancers required for the development of
penis, claspers and posterior lobes that are different from the leg/antennal
enhancer argues that these structures are not homologous to the leg/antennal
segments and that the genital and leg/antennal discs may exhibit only a
distant evolutionary relationship (Hadorn,
1978
). A similar argument can be made for the Poxn
enhancer, the function of which is required in the wing disc
(Fig. 1C) for proper
development of the wing hinge (Awasaki and
Kimura, 2001
) (W. B. and M. N., unpublished). It therefore appears
that the wing is not homologous to the leg or antenna, but only distantly
related to it, a notion in agreement with the current model
(Wigglesworth, 1973
;
Kukalová-Peck, 1983
;
Averof and Cohen, 1997
).
Nevertheless, it is intriguing that Poxn has acquired during
evolution enhancers that regulate functions in leg/antennal, wing and genital
discs. Rather than homology of the structures derived from the different parts
of the discs expressing Poxn, Poxn activity may reflect the close
relationship of the gene networks in which Poxn participates, in
agreement with the gene network hypothesis
(Noll, 1993
).
Role of Poxn in the reception of pheromone signals through
taste bristles
Our analysis of enhancer functions was not limited to their more direct
effects such as, for example, the regulation of Poxn expression in developing
taste bristles and specification of their chemosensory fate. Our main interest
in this study was rather to assess and measure the indirect effects of
separate Poxn enhancers and functions on male fertility and courting
behavior. We were able to test primarily the first step in this communication
system, by which females arouse the interest of males, which react by
extending and vibrating a wing and thus initiate courting the female
(Greenspan and Ferveur, 2000).
The male receives through its sensory organs, and reacts to, many types of
signals, which are additive and, at least under laboratory conditions,
redundant, i.e. as long as the combined input signal exceeds a threshold, the
male begins courtship (Hall,
1994
). Our approach to analyze the various Poxn functions
by a dissection of its enhancers allows us to answer which Poxn
functions are involved in the reception and processing of these signals. These
partially characterized courtship functions of Poxn can be divided
into primary functions of the peripheral nervous system, which receives and
propagates the different signals, and secondary functions of the CNS and
brain, on which processing and integration of the signals depends.
The following sensory modalities play a role in courtship
(Greenspan and Ferveur, 2000):
(1) visual input received by the photoreceptors of the eye; pheromone signals
received (2) by gustatory receptors in the neurons of the taste bristles on
legs, wing and labellum, and (3) by olfactory receptors in the neurons of the
olfactory sensilla in the third antennal segment and maxillary palp; (4)
auditory signals innervating neurons of the chordotonal organs of Johnston's
organ in the second antennal segment; and (5) mechanosensory input innervating
neurons of mechanosensory bristles. Courting tests with Poxn mutant
males some of whose Poxn functions have been rescued showed that
these are important only for the reception of signals by neurons of taste
bristles. As Poxn is never expressed in developing and adult
olfactory or chordotonal organs and mechanosensory bristles, it might have
functions in the reception of only the first two types of signals. However, an
essential function of Poxn in the reception of light input is ruled
out by two observations: Poxn
M22-B5 males are able
to initiate courtship at daylight, but not under red light
(Fig. 4), and w;
Poxn
M22-B5 double mutants also fail to initiate
courtship at daylight. This result further supports the notion that visual and
chemosensory taste and olfactory inputs play the major role in the initiation
of male courtship behavior, while mechanosensory and auditory inputs play a
subordinate role (Cook, 1980
;
Tompkins et al., 1980
;
Gailey et al., 1986
;
Markov, 1987
;
Heimbeck et al., 2001
).
Moreover, it demonstrates that the visual input alone is sufficient to trigger
male courtship, though at a much reduced efficiency when compared with the use
of all sensory modalities affecting courtship initiation. Finally, it follows
that Poxn plays an important role in the reception of pheromones by
gustatory receptors as evident from the observation that the latency times of
courtship initiation at daylight and under red light are considerably
prolonged by the selective removal of taste bristle functions (compare with
rescue of Poxn
M22-B5 by
XBs and
Full in Fig. 4). The
fact that the selective removal of all taste bristles does not eliminate
courtship in the dark strongly suggests that Poxn has functions
crucial for the processing of signals elicited by female pheromones in the
olfactory receptors (see below). It further follows that not only visual, but
also olfactory input alone is sufficient to trigger male courtship, though
also at reduced efficiency, illustrating the redundancy of the system.
At present, we cannot answer what the contributions of leg, wing and labellar taste bristles are in the reception of the female pheromone signals. We do not know, for example, if wing taste bristles recognize pheromones or are redundant for this function because no significant change in courtship behavior is noticed between males rescued by SaK or BaK despite a considerable difference in the number of wing, but not leg or labellar, taste bristles (Fig. 4). These and related questions are now amenable to an experimental approach if future analysis of the taste bristle enhancers permits a selective removal of the different taste bristle functions.
It may be important that Poxn is expressed in chemosensory neurons of
prothoracic legs that connect contralaterally in the male, but not in the
female (Possidente and Murphey,
1989). This and the additional sexual dimorphism that males have
about 50% more chemosensory bristles on their forelegs than females
(Nayak and Singh, 1983
)
suggest that pheromone receptors on male leg taste bristles are restricted to
the forelegs.
Role of Poxn in processing olfactory signals in defined
neurons of the brain
We have identified two Poxn enhancers that regulate secondary
courtship functions of Poxn, one active in the developing brain
(brain enhancer), the other in the embryonic ventral CNS (ventral cord
enhancer). Our results suggest that Poxn expression under control of the brain
enhancer in the developing and adult brain is crucial for the proper
processing of courtship signals elicited by female pheromones in the olfactory
receptors (see above). This conclusion is further supported by comparing
Poxn males without taste bristle and ventral cord functions in the
presence (PoxnM22-B5; XK) and absence of the brain
function (Poxn
M22-B5; E77). In the absence of the
brain function, these males do not initiate courtship under red light, while
no difference in courting between the two types of males is apparent in
daylight (Fig. 4). Similarly,
if we compare Poxn males without taste bristle functions, but with
the ventral cord function, in the presence (Poxn
M22-B5;
XK) and absence of the brain function (Poxn
M22-B5;
C1), we find that the brain function is crucial for courting under red
light, but not in daylight (Fig.
4). As these males have no taste bristle input, they are able to
court only in the absence of proper visual input if the olfactory input is
processed by the brain function of Poxn. Our results, therefore,
demonstrate that the Poxn brain function is necessary for the
processing of olfactory input, to which the ventral cord function does not
contribute. This conclusion is also consistent with the observation that the
antennal lobe, which receives the olfactory signal through the antennal nerve,
is targeted by the Poxn-expressing ventral and ventrolateral neuronal clusters
in the brain (Fig. 7D,F,G). In
the Poxn mutant, however, the Poxn-expressing neurons in the brain
fail to make their proper connections (Fig.
7E).
In summary, Poxn includes two courtship functions involved in the reception and processing of sensory input: (1) the reception and propagation of female pheromone signals through taste bristles, and (2) the processing in the brain of olfactory signals elicited by female pheromones. A third function of Poxn during copulation is discussed below. Because the brain enhancer has not been removed completely in any of the Poxn rescue constructs that include taste bristle enhancers, it is possible that the brain enhancer is also required for the processing of signals received from taste bristles. Similarly, we cannot exclude a role of the brain enhancer in the processing of mechanosensory and auditory input.
Role of Poxn in the processing of courtship signals during
copulation
Also the function of the ventral cord enhancer appears to be required for
the processing of sensory signals that affect male courting behavior. However,
in contrast to the brain enhancer, the ventral cord enhancer controls a
Poxn function that does not influence the initiation of courtship,
but somehow affects the success of copulation. In the absence of the ventral
cord function, males attempt to copulate, but are unable to attach themselves
to the female genitalia or soon fall off after copulation and remain on their
back shivering for several minutes before they recover. By contrast, the
ventral cord function is dispensable for the processing of input received
through taste bristles. This is evident from the courtship behavior of males
that lack the ventral cord but not the brain function:
PoxnM22-B5; SaK males, many of whose leg and wing
taste bristles are rescued, initiate courtship at a considerably enhanced
frequency both at daylight and under red light when compared with
Poxn
M22-B5; XK males, which have no taste bristles
(Fig. 4). It follows that the
ventral cord function is not primarily required for the reception of signal(s)
received from sensory organs, but rather for their processing and input into
the efferent nervous system, such as for the activation of certain
motorneurons required to initiate and maintain copulation.
To evaluate how the ventral cord function influences copulation, we
compared PoxnM22-B5;
SH males, whose
genitalia are rescued and thus are able to copulate, in the presence and
absence of the ventral cord function carried by C1, which cannot
contribute to taste bristle development in trans
(Fig. 1B,C). As evident from
Fig. 4, the ventral cord
function dramatically enhances successful copulation and hence male fertility.
Thus, the ventral cord function of Poxn is not responsible for the
processing of courtship signals, except during its last phase to initiate and
maintain copulation.
What could the ventral cord function of Poxn be? Most probably it is required to orchestrate the complex movements of copulation. Thus, the male initiates copulation by bending the abdomen forward, attaching its end to the female genitalia through its claspers, mounting the female and inserting the penis into the vagina. It maintains this position by anchoring the penis within the vagina. Apparently, this complex coordinated movement of male abdomen and genitalia, which is regulated by efferents of motorneurons, is disturbed by the absence of the ventral cord function of Poxn. Specifically, mechanosensory signals in the male genital region may not be properly processed and thus impair the coordinated movement regulated by motorneurons.
An intriguing feature of the ventral cord function of Poxn is its
early expression during embryogenesis, whereas its mutant phenotype becomes
apparent only in the adult. A probable explanation is that many of the larval
ventral cord neurons specified during embryogenesis persist to the adult stage
(Truman, 1990). Although they
constitute only a small fraction of about 5-10% of the adult CNS, they
contribute disproportionately to certain neuronal classes and thus may provide
clues that are important for the proper organization of the adult CNS
(Truman et al., 1993
). Future
studies investigating the function of Poxn in these neurons might not
only shed light on their role in adult courtship behavior, but also on the
complex developmental changes in the neurons of the larval CNS that are
conserved during metamorphosis.
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ACKNOWLEDGMENTS |
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