1 Molecular Biology, Cell Biology, and Biochemistry Department, Brown
University, Providence, RI 02912, USA
2 Molecular Biology and Virology Laboratory, The Salk Institute for Biological
Studies, La Jolla, CA 92037, USA
3 Department of Biology, University of California, San Diego, La Jolla, CA
92093, USA
4 Department of Zoology, Oregon State University, Corvallis, OR 97331, USA
Author for correspondence (e-mail:
michael_mckeown{at}brown.edu)
Accepted 4 November 2004
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SUMMARY |
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Key words: Courtship, Behavior, retained, fruitless, Neuronal pathfinding, Drosophila
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Introduction |
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This binary behavioral system is controlled by the sex differentiation
cascade (Hall, 1994;
Yamamoto et al., 1998
;
O'Kane and Asztalos, 1999
;
Goodwin, 1999
;
Christiansen et al., 2002
).
Sex-lethal (Sxl), transformer (tra) and
transformer 2 (tra2) catalyze splicing of the next step of
the pathway, leading to the activation of sex-specific forms of
doublesex (dsx) and fruitless (fru).
dsx controls external differentiation, yolk protein synthesis,
aspects of male song production (Villella
and Hall, 1996
) and potentially some aspects of female neural
differentiation (Waterbury et al.,
1999
). fru determines many aspects of male courtship and
copulatory behaviors, but has no apparent role in female sexual development
(Ryner et al., 1996
;
Ito et al., 1996
;
Gailey et al., 1991
;
Villella et al., 1997
).
dissatisfaction (dsf) females resist males during courtship,
whereas dsf males are bisexual
(Finley et al., 1997
;
Finley et al., 1998
). Many
male courtship mutants have been identified, while few mutations linked to
female receptivity have been characterized
(Yamamoto et al., 1997
).
We identified retained/dead ringer (retn) from a genetic screen for female behavioral mutations. retn females are resistant to courtship, and show fru-independent male-like courtship behaviors, while retn males are behaviorally normal. These sex-specific effects on behavior do not correlate with sexually distinct expression or splicing patterns in the CNS. Examination of retn cells in retn mutant backgrounds reveals aberrant projections by mushroom body, photoreceptor and subesophageal neurons. retn affects development of sex-specific neurons, and may repress male behavior patterns in the female CNS.
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Materials and methods |
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Female resistance and male courtship indices were tested as previously
described (Finley et al.,
1997). Male-male courtship and female bisexuality were tested in
groups of 10 animals and quantitated as number of courtship events per
5-minute interval. A courtship event was counted as one fly following, tapping
or singing to a target fly for a minimum of 2 seconds. Multiple trials were
carried out for each genotype and age. All P-values are derived from
two-tailed paired t-tests. Multiple retn-Gal4 lines were
used to drive UAS-retn, UAS-TraF and
UAS-GFP. All generated the same pattern and had similar effects.
retn-Gal489, a lethal insertion, showed the most complete
rescue of female resistance behavior and egg laying, and was primarily used
for studies of retn function and expression. Rescue of male-like
behaviors in females was complicated by inconsistency of these behaviors in
retn-Gal4/retn mutants.
Sequencing
Genomic DNA from retnz2-428,
retnRO44, retnRU50,
retndri1 and retndriB142 flies was
amplified by PCR. Purified PCR product was sequenced at the Salk Sequencing
Facility (La Jolla, CA). Sequences were assembled using DNA Sequencher (Gene
Codes Corp, Ann Arbor, MI). Sequence comparison and database searches utilized
BLAST (Altschul et al., 1990)
and/or FASTA (Pearson and Lipman,
1988
).
RETN fusion and mutant expression: EMSA
Full-length retn cDNA was generated by PCR, using genomic DNA from
UAS-retn flies. The cDNA product was cloned into pBluescript-SK+
(pBS, Stratagene) and sequenced on both strands.
To produce ARID-box subclones, the pBS-retn plasmid was used as template for further PCR. The subsequent retnARID product encodes amino acids 230-500 of RETN, and includes the ARID domain plus flanking sequence. This was subcloned into pBS and sequenced on both strands. pGEX-retnARID was produced by inserting a BamHI-XhoI fragment of pBS-retnARID into the BamHI and SalI sites of pGEX-KG.
BS-retnRO44ARID and BS-retnz2-428ARID vectors were generated using PCR-based site-directed mutagenesis of the pBS-retnARID template. Positive clones were confirmed by sequencing and transferred into pGEX-KG.
DNA-binding analysis was performed as described by Pitman et al.
(Pitman et al., 2002). RETN
wild-type and mutant fragments were expressed as GST-RETN fusion proteins in
BL21 pLysS bacteria. Proteins were purified and eluted
(Kaelin et al., 1992
). EMSA
analysis used 2 µl of eluted protein. Proteins were tested for relative
expression on a western blot, using rabbit anti-GST antibodies.
RT-PCR
For examination of retn RNA, CNS tissue (sans imaginal discs) was
isolated from both sexes of late third instar larvae or mid-stage pupae. Total
RNA was extracted using RNeasy Mini Kit (Qiagen). An antisense primer targeted
to either exon 11 or 12 of retn was used to prime DNA synthesis by
M-MLV Reverse Transcriptase (Sigma). A first round of PCR was carried out
using primers against exons 1 and 4, 4 and 8, 8 and 11, 8 and 12, 1 and 8, and
4 and 11. A second round of PCR was then carried out using primers internal to
those used in the first round. For examination of fru P1-derived RNAs
in retn mutants, RNA was isolated from mid-pupal CNS tissue and from
adult heads. For analysis of fru P1 RNAs in retn fru double
mutants, RNA was isolated from adult heads. The RT-PCR procedure was as above
with fru primers. For all fru RNA tests, reverse
transcription was primed from within exon 3, which is common to all
fru RNAs. The 3' primer for both first and second round PCR was
placed just inside (more 5' on the RNA) to the RT primer. For analysis
of the fruM RNA, first round PCR was primed at the 5' side from
within promoter P1-derived exon 2. Second round PCR used a primer just
3' of this. For analysis of the fruF RNA, first and second
round 5' primers were just upstream of the TRA/TRA2 activated splice
site of fru.
Microscopy
Confocal images were obtained on Zeiss LSM 480 and LSM510 Meta microscopes,
using Renaissance 410 (Microcosm, Columbia, MD) software. Antibodies to Fas2
were obtained from the Developmental Studies Hybridoma Bank (University of
Iowa). The brains of mutant and wild-type males and females were labeled with
anti-Fasciclin 2 (Fas2) (1:20) and then with an anti-mouse secondary Alexa 488
(1:200; Molecular Probes) using standard methods
(Finley et al., 1997).
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Results |
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Alleles of dead ringer (dri), an extended ARID (AT-rich
interaction domain) Box-Family embryonic DNA-binding factor
(Gregory et al., 1996;
Iwahara et al., 2002
) also
fail to complement retn. We sequenced the exons and exon/intron
boundaries of dri in z2-428, retnRO44 and
retnRU50 (Fig.
1). Each allele has a single nucleotide change in the
dri-coding region, corresponding to an ARID box amino acid
substitution. Two dri lethal alleles, dri1 and
driB142, encode premature stop codons, truncating the
protein (Fig. 1). Thus,
missense alleles retnz2-428, retnRU50
and retnRO44 encode a protein with sufficient function
that mutant progeny survive to adulthood, while nonsense alleles are
lethal.
|
ARID box point mutants affect viability
retn mutant proteins have residual DNA-binding ability (data not
shown) consistent with survival of some mutant individuals to adult stages. We
asked whether these mutations alter the vital function of retn and to
what extent phenotypes may be limited to later functions. In examining
viability of retn heteroallelic combinations, we found variability in
eclosion rates (Fig. 2A) with
most lethality in the larval stages. Allelic strength in terms of pre-adult
mortality is retndri2 > retndri1
> retn-Gal489 > retndri8 >
retnz2-428 > retnRU50 >
wild-type. retnz2-428/retndri2 flies
eclose with only 8% of expected rates, while
retnz2-428/retndri1 flies eclose with
25% of expected rates.
retnRU50/retndri1 and
retnRU50/retndri2 eclose with 65% and
68%, respectively, of expected numbers. P-element insertion alleles show full
or nearly full viability with retn-class alleles. retn
lethal alleles show no dominant lethality. Thus, all retn-class
alleles at least partially complement the vital functions of retn. In
addition, the retn cDNA rescues the partial lethality of
retnz2-428/retn-Gal489
(Fig. 2B). retnz2-428/retn-Gal489 flies eclose
with 33% of expected numbers, while
retnz2-428/retn-Gal489;
UAS-retn flies eclose with 100% of expected numbers.
|
|
retn females show male courtship behaviors
retn females show one behavior not shown by dsf, dsx or
fru females: male-like courtship of females and males, especially as
they age (Fig. 3C-F).
retn females follow, tap and appear to sing. Although not as robust
as male courtship - following is not as sustained, full wing extension and
vibration are not seen, and copulatory bending is weak or absent - these
behaviors highly resemble courtship. Fig.
3 shows still frames of this behavior, directed towards females
(Fig. 3C,D) or a courting male
(Fig. 3E). These behaviors vary
between and within allelic combinations, but when the behaviors are seen they
are striking and continue for hours.
retnz2-428/retndri8 females, which
show the most consistent behaviors, with maximum penetrance at 3-4 weeks
post-eclosion, averaged 42 courtship events per 5-minute observation period
(Fig. 3F), while control
females display fewer than three courtship-like events in the same period.
Although male behaviors are evident, the fruM-dependent Muscles of
Lawrence are not seen in retn females (not shown and L. M. Ditch, PhD
thesis, University of California, 2002).
Aspects of the retn female behaviors are similar to wild-type
female defenses of food and egg-laying resources. One study on
Drosophila aggressive behaviors
(Ueda and Kidokoro, 2002)
indicated that aggression in wild-type females increases if females are raised
individually before pairing for observation. We found no increase in male-like
behaviors in females kept separately from eclosion until testing (not shown;
L. M. Ditch, PhD thesis, University of California, 2002). This suggests that
these behaviors are not an exaggerated defense response. Other indications
that these behaviors are not based on access to food come from observations of
wild-type females starved overnight on moistened filter paper and transferred
back onto food. These females showed short head-to-head and head-to-side
interactions, but did not show behavior resembling male courtship. Courting
retn females, by contrast, primarily show posterior orientation
(Fig. 3C,D), and will follow
other females on and off a food source for minutes at a time.
Male-like behaviors in retn females are not dependent on fru
Genetic data indicate that males lacking fruM (P1 derived)
transcripts show a `complete absence of sexual behavior'
(Anand et al., 2001). However,
we observe male-like courtship by retn mutant females, which should
lack fruM (Ryner et al.,
1996
). This suggests three possibilities: (1) retn
mutants could lead to an up regulation of fruM in females; (2) there
could be a very low level of fruM in wild-type and retn
females, which, in the absence of retn, is sufficient to induce some
male behavior; or (3) there could be an intrinsic, but weak,
fru-independent pathway for male behavior that is repressed by
retn or retn-expressing neurons (see Discussion for a model
incorporating this idea). We have tested these possibilities.
As fruM RNA expression is male specific and is eliminated in females by TRA- and TRA2-mediated splicing of P1 transcripts into the fruF RNA form, we expect no increase in fruM in retn females. We addressed whether retn loss-of-function leads to upregulation of fruM in females. RT-PCR with one round of amplification using primers against fruM gave no detectable fruM product in Canton S or retn- midpupal or aged-adult female CNS tissue (data not shown). A second round of amplification showed an extremely low signal for fruM in equal amounts in both wild-type and retn- CNS tissue (data not shown). These results indicate that fruM is not upregulated in retn- CNS tissue, although the small amount of fruM detected in the second round of amplification might be responsible for the male-like behaviors in retn females.
We tested the dependence of the male-like behaviors in retn
females upon the observed amount of fruM.
Df(3R)fru4-40 removes the P1 (responsible for transcripts
under tra/tra2 control) and P2 promoters, leaving the P3 and
P4 promoters intact. Df(3R)fruAJ96u3 removes P4 and the
entire fru protein coding region
(Song et al., 2002).
fru4-40/fruAJ96u3 flies lack P1
derived transcripts, but are healthy because of P3 and P4 activity
(Song et al., 2002
). RT-PCR
analysis with two rounds of amplification upon CNS tissue from these females
indicated a complete absence of fruF and fruM (data not
shown), as expected. We tested for male-like behaviors by
retn-; fru- females
(retnz2-428/retndri8;
fru4-40/fruAJ96u3). Such females aged
for
2.5 weeks, produced retn-like male behaviors
(Fig. 3G,H), indicating an
independence of such behaviors from fruM. In addition, similarly aged
retn- females carrying a different fruM null
allelic combination
[Df(3R)frusat15/Df(3R)fru4-40
(Anand et al., 2001
)] also
display substantial male-like courtship behavior (not shown). Taken together,
these data indicate that the male-like behaviors observed in retn
females are specified by a means independent of fruM.
retn does not alter male behaviors
We tested if retn alters male behaviors or functions.
retn males court females, are not delayed in copulation
(Fig. 4A), do not show
significant courtship of other males (Fig.
4B) and have normal Muscles of Lawrence. retn males
produce motile sperm and copulate normally, but show defects in sperm transfer
and are partially sterile (L. M. Ditch, PhD thesis, University of California,
2002).
|
Alternative splicing of retn transcripts does not show sex specificity
As retn has female-specific phenotypes, we asked if it is a direct
target of regulation by Tra/Tra2-mediated alternative splicing focusing on
central nervous system RNAs, as retn has non-sex-specific functions
in other tissues (Gregory et al.,
1996; Shandala et al.,
1999
; Shandala et al.,
2002
; Bradley et al.,
2001
; Iwaki et al.,
2001
). We analyzed RNA from the larval CNS, prior to the most
sensitive period for sexual nervous system differentiation, and the early/mid
pupal CNS, the primary period of sex-specific nervous system determination
(Belote and Baker, 1987
;
Arthur et al., 1998
).
retn has 12 exons, most of which are separated by small (fewer than 100 nucleotides) introns (Fig. 1). Exons 1 and 2, 4 and 6, and 6 and 7 are separated by large (multiple kb) introns, while exons 11 and 12 are separated by a 182 base intron. We used RT-PCR to analyze alternative processing between exons 1 and 4, 1 and 8 (pupal only), 4 and 8, 4 and 11 (pupal only), 8 and 11, and 8 and 12 (not shown). The data (Fig. 5) show the expected products, and two novel variants. None of these is sex-specific, which is completely consistent with the rescue of retn female behavioral (Fig. 3A,B) and egg-laying phenotypes using a common form cDNA.
|
retn is expressed in the CNS during pupal stages when sexual behavior is hardwired
To map retn expression in the CNS, we examined
retn-driven GFP expression using retn-Gal4 insertions that
rescue retn phenotypes with the retn cDNA. These Gal4
enhancer traps, in addition to rescuing retn viability and behaviors,
exactly reproduce Retn antibody patterns in embryos and larval eye tissue
(Shandala et al., 1999) (J.
Sibbons, personal communication); therefore, they should represent the later
CNS expression to a high degree of accuracy. Expression and projections were
monitored using membrane-associated UASCD8::GFP (UAS-mGFP). retn
expression in the CNS begins in the embryo
(Gregory et al., 1996
;
Shandala et al., 2002
), and
continues through adulthood, in specific subsets of neurons. As we were
primarily interested in neurons involved in adult behaviors, we focused on
expression of retn in the periods before and during metamorphosis,
when adult neurons are born and larval neurons are remodeled into
adult-specific forms. Notably, we see expression in the mushroom bodies,
subesophageal ganglion, ventral ganglion and developing photoreceptors. These
patterns are essentially the same in both sexes.
Mushroom body (MB)
In the third instar, MB expression is seen in the Kenyon cell (KC) bodies
lying in the dorsoposterior of the central brain, with staining in the calyx,
containing KC dendrites, and the pedunculus and lobes, containing KC axons
(Fig. 6D). Between 12 and 18
hours after puparium formation (APF), the calyx retracts, the and
ß lobes narrow and what appears to be axonal debris can be seen at the
lobe tips (arrow, Fig. 6E). At
this stage there are slightly more retn cells in females than in
males, perhaps reflecting the greater axon number in female MBs
(Technau, 1984
). By 36 hours
APF, the adult
,
', ß, ß', and
lobe projections are visible, although retn expression is stronger in
/ß projections (Fig.
6F). Between 24 and 48 hours APF, expression in all lobes except
/ß gradually fades, and by 48 hours only the
/ß lobes
can be seen. This pattern remains through the rest of metamorphosis.
|
Ventral ganglion
In the larval ventral nerve cord (VNC), 18 paired dorsal lateral neurons,
nine per side, send projections towards the midline
(Fig. 6J). These may mediate
signaling to or from the nine larval abdominal segments. By 24 hours APF, the
abdominal neurons are now six pairs, residing at the abdominal tip
(Fig. 6K). Beyond 36 hours APF
and continuing into adulthood, three sets of paired abdominal neurons are
visible (Fig. 6L). These final
neurons may project outwards from the CNS. A small subset of adult peripheral
sensory neurons that innervate the female reproductive structures also send
their
Eye
retn-Gal489 is expressed posterior to the morphogenetic
furrow, in photoreceptor cells R1-R6, which project to the lamina and R8,
which projects to the medulla (not shown), as is also seen with Retn antibody
staining (J. Sibbons, personal communication). Beyond 48 hours APF, R8
expression and projections fade, although lamina projections remain (48 hour
pupal eye, Fig. 7J). Expression
in the eye, MB, SOG and ventral nerve cord is still visible post-eclosion
(Fig. 6C, early adult).
|
Neuronal birthdates and pathfinding errors in mutant clones
To determine retn neuronal birth dates and the neural phenotypes
of dri-class alleles, we used the MARCM system
(Lee and Luo, 1999), which can
simultaneously create homozygous mutant cells and allow them to express
Gal4-regulated marker genes. retn-expressing MB neurons are born
throughout the larval and pupal stages and eye clones appear at all embryonic
and larval stages. The VNC neurons are born only within 48 hours of egg
laying, and SOG retn neurons are born in 8-hour-old or younger
embryos.
Homozygous retn-Gal489 clones show striking mis-projection phenotypes in SOG neurons. The normal elaboration and symmetry of arbors in mid-pupae is diminished; ventral dendritic branches do not show normal density (compare arrowhead in Fig. 7F with arrowhead in Fig. 7G), and anterior projections wander and fail to extend (compare arrows in Fig. 7F and Fig. 7G). Neurons also fail to fasciculate normally. A central SOG midline-crossing tract, visible throughout metamorphosis, contains tightly bundled projections (arrow, Fig. 7H). In mutant clones, projections stray from this tract, apparently losing some adherent ability (arrow, Fig. 7I). Photoreceptor neurons also mis-project. In retndri clones, induced in the embryo, R1-R6 cells overshoot the lamina, and a number now target the medulla (ME, arrows; Fig. 7J, wild type; Fig. 7K, mutant). Although retn mutations alter neuronal projection patterns, and projection differences are consistent with changes in behavior, we have not yet mapped retn behavioral functions to a particular set of neurons, nor have we demonstrated that the projection differences, as opposed, for example, to retn-induced reductions in neural activity, are responsible for behavioral changes.
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Discussion |
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retn neural and behavioral phenotypes are substantially different
from those of dsf or fru. dsf females, like
retn-females, are sterile and resist male courtship
(Finley et al., 1997). For
dsf, sterility results from loss of motor synapses on the circular
muscles of the uterus (Finley et al.,
1997
). By contrast, these synapses are intact in retn
females. dsf females show no male behaviors
(Finley et al., 1997
), while
retn females do. dsf males are bisexual and slow to
copulate, owing to inefficient abdominal bending, correlated with abnormal
synapses on the muscles of ventral abdominal segment 5
(Finley et al., 1997
).
retn males court and mate with normal kinetics and have normal A5
synapses. This suggests that retn and dsf have largely
separate functions.
retn and fru also have different phenotypes. In a
wild-type background retn behavioral phenotypes are restricted to
females. fru behavioral phenotypes are restricted to males and
include failure to attempt copulation, bisexual and homosexual courtship, and,
in the strongest allelic combinations, complete lack of male courtship. In
addition, fru males lack the male-specific muscles of Lawrence in
dorsal abdominal segment 5. retn males have normal muscles of
Lawrence, and retn females do not have muscles of Lawrence. In
addition, the larval and pupal expression patterns of retn (this
paper) and the sex-specific products of the fru P1 promoter
(Lee et al., 2000), notably
the active male-specific fru proteins, show little or no overlap.
This all suggests that fru and retn are unlikely to interact
intracellularly and would be expected to be involved in different aspects of
behavioral control.
The latter conclusion seems to be contradicted by the male-like courtship
generated by retn females, as previous work demonstrates that
otherwise wild-type males require FRU-M to generate male behavior
(Anand et al., 2001). We have
operationally and molecularly shown that the male behavior generated by
retn females occurs even in the absence of fru P1
transcripts (Fig. 3G,H).
A model for the roles of fru and retn in male sexual behavior
We have developed a plausible working model that reconciles the data on the
necessity of fruM in males and male-like courtship by retn
females. The largely non-overlapping expression patterns of fru and
retn suggests that the formal interactions of this model will result
from interactions between networks of fru- and
retn-influenced neurons rather than by intracellular regulatory
interactions involving FRU-M and RETN, although the model can accommodate
either situation.
Our model posits that in the absence of fruM and retn the nervous system has an inherent tendency to set down some rudiments of neural pathways for male courtship behavior (Fig. 8A).
|
Finally, in wild-type males, fruM or cells expressing fruM, perhaps along with other factors such as dsxM, act to strengthen the male courtship pathway such that the repressive action of retn-expressing cells is overpowered (Fig. 8C). This makes fru the switch that results in male behavior and captures both the requirement for fru+ in males, and the male-like courtship by retn females.
This model does not rule out involvement of other components. For example,
work by Waterbury et al. (Waterbury et
al., 1999) suggests that dsxF can suppress male behaviors
in a retn+ background. This can be fitted into the model
as an additional female-specific block to male behavior in both
Fig. 8A and
8B. A simple prediction of such
a role for dsx is that reduction of dsx expression in a
retn mutant background will enhance the retn phenotype.
Recent work involving expression of fru RNAi in a subset of
fru neurons suggests a role for temporally repression in the
sequencing of male behaviors in courtship
(Manoli and Baker, 2004
).
An extensive series of experiments is in progress to test predictions of this model. Experiments are also in progress to determine if dsx participation fits within the context of the model, and to identify the molecules and mechanisms downstream of retn in the control of behavior.
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ACKNOWLEDGMENTS |
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![]() |
Footnotes |
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Present address, Ambit Biosciences, San Diego, 4215 Sorrento Valley
Boulevard, San Diego, CA 92121, USA
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