Fruitless Splicing Specifies Male Courtship Behaviour in Drosophila Ebru Demir and Barry J' Dickson

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Fruitless Splicing Specifies Male Courtship
Behaviour in DrosophilaEbru Demir and Barry J.
Dickson

• Male-specific fruitless specifies the neural
  substrates of Drosophila courtship behaviour
• Devanand S. Manoli, Margit Foss, Adriana
  Villella, Barbara J. Taylor, Jeffrey C. Hall and
  Bruce S. Baker

Danna Loder March 27th, 2006
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Overview

• Background Introduction
• Previous Research
• Purpose
• Results and Methods
• Discussion
• Conclusions
• Future Directions

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Background Introduction

• Animals are born not only with their
  characteristic body plan and morphology, but also
  a set of innate behaviours
• Our understanding of how innate behaviours are
  specified is limited
• Morphological development is often specified by
  switch or selector genes
• A switch gene turns developmental pathways on or
  off, and is both necessary and sufficient to
  trigger development

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• An example of a switch gene in Drosophila sex
  determination
• Regulatory switch gene is Sex lethal (Sxl)
• If Sxl is on, the female sex-differentiation
  pathway is turned on
• If Sxl is off, the sex-differentiation pathway is
  not activated and the default male sex results

Griffiths et al. (2000)
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Switch Genes Behaviour

• Are instinctual behaviours controlled by switch
  genes? Or, do instincts result from the combined
  action of many genes?
• If behavioural switch genes exist, they are
  likely to be found in the specification of sexual
  behaviours
• A switch gene for sexual behaviour should specify
  either male of female behaviour irrespective of
  the sexual phenotype
• A candidate switch gene is the fruitless (fru)
  gene in Drosophila
• Fru is linked to male sexual orientation and
  behaviour

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Male Courtship

• An elaborate ritual involving visual, olfactory,
  gustatory, tactile, acoustic and mechanosensory
  stimuli
• The male orients toward the female, extends a
  wing and vibrates it, serenading the female with
  a species-specific love-song, licks the females
  genitalia and copulates
• A sex-specific behaviour males only court
  females, and females do not court at all

Charalambos P. Kyriacou (2005)
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Fruitless gene (fru)

• Fru is linked to male sexual orientation and
  behaviour
• Tra protein is responsible for sex-specific
  splicing of fru mRNA

Ryner et al. (1996)
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Fruitless gene (fru)

• Fru locus spans 130kb, and includes 4 promoters
  (P1-P4)
• P2-P4 transcripts are not sex-specifically
  spliced
• P1 transcripts include the S exon, which is
  sex-specifically spliced by Tra and Tra-2
• Tra is absent in males S exon spliced at
  default male-specific donor site, resulting in
  FruM
• In females, Tra binds to fru P1 pre-mRNA to
  promote splicing at more 3 site, blocking
  production of FruM
• Male FruM
• Female FruM

Demir et al. (2005)
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Previous Research

• Certain loss-of-function alleles of fru disrupt
  male courtship behaviour and sexual orientation
• Performance of courtship ritual is significantly
  decreased and is directed at either sex
• Strong fru alleles completely block courtship
• Weaker fru alleles disrupt individual steps
• Studies suggest that fru is required for every
  step of courtship ritual

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Purpose

• To demonstrate that male-specific splicing of
  the fru P1 transcripts specifies male courtship
  behaviour and sexual orientation

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Results Methods

• Fruitless Splicing Mutants
• Generated four fru alleles by gene targeting
• 1) fruF prevents FruM
• 2) fruM forces FruM
• 3) fru?tra forces FruM
• 4) fruC control (splicing
  unchanged)

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1. fru regulates sexual behaviour but not gross
sexual anatomy

• Like fru, dsx is sex-specifically spliced by Tra,
  producing either male (DsxM) or female (DsxF)
• Dsx proteins direct morphological development but
  not behaviour
• Males lacking DsxM resemble females but still
  court
• Females with DsxM resemble males but do not court

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1. fru regulates sexual behaviour but not gross
sexual anatomy

• In this study Fru P1 splice mutants do not
  generally alter external or internal sexual
  anatomy
• fruF males have normal male anatomy
• fruM and fru?tra females have normal female
  anatomy
• One exception fruM and fru?tra females have a
  male-specific muscle of Lawrence

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2. Male courtship behaviour and sexual
orientation require male splicing

• Does male behaviour require male splicing?
• If yes fruF males should display little or no
  courtship
• - fruM and fru?tra males should behave
  normally
• To test prediction, used courtship, fertility,
  and chaining assays

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2. Male courtship behaviour and sexual
orientation require male splicing

• Male-Female Courtship Assays
• Test male paired with wild-type virgin female
• Percentage of time male courts female is recorded
  as his courtship index (CI)
• Wild-type, fruC, fruM and fru?tra males are avid
  courters (CI gt 70)
• fruF males barely court at all (CI lt 5)

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2. Male courtship behaviour and sexual
orientation require male splicing

• Competitive Mating Assays
• Wild-type virgin female placed with 2 males a
  test male and a wild-type competitor
• Observed which male succeeds in copulating with
  female
• fruF males always lost to wild-type male
• fruC, fruM and fru?tra males were as successful
  as wild-type competitor
• fruF males sterile
• fruC, fruM and fru?tra males fertile

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2. Male courtship behaviour and sexual
orientation require male splicing

• Sexual Orientation Assay
• Test male paired with wild-type male Male-male
  courtship is low for all genotypes
• However, fruF males court other males more
  actively than controls

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2. Male courtship behaviour and sexual
orientation require male splicing

• Chaining behaviour is a more reliable assay if
  leave test males on food plates, will form
  courtship chains in which each male courts the
  male ahead of him
• Movie of chaining behaviour
• Observed dramatically elevated levels of
  male-male courtship amongst fruF males compared
  to wild-type, fruC, fruM and fru?tra males

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3. FruM inhibits female reproductive behaviours
copulation and egg laying

• Examined sexual behaviours of females
• Virgin females placed with males and scored as
  sterile if flies were still alive but no progeny
  present after 20 days
• fruC and fruF females are as fertile as fru
  controls (gt99)
• Less than 25 of fruM and fru?tra females are
  fertile
• Reduced fertility may be due to behavioural
  rather than anatomical defects

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3. FruM inhibits female reproductive behaviours
copulation and egg laying

• Female Receptivity
• Virgin test female was paired with wild-type male
• fruC and fruF females almost always copulated
  within 60 min (gt94)
• lt16 of fruM and fru?tra females copulated

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3. FruM inhibits female reproductive behaviours
copulation and egg laying

• Female Receptivity
• In competition assays, wild-type male offered
  choice of two virgin females (test and wild-type)
• fruC and fruF females competed equally with
  wild-type females
• fruM and fru?tra females were never chosen

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3. FruM inhibits female reproductive behaviours
copulation and egg laying

• Female Fertility
• Took females that did mate and counted number of
  eggs laid over next three days
• fruC and fruF females laid gt65 eggs
• fruM and fru?tra females laid lt2 eggs

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4. Females behaving like males

• If fru is a behavioural switch gene, then fruM
  and fru?tra females should gain male behaviours,
  ie. Females should court other females
• Tested hypothesis using courtship and chaining
  assays
• fruM and fru?tra females court wild-type females

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4. Females behaving like males

• fruM and fru?tra females formed courtship chains
  similar to those formed by fruF males
• Neither fruC and fruF females show any
  female-female courtship

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5. Reversing the sex roles

• Males normally court females, not the other way
  around
• Courtship is driven in part by female pheromones
  produced in oenocytes
• Feminized oenocytes of male by ectopic expression
  of tra (oe-GAL4/UAS-tra)
• fruM and fru?tra females with oe-GAL4/UAS-tra
  males resulted in reversal of courtship roles
  females courted the males

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Discussion

• Fruitless (fru) gene of Drosophila is a switch
  gene for a complex innate behaviour the
  elaborate ritual of male courtship
• fru as a Switch Gene for Male Courtship
  Behaviour
• Confimed key predictions of hypothesis by showing
  that male splicing is necessary for male
  courtship behaviour and is also sufficient to
  generate male behaviour by an otherwise normal
  female
• Male courtship performed by fruM and fru?tra
  females is remarkable mimic of courtship by
  wild-type or control fruC males

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Discussion

• The distinct roles of fru and dsx in sexual
  development clearly illustrated
• Animals expressing i)DsxM but not FruM
  resemble normal males but do not court
• ii) FruM but not DsxM resemble
  normal females but do court
• FruM is both necessary and sufficient for male
  courtship, whereas DsxM is neither necessary nor
  sufficient

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Discussion

• How does fru specify male courtship behaviour?
• Constructed a fruP1-GAL4 construct which is null
  for P1-fru function
• By driving UAS-GFPnls with fruP1-GAL4, confirmed
  CNS expression patterns coincided with FruM

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Discussion

• mCD8GFP expression driven by fruP1-GAL4 revealed
  complex pattern of neuronal projections with many
  nerve bundles and neuropil structures no marked
  difference seen in males and females
• Suggests that FruM proteins do not specify
  distinct neural structures of function at level
  of pathfinding and early development
• Rather, FruM likely specify fine connectivity
  and/or physiology

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Discussion

• Examined fruP1-GAL4 expression throughout the
  body
• Expression found in all peripheral sensory
  systems implicated in courtship

• That FruM is expressed in subsets of sensory
  neurons suggests males and females detect
  distinct sensory stimuli at the level of sensory
  neurons themselves, or perceive sensory
  information in different ways

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Discussion

• Does FruM function in the primary and/or
  secondary olfactory neruons involved in male-male
  habituation?
• Inhibited FruM in majority of olfactory receptor
  neurons by expressing RNA-mediated interference
  transgene (UAS-fruMIR)
• Inhibition resulted in sustained male-male
  courtship compared to controls which showed a
  decrease in male-male courtship
• Thus, FruM function in olfactory receptor neurons
  and/or secondary neurons required for male-male
  habituation

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Discussion

• Second-order olfactory projection neurons project
  to mushroom bodies
• Male mushroom body ?-lobes are necessary for
  courtship conditioning to mated females
• Is FruM function in mushroom body neurons
  necessary for such conditioning?

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Discussion

• Analyzed conditioning in males in which FruM
  expression was inhibited in mushroom body neurons
  by UAS-fruMIR expression
• Reduced conditioning response
• Thus, FruM functions in mushroom bodies to
  regulate courtship conditioning to mated females

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Discussion

• Minimal fruP1-GAL4 expression in higher-order
  centers
• Suggests that FruM neurons are unlikely to be
  involved in general processing and coordination
  of behaviour
• fruP1-GAL4 expression is not detected in most
  motor neurons in ventral nerve cord
• Suggests that FruM expressing neurons might
  modulate, rather than directly mediate,
  behavioural output
• Examples in courtship song and muscle of Lawrence
• Thus, FruM-expressing neurons might directly
  mediate output through male-specific structures,
  and indirectly modulate output dependent on
  structures common to both sexes

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Discussion

• Findings offer new insights into neuronal
  circuitry underlying complex behavioural
  programmes
• FruM expression in peripheral sensory systems
  involved in courtship, and in second and
  third-order neurons in the sensory systems
  suggest they mediate the detection and initial
  processing of sensory cues relevant to courtship
• Lack of overt sexual dimorphism in
  FruM-expressing neurons suggests FruM proteins
  alter fine neuronal connectivity and/or
  physiology in order to process and transmit
  information relevant to courtship arousal

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Conclusions

• FruM is necessary and sufficient for male
  courtship behaviour
• FruM-expressing neurons might directly mediate
  output through male-specific structures, and
  indirectly modulate output dependent on
  structures common to both sexes, resulting in
  male courtship behaviour

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Future Directions

• Does fru specify male-like behavioural patterns
  more generally or is it exclusively involved in
  male courtship behaviour?
• Are there other behavioural switch genes like
  fru, and if so, how will we find them?
• Address how specific neurons function to detect
  or transmit behaviourally relevant sensory cues,
  and integrate and process information to generate
  meaningful behavioural output
• Given the appropriate genetic tools, behavioral
  instincts should ultimately succumb to the same
  kind of molecular genetic analysis that has so
  successfully revealed the principles of
  morphological development

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Literature Cited

• Aigaki. GFP-labeling of Drosophila adult brain
  with staining of mushroom bodies. Tokyo
  Metropolitan University, Science Dept.
  Cytogenetics. Online. 26 Mar 2006. Available
  microscope.olympus.com.
• Demir, E. and Dickson, B.J. 2005. Fruitless
  Splicing Specifies Male Courtship Behaviour in
  Drosophila. Cell 121, 5 785-794.
• Griffiths, A.J.F., Miller, J.H., Susuki, D.T.,
  Lewontin, R.C., Gelbart, W.M. 2000. An
  Introduction to Genetic Analysis. W.H. Freeman
  and Company 678-681.
• Kyriacou, C.P. 2005. Behavioural genetics Sex in
  fruitflies is fruitless. Nature 436 334-335.
• Manoli, D.S. 2005. Male-specific fruitless
  specifies the neural substrates of Drosophila
  courtship behaviour. Nature 436 395-400.
• Ryner, L. C. et al. Cell 87, 1079-1089 (1996).

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Questions?