The Drosophila FoxP gene is necessary for operant self

LP-T25-1B
The Drosophila FoxP gene is necessary for operant self-learning:
Implications for the evolutionary origins of language
Björn Brembs1, Diana Pauly2, Rüdiger Schade3, Ezequiel Mendoza1, Hans-Joachim Pflüger1, Jürgen Rybak4, Constance Scharff1, Troy Zars5
Institut für Biologie - Neurobiologie, Freie Universität Berlin; 2 Robert Koch-Institut, Berlin; 3 Institut für Pharmakologie, Charité, Berlin;
4
Max Planck Institute for Chemical Ecology, Jena, Germany; 5 Division of Biological Sciences, University of Missouri, Columbia, Mo, USA
1
bjoern@brembs.net, http://brembs.net
1. Abstract
CS
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40
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20
invertebrates
FoxP1
vertebrates
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Mu otis rick
ypus
My nycteris pach
Tylo erma spasma
Megad
Rhinolophus luctus
Rhinolophus ferrumeq
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Hippo
Asellissideros armiger
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Tapelops fri oliczkanus
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Ch iniop ous m
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Ca us us a phon schre pogon
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Cy ous arro s
R im tu
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Ch lis cayx col s
Fe ton
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Arc nis fam s cuniculu
Ca olagu
Oryct ca mulatta
Maca
Papio anubis
Gorilla gorilla
FoxP2
FoxP3
FoxP4
0.3
Fig. 1: The insect FoxP orthologues suggest the ancestral form
4
The bilaterian FoxP gene family arose from a single FoxP gene. The ancestral variant, conserved in
the invertebrate lineage, later underwent two subsequent duplications, leading to the four vertebrate genes, FoxP1, FoxP2, FoxP3 and FoxP4.
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3. The Drosophila FoxP gene locus
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Fraction of segmented neuropil [%]
CS
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dra us k ano yx
ma ow ga mo
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ipa vs r
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Macac sapiens
a mula
Felis catuttsa
Sus scrofa
Bos taurus
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Mus musc icus
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e
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Rattus o sapienelii
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go a tes
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Rattus norvegicus
Bos taurus
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Canistolagus cunic
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Cal uus ca iliaris
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Pa acac rix jac lus
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te
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3955
2. The FoxP gene family tree
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9. No obvious brain defects in FoxP3955
mutants
FoxP
FoxP
A
B
In humans, mutations of the transcription factor Forkhead box protein
P2 (FoxP2) cause a severe speech and language disorder. Downregulating the Zebrafinch FoxP2 orthologue in development results in incomplete and inaccurate song imitation. These forms of vocal learning exhibit two common characteristics: 1. Spontaneous initiation of behavior (‘trying out’); 2. Evaluation of sensory feedback shaping behavior.
Using a torque learning essay in which both characteristics have been
realized, we investigated the involvement of the fly orthologue, FoxP,
in operant self-learning in the fruit fly Drosophila. The experiments
were performed using stationary flying Drosophila at the torque compensator with heat as punishment. Both a P-Element insertion and
RNAi-mediated knockdown of the isoform B of the Drosophila FoxP
gene did not lead to alterations of the gross brain anatomy, nor to an
impairment in operant world-learning, i.e., color-learning, compared
to control flies. However, both fly strains were impaired in operant
self-learning, i.e., yaw-torque learning without any environmental predictors. Neither the FoxP intron retention isoform nor isoform A appear
to be involved in this form of learning. These results suggest a specific
involvement of isoform B of the Drosophila FoxP gene in the neural
plasticity underlying operant self-learning but not in other forms of
learning. To investigate the effects of RNAi knockdown and P-Element
insertion on FoxP abundance and localization in the fly central nervous
system, we have generated polyclonal chicken antibodies against four
different regions of the putative FoxP protein.
Perhaps not surprisingly, these results are consistent with the hypothesis that one of the evolutionary roots oflanguage is the ability to directly modify the neural circuits controlling behavior. It is noteworthy
that these roots can apparently be traced back to the Ur-bilaterian, the
last common ancestor of vertebrates and invertebrates.
Fig. 8: Neither qualitative nor quantitative anatomical comparison reveals
nany major differences between wildtype CS and FoxP mutant brains.
A - Frontal sections of one typical wildtype and mutant fly brain, respectively.
B - Volume rendering of a wildtype and a mutant fly brain. C - Quantitative
study comparing the relative volumes of ten registered neuropil regions.
Number of animals: FoxP: 7; CS: 5.
1
2
3 4 5
c03619
f03746
3955
7
6
8
A
A
8. FoxP protein expression
2 3 45 6 I
1
2 3 45 6
1
2 3 45 6
B
100bp
FH
1
B
7
7
8
Intron retention
isoform A
isoform B
Fig. 2: The Drosophila FoxP gene locus and putative isoform mRNA
structure.
Triangles indicate insertions, grey arrows indicate the two (A, B) primer pairs used in our rtPCR. FH
- Forkhead Box.
4. Characterizing three insertion lines
only self-learning
FoxPc03619
FoxPf03746
Median
25%-75%
15%-85%
2000
1718bp
IR
1347bp
A
532bp
B
p<0.05
2500
Total Flight Time [s]
7. Drosophila FoxP isoform B
is required for self-learning
FoxP3955
B
Canton S
A
1500
1000
500
*
*
f03746
c03619
0
CS
Fig. 3: The three insertion mutants differ in isoform expression patterns and
only one insetion line shows normal flight performance.
0.6
A - rtPCR results using the primers as described in Fig. 2. The three lines show marked differences in
the expression patterns of the three isoforms. B - Flight performace tests show that only line 3955 is
suitable for behavioral experiments at the torque meter. Number of animals: CS: 18, 3955: 30, f03746:
34, c03619: 37
0.4
Fig. 7: Raising polyclonal chicken IgY antibodies against
Drosophila FoxP protein.
0.2
PI [rel. units]
0.0
rtPCR
30
37
6. Insertion 3955 in the FoxP
gene affects self-learning
only self-learning
self- and world-learning
5. PKC activity is required specifically for
self-learning
p<0.02
0.6
IR laser diode
0.6
torque meter
yaw torque signal
Behavior
0.4
self l.
0.2
0.2
0.0
Control
heat
light source
0.4
29
36
genetic
FoxP-RNAi
controls
Fig. 6: Self-learning requires isoform B.
Targeting isoform B with with an RNAi construct directed against
the last exon of the FoxP gene yields a phenocopy of the FoxP3955
insertion: self-learning is abolished, while world-learning is
unaffected.
PI [rel. units]
A Peptide regions used for BSA-conjugates to immunize chicken. Peptide 1
(IgY1), peptide 2 (IgY2) and peptide 3 (IgY) are sequences of CG16899
(isoform A) and peptide 4 is located in CG32937. All IgY except IgY 3 could
bind to a putative fusionprotein of CG16899 and CG32937 (isoform B). B
Indirect ELISA-Titer after eight boosts. Only IgY 1 and IgY 2 specficially
detect their peptide. All IgY bind to extracts of Drosophila heads from
FoxP3955 or wildtype Canton S. The detection of BSA is shown as a positive
control. C Immunoblot using IgY2, IgY3 and IgY4 binding to head extracts
from FoxP3955 or wildtype Canton S. Different polyclonal antibodies show
different positive protein bands.
3955
rtPCR
light guides
0.0
22
20
diffusor
WT
cAMP
PKC
MB
OK
OK
Impaired
OK
self- and world-learning
0.6
Behavior
heat
0.4
self l.
0.2
0.0
Control
22
CS
21
FoxP3955
Fig. 5: FoxP function dissociates between self- and
world-learning.
Canton S wild-type flies perform well in both learning situations,
whereas a FoxP insertion mutant line (3955) how significantly
reduced learning scores specifically in the self-learning task.
Reverse transcriptase PCR shows that the insertion affects both
FoxP isoforms, but while small amounts of isoform A can still be
detected, isoform B appears to be entirely absent
color
world l.
WT
cAMP
PKC
MB
OK
Impaired
OK
OK
Fig. 4: Two operant conditioning experiments, distinguished by the
presence or absence of predictive stimuli.
Above: Flies learn to avoid the heat by trying out different behavioral programs and evaluating
the resulting sensory feedback. No sensory predictors are present. Manipulating PKC activity,
but not cAMP levels abolishes learning in this task. Below: Adding predictive color stimuli allows
the animal to also learn which colors are predicting the heat punishment. Manipulating cAMP
levels abolishes learning in this task, while reducing PKC activity has no effect. Brembs & Plendl,
Curr. Biol. 2008
Presented at the ninth Göttingen Meeting of the German Neuroscience Society, March 25, 2011