The Drosophila FoxP gene is necessary for operant self-learning:
Implications for the evolutionary origins of language
Björn Brembs 1 , Diana Pauly 2 , Rüdiger Schade 3 , Ezequiel Mendoza 1 , Hans-Joachim Pflüger 1 , Jürgen Rybak 4 , Constance Scharff 1 , Troy Zars 5
1
Institut für Biologie - Neurobiologie, Freie Universität Berlin;
2Robert Koch-Institut, Berlin;
3Institut für Pharmakologie, Charité, Berlin;
4
Max Planck Institute for Chemical Ecology, Jena, Germany;
5Division of Biological Sciences, University of Missouri, Columbia, Mo, USA
bjoern@brembs.net, http://brembs.net
1. Abstract
In humans, mutations of the transcription factor Forkhead box protein P2 (FoxP2) cause a severe speech and language disorder. Downregula- ting the Zebrafinch FoxP2 orthologue in development results in incom- plete and inaccurate song imitation. These forms of vocal learning ex- hibit two common characteristics: 1. Spontaneous initiation of behavi- or (‘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 com- pensator 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 pre- dictors. 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 hypo- thesis that one of the evolutionary roots oflanguage is the ability to di- rectly 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.
LP-T25-1B
Presented at the ninth Göttingen Meeting of the German Neuroscience Society, March 25, 2011
5. PKC activity is required specifically for self-learning
5. PKC activity is required specifically for self-learning
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
Control Behavior
heat
WT cAMP PKC MB
OK OK Impaired OK self l.
torque meter
yaw torque signal
diffusor light guides light source
IR laser diode
Control
WT cAMP PKC MB
OK Impaired OK OK Behavior
heat color
world l.
self l.
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
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
6. Insertion 3955 in the FoxP gene affects self-learning
6. Insertion 3955 in the FoxP gene affects self-learning
0.0 0.2 0.4 0.6
22 21
CS FoxP
39550.0 0.2 0.4 0.6
p<0.02
22 20
PI [rel. units]
only self-learning
rtPCR
self- and world-learning
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.
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.
7. Drosophila FoxP isoform B is required for self-learning 7. Drosophila FoxP isoform B is required for self-learning
0.0 0.2 0.4 0.6
36 29
FoxP-RNAi genetic
controls
0.0 0.2 0.4 0.6
p<0.05
30 37
PI [rel. units]
self- and world-learning only self-learning
rtPCR
8. FoxP protein expression 8. FoxP protein expression
Fig. 7: Raising polyclonal chicken IgY antibodies against Drosophila FoxP protein.
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.
2. The FoxP gene family tree 2. The FoxP gene family tree
Fig. 1: The insect FoxP orthologues suggest the ancestral form
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 verte- brate genes, FoxP1, FoxP2, FoxP3 and FoxP4.
Hydra magnipapillata Saccoglossus kowalevskii
Danio rerio Xenopus laevis
Equus asinus Sus scrofa Capra hircus Bos taurus Chimarrogale himalayica Felis catus
Arctonyx collaris Canis familiaris
Cynopterus sphinx Rousettus leschenaultii Oryctolagus cuniculus
Gorilla gorilla
Homo sapiens Pan troglodytes
Macaca mulatta Papio anubis
Taphozous melanopogon Miniopterus schreibersii Chaerephon plicatus
Rhinolophus luctus
Rhinolophus ferrumequinu Hipposideros armiger Aselliscus stoliczkanus Coelops frithii
Myotis ricketti
Tylonycteris pachypus Megaderma spasma Rattus norvegicus
Mus musculus Trachemys scripta
Gallus gallus
Melopsittacus undulatus Taeniopygia guttata Xenopus laevis
Bos taurus Oryctolagus cuniculus
Callithrix jacchus Macaca mulattaPan troglodytesHomo sapiens Canis familiarisEquus caballus
Mus musculus
Rattus norvegicus Gallus gallus
Taeniopygia guttata Ornithorhynchus anatinus
Monodelphis domestica
Danio rerio Taeniopygia guttata Bos taurus Equus caballus Canis familiaris
Sus scrofa Pan troglodytes Homo sapiens
Pongo abelii
Macaca mulatta Papio anubis
Rattus norvegicus Mus musculus Felis catus
Macaca mulatta Homo sapiens
Bos taurus Sus scrofa
Rattus norvegicus Mus musculus
Xenopus tropicalis Xenopus laevis Drosophila melanogaster
Anopheles gambiaeNasonia vitripennis Apis mellifera
Bombyx mori Tribolium castaneum
invertebrates
FoxP2 FoxP1
FoxP4 FoxP3
vertebrates
3. The Drosophila FoxP gene locus
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.
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.
100bp
Intron retention isoform A
isoform B
2 3 4 5 6 7 8
1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6
7 7
8 I
3955 f03746 c03619
A A B B
FH
1
CS 3955
0 500 1000 1500 2000 2500
T o ta l F lig h t T im e [s]
Median 25%-75%
15%-85%
f03746
*
c03619
*
Canton S Fo xP
3955IR A B
Fo xP
c03619Fo xP
f037461347bp 532bp 1718bp
A B
4. Characterizing three insertion lines
Fig. 3: The three insertion mutants differ in isoform expression patterns and only one insetion line shows normal flight performance.
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
Fig. 3: The three insertion mutants differ in isoform expression patterns and only one insetion line shows normal flight performance.
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
9. No obvious brain defects in FoxP 3955 mutants
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.
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.
ellipsoid-body
noduli
protocerebral b ridge 0
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8
mushroom-bodies
fan-shaped body a sontgennal lobes
0 2 4 6 8 10 12
medulla
lobula
lobula plate 0
10 20 30 40 50
Fr action of segmented neuropil [%]
FoxP3955
CS
A
FoxP3955CS
