A humanized version of Foxp2 affects cortico-basal ganglia circuits in mice.

Abstract

It has been proposed that two amino acid substitutions in the transcription factor FOXP2 have been positively selected during human evolution due to effects on aspects of speech and language. Here, we introduce these substitutions into the endogenous Foxp2 gene of mice. Although these mice are generally healthy, they have qualitatively different ultrasonic vocalizations, decreased exploratory behavior and decreased dopamine concentrations in the brain suggesting that the humanized Foxp2 allele affects basal ganglia. In the striatum, a part of the basal ganglia affected in humans with a speech deficit due to a nonfunctional FOXP2 allele, we find that medium spiny neurons have increased dendrite lengths and increased synaptic plasticity. Since mice carrying one nonfunctional Foxp2 allele show opposite effects, this suggests that alterations in cortico-basal ganglia circuits might have been important for the evolution of speech and language in humans.

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A Humanized Version of Foxp2 Affects
Cortico-Basal Ganglia Circuits in Mice
Wolfgang Enard,1,* Sabine Gehre,1Kurt Hammerschmidt,2Sabine M. Ho ¨lter,3Torsten Blass,1Mehmet Somel,1,25
Martina K. Bru ¨ckner,4Christiane Schreiweis,1Christine Winter,5Reinhard Sohr,6Lore Becker,7,8Victor Wiebe,1
Birgit Nickel,1Thomas Giger,1Uwe Mu ¨ller,9Matthias Groszer,10,26Thure Adler,8,11Antonio Aguilar,12Ines Bolle,13
Julia Calzada-Wack,14Claudia Dalke,3Nicole Ehrhardt,8,15Jack Favor,16Helmut Fuchs,8Vale ´rie Gailus-Durner,8
Wolfgang Hans,8Gabriele Ho ¨lzlwimmer,14Anahita Javaheri,8,12Svetoslav Kalaydjiev,11,27Magdalena Kallnik,3
Eva Kling,7,8Sandra Kunder,14,28Ilona Moßbrugger,14Beatrix Naton,8Ildiko ´ Racz,17Birgit Rathkolb,8,18Jan Rozman,8,20
Anja Schrewe,8,19Dirk H. Busch,11Jochen Graw,3Boris Ivandic,19Martin Klingenspor,20Thomas Klopstock,7
Markus Ollert,12Leticia Quintanilla-Martinez,14,29Holger Schulz,13Eckhard Wolf,18Wolfgang Wurst,3,21
Andreas Zimmer,17Simon E. Fisher,10Rudolf Morgenstern,6Thomas Arendt,4Martin Hrabe ´ de Angelis,8,22
Julia Fischer,2Johannes Schwarz,23,24and Svante Pa ¨a ¨bo1
1Max-Planck Institute for Evolutionary Anthropology, Deutscher Platz 6, D-04103 Leipzig, Germany
2Department of Cognitive Ethology, German Primate Center, Kellnerweg 4, D-37077 Goettingen, Germany
3Institute of Developmental Genetics, Helmholtz Zentrum Mu ¨nchen - German Research Center for Environmental Health (GmbH),
Ingolstaedter Landstrasse 1, D-85764 Munich/Neuherberg, Germany
4Department of Neuroanatomy, Paul Flechsig Institute of Brain Research, Universita ¨t Leipzig, D-04109 Leipzig, Germany
5Department of Psychiatry and Psychotherapy, University Medicine Berlin, Charite ´ Campus Mitte, Berlin, Germany
6Institute of Pharmacology and Toxicology, University Medicine Berlin, Charite ´ Campus Mitte, Berlin, Germany
7Department of Neurology, Friedrich-Baur-Institute, Ludwig-Maximilians-Universita ¨t Mu ¨nchen, Ziemssenstrasse 1a,
D-80336 Munich, Germany
8Institute of Experimental Genetics, Helmholtz Zentrum Mu ¨nchen - German Research Center for Environmental Health (GmbH),
Ingolstaedter Landstrasse 1, D-85764 Munich/Neuherberg, Germany
9BBZ, Institute of Immunology, University of Leipzig, Deutscher Platz 5, D-04103 Leipzig, Germany
10Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Headington, Oxford, OX3 7BN, UK
11Institute for Medical Microbiology, Immunology and Hygiene, Technische Universita ¨t Mu ¨nchen, Trogerstrasse 9,
D-81675 Munich, Germany
12DivisionofEnvironmentalDermatologyandAllergyTUM/HMGU,DepartmentofDermatologyandAllergy,TechnischeUniversita ¨tMu ¨nchen,
Biedersteiner Strasse 29, D-80802 Munich, Germany
13Institute of Lung Biology and Disease, Helmholtz Zentrum Mu ¨nchen - German Research Center for Environmental Health (GmbH),
Ingolstaedter Landstrasse 1, D-85764 Munich/Neuherberg, Germany
14Institute of Pathology, Helmholtz Zentrum Mu ¨nchen - German Research Center for Environmental Health (GmbH),
Ingolstaedter Landstrasse 1, D-85764 Munich/Neuherberg, Germany
15Faculty of Biology, Philipps University of Marburg, Karl-von-Frisch-Strasse 8, D-35032 Marburg, Germany
16Institute of Human Genetics, Helmholtz Zentrum Mu ¨nchen - German Research Center for Environmental Health (GmbH),
Ingolstaedter Landstrasse 1, D-85764 Munich/Neuherberg, Germany
17Department of Molecular Psychiatry, Life & Brain Center, University of Bonn, Sigmund Freud Strasse 25, D-53105 Bonn, Germany
18Institute of Molecular Animal Breeding and Biotechnology, Gene Center, Ludwig-Maximilians-Universita ¨t Mu ¨nchen,
Feodor-Lynen-Strasse 25, D-81377 Munich, Germany
19Department of Medicine III, Div. of Cardiology, University of Heidelberg, Im Neuenheimer Feld 410, D-69120 Heidelberg, Germany
20Molecular Nutritional Medicine, Technische Universita ¨t Mu ¨nchen, Else Kro ¨ner-Fresenius Center and ZIEL-Research Center for Nutrition
and Food Science, Am Forum 5, D-85350 Freising – Weihenstephan, Germany
21Max Planck Institute of Psychiatry, Kraepelinstrasse 2-10, D-80804 Munich, Germany
22Lehrstuhl fu ¨r Experimentelle Genetik, Technische Universita ¨t Mu ¨nchen, Am Hochanger 8, D-85350 Freising - Weihenstephan
23Department of Neurology, University of Leipzig, Liebigstr. 22a, D-04103 Leipzig, Germany
24Division of Biology 156-29, California Institute of Technology, 1200 E. California Blvd, Pasadena, CA 91125-2900, USA
25Present address: CAS-MPG Partner Institute for Computational Biology, SIBS, 320 Yue Yang Road, Shanghai, 200031, China
26Present address: INSERM U 839, Institut du Fer a ` Moulin, University Pierre&Marie Curie, 17 rue du Fer a ` Moulin, 75005 Paris, France
27Present address: Faculty of Life Sciences, AV Hill Building, University of Manchester, Oxford Road, Manchester M13 9PT, UK
28Present address: Infectious Diseases Program, Lovelace Respiratory Research Institute. 2425 Ridgecrest Dr. SE. Albuquerque,
NM 87108, USA
29Present address: Institut fu ¨r Pathologie, University of Tuebingen, Liebermeisterstr. 8, 72076 Tuebingen, Germany
*Correspondence: enard@eva.mpg.de
DOI 10.1016/j.cell.2009.03.041
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SUMMARY
It has been proposed that two amino acid substitu-
tions in the transcription factor FOXP2 have been
positively selected during human evolution due to
effects on aspects of speech and language. Here,
we introduce these substitutions into the endoge-
nous Foxp2 gene of mice. Although these mice are
generally healthy, they have qualitatively different
ultrasonic vocalizations,
behavior and decreased dopamine concentrations
in the brain suggesting that the humanized Foxp2
allele affects basal ganglia. In the striatum, a part of
the basal ganglia affected in humans with a speech
deficit due to a nonfunctional FOXP2 allele, we find
that medium spiny neurons have increased dendrite
lengths and increased synaptic plasticity. Since
mice carrying one nonfunctional Foxp2 allele show
oppositeeffects,thissuggeststhatalterationsincor-
tico-basalgangliacircuitsmighthavebeenimportant
for the evolution of speech and language in humans.
For a video summary of this article, see the Paper-
Flick file available with the online Supplemental Data.
decreasedexploratory
INTRODUCTION
Identification ofthegenomicdifferencesthatunderliethepheno-
typic traits that set humans apart from their closest relatives
among the primates is important from an evolutionary, medical
and a cultural perspective (Enard and Pa ¨a ¨bo, 2004; Varki et al.,
2008). The sequencing of human, chimpanzee and rhesus
macaque genomes has accomplished the first step toward this
goal by cataloguing the ?20 million genomic changes that
occurred on the human evolutionary lineage (Mikkelsen et al.,
2005). The current challenge, therefore, is not to identify
human-specific genomic features, but to distinguish the small
number of features that may have phenotypic consequences
from the vast majority of functionally neutral features. Since
crosses between humans and chimpanzees are not possible,
this can only be achieved by the analysis of additional informa-
tion such as the extent of evolutionary conservation among
primates,populationvariationamonghumansanddiseaseasso-
ciations. Examples of such work include the identification of
a noncoding RNA gene that evolved rapidly on the human
lineage (Pollard et al., 2006), evidence for positive selection in
genes involved in primary microcephaly (Gilbert et al., 2005)
and evidence for positive selection in FOXP2 (Enard et al.,
2002; Zhang et al., 2002), a gene involved in speech and
language disorders (Lai et al., 2001; MacDermot et al., 2005).
Once such candidate features are identified, the next challenge
is to test whether they are indeed involved in any human-specific
phenotype. This is obviously a difficult task since genetic
manipulations of humans or chimpanzees are impossible and
the mouse is the only mammal into which genetic changes can
currently be efficiently introduced and their effects tested.
Although human disease mutations have been successfully
modeled in the mouse (Cox and Brown, 2003) it is unclear
whether the supposedly slight phenotypic effects of human-
specific evolutionary changes can be modeled and recognized
in the mouse. Here we establish a mouse model and study the
functional consequences of evolutionary changes that affected
the transcription factor FOXP2 in humans.
Individuals that are heterozygous for FOXP2 alleles that carry
a missense mutation (R553H) affecting the forkhead DNA
binding domain of the protein, a nonsense mutation (R328X) or
disruptions of the gene by a chromosomal rearrangement suffer
from a developmental impairment especially affecting speech
and language (Lai et al., 2001; MacDermot et al., 2005; Var-
gha-Khadem et al., 2005). Analyses of the evolution of the
FOXP2 gene in primates identified two amino acid substitutions
(T303N, N325S), which became fixed on the human lineage after
its separation from the chimpanzee and which appear to have
been subject to positive selection (Enard et al., 2002; Zhang
et al., 2002). It has been hypothesized that these substitutions
underwent selection due to effects on some aspects of speech
and language (Enard et al., 2002; Zhang et al., 2002). Conve-
niently, FoxP2 in chimpanzees differs from Foxp2 in mice by
only one conservative amino acid substitution (D80E). Thus,
the wild-type mouse Foxp2 protein can be regarded as a model
for the ancestral version of the human FOXP2 protein and
compared to a partly ‘‘humanized’’ version in which the two
amino acid replacements have been introduced (Figure 1A).
RESULTS
Generation of Mice
The two nucleotide substitutions that occurred during human
evolution are both located in exon seven of the FOXP2 gene.
We introduced these two substitutions into the orthologous
exon of the mouse Foxp2 gene by homologous recombination
in embryonic stem (ES) cells derived from C57BL/6 mice
(Figure 1A and Figure S1 available with this article online). This
‘‘humanized’’ mouse allele (Foxp2hum) segregates in Mendelian
ratios (chi-square = 1.44, n = 636, p = 0.49) and Foxp2hum/hum
mice seem healthy, and are as fertile and as long-lived (data
not shown) as their wild-type littermates. This is in stark contrast
to mice homozygous for knock-out or nonfunctional mutant
alleles of Foxp2, which die 3–4 weeks after birth (French et al.,
2007; Fujita et al., 2008; Groszer et al., 2008; Shu et al., 2005).
Thus, the Foxp2humallele is generally functional in mice.
We also crossed mice carrying Foxp2humto mice ubiquitously
expressing Cre recombinase to generate a Foxp2koallele in
which exon 7 is deleted (Figure 1B). This is expected to lead to
a truncated Foxp2 protein of 291 amino acids. We did not
analyze homozygous Foxp2ko/komice in any detail, but similar
to mice homozygous for an allele with a nonsense mutation
(S321X) in exon 7 (Groszer et al., 2008) they show reduced
Foxp2 mRNA levels and an absence of truncated Foxp2 protein
(FigureS2).Thisislikelyduetoacombinationofnonsense-medi-
ated RNA decay (Groszer etal., 2008) and instability of truncated
proteins (Vernes et al., 2006). Heterozygous Foxp2wt/komice
show intermediate levels of Foxp2 protein (Figure S2) and can
thus be used to assess the consequences of reduced Foxp2
expression. This may be useful since effects caused by the
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Foxp2humallele mayconceivably bedueto impairedFoxp2func-
tioninthemousebackgroundthatcouldbefunctionallysimilarto
a reduced amount of protein. Furthermore, since humans that
are heterozygous for nonfunctional FOXP2 variants exhibit
speech and language deficits (Lai et al., 2001; MacDermot
et al., 2005), these mice may model FOXP2 effects that lead to
speech and language impairments in humans (Groszer et al.,
2008).Hence, these micecan beused to assesswhetherpheno-
typic changes in Foxp2hum/hummice might be related to the
evolution of speech and language.
A Comprehensive Phenotypic Screen
Foxp2 is expressed in the brain (Campbell et al., 2009; Ferland
et al., 2003; Lai et al., 2003; Takahashi et al., 2003) as well as
in a wide variety of other tissues (Lai et al., 2001; Shu et al.,
2005, 2007). For example, it has been proposed to play a role
in combination with Foxp1 during the development of the lung
and the esophagus (Shu et al., 2007). Thus, the Foxp2humallele
may have effects in multiple organs. To investigate which phys-
iological systems might be affected by the human amino acid
substitutionsweanalyzedatotalof60animals,15ofeachhomo-
zygous genotype and sex, derived from 17 litters of heterozy-
gous parents in a large standardized phenotypic screen at the
German Mouse Clinic (Gailus-Durner et al., 2005) (see Supple-
mentalDataS2foradetaileddescription)thatassessesmorpho-
logical traits (Fuchs et al., 2000), hearing, vision, bone
morphology and density (Fuchs et al., 2006), various clinical-
chemical parameters and hematological parameters, including
leukocyte subpopulations and classes of immunoglobulins,
metabolism, lung function, blood pressure and heart function.
We also studied 24 neurological parameters (Schneider et al.,
2006) including forepaw grip strength and nociception as well
as motor coordination and motor learning, which were assessed
on an accelerating rotarod over 3 consecutive days. Sensori-
motor behavior was examined by the acoustic startle response
and the prepulse inhibition of the startle response. Locomotor
activity, exploration, novel object recognition and frequency of
contact with group members was assessed using the modified
hole board (Ohl et al., 2001). Finally, 31 tissues were analyzed
histologically. Many known sex differences were identified in
thesetests.Forthevastmajorityoftests,nosignificantFoxp2hum
effects were found (Table S1). There were, however, two excep-
tions: First, several measurements indicated a reduced explor-
atory behavior on the modified hole board (see below) and this
was also evident from a reduced forward locomotor activity in
the neurology screen (ANOVA, n = 60, p < 0.05). Second, in elec-
trocardiograms Foxp2hum/hummice had lower R-wave ampli-
tudes (ANOVA, n = 39, p < 0.01). To test if these results are
robust, we analyzed heart function and behavior on the modified
hole board in a second batch of 60 mice. These animals were
derived from a different ES cell clone and had the Neomycin
cassette (Figure 1A) removed by FLPe recombination between
FRT-sites flanking the Neomycin cassette (Supplemental Data
S1). No effect of the Foxp2humallele on R-amplitude (ANOVA,
n = 59, p = 0.7) was seen in these animals suggesting that
Foxp2humhas little or no effect on heart function. However, we
A
B
Figure 1. Introduction of Human FOXP2 Substitutions into Mice
(A) Since the human and chimpanzee lineages diverged, human FOXP2 changed at two amino acid positions (T303N and N325S). Only one other amino acid
substitution separates humans and chimpanzees from mice (D80E). We generated a Foxp2 knock-in allele (Foxp2hum) of the endogenous mouse Foxp2 gene.
Foxp2humcarries the substitutions T302N and N324S which are orthologous to the human substitutions. Its neomycin resistance cassette is flanked by FRT sites
(green arrows) and exon 7 which carries the substitutions is flanked by loxP sites (blue arrows). Equal amounts of Foxp2 protein are detected in embryonic brains
of mice homozygous for Foxp2hum(h/h) and homozygous for the Foxp2 wild-type allele (+/+). See Figure S1 for further details.
(B) A nonfunctional Foxp2koallele was generated from the Foxp2humallele using Cre-mediated recombination in order to model the R328X nonsense mutation
(MacDermot et al., 2005) and the R553H missense mutation (Lai et al., 2001) in humans with speech impairment. This leads to an absence of Foxp2 protein in
Foxp2ko/koembryos and intermediate levels of Foxp2 in Foxp2ko/wtmice (Figure S2).
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again found several measurements indicating reduced explor-
atory behavior of Foxp2hum/hummice (Table S2). Importantly,
we found no significant interaction between genotype and batch
foranyparameter,suggestingthattheeffectofFoxp2humisinde-
pendent of the ES cell clone and the presence of the Neomycin
cassette (Table S3).
Onthemodifiedholeboard(FigureS3),Foxp2hum/hummicefrom
both batches traveled significantly shorter distances (ANOVA, n =
109, p < 0.001) at a significantly lower mean velocity (p < 0.001),
madefewerturns(p<0.01),crossedfewerlines(p<0.01),entered
theholeboardlessoften(p<0.01),exploredfewerholes(p<0.01)
and stayed closer to the wall (p < 0.05) than their wild-type litter-
mates(FigureS3andTableS5).SinceFoxp2hum/hummiceperform
as well as their wild-type littermates on the rotarod and show no
other signs of motoric impairments (Table S1), their altered
behavior can be interpreted as a slightly reduced exploratory
activity in a novel environment. The effect also seems to be rela-
tively specific for exploratory behavior since we did not find any
behavioral effects in a light-dark box or in an elevated plus maze,
two tests assessing anxiety-related behavior (Table S2).
To put these results into perspective, we compared 25 mice
heterozygous for the Foxp2koallele to 23 wild-type littermates
in the primary phenotypic screen (Table S4). We found that
Foxp2wt/komice reacted less to a clicking sound (p < 0.001),
show slightly impaired motor learning on the rotarod (less
improvement over trials at day 1: p < 0.01), had a higher amount
of fat mass (p < 0.05) and a lower amount of lean mass (p < 0.05),
consumed more food (p < 0.05), assimilated more energy (p <
0.05), had a higher respiratory rate during activity and at rest
(p < 0.05), a lower pulse rate (p < 0.05), slightly higher plasma
Ferritin (p < 0.05) and lower plasma inorganic phosphorus (p <
0.05) concentrations and higher proportions of CD62L CD8a+
and CD4+ and CD8+ T cells (p < 0.05). Although we did not repli-
cate these results in a second batch of animals, these results
indicate that reduced expression of Foxp2 has several subtle,
but significant effects on multiple organs other than the brain.
Interestingly, although measures of forward locomotor activity
of Foxp2wt/komice were reduced on the modified hole board,
other parameters, such as a longer exploration of an unfamiliar
object (ANOVA, n = 48, p < 0.05), suggest a slightly increased
exploratory behavior of Foxp2wt/komice (Table S4). Indeed,
several parameters differ significantly between Foxp2wt/komice
and Foxp2hum/hummice, indicating increased exploratory be-
havior in Foxp2wt/komice and decreased exploratory behavior
in Foxp2hum/hummice (Figure 2 and Table S5).
In summary, we find a reproducible and specific effect of the
Foxp2humallele on mouse exploratory behavior, but no signifi-
cant effect on almost 300 other measurements assessing
a variety of physiological systems. This indicates that the
Foxp2humallele affects predominantly the brain. Furthermore,
this does not seem to be a simple loss-of-function effect of the
Foxp2humallele in the mouse since Foxp2wt/komice show
different and partly opposite effects.
Foxp2humReduces Dopamine Levels
Given that the phenotypic screen indicated that the Foxp2hum
allele affects the brain rather than other organ systems, we
further investigated the effects of the humanized Foxp2 in this
organ. We first compared the brains of at least one Foxp2wt/wt
and one Foxp2hum/hummouse at embryonic day 16.5 (E16.5),
postnatal day (P) 1, P10, P20 and 3 months with respect to gross
anatomyandFoxp2proteinexpressionandfoundnodifferences
(Figures S4–S6). Expression patterns agreed well with published
results, i.e., expression and nuclear localization of Foxp2 was
observed in a subset of postmitotic neurons in the striatum,
the thalamus, cortical layer VI, the cerebellum (Purkinje cells),
and several other areas (Ferland et al., 2003; Lai et al., 2003;
Takahashi et al., 2003).
Next, we analyzed the tissue concentrations of four major
neurotransmitters (glutamate, serotonin, dopamine and GABA)
in five brain regions (frontal cortex, cerebellum, caudate-puta-
men, nucleus accumbens and globus pallidus) from 10 male
Foxp2hum/hummiceand10maleFoxp2wt/wtlittermates.Wefound
a reduction in dopamine concentrations in all regions (repeated-
measure ANOVA, p < 0.001) in Foxp2hum/hummice but no effects
for any of the other neurotransmitters (Table S6). When com-
paring neurotransmitter levels between 10 male Foxp2wt/koand
10 male Foxp2wt/wtlittermates in the same way, we find an
increase in dopamine levels in all regions (repeated-measure
ANOVA, p < 0.05; Figure 3) as well as an increase in serotonin
levels especially in the nucleus accumbens (Table S6).
Hence, similar to the patterns observed for exploratory
behavior, Foxp2hum/humand Foxp2wt/komice show opposite
effects on their dopamine levels. These effects could be causally
linked since increasing extracellular dopamine levels pharmaco-
logically or genetically tends to increase exploratory behavior
and vice versa (David et al., 2005; Viggiano et al., 2003).
However, tissue levels of dopamine largely reflect the storage
pool of dopamine (Gainetdinov et al., 1998). Further studies are
Figure 2. Different Exploratory Behavior of Foxp2hum/humand
Foxp2wt/koMice on the Modified Hole Board
All parameters that differ significantly between Foxp2wt/ko(striped bars) and
Foxp2hum/hummice (solid bars) are shown (see Table S5 and Supplemental
Data S3 for details). Means (±SEM) are normalized to wild-type levels of the
respective littermates (dashed line). Asterisks represent significance levels of
0.05 (*) and 0.01 (**).
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needed to clarify effects on extracellular dopamine levels in
Foxp2hum/humand Foxp2wt/komice as well as the underlying
mechanism. However, since Foxp2 is not expressed in dopami-
nergicneurons(Wijchersetal.,2006)anymechanismisprobably
indirect. In this regard, it is interesting that Foxp2 is expressed in
many medium spiny neurons (Ferland et al., 2003; Lai et al.,
2003; Scharff and Haesler, 2005; Takahashi et al., 2003), which
are the major targets of dopaminergic neurons, and make up
over 90% of neurons in the striatum. For several reasons they
may be of relevance with respect to the role of Foxp2 in speech
and language development. First, the striatum is structurally and
functionally affected in patients heterozygous for the FOXP2-
R553H substitution that impairs speech and language (Liegeois
et al., 2003; Vargha-Khadem et al., 1998; Watkins et al.,
2002b). Second, mediumspiny neurons show abnormal function
in mice that are heterozygous for a substitution equivalent to this
human mutation (Foxp2-R552H) (Groszer et al., 2008). Third, in
birds, a knock-down of FoxP2 in a striatal nucleus (A, area X)
impairs vocal imitation (Haesler et al., 2007). Therefore, we
decidedtofurtherinvestigatetheeffectsofFoxp2humonmedium
spiny neurons.
Foxp2humIncreases Dendritic Length
We isolated striatal neural precursor cells from Foxp2hum/hum
embryos and their Foxp2wt/wtlittermates and compared their
proliferation and differentiation in vitro. The two genotypes did
not differ significantly in cell growth or survival rate during proli-
feration (Supplemental Data S6). However, 7 days after differen-
tiation, Foxp2hum/humcells positive for the neural marker TUBB3
had neurites, i.e., outgrowths from the cell body that may repre-
sent axons as well as dendrites, that were on average 80%
longer than in Foxp2wt/wtcells (Mann-Whitney U, p < 0.01;
Figure 4A). In contrast, the size of the cell bodies was not signi-
ficantly different between genotypes (data not shown).
In order to test the in vivo relevance of this finding, we stained
adult brains using the Golgi-Cox method and measured the total
dendrite length of three medium spiny neurons each in nine
Foxp2wt/wt, six Foxp2hum/humand six Foxp2wt/komice. We found
that Foxp2hum/hummice had dendritic trees that were on average
22% longer than Foxp2wt/wtmice (Mann-Whitney U, p < 0.05)
and Foxp2wt/komice (p < 0.05; Figure 4B). Foxp2wt/komice
tended to have neurons with shorter dendrites when compared
to their Foxp2wt/wtlittermates, but this difference was not signi-
ficant (Mann-Whitney U, p = 0.145). Thus, Foxp2humincreases
the length of the dendritic trees of medium spiny neurons in vitro
as well as in vivo.
Foxp2humIncreases Long-Term Synaptic Depression
Medium spiny neurons integrate glutamatergic input from the
cortex and dopaminergic input from the midbrain and generate
output that contributes to the control and selection of appro-
priate behaviors via cortico-basal ganglia circuits (Graybiel,
2008) and the strength of corticostriatal synapses are important
for acquiring and altering behaviors (Berretta et al., 2008). In
order to investigate whether Foxp2huminfluences plasticity of
corticostriatal synapses, we recorded membrane potentials of
medium spiny neurons in acute tissue slices and studied the
long-term depression (LTD) of their depolarization after high-
frequency stimulation of cortical fibers. Current-voltage relation-
ships and resting membrane potentials did not differ between
Foxp2hum/humand Foxp2wt/wtneurons (Figure S7), indicating
that they do not differ grossly in their physiology. However,
LTD was almost twice as strong in Foxp2hum/humneurons
compared to Foxp2wt/wtneurons (repeated-measures ANOVA,
n = 17, p < 0.05; Figure 5). In stark contrast, it has been reported
(Groszer et al., 2008) that LTD in mice heterozygous for the
R552H mutation, which corresponds to the R553H mutation
implicated in human speech and language deficits, is almost
absent in striatal neurons. Thus, whereas Foxp2humincreases
synaptic plasticity in medium spiny neurons, a nonfunctional
Foxp2 allele has the opposite effect.
Foxp2humand Striatal Gene Expression
Genome-wide gene expression patterns were analyzed in stria-
tal biopsies from embryos (E16.5), young animals (P15-P21) and
adults (3 month old) in a total of 30 Foxp2hum/hummice and their
23 Foxp2wt/wtlittermates using high-density oligonucleotide
arrays (see Supplemental Data S8 for details). The significance
of results was assessed by generating 1,000 data sets where
genotype labels were randomly permutated. This is a conserva-
tive approach that is robust against violations of analysis
assumptions, for example the assumption that the expression
levels of genes are independent of each other (Tusher et al.,
2001). At a threshold of p < 0.001 (F-test after correcting for
batch, age and sex effects), the expression of 34 genes differs
between Foxp2hum/humand Foxp2wt/wtmice in the observed data
(Figure S8A), while on average four genes differ at this threshold
Figure 3. Brain Dopamine Concentrations in Foxp2hum/humand
Foxp2wt/koMice
Meanpostmortemdopamineconcentrations(±SEM)relativetowild-typelitter-
mates (dashed line) are given for Foxp2hum/hum(solid bars) and Foxp2wt/ko
(striped bars) mice in different brain regions. Asterisks above columns indicate
asignificantdifferencetowild-typelevels(Student’sttest),andasterisksabove
bracketsindicateasignificantdifferencebetweenFoxp2hum/humandFoxp2wt/ko
mice (see also Table S6).
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inthepermutations(i.e.,afalsediscoveryrateof12%).Amongthe
1,000 permutated data sets, 36 show 34 or more genes to differ
(i.e., permutation test p < 0.05). In the promoters of the genes
that are higher expressed in Foxp2hum/hummice, Foxp2 binding
motifs (Wang et al., 2003) are enriched (permutation test p <
0.05; e.g., the motif TATTTAT occurs on average 2.6 times in
such genes and 1.8 times in other genes), indicating that some
of these genes may be primary targets of the humanized version
of Foxp2. In conclusion, the analysis shows that Foxp2humhas
a significant effect on gene expression patterns in the striatum. It
should be noted, however, that although many animals were
analyzed, the expression of relatively few genes were found to
be significantly affected by Foxp2humand the expression of those
thatare affecteddiffer byno more than 30% from wild-type levels
(Figure S8A). Two evolutionaryamino acid substitutions in a tran-
scription factor are not necessarily expected to cause major
changes in gene expression and it is certainly possible that the
subtle expression changes observed is sufficient to cause the
phenotypic effects seen in the animals. However, we can obvi-
ously not exclude that the Foxp2humallele causes more
pronounced changes in expression patterns at other time points
during striatal development, or in other parts of the brain.
We also analyzed gene expression in striatal biopsies from 12
Foxp2wt/koand their 6 Foxp2wt/wtlittermates (P15–P21). Foxp2
expression in the former mice is reduced to 68% of that in their
wild-type littermates in agreement with what is seen in embry-
onic brains (Figure S2). When Foxp2wt/koand Foxp2hum/hum
mice (P15-P21) are compared to their wild-type littermates,
gene expression tends to be affected in opposite directions
(Spearman rank correlation of effect sizes across genes =
A
B
Figure
Dendritic Trees
(A) Foxp2hum/humand Foxp2wt/wtstriatal neurons stained
for the neuronal marker beta–III-tubulin (green) after
7 days of differentiation. Plotted are averages (±SEM) of
three individuals for each of which the total neurite length
of five neurons was measured. Asterisks indicate a signifi-
cant difference (Mann-Whitney U, p < 0.01) between the
15 neurons of each genotype.
(B) Golgi-Cox staining and representative drawings of
medium spiny neurons in vivo. Plotted are averages
(±SEM) of six (nine for Foxp2wt/wt) individuals for each
genotype of which the total dendritic tree of three neurons
has been measured. Asterisks indicate a significant differ-
ence (Mann-Whitney U, p < 0.05) between the neurons of
each genotype. Scale bars represent 25 mm.
4. Foxp2hum
IncreasestheLength of
?0.18; permutation test p = 0.2). Interestingly,
among the 106 genes with higher expression in
Foxp2wt/koandlowerexpressioninFoxp2hum/hum,
24 are known to be preferentially expressed in
medium spiny neurons expressing the D1 dopa-
mine receptor (Heiman et al., 2008), whereas
only one would be expected by chance (permu-
tation test p < 0.01; Figure S8B). In contrast, no
such effect is seen for genes preferentially
expressed in medium spiny neurons expressing
the D2 dopamine receptor (Supplemental Data
S8). Since Foxp2 itself is preferentially expressed in the former
cells (Heiman et al., 2008), this suggests that among the two
major known subtypes of medium spiny neurons that differ
e.g., in their axonal projections and their electrophysiological
properties (Kreitzer and Malenka, 2008), Foxp2 primarily affects
D1 positive medium spiny neurons .
Foxp2humAlters Vocalization
Finally, to assess whether Foxp2humimpacts vocalization, we re-
corded ultrasonic vocalizations emitted by pups when placed
outside the nest (Ehret, 2005) at day P4, P7, P10, and P13
from 32 Foxp2hum/hummice and 39 Foxp2wt/wtlittermates. Using
asemi-automatedproceduretoextractsinglecallsandageneral
linear model (GLM) with the variables postnatal day, genotype,
sex, litter and weight we found no significant differences in the
number of calls emitted per minute or in the duration of intervals
between calls (GLM, n = 71, p > 0.4; Figure S9A). To analyze the
structure of calls (Figure 6A), we assigned them to one of four
categories: (1) calls shorter than 50 ms with no frequency jumps;
(2) calls longer than 50 ms with no frequency jumps; (3) calls with
frequency jumps; and (4) remaining sounds, which were not
analyzed (see Figure S9B for spectrographic displays of call
types). The first call type, which was the most frequent, showed
no difference between the two genotypes with regard to the
number of calls, the duration of calls and five other parameters
(Table S7). However, Foxp2hum/humanimals, they had a signifi-
cantly lower start peak frequency (p < 0.001), and lower mean
(p < 0.01), minimum (p < 0.01) and maximum (p < 0.001) peak
frequencies (Figure 6B). In addition, the slope of the calls
declined less in frequency (p < 0.01, Figure 6B) and were locally
966 Cell 137, 961–971, May 29, 2009 ª2009 Elsevier Inc.

Page 7

less modulated (p < 0.01). Analyses using a second batch of
animals that originated from a different ES cell clone, confirmed
these findings except for the parameter local modulation (Table
S8 and Supplemental Data S9). Further, similar nonsignificant
tendencies were observed for calls longer than 50 ms (data not
shown), whereas calls with frequency jumps lasted longer
(ANOVA, n = 149, p < 0.05), had longer gaps (p < 0.05) and
started (p < 0.01) and ended (p < 0.05) with higher peak frequen-
cies in Foxp2hum/hummice than in their wild-type littermates
(Table S9 and Figure S9C).
Mice homozygous for nonfunctional Foxp2 alleles produce
much fewer isolation calls than their wild-type littermates (Fujita
et al., 2008; Groszer et al., 2008; Shu et al., 2005), but given that
these animals suffer from severe developmental deficits and die
around 3 weeks after birth, this finding may not represent
specific effects of Foxp2 on mouse vocalizations (Groszer
et al., 2008). It has been suggested that mouse pups heterozy-
gous for nonfunctional Foxp2 alleles have mild developmental
delays and produce fewer ultrasonic calls (Fujita et al., 2008;
Shu et al., 2005), but these observations could not be verified
in another study (Groszer et al., 2008). Notably, studies of mouse
pups with nonfunctional Foxp2 alleles have not identified differ-
ences in the structural properties of calls (Groszer et al., 2008;
Shu et al., 2005). Hence, the Foxp2humallele affects ultrasonic
isolation calls of mice subtly but specifically and does so in
a way different from nonfunctional Foxp2 alleles.
DISCUSSION
A Mouse Model for Human Evolution?
To the best of our knowledge, our analysis of Foxp2hummice
represents the first investigation of amino acid substitutions of
potential relevance for human evolution in an animal model.
This raises the question whether such genetic changes can
be reasonably modeled in a mouse. One concern is that pheno-
typic effects elicited in the mouse could simply represent an
inability of the human gene product to function in the mouse
background. A complete inability of the Foxp2humallele to
function in the mouse would be equivalent to a knockout of
Foxp2. Since Foxp2hum/hummice are fertile and healthy, whereas
mice homozygous for nonfunctional Foxp2 alleles die within
3–4 weeks after birth (French et al., 2007; Fujita et al., 2008;
Groszer et al., 2008; Shu et al., 2005), Foxp2humcertainly func-
tions in the mouse, at least with respect to major effects with
obvious phenotypic consequences. Furthermore, when the
effects seen in Foxp2hum/hummice are compared to mice hetero-
zygous for nonfunctional Foxp2 alleles they are either not
observed in the latter mice (e.g., altered vocalization) or show
opposite effects to those seen in such mice (e.g., exploratory
behavior, dopamine levels, long-term depression). Hence, it
seems unlikely that the effects seen in Foxp2hum/hummice are
caused by a simple reduction in biological activity of Foxp2hum
in the mouse background.
Given that Foxp2hum/hummice are generally healthy, how can
any specific phenotypic effects be found if they exist? Since
FOXP2 is expressed in many organs (Lai et al., 2001; Shu
et al., 2001) and Foxp2humcould have effects in any number of
these, it is crucial to perform a comprehensive phenotypic
screenwherethefunctions of manyorgansystemsareassessed
in order not to bias results to organs or behaviors which may
a priori be deemed interesting. An analysis of many organ
systems is also crucial in order to assess the extent to which
effects detected are specific to an organ system or may be
secondary, especially if they are subtle as may be expected for
evolutionary innovations that occurred over short time scales.
Thus, we analyzed almost 300 different phenotypic parameters
in the mice. None of them produced any evidence for effects
of the Foxp2humallele in any organ system except the central
nervous system. This suggests that the two amino acid substitu-
tions that occurred on the human evolutionary lineage specifi-
cally affected the brain in the mouse. We therefore focused the
further analyses on this organ.
Of special interest is obviously if any of the effects detected
in the Foxp2hum/hummice might have something to do with any
aspect of speech and language in humans. Since humans
heterozygous for a nonfunctional FOXP2 allele show speech
and language impairments (Lai et al., 2001; MacDermot et al.,
2005; Vargha-Khadem et al., 2005), a comparison with
Foxp2wt/komice may be helpful as they may recapitulate
aspects of speech and language impairment. Thus, traits
affected in opposite directions in Foxp2wt/koand Foxp2hum/hum
mice are of potential interest as candidates for being involved
in aspects of speech and language evolution. We find that
exploratory behavior, dopamine levels, striatal gene expression
patterns and striatal synaptic plasticity are all affected in oppo-
site directions in Foxp2hum/humand Foxp2wt/komice respec-
tively in mice heterozygous for a nonfunctional Foxp2 allele
(Groszer et al., 2008). As argued in detail below, some of these
effects could model aspects relevant for speech and language
in humans.
Figure 5. Foxp2humIncreases Long-Term Depression in Medium
Spiny Neurons
Mean ± SEM amplitudes normalized to baseline levels at time 0 are shown
from Foxp2hum/hum(n = 8) and Foxp2wt/wtneurons (n = 9). Following 20 min
baseline stimulation, three high-frequency tetani were applied (100 Hz, 3 s)
separated by 30 s. Asterisks indicate significantly different means (Student’s
t test) between Foxp2hum/hum(open squares) and Foxp2wt/wt(filled squares)
neurons.
Cell 137, 961–971, May 29, 2009 ª2009 Elsevier Inc. 967

Page 8

Relevance of Ultrasonic Vocalization
The fact that Foxp2huminfluences ultrasonic vocalization of pups
in a specific and reproducible way is of obvious interest.
However, it is important to note that this influence is subtle and
within the range of normal variation among mice. A relevant
question is also to what extent mouse vocalization can be
compared to human speech. All terrestrial mammals produce
their vocalizations by an air stream from the lungs that passes
the larynx and generates sounds. In most animals, oscillations
of the vocal folds and/or specific structures of the vocal folds
generate the sound through oscillations (Fitch, 2000; Ham-
merschmidt and Fischer, 2008; Lieberman, 2006). Ultrasonic
vocalizations in rodents are also produced by the larynx (Rob-
erts, 1975a) but they are thought to derive from an aerodynamic
whistle rather than vibrations of vocal cords (Roberts, 1975b).
Nevertheless, the basic neurological and muscular systems
necessary for vocalizations probably overlap to a large degree
in mice and humans (Hammerschmidt and Fischer, 2008; Ju ¨r-
gens, 2002). Indeed, some neural circuits important for vocaliza-
tion areevenconservedinfish(Bassetal.,2008).Hence, thefact
that Foxp2huminfluences the structure of isolation calls in the
mouse, especially since this effect is not accompanied by phys-
iological effects outside the brain, supports the hypothesis that
the two amino acid substitutions that occurred during human
evolution affect aspects of speech and/or language.
However, we currently cannot exclude that very subtle
changesinlungfunctionorlarynxmorphologythatcouldbeirrel-
evant for speech and language evolution can be responsible for
theeffectsoncallstructures,despitethefactthatwefindnoindi-
cationthate.g.,lungfunctionisinfluencedbyFoxp2huminadults.
Furthermore, it is important to remember that vocalizations of
mice as well as most other terrestrial mammals are considered
to be innate. Humans share innate vocalizations like grunts,
cries, and screams with other animals, but in addition humans
have an unmatched ability to learn vocalizations (Egnor and
Hauser, 2004; Hammerschmidt and Fischer, 2008). The acquisi-
tion and extensions of neural circuits making voluntary control of
vocalizations possible is thought to be a hallmark in the evolution
of human speech (Ju ¨rgens, 2002; Krubitzer, 2007). Since little is
known about the neurological and anatomical basis of mouse
vocalizations, it is an open question if some neural circuits
homologous tothe onesmakingvoluntary vocalizations possible
in humans would be affected in the Foxp2hum/hummice. Hence,
more studies will be needed to clarify to what extent mouse
vocalizations can model aspects of human speech evolution.
As argued below, it will be especially important to clarify any
functional relationship to Foxp2hum-dependent effects on cor-
tico-basal ganglia circuits.
Cortico-Basal Ganglia Circuits and Speech
and Language
The fact that Foxp2humaffects dopamine levels, dendrite
morphology, gene expression and synaptic plasticity of medium
spiny neurons indicates that it impacts cortico-basal ganglia
circuits where medium spiny neurons in the striatum receive
contextual information from the cortex and reward signals from
dopaminergic neurons and send integrated signals to brain
stem structures and the cortex (Graybiel, 2008). Several lines
of evidence indicate that cortico-basal ganglia circuits could
be relevant for speech and language (Lieberman, 2002; Lieber-
man, 2006; Ullman, 2001). For example, reduced dopamine
release in the striatum is positively correlated with speed and
accuracy of phonological processing (Tettamanti et al., 2005),
activation of a part of the striatum plays a crucial role in lexical-
semanticcontrol(Crinionetal.,2006),andpatients withHunting-
ton’s disease show - dependent on the striatal subregions
affected - impairments in the retrieval of lexical information and
the application of combinatorial rules (Teichmann et al., 2008).
Furthermore, cortico-basal ganglia circuits and their dopami-
nergic modulations are crucial for song learning in birds, which
is thought to resemble aspects of vocal learning in humans
(Hara et al., 2007; Jarvis, 2004). This is supported by the recent
finding that when FoxP2 expression is knocked down in basal
ganglia of songbirds, vocal imitation is impaired (Haesler et al.,
2007).Furthermore, individuals heterozygous for anonfunctional
FOXP2 allele show structural effects and functional impairments
in the striatum (Vargha-Khadem et al., 2005; Vargha-Khadem
et al., 1998) supporting a role for FOXP2 in cortico-basal ganglia
circuits with respect to speech and language.
In conclusion, it is possible that the effects on cortico-basal
ganglia circuits seen in the Foxp2hum/hummice model aspects
of speech and language evolution in humans. It will now be
important to further explore the mechanistic basis of these
effects and their possible relationship to phenotypic differences
between humans and apes. Currently, one can only speculate
about the role these effects may have played during human
evolution. However, since patients that carry one nonfunctional
FOXP2 allele show impairments in the timing and sequencing
of orofacial movements (Alcock et al., 2000; Watkins et al.,
2002a), one possibility is that the amino acid substitutions in
A
B
Figure 6. Foxp2humAffects the Structure of
Pup Isolation Calls
(A) Examples of acoustic parameters analyzed
illustrated for typical call.
(B) Plots of acoustic parameters differing between
genotypes. Measurements are averaged across
days and individuals (±SEM). The peak frequency
(PF) refers to the frequency with the maximum
amplitude in each analyzed time window of
0.21 ms. Asterisks indicate significant differences
between genotypes (**: < 0.01; ***: < 0.001).
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FOXP2 contributed to an increased fine-tuning of motor control
necessary for articulation, i.e., the unique human capacity to
learn and coordinate the muscle movements in lungs, larynx,
tongue and lips that are necessary for speech (Lieberman,
2006). We are confident that concerted studies of mice, humans
and other primates will eventually clarify if this is the case.
EXPERIMENTAL PROCEDURES
Generation of Mice
Mice carrying the Foxp2humallele were generated by Ozgene (Bentley,
Australia) from two C57BL/6 ES cell clones that had integrated the vector via
homologous recombination. Whereas the first line (clone 5H10) was used for
initial analyses, the second one (clone 5H11) was used for testing the repro-
ducibility of the results from the first line. To this end, some mice were gener-
ated that carried a Foxp2humallele in which the Neomycin resistance cassette
had been removed by crossings with a FLPe deleter strain. Mice carrying the
Foxp2koallele were generated by crossing chimeric mice (clone 5H11) to a B6
Cre deleter strain (Ozgene, Bentley, Australia). and subsequent crosses to re-
move the Cre transgene. All animal work was performed in accordance with
governmental and institutional ethical guidelines.
Immunohistochemistry
Mice were transcardially perfused with 4% paraformaldehyde in PBS and
brains were either paraffin embedded (E16.5) or cryopreserved (P1, P10,
P20, and adults). Sections were immunostained with antibodies against
FOXP2 (HPA000382 Atlas Antibodies, Stockholm, Sweden or ab16046,
Abcam, Cambridge, UK).
Neurotransmitter Measurements
Biopsiesweretakenfromfrozenbrainslicesof10maleFoxp2hum/hummiceand
10 male Foxp2wt/wtlittermates as well as from 10 male Foxp2wt/komice and 10
male Foxp2wt/wtlittermates. Neurotransmitter levels were measured by high-
performance liquid chromatography and normalized to the protein content
ofthesample todeterminetheirconcentrations.WeusedanANOVAwithbrain
regions treated as repeated-measures and genotype as factor to determine
significant differences between genotypes separately for each strain and
neurotransmitter. Differences between Foxp2wt/komice and Foxp2hum/hum
mice were identified by a significant interaction between strain and genotype
in a combined analysis, again using brain regions as repeated-measures.
Neuronal Cell Culture
Striatal neural precursor cells were dissected from ?E14-old embryos,
expanded for 4 weeks as three-dimensional (neurosphere) cultures during
which proliferation and cell survival was measured. Differentiation was
induced via replacement of expansion media by media containing 1% FCS
and 5 mM forskoline on precoated poly L-lysine coverslips. After 7 days cells
were stained with DAPI and antisera against the neural marker TUBB3. For
three cultures per genotype, we measured neurite length of five neurons each.
Golgi Staining
Brainswereisolatedfromsix12-week-oldFoxp2hum/hummiceandsixwild-type
littermates (derived from the 5H11 line in which the Neomycin cassette had
been removed) and six 12-week-old Foxp2wt/komice and three wild-type litter
mates.Golgi-CoxstainingwasperformedusingtheFDRapidGolgiStainkit(FD
NeuroTechnologies, Ellicott City, MD, USA) according to the manufacturer’s
protocol. Stained slices were sectioned at a thickness of 200 mm using a cryo-
microtome (Microm Thermo Scientific, Walldorf, Germany). Medium spiny
neurons were identified by their morphology using a Axiovert 200 microscope
equipped with a xyz motorized stage and their dendrites were traced using
Mercator software (Explora Nova, La Rochelle, France).
Electrophysiology
Acute brain slices were prepared from the brains of 15- to 27-day-old
Foxp2hum/hummice and their wild-type littermates (all derived from the 5H11
line in which the Neomycin cassette had been removed) and medium spiny
neurons in the striatum were recorded at a holding potential of ?70 mV. Excit-
atory medium spiny neuron afferents were stimulated to yield largest EPSC
amplitudes without eliciting an action potential or inducing direct stimulation.
Continuous stimulation was performed at a frequency of 0.5 Hz. Following
20 min. of baseline stimulation, we applied 3 tetani of 3 s duration and
a frequency of 100 Hz separated by 30 s. LTD was then measured for
30min.Datapointswerecalculatedevery30sbyaveragingthelast15EPSCs.
LTD data was analyzed using a repeated-measures ANOVA with genotype as
fixed factor.
Gene Expression Analysis
Total RNA from striatal biopsies of 13 Foxp2hum/humembryos (E16.5) and 12
Foxp2wt/wtlittermates (prepared in two separate batches), 11 Foxp2hum/hum
mice (P15, P18, P21) and 6 Foxp2wt/wtlittermates, 12 Foxp2wt/komice (P15,
P18,P21)and 6Foxp2wt/wtlittermates aswell as6Foxp2hum/hummice(3month
old) and 6 Foxp2wt/wtlittermates was labeled and hybridized to Affymetrix
Mouse Genome 430 2.0 arrays. Expression levels were calculated using Bio-
conductor (Gentleman et al., 2004) and custom CDF files (Dai et al., 2005).
Genotype-dependent effects were assessed after correcting for batch, age
and sex effects using multiple regression. Using Cohen’s D estimate of effect
size, we correlated genotype-dependent effects with functional annotations
(Pru ¨fer et al., 2007), putative FOXP2 targets in a human neuroblastoma cell
line (Vernes et al., 2007), the number of putative Foxp2 binding motifs (Wang
et al., 2003) 5 kbp upstream and 2 kbp downstream of the transcription start
site and genes differently expressed in D1 and D2 positive striatal cells (Hei-
man et al., 2008). The significance of the genotype-dependent effects was in
each case assessed against at least 300 permutations of genotype labels.
All primary expression data are available at the NCBI GEO database (acces-
sion number GSE13588).
Vocalization
For each recording at P4, P7, P10, and P13, a pup was selected randomly,
placed on a cotton pad in a plastic beaker, weighed and recorded for 2 min
(3 min for P13) in a soundproof plexiglas box. Calls were counted using the
AVISOFT Recorder 2.97 (Avisoft Bioacoustics, Berlin). For the analysis of
call structure, we visually inspected all recordings to ensure that the auto-
mated sampling routine selected only calls of mouse pups and calculated
spectrograms. Wesubmittedthe resulting spectrogramstoa custom software
to extract a set of acoustic parameters (Table S7). To eliminate an overrepre-
sentation of subjects with high vocal activity, we randomly selected a
maximum of 10 calls per subject and recording day. The mean values per
subject and recording day were analyzed using a general linear mixed model
(SPSS 13.0), with day (P4, P7, P10, and P13) as within-subject factor, weight
as covariate and genotype, sex, and litter as fixed between-subject factors.
SUPPLEMENTAL DATA
Supplemental Data include Supplemental Experimental Procedures, nine
tables,ninefigures,andavideosummaryandcanbefoundwiththisarticleon-
line at http://www.cell.com/supplemental/S0092-8674(09)00378-X.
ACKNOWLEDGMENTS
We are grateful to Ozgene Inc. for generating mice; to Uta Zirkler (MPI-EVA) for
animal care; to Reinhard Seeliger and the German Mouse Clinic technician
team (Maria Kugler, Tamara Halex, Claudia Zeller, Sandra Scha ¨dler, Regina
Kneuttinger, Bettina Sperling, Elfi Holupirek, Susanne Wittich, Elisabeth
Schwarz, Miriam Backs, Eleonore Samson, Christine Fu ¨hrmann-Franz, and
Kerstin Kutzner) and the animal caretaker team for expert technical help; to
Eunjong Park (MPI-EVA, Neurology Leipzig) for assistance in neuronal cell
culture; to Sven-Holger Puppel and Sabrina Reimers (MPI-EVA) for assistance
ingeneratingexpressiondata;andtoChristineGreen(MPI-EVA)forcomments
on the manuscript. This work was supported by NGFNplus grants from the
Bundesministerium fu ¨r Bildung und Forschung (01GS0850 (I.B., C.C.-W.,
C.D., J.F., H.F., V.G.-D., W.H., G.H., M.K., S.K., I.M., B.,N., J.G., L.Q.-M.,
H.S., W.W., and M.H.A), 01GS0851 (L.B., B.R. C.M., E.K., E.W. and Th.K.),
Cell 137, 961–971, May 29, 2009 ª2009 Elsevier Inc. 969

Page 10

01GS0852 (T.A., S.K., and D.B.), 01GS0869 (N.E., J.R., and M.K.), 01GS0853
(I.R. and A.Z.), 01GS0854 (A.S. and B.I.), 01GS0868 (A.A., A.J., and M.O.), by
an EU grant (EUMODIC LSHG-2006-037188, German Mouse Clinic), grants
from the Deutsche Forschungsgemeinschaft (S.G. and J.S., M.K.B. and
T.A., Heisenberg grant to J.F.), the Saxonian Staatsministerium fu ¨r Wissen-
schaft und Kunst (M.K.B. and T.A.), the Interdisziplina ¨rem Zentrum fu ¨r Klini-
sche Forschung in Leipzig (TP C27 to S.G. and J.S.), the Royal Society
(research fellowship to S.E.F.), and the Max Planck Society (W.E. and S.P.).
Received: April 30, 2008
Revised: January 27, 2009
Accepted: March 17, 2009
Published: May 28, 2009
REFERENCES
Alcock, K.J., Passingham, R.E., Watkins, K.E., and Vargha-Khadem, F. (2000).
Oral dyspraxia in inherited speech and language impairment and acquired
dysphasia. Brain Lang 75, 17–33.
Bass, A.H., Gilland, E.H., and Baker, R. (2008). Evolutionary origins for social
vocalization in a vertebrate hindbrain-spinal compartment. Science 321,
417–421.
Berretta, N., Nistico, R., Bernardi, G., and Mercuri, N.B. (2008). Synaptic plas-
ticity in the basal ganglia: a similar code for physiological and pathological
conditions. Prog. Neurobiol. 84, 343–362.
Campbell, P., Reep, R.L., Stoll, M.L., Ophir, A.G., and Phelps, S.M. (2009).
Conservation and diversity of Foxp2 expression in muroid rodents: functional
implications. J. Comp. Neurol. 512, 84–100.
Cox, R.D., and Brown, S.D. (2003). Rodent models of genetic disease. Curr.
Opin. Genet. Dev. 13, 278–283.
Crinion, J., Turner, R., Grogan, A., Hanakawa, T., Noppeney, U., Devlin, J.T.,
Aso, T., Urayama, S., Fukuyama, H., Stockton, K., et al. (2006). Language
control in the bilingual brain. Science 312, 1537–1540.
Dai, M., Wang, P., Boyd, A.D., Kostov, G., Athey, B., Jones, E.G., Bunney,
W.E., Myers, R.M., Speed, T.P., Akil, H., et al. (2005). Evolving gene/transcript
definitionssignificantlyaltertheinterpretationofGeneChipdata.NucleicAcids
Res. 33, e175.
David, H.N., Ansseau, M., and Abraini, J.H. (2005). Dopamine-glutamate reci-
procal modulation of release and motor responses in the rat caudate-putamen
and nucleus accumbens of ‘‘intact’’ animals. Brain Res. Brain Res. Rev. 50,
336–360.
Egnor, S.E., and Hauser, M.D. (2004). A paradox in the evolution of primate
vocal learning. Trends Neurosci. 27, 649–654.
Ehret, G. (2005). Infant rodent ultrasounds–a gate to the understanding of
sound communication. Behav. Genet. 35, 19–29.
Enard, W., and Pa ¨a ¨bo, S. (2004). Comparative Primate Genomics. Annu. Rev.
Genomics Hum. Genet. 5, 351–378.
Enard, W., Przeworski, M., Fisher, S.E., Lai, C.S., Wiebe, V., Kitano, T.,
Monaco, A.P., and Pa ¨a ¨bo, S. (2002). Molecular evolution of FOXP2, a gene
involved in speech and language. Nature 418, 869–872.
Ferland, R.J., Cherry, T.J., Preware, P.O., Morrisey, E.E., and Walsh, C.A.
(2003). Characterization of Foxp2 and Foxp1 mRNA and protein in the devel-
oping and mature brain. J. Comp. Neurol. 460, 266–279.
Fitch, W.T. (2000). The evolution of speech: a comparative review. Trends
Cogn. Sci. 4, 258–267.
French, C.A., Groszer, M., Preece, C., Coupe, A.M., Rajewsky, K., and Fisher,
S.E.(2007).Generationofmicewithaconditional Foxp2nullallele.Genesis45,
440–446.
Fuchs, H., Lisse, T., Abe, K., and Hrabe ´ de Angelis, M. (2006). Screening for
bone and cartilage phenotypes in mice. In Phenotyping of the Laboratory
Mouse, M. Hrabe de Angelis, P. Chambon, and S. Browns, eds. (Weinheim:
Wiley-VCH), pp. 35–86.
Fuchs, H., Schughart,K., Wolf, E.,Balling,R., and Hrabe ´ deAngelis,M.(2000).
Screening for dysmorphological abnormalities–a powerful tool to isolate new
mouse mutants. Mamm. Genome 11, 528–530.
Fujita, E., Tanabe, Y., Shiota, A., Ueda, M., Suwa, K., Momoi, M.Y., and Mo-
moi, T. (2008). Ultrasonic vocalization impairment of Foxp2 (R552H) knockin
mice related to speech-language disorder and abnormality of Purkinje cells.
Proc. Natl. Acad. Sci. USA 105, 3117–3122.
Gailus-Durner, V., Fuchs, H., Becker, L., Bolle, I., Brielmeier, M., Calzada-
Wack, J., Elvert, R., Ehrhardt, N., Dalke, C., Franz, T.J., et al. (2005). Intro-
ducing the German Mouse Clinic: open access platform for standardized phe-
notyping. Nat. Methods 2, 403–404.
Gainetdinov, R.R., Jones, S.R., Fumagalli, F., Wightman, R.M., and Caron,
M.G.(1998).Re-evaluationoftheroleofthedopaminetransporterindopamine
system homeostasis. Brain Res. Brain Res. Rev. 26, 148–153.
Gentleman,R.C.,Carey,V.J.,Bates,D.M.,Bolstad,B.,Dettling,M.,Dudoit,S.,
Ellis, B., Gautier, L., Ge, Y., Gentry, J., et al. (2004). Bioconductor: open soft-
ware development for computational biology and bioinformatics. Genome
Biol. 5, R80.
Gilbert, S.L., Dobyns,W.B., and Lahn, B.T. (2005).Genetic links betweenbrain
development and brain evolution. Nat. Rev. Genet. 6, 581–590.
Graybiel, A.M. (2008). Habits,rituals,and theevaluative brain.Annu. Rev. Neu-
rosci. 31, 359–387.
Groszer, M., Keays, D.A., Deacon, R.M., de Bono, J.P., Prasad-Mulcare, S.,
Gaub, S., Baum, M.G., French, C.A., Nicod, J., Coventry, J.A., et al. (2008).
Impaired synaptic plasticity and motor learning in mice with a point mutation
implicated in human speech deficits. Curr. Biol. 18, 354–362.
Haesler, S., Rochefort, C., Georgi, B., Licznerski, P., Osten, P., and Scharff, C.
(2007). Incompleteand inaccurate vocal imitationafter knockdown of FoxP2 in
songbird basal ganglia nucleus Area X. PLoS Biol. 5, e321. 10.1371/journal.
pbio.0050321.
Hammerschmidt, K., and Fischer, J. (2008). Constraints in primate vocal
production. In The evolution of communicative creativity: From fixed signals
to contextual flexibility, U. Griebel and K. Oller, eds. (Cambridge, MA: The
MIT Press).
Hara, E., Kubikova, L., Hessler, N.A., and Jarvis, E.D. (2007). Role of the
midbrain dopaminergic system in modulation of vocal brain activation by
social context. Eur. J. Neurosci. 25, 3406–3416.
Heiman, M., Schaefer, A., Gong, S., Peterson, J.D., Day, M., Ramsey, K.E.,
Suarez-Farinas, M., Schwarz, C., Stephan, D.A., Surmeier, D.J., et al. (2008).
A translational profiling approach for the molecular characterization of CNS
cell types. Cell 135, 738–748.
Jarvis, E.D. (2004). Learned birdsong and the neurobiology of human
language. Ann. N Y Acad. Sci. 1016, 749–777.
Ju ¨rgens, U. (2002). Neural pathways underlying vocal control. Neurosci. Bio-
behav. Rev. 26, 235–258.
Kreitzer, A.C., and Malenka, R.C. (2008). Striatal plasticity and basal ganglia
circuit function. Neuron 60, 543–554.
Krubitzer, L. (2007). The magnificent compromise: cortical field evolution in
mammals. Neuron 56, 201–208.
Lai, C.S., Fisher, S.E., Hurst, J.A., Vargha-Khadem, F., and Monaco, A.P.
(2001). A forkhead-domain gene is mutated in a severe speech and language
disorder. Nature 413, 519–523.
Lai,C.S.,Gerrelli,D.,Monaco,A.P.,Fisher,S.E.,andCopp,A.J.(2003).FOXP2
expression during brain development coincides with adultsites of pathologyin
a severe speech and language disorder. Brain 126, 2455–2462.
Lieberman,P.(2002).Onthenatureandevolutionoftheneuralbasesofhuman
language. Am. J. Phys. Anthropol. (Suppl 35), 36–62.
Lieberman,P.(2006).Towardanevolutionarybiology oflanguage(Cambridge,
MA: Harvard Univ. Press).
Liegeois, F., Baldeweg, T., Connelly, A., Gadian, D.G., Mishkin, M., and Var-
gha-Khadem, F. (2003). Language fMRI abnormalities associated with
FOXP2 gene mutation. Nat. Neurosci. 6, 1230–1237.
970 Cell 137, 961–971, May 29, 2009 ª2009 Elsevier Inc.

Page 11

MacDermot, K.D., Bonora, E., Sykes, N., Coupe, A.M., Lai, C.S., Vernes, S.C.,
Vargha-Khadem, F., McKenzie, F., Smith, R.L., Monaco, A.P., and Fisher, S.E.
(2005). Identification of FOXP2 truncation as a novel cause of developmental
speech and language deficits. Am. J. Hum. Genet. 76, 1074–1080.
Mikkelsen, T., Hillier, L., Eichler, E., Zody, M., Jaffe, D., Yang, S., Enard, W.,
Hellmann, I., Lindblad-Toh, K., Altheide, T., et al. (2005). Initial sequence of
the chimpanzee genome and comparison with the human genome. Nature
437, 69–87.
Ohl, F., Holsboer, F., and Landgraf, R. (2001). The modified hole board as
a differential screen for behavior in rodents. Behav. Res. Methods Instrum.
Comput. 33, 392–397.
Pollard, K.S., Salama, S.R., Lambert, N., Lambot, M.A., Coppens, S., Peder-
sen, J.S., Katzman, S., King, B., Onodera, C., Siepel, A., et al. (2006). An
RNA gene expressed during cortical development evolved rapidly in humans.
Nature 443, 167–172.
Pru ¨fer, K., Muetzel, B., Do, H.H., Weiss, G., Khaitovich, P., Rahm, E., Pa ¨a ¨bo,
S., Lachmann, M., and Enard, W. (2007). FUNC: a package for detecting sig-
nificant associations between gene sets and ontological annotations. BMC
Bioinformatics 8, 41.
Roberts, L.H. (1975a). Evidence for the larynx as the source of both ultrasonic
and audible cries of rodents. J. Zool. 175, 243–257.
Roberts, L.H. (1975b). The rodent ultrasound production mechanism. Ultra-
sonics 13, 83–88.
Scharff, C., and Haesler, S. (2005). An evolutionary perspective on FoxP2:
strictly for the birds? Curr. Opin. Neurobiol. 15, 694–703.
Schneider, I., Tirsch, W.S., Faus-Kessler, T., Becker, L., Kling, E., Busse, R.L.,
Bender, A., Feddersen, B., Tritschler, J., Fuchs, H., et al. (2006). Systematic,
standardized and comprehensive neurological phenotyping of inbred mice
strains in the German Mouse Clinic. J. Neurosci. Methods 157, 82–90.
Shu, W., Cho, J.Y., Jiang, Y., Zhang, M., Weisz, D., Elder, G.A., Schmeidler, J.,
De Gasperi, R., Sosa, M.A., Rabidou, D., et al. (2005). Altered ultrasonic vocal-
ization in mice with a disruption in the Foxp2 gene. Proc. Natl. Acad. Sci. USA
102, 9643–9648.
Shu, W., Lu, M.M., Zhang, Y., Tucker, P.W., Zhou, D., and Morrisey, E.E.
(2007). Foxp2 and Foxp1 cooperatively regulate lung and esophagus develop-
ment. Development 134, 1991–2000.
Shu, W., Yang, H., Zhang, L., Lu, M.M., and Morrisey, E.E. (2001). Character-
ization of a new subfamily of winged-helix/forkhead (Fox) genes that are ex-
pressed in the lung and act as transcriptional repressors. J. Biol. Chem. 276,
27488–27497.
Takahashi, K., Liu, F.C., Hirokawa, K., and Takahashi, H. (2003). Expression of
Foxp2, a gene involved in speech and language, in the developing and adult
striatum. J. Neurosci. Res. 73, 61–72.
Teichmann, M., Gaura, V., Demonet, J.F., Supiot, F., Delliaux, M., Verny, C.,
Renou, P., Remy, P., and Bachoud-Levi, A.C. (2008). Language processing
within the striatum: evidence from a PET correlation study in Huntington’s
disease. Brain 131, 1046–1056.
Tettamanti, M., Moro, A., Messa, C., Moresco, R.M., Rizzo, G., Carpinelli, A.,
Matarrese, M., Fazio, F., and Perani, D. (2005). Basal ganglia and language:
phonology modulates dopaminergic release. Neuroreport 16, 397–401.
Tusher, V.G., Tibshirani, R., and Chu, G. (2001). Significance analysisof micro-
arrays applied to the ionizing radiation response. Proc. Natl. Acad. Sci. USA
98, 5116–5121.
Ullman, M.T. (2001). A neurocognitive perspective on language: the declara-
tive/procedural model. Nat. Rev. Neurosci. 2, 717–726.
Vargha-Khadem, F., Gadian, D.G., Copp, A., and Mishkin, M. (2005). FOXP2
and the neuroanatomy of speech and language. Nat. Rev. Neurosci. 6,
131–138.
Vargha-Khadem, F., Watkins, K.E., Price, C.J., Ashburner, J., Alcock, K.J.,
Connelly, A., Frackowiak, R.S., Friston, K.J., Pembrey, M.E., Mishkin, M.,
et al. (1998). Neural basis of an inherited speech and language disorder.
Proc. Natl. Acad. Sci. USA 95, 12695–12700.
Varki, A., Geschwind, D.H., and Eichler, E.E. (2008). Explaining human unique-
ness: genome interactions with environment, behaviour and culture. Nat. Rev.
Genet. 9, 749–763.
Vernes, S.C., Nicod, J., Elahi, F.M., Coventry, J.A., Kenny, N., Coupe, A.M.,
Bird, L.E., Davies, K.E., and Fisher, S.E. (2006). Functional genetic analysis
of mutations implicated in a human speech and language disorder. Hum.
Mol. Genet. 15, 3154–3167.
Vernes, S.C., Spiteri, E., Nicod, J., Groszer, M., Taylor, J.M., Davies, K.E.,
Geschwind, D.H., and Fisher, S.E. (2007). High-throughput analysis of
promoter occupancy reveals direct neural targets of FOXP2, a gene mutated
in speech and language disorders. Am. J. Hum. Genet. 81, 1232–1250.
Viggiano, D., Ruocco, L.A., and Sadile, A.G. (2003). Dopamine phenotype and
behaviour in animal models: in relation to attention deficit hyperactivity
disorder. Neurosci. Biobehav. Rev. 27, 623–637.
Wang, B., Lin, D., Li, C., and Tucker, P. (2003). Multiple domains define the
expressionandregulatorypropertiesofFoxp1forkheadtranscriptionalrepres-
sors. J. Biol. Chem. 278, 24259–24268.
Watkins, K.E., Dronkers, N.F., and Vargha-Khadem, F. (2002a). Behavioural
analysis of an inherited speech and language disorder: comparison with
acquired aphasia. Brain 125, 452–464.
Watkins,K.E.,Vargha-Khadem,F.,Ashburner,J.,Passingham,R.E.,Connelly,
A., Friston, K.J., Frackowiak, R.S., Mishkin, M., and Gadian, D.G. (2002b). MRI
analysis of an inherited speech and language disorder: structural brain abnor-
malities. Brain 125, 465–478.
Wijchers, P.J., Hoekman, M.F., Burbach, J.P., and Smidt, M.P. (2006). Identi-
fication of forkhead transcription factors in cortical and dopaminergic areas of
the adult murine brain. Brain Res. 1068, 23–33.
Zhang, J., Webb, D.M., and Podlaha, O. (2002). Accelerated protein evolution
and origins of human-specific features: Foxp2 as an example. Genetics 162,
1825–1835.
Cell 137, 961–971, May 29, 2009 ª2009 Elsevier Inc. 971