Learning curves of wildtype (a), heterozygous R552H mutant (b), and heterozygous S321X mutant (c) mice.

<p>For all 20 training days (one training session per day) the mean numbers of jumps across the hurdle averaged from the performances of the 11 animals per experimental group are shown. The animals could show hits (CR+) in the presence of 12 kHz tones or false alarms (CR−) in the presence of 7 kHz tones. Since each training session consisted of 60 trials with 30 randomized presentations of both CS+ and CS− a maximum of 30 hits and 30 false alarms could be reached if the animals responded to each tone with a jump, irrespective of the tone frequency. The larger the distance is between the CR+ and CR− curves the better is the learning performance. Standard deviations of the means are shown only for one side to improve readability of the data. Statistically significant differences between the CR+ and CR− rates calculated for each training session are indicated as ** p<0.01; *** p<0.001.</p

Latencies of jumps across the hurdle.

<p>Mean latencies of jumps across the hurdle of the shuttle-box after tone onset are plotted as a function of the training sessions. There are no systematic significant differences of latencies between the experimental groups of animals. At training session 15, R552H mutants are significantly different from WTs and S321X mutants (p<0.05; F = 4.42) and at training session 18, WTs differ from R552H mutants (p<0.05; F = 5.41). At any other training session, significant differences did not occur (F-values<2.50). Standard deviations are shown only for the WTs for better visibility. They are of the same order of magnitude for both groups of heterozygous mutants.</p

Logistic growth functions modeling the increase of the discrimination index d′ as function of the training day.

<p>d′ expresses the achieved average performance level of tone discrimination of the animals in each experimental group (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033130#s3" target="_blank">Methods</a>). Discrimination performance of the WTs increases rapidly and stays at a maximum level already from day 2 onwards. Discrimination performance of the R552H heterozygotes increases slowly but finally reaches the level of the WTs. Discrimination performance of the S321X heterozygotes increases very slowly and does not reach the levels of WTs and R552H heterozygotes. The correlation coefficients of the growth functions are statistically significant (p<0.01 in each case).</p

Spontaneous motor behavior.

<p>Mean numbers of spontaneous jumps across the hurdle of the shuttle-box during the three minutes before the beginning of the daily training session. At the first training day, WTs show significantly more spontaneous jumping compared to both types of heterozygous mutants (*** p<0.001 in each case; F-value of the ANOVA = 14.92). At the second training day, the WTs show significantly more jumps than the heterozygous R552H mutants (p<0.02 **; F-value of the ANOVA = 5.12). For training days 3–13, 18 and 20 the ANOVA-tests did not lead to significant differences, F<3.42). WTs showed more spontaneous jumps compared to both mutants (p<0.05*; F>5.30) on days 17 and 19, and compared to R552H mutants (p<0.05*; F>4.40) on days 14–16. Standard deviations of the means are shown only for one side to improve readability of the data.</p

Table_2.pdf

<p>Mutations of the FOXP2 gene cause a severe speech and language disorder, providing a molecular window into the neurobiology of language. Individuals with FOXP2 mutations have structural and functional alterations affecting brain circuits that overlap with sites of FOXP2 expression, including regions of the cortex, striatum, and cerebellum. FOXP2 displays complex patterns of expression in the brain, as well as in non-neuronal tissues, suggesting that sophisticated regulatory mechanisms control its spatio-temporal expression. However, to date, little is known about the regulation of FOXP2 or the genomic elements that control its expression. Using chromatin conformation capture (3C), we mapped the human FOXP2 locus to identify putative enhancer regions that engage in long-range interactions with the promoter of this gene. We demonstrate the ability of the identified enhancer regions to drive gene expression. We also show regulation of the FOXP2 promoter and enhancer regions by candidate regulators – FOXP family and TBR1 transcription factors. These data point to regulatory elements that may contribute to the temporal- or tissue-specific expression patterns of human FOXP2. Understanding the upstream regulatory pathways controlling FOXP2 expression will bring new insight into the molecular networks contributing to human language and related disorders.</p

Table_1.pdf

<p>Mutations of the FOXP2 gene cause a severe speech and language disorder, providing a molecular window into the neurobiology of language. Individuals with FOXP2 mutations have structural and functional alterations affecting brain circuits that overlap with sites of FOXP2 expression, including regions of the cortex, striatum, and cerebellum. FOXP2 displays complex patterns of expression in the brain, as well as in non-neuronal tissues, suggesting that sophisticated regulatory mechanisms control its spatio-temporal expression. However, to date, little is known about the regulation of FOXP2 or the genomic elements that control its expression. Using chromatin conformation capture (3C), we mapped the human FOXP2 locus to identify putative enhancer regions that engage in long-range interactions with the promoter of this gene. We demonstrate the ability of the identified enhancer regions to drive gene expression. We also show regulation of the FOXP2 promoter and enhancer regions by candidate regulators – FOXP family and TBR1 transcription factors. These data point to regulatory elements that may contribute to the temporal- or tissue-specific expression patterns of human FOXP2. Understanding the upstream regulatory pathways controlling FOXP2 expression will bring new insight into the molecular networks contributing to human language and related disorders.</p

Table_4.xlsx

<p>Mutations of the FOXP2 gene cause a severe speech and language disorder, providing a molecular window into the neurobiology of language. Individuals with FOXP2 mutations have structural and functional alterations affecting brain circuits that overlap with sites of FOXP2 expression, including regions of the cortex, striatum, and cerebellum. FOXP2 displays complex patterns of expression in the brain, as well as in non-neuronal tissues, suggesting that sophisticated regulatory mechanisms control its spatio-temporal expression. However, to date, little is known about the regulation of FOXP2 or the genomic elements that control its expression. Using chromatin conformation capture (3C), we mapped the human FOXP2 locus to identify putative enhancer regions that engage in long-range interactions with the promoter of this gene. We demonstrate the ability of the identified enhancer regions to drive gene expression. We also show regulation of the FOXP2 promoter and enhancer regions by candidate regulators – FOXP family and TBR1 transcription factors. These data point to regulatory elements that may contribute to the temporal- or tissue-specific expression patterns of human FOXP2. Understanding the upstream regulatory pathways controlling FOXP2 expression will bring new insight into the molecular networks contributing to human language and related disorders.</p

Image_1.tif

<p>Mutations of the FOXP2 gene cause a severe speech and language disorder, providing a molecular window into the neurobiology of language. Individuals with FOXP2 mutations have structural and functional alterations affecting brain circuits that overlap with sites of FOXP2 expression, including regions of the cortex, striatum, and cerebellum. FOXP2 displays complex patterns of expression in the brain, as well as in non-neuronal tissues, suggesting that sophisticated regulatory mechanisms control its spatio-temporal expression. However, to date, little is known about the regulation of FOXP2 or the genomic elements that control its expression. Using chromatin conformation capture (3C), we mapped the human FOXP2 locus to identify putative enhancer regions that engage in long-range interactions with the promoter of this gene. We demonstrate the ability of the identified enhancer regions to drive gene expression. We also show regulation of the FOXP2 promoter and enhancer regions by candidate regulators – FOXP family and TBR1 transcription factors. These data point to regulatory elements that may contribute to the temporal- or tissue-specific expression patterns of human FOXP2. Understanding the upstream regulatory pathways controlling FOXP2 expression will bring new insight into the molecular networks contributing to human language and related disorders.</p

Supplementary material for Heim et al.: 1. Auditory discrimination learning and acoustic cue weighing in female zebra finches with localised FoxP1 knockdowns

Rare disruptions of the transcription factor FoxP1 are implicated in a human neurodevelopmental disorder characterised by autism and/or intellectual disability with prominent problems in speech and language abilities. Avian orthologues of this transcription factor are evolutionarily conserved and highly expressed in specific regions of songbird brains, including areas associated with vocal production learning and auditory perception. Here, we investigated possible contributions of FoxP1 to song discrimination and auditory perception in juvenile and adult female zebra finches. They received lentiviral knockdowns of FoxP1 in one of two brain areas involved in auditory stimulus processing, HVC (proper name) or CMM (caudomedial mesopallium). Ninety-six females, distributed over different experimental and control groups were trained to discriminate between two stimulus songs in an operant Go/Nogo paradigm and subsequently tested with an array of stimuli. This made it possible to assess how well they recognised and categorised altered versions of training stimuli and whether localised FoxP1 knockdowns affected the role of different features during discrimination and categorisation of song. Although FoxP1 expression was significantly reduced by the knockdowns, neither discrimination of the stimulus songs nor categorisation of songs modified in pitch, sequential order of syllables or by reversed playback were affected. Subsequently, we analysed the full dataset to assess the impact of the different stimulus manipulations for cue weighing in song discrimination. Our findings show that zebra finches rely on multiple parameters for song discrimination, but with relatively more prominent roles for spectral parameters and syllable sequencing as cues for song discrimination.</p

Confluent growth arrest is associated with increased FOXP2 expression.

<p>(A) Cell cycle analysis of 143B subjected to increasing confluence (approximate percentage confluence indicated top), numbers within plots from left to right indicate percentage of cells in G1/G0, S, and G2/M phase respectively, representative of three experiments; (B) Quantitation of apoptotic cell death in 143B cell populations by flow cytometric analysis of Annexin V positivity, in cultures either exponentially growing (Exp. Growth), subjected to overnight culture with 20μg/ml cyclohexmide as a positive control that induces apoptosis (cycloheximide), or subjected to 4 days growth arrest at confluence (late arrest, as per <i>A</i>). Numbers represent mean % annexin positive ± SD from three experiments; (C) Real-time PCR analyses of <i>FOXP</i> expression in MG-63, 143B and U2-OS cultured to increasing confluence, expressed as 2^-δβCT, relative to growing culture, <i>N</i> = 3 ± SD; (D) Immunoblot analyses of nuclear extracts from cells cultured as in <i>C</i>, including nucleophosmin (NPM) as a loading and transfer control, representative of two experiments, (E) Real-time PCR analyses of <i>p21</i>, <i>p27</i> and <i>IL-6</i> expression in 143B cultured to increasing confluence, expressed as 2^-δδCT, relative to growing culture, <i>N</i> = 3 ± SD.</p