A Diagnostic Marker to Discriminate Childhood Apraxia of Speech From Speech Delay: I. Development and Description of the Pause Marker

Rare mutations of the FOXP2 transcription factor gene cause a monogenic syndrome characterized by impaired speech development and linguistic deficits. Recent genomic investigations indicate that its downstream neural targets make broader impacts on common language impairments, bridging clinically distinct disorders. Moreover, the striking conservation of both FoxP2 sequence and neural expression in different vertebrates facilitates the use of animal models to study ancestral pathways that have been recruited towards human speech and language. Intriguingly, reduced FoxP2 dosage yields abnormal synaptic plasticity and impaired motor-skill learning in mice, and disrupts vocal learning in songbirds. Converging data indicate that Foxp2 is important for modulating the plasticity of relevant neural circuits. This body of research represents the first functional genetic forays into neural mechanisms contributing to human spoken language.

Introduction Forkhead-box protein P2 is a highly conserved vertebrate protein, belonging to an important group of transcription factors [1]. By modulating the expression of downstream target genes, forkhead-box proteins influence a diverse array of processes, including cell-cycle regulation, signal transduction, differentiation, patterning and metabolism [2]. They thereby play crucial roles during embryogenesis, in postnatal development and in the mature organism, and many have been linked to disease states [3]. The P subgroup is a divergent branch of forkhead-box proteins that share a distinctive DNA-binding domain located near the C-terminal end of the protein, as well as zinc-finger/leucine-zipper motifs that mediate dimerization, and a glutamine-rich region towards the N-terminus [4], [5]. Functional evidence from multiple species implicates Forkhead-box protein P2 in particularly intriguing aspects of brain development and function [1]. (Here we adopt the standard accepted nomenclature to refer to the protein in different species: FOXP2 in humans, Foxp2 in mice, FoxP2 in other chordates, with the corresponding gene names in italics [6].) In humans, damage to one copy of the FOXP2 gene causes a rare neurodevelopmental disorder, characterised by difficulties mastering sequences of mouth movements during speech, as well as impaired language processing [4], [7], [8]. Heterozygous disruptions of the mouse orthologue (Foxp2) yield dramatic reductions in synaptic plasticity of cortico-striatal brain circuits, associated with deficits in learning of rapid motor skills [9]. Mouse pups with homozygous Foxp2 mutations show more severe neural effects – gross motor impairments, delayed postnatal maturation of the cerebellum and dramatic reductions in emission of ultrasonic vocalisations – against a background of reduced weight-gain and postnatal lethality [9]-[11]. In addition, the avian orthologue (FoxP2) is required for normal vocal learning in songbirds [12], [13]. Selective knockdown of the gene in a key striatal nucleus in juvenile zebrafinches leads to incomplete and inaccurate imitation of tutor songs [14]. Studies of both human FOXP2 and mouse Foxp2 identified similarly strong CNS (central nervous system) expression during embryogenesis, which is confined to neurons (absent from glial cells) and enriched in various brain structures, including deep layers of the developing cortical plate, and parts of the striatum, thalamus and cerebellum [15],[16]. These embryonic expression patterns appear highly concordant in the different species, and show remarkable overlaps with sites of pathology identified by neuroimaging of human children and adults carrying FOXP2 mutations [16], [17]. Neural expression of the gene continues postnatally and into adulthood [4], [15], and is also observed in certain non-neural tissues, most notably the distal alveolar lung epithelium, and the outflow tract and atrium of the cardiovascular system [18]. The above observations of well-conserved and specific CNS expression patterns [15], [16] suggest that Foxp2 is likely to have important functions in neurodevelopment. Nevertheless, as data continue to accumulate regarding its impacts on the postnatal brain [9], [11], [14], the specific roles of Foxp2 in the developing CNS remain largely elusive. One route for gaining insights into the biological processes controlled by a transcription factor is to define the regulatory networks that are directly downstream of it [1]. An efficient strategy for identifying direct targets exploits chromatin immunoprecipitation (ChIP) methods to screen the tissue of interest [19]. Two previous investigations have coupled ChIP with hybridisation to promoter microarrays (ChIP-chip) in order to uncover binding sites of FOXP2 in human foetal brain tissue [20] and in human neurons grown in culture [21]. Both screens were of limited scope – the microarrays in these studies comprised fragments from the 5′ ends of ∼5,000 loci [20], [21], representing a small percentage of the known gene promoters in the genome. Neither study combined ChIP data with large-scale expression analyses. A more recent report used mRNA expression profiling in human neuronal models transfected with different versions of FOXP2 to explore regulatory differences between the human and chimpanzee orthologues, but did not include any ChIP screening for direct targets [22]. In the present study, we performed a systematic large-scale in vivo ChIP-chip screen of the embryonic mouse brain, coupling Foxp2-ChIP to high-density arrays with oligonucleotides tiled across >17,000 promoters. We robustly established the empirical significance of our ChIP results in wild-type brains by determining the null distribution of signals generated by matched control tissue from littermates that expressed no Foxp2 protein. Under strict empirical thresholds that minimised false positive signals, we isolated a set of 264 high-confidence in vivo targets. Gene ontology (GO) analyses of the ChIP-chip data, as well as genome-wide expression profiling and in situ hybridisations of wild-type and mutant mice, converged on neurite outgrowth as one of the most prominent biological themes associated with Foxp2 function in the embryonic CNS. We went on to directly demonstrate, using neuronal cell models and primary neurons from the embryonic mouse brain, that Foxp2 alters expression of neurite-outgrowth targets and thereby influences neurite process length and branch number. Results Genome-wide identification of in vivo Foxp2 targets in embryonic mouse brain In vivo Foxp2-ChIP screening was carried out using brains harvested from embryonic mice. Experiments were performed with mice that were wild-type for Foxp2, as well as homozygous littermates that do not express any Foxp2 protein (Foxp2-S321X mutants; see Materials and Methods) [9]. The different types of sample were screened in parallel, undergoing identical experimental manipulations and data processing. In this context, the homozygous mutant mouse tissue acts as an ideal negative control [21]. Since such samples completely lack Foxp2 protein (see Figure S1 and [9]), fragments that are pulled down by Foxp2-ChIP in these cases give an unbiased empirical indication of background noise and false positive rates yielded by the procedure. Whole mouse brains from wild-type or mutant mice were harvested at embryonic day 16 (E16), corresponding to a timepoint at which particularly high levels of Foxp2 expression are observed in the developing CNS [16]. Chromatin isolated by Foxp2-ChIP was labelled and hybridised to DNA microarrays covering the promoter regions of ∼17,000 mouse transcripts (Agilent Technologies), using total input DNA as a reference sample. Each promoter on these arrays is represented by an average of twenty-five 60-mer probes spanning ∼5.5 kb upstream and ∼2.5 kb downstream of the transcription start site, allowing peak regions of binding to be precisely defined (Figure 1). Moreover, the presence of multiple probes for each promoter scattered throughout the array gives independent enrichment values within the same promoter, which aids discrimination of real biological targets from false positive events. Specifically, since the shearing process during ChIP produces overlapping fragments of chromatin, true targets should show evidence of enrichment for multiple probes across the promoter region, while promoters with only a single enriched probe are most likely to be false positive results. 10.1371/journal.pgen.1002145.g001 Figure 1 In vivo Foxp2 promoter occupancy in embryonic mouse brain. (A) Foxp2-ChIP window enrichment scores of probes across promoters of a subset of putative targets from the neurite outgrowth and axon guidance pathways. The window score (Y axis) is given versus the distance across the promoter region (X axis) - each cross bar represents 1000 bp and each data point represents a single probe (chromosome and position in bp are given below the X axis of each graph). The enrichment in the wild-type experiments is shown by the blue trace, and the pink trace indicates the corresponding values in null mutant controls that lack Foxp2 protein. The predicted start site of the gene (as annotated on UCSC genome browser: http://genome.ucsc.edu/) is given by the black box and arrows denote the direction of transcription. Grey shading indicates the peak area of enrichment and the most likely region for Foxp2 binding to occur. (B) Analysis of in vivo promoter occupancy. DNA isolated via Foxp2-ChIP was PCR amplified using primers directed towards the promoter regions of Nrp2, Sema3a, Nrn1 or the β-actin control. The position of these amplicons within the target promoter region is given by red bars in part A. Results from E16 wild type mice were compared to those from homozygous null mutant littermates. Lane 1 =  wild-type Foxp2-ChIP, lane 2 =  mutant null Foxp2-ChIP, lane 3 =  wild-type total DNA, lane 4 =  mutant total DNA. Target gene promoters were found to be specifically enriched in Foxp2-ChIP samples isolated from wild-type brains compared to those from null mutant brains, unlike the β-actin control promoter. Gels are representative of results from triplicate experiments. In order to identify enriched promoters, Foxp2-ChIP data were analysed as per Materials and Methods. Briefly, array data from independent biological replicates (three independent ChIP experiments hybridised to one each of three array sets) were normalised for each genotype (wild-type or mutant control) separately. Normalised array data (excluding probes with a negative average enrichment across replicate experiments) were subjected to a sliding window analysis, using a similar method to that employed in genome-wide ChIP-chip studies of other forkhead transcription factors [23]. Each probe was assigned a value (window-adjusted score) based on the median fold enrichment of itself and its neighbouring probe on either side (within 500 bp upstream and 500 bp downstream), and then probes were ranked based on this window score. By analysing the distribution of window scores observed in the mutant null control experiments we were able to derive an empirical threshold for significance, which could then be applied to the wild-type data. We found that window scores greater than or equal to 0.974 (corresponding to ∼2-fold enrichment) excluded 99% of the data-points in the mutant null control experiments. When we applied this threshold to data from wild-type experiments, we identified a set of 1,217 promoter regions that were consistently enriched by Foxp2-ChIP over 3 replicates in wild-type mouse brains (Table S1). On inspection of the locations of the enriched probes throughout the mouse genome, no positional bias was observed (Figure S2). Since some of the enriched regions lay close to the transcriptional start site (TSS) of more than one gene, the 1,217 promoter regions corresponded to 1,253 genes. Of note, using the same analysis parameters, only 147 genes were enriched in the mutant null controls, suggesting a low false discovery rate. Nevertheless, in order to minimize false-positive findings, we excluded any enriched genes from the wild-type dataset that also had window scores exceeding the 99% threshold in the mutant control dataset. This filtering process yielded a slightly smaller set of 1,164 putative targets (Table S2). When we applied stricter thresholds to the wild-type data, selecting only those promoters in which at least one probe gave a window score of ≥ 1.5, we identified a shortlist of 259 promoter regions. Since a small number of peak regions lay directly between the TSSs of two different genes, these 259 promoters corresponded to a slightly higher total of 266 genes. Crucially, the same analyses of the entire mutant null control dataset identified only a single gene in the genome with a window score of ≥ 1.5 (the Pigt gene), indicating an extremely low rate of false positives under these stricter selection criteria. We excluded two genes from the strict wild-type shortlist (Pigt and Zfp496) since they contained probes that exceeded the 99% threshold (i.e. window score of >0.974) in mutant null controls (Figure S3). The outcome of these analyses was a final curated shortlist of 264 high-confidence in vivo targets (Table S3). Given that DNA is sheared randomly during the ChIP process, we would expect a true Foxp2 binding event to be represented by a peak of enrichment at a target promoter. This peak would result from the sheared DNA forming a series of overlapping fragments, with the region closest to the binding site showing the highest degree of enrichment (i.e. highest number of fragments pulled down during immunoprecipitation) and with progressively less enrichment observed as the distance to the binding site increases on either side. Figure 1A gives typical examples of the enrichment peaks observed for putative targets from our Foxp2-ChIP dataset. Examination of corresponding data from mutant control experiments emphasises the relative lack of enrichment in nulls that lack Foxp2 protein, indicating that the enrichment in wild-type samples results from highly specific Foxp2-mediated interactions. Furthermore, we followed up a subset of candidates with qPCR, consistently confirming their enrichment (Figure 1B). Enriched regions represented in the shortlist of high-confidence targets were assessed in silico for any over-represented sequence motifs (see Text S1). This analysis did not enforce a priori conditions of motif sequence, other than a length restriction of 8 bases. This meant that rather than limiting our search to occurrences of known patterns in the promoters, we obtained an unbiased list of motifs that were characteristic of the Foxp2-ChIP target promoters. Eight sequences (motifs A-H) were found to be significantly over-represented (p 0.974) are highlighted on the WebGestalt KEGG pathway in red, with those from expression analysis (p 15 images taken for three independent clones of each cell type (Scale bar, 100 µm). To further assess the in vivo relevance of these findings, we examined whether there were corresponding phenotypic effects mediated by functional Foxp2 in neurons of the developing brain. We isolated primary neurons from the ganglionic eminences of E16 mouse brains, matching the region and timepoint used for our original target screening. Here, we aimed to directly test whether the Foxp2-positive neurons derived from the developing basal ganglia show altered neurite outgrowth when the gene is mutated. The assay was facilitated by availability of a mouse model (Foxp2-R552H) in which the protein is expressed at normal levels, but is nevertheless dysfunctional [9]. R552H mice recapitulate an aetiological mutation that causes speech and language deficits in a large human family. This change yields a substitution in the DNA-binding domain which severely impairs the transcription factor function of the mutant protein [30], such that the overall phenotype of homozygous R552H animals is very similar to that observed for mice which completely lack Foxp2 [9]–[11]. However, unlike the Foxp2-null mice, R552H homozygotes still express detectable levels of the protein, allowing us to clearly identify Foxp2-positive cells in our primary cultures via antibody staining. This represents an important measure, given the heterogeneous nature of the dissected material used to generate the primary culture. We again observed obvious differences in neurite outgrowth associated with presence of functional Foxp2 (Figure 6A). A blinded analysis revealed statistically significant changes in quantitative measures of neurite outgrowth for Foxp2-expressing neurons from wild-type embryos as compared to those from homozygous Foxp2-R552H littermates (Figure 6B). In particular, the latter showed significant reductions in total outgrowth (p = 0.001); mean (p 1.5) in wild-type Foxp2-chip. (TIF) Click here for additional data file. Figure S4 EMSA analysis of a novel Foxp2 DNA-binding motif. (TIF) Click here for additional data file. Figure S5 Differential expression of Foxp2 target genes in E16 developing mouse ganglionic eminences. (TIF) Click here for additional data file. Figure S6 Co-expression of Foxp2 and putative target genes in the E16 mouse brain. (TIF) Click here for additional data file. Figure S7 Expression of Foxp2 in medium spiny neurons (MSNs). (TIF) Click here for additional data file. Figure S8 In vivo binding of mouse Foxp2 to intron 1 of Cntnap2 in embryonic brain tissue. (TIF) Click here for additional data file. Table S1 Foxp2-ChIP (window score ≥0.97) complete dataset. (XLS) Click here for additional data file. Table S2 Foxp2-ChIP putative target promoters (curated long list n = 1164, window score ≥0.97). (XLS) Click here for additional data file. Table S3 Foxp2-ChIP putative target promoters (curated short list n = 264, at least 1 window score ≥1.50). (XLS) Click here for additional data file. Table S4 Foxp2-dependent gene expression changes in the E16 mouse brain (p<0.01). (XLS) Click here for additional data file. Table S5 Overlapping target genes from Foxp2-ChIP and expression analysis studies. (XLS) Click here for additional data file. Table S6 Significant over-represented KEGG pathways identified in the Foxp2-ChIP and gene expression datasets. (XLS) Click here for additional data file. Table S7 Cntnap2 semi-quantitative PCR primers. (XLS) Click here for additional data file. Text S1 Supporting Materials and Methods. (DOC) Click here for additional data file.

Any account of "what is special about the human brain" (Passingham 2008) must specify the neural basis of our unique ability to produce speech and delineate how these remarkable motor capabilities could have emerged in our hominin ancestors. Clinical data suggest that the basal ganglia provide a platform for the integration of primate-general mechanisms of acoustic communication with the faculty of articulate speech in humans. Furthermore, neurobiological and paleoanthropological data point at a two-stage model of the phylogenetic evolution of this crucial prerequisite of spoken language: (i) monosynaptic refinement of the projections of motor cortex to the brainstem nuclei that steer laryngeal muscles, presumably, as part of a "phylogenetic trend" associated with increasing brain size during hominin evolution; (ii) subsequent vocal-laryngeal elaboration of cortico-basal ganglia circuitries, driven by human-specific FOXP2 mutations.;>This concept implies vocal continuity of spoken language evolution at the motor level, elucidating the deep entrenchment of articulate speech into a "nonverbal matrix" (Ingold 1994), which is not accounted for by gestural-origin theories. Moreover, it provides a solution to the question for the adaptive value of the "first word" (Bickerton 2009) since even the earliest and most simple verbal utterances must have increased the versatility of vocal displays afforded by the preceding elaboration of monosynaptic corticobulbar tracts, giving rise to enhanced social cooperation and prestige. At the ontogenetic level, the proposed model assumes age-dependent interactions between the basal ganglia and their cortical targets, similar to vocal learning in some songbirds. In this view, the emergence of articulate speech builds on the "renaissance" of an ancient organizational principle and, hence, may represent an example of "evolutionary tinkering" (Jacob 1977).