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.

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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.

https://doi.org/10.1371/journal.pgen.1002145.g001

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<0.05) in the shortlist of high confidence target promoter sequences (Table 1). Importantly, the three most commonly identified over-represented motifs from this unbiased search (A–C) were partial or complete matches to well established FOX/FOXP/FOXP2 binding motifs (RYMAAYA/TATTTRT/AATTTGT), providing additional strong support for the biological relevance of our findings. A further over-represented motif (motif D) did not match the known consensus motifs and was detected in 182 promoters out of the 247 promoter regions that could be surveyed from the Foxp2-ChIP shortlist (See Text S1; Figure S4A). Thus, we reasoned that it may represent a novel putative Foxp2 binding sequence. EMSA (Electrophoretic Mobility Shift Assay) experiments demonstrated strong specific binding of FOXP2 to this motif (Figure S4B), when located in putative Foxp2 target promoter sequences, such as those for Nrn1, Nfat5 and Sema6d. However, not all occurrences of this motif were strongly bound by Foxp2, suggesting that while the site is capable of being bound by Foxp2 protein, the binding is context specific – as is regularly seen for other FOX family binding sites [24].

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Table 1. Over-represented motifs identified in the shortlist of Foxp2-ChIP target promoters.

https://doi.org/10.1371/journal.pgen.1002145.t001

In addition to the use of in vivo ChIP to uncover target genes that are directly bound by Foxp2 (direct targets), we assessed regulatory cascades further downstream (indirect targets) via an expression profiling approach. Again we focused on E16 mouse brain tissue, analysing the same genotypes (wild-type mice and their homozygous Foxp2-S321X littermates, 5 and 6 biological replicates, respectively) on the same genomic background as used for the ChIP experiments. While ChIP identifies DNA-binding events of Foxp2-positive cells, expression profiling is expected to be more sensitive to tissue heterogeneity. Therefore we selected a key site of high Foxp2 expression with considerable prior evidence of functional relevance [9], [14]–[17], the ganglionic eminences (developing striatum and pallidum). Analysis of genome-wide expression data (see Materials and Methods for details) identified 340 genes that were differentially expressed (p<0.01) between wild-type and Foxp2-S321X homozygous mutant samples (Table S4). 180 of these genes showed reduced expression in absence of Foxp2 protein, while the remaining 160 genes showed increases (Table S4). Of these 340 genes, 19 genes (5.6%) were found in common with the ChIP-chip target gene list (Table S5), including those with known CNS functions, such as Nell2 (neural epidermal growth factor-like like 2), Nrn1 (neuritin), Cck (cholecystokinin), and Alcam (activated leukocyte cell adhesion molecule). Notably, the human orthologues of Nrn1 and Cck have been independently proposed as top direct targets in small-scale ChIP screens of human foetal tissue [20].

We went on to determine whether any biological themes were over-represented within the direct targets (promoter bound by Foxp2) and indirect targets (not bound by, but regulated downstream of Foxp2), using unbiased GO analyses. The Foxp2-ChIP and expression profiling datasets were each assessed independently using the WebGestalt program [25], identifying functional categories that were significantly enriched (Figure 2 and Figure S5). In the Foxp2-ChIP dataset we observed significant over-representation of genes involved in processes including cell motility and migration, chromatin architecture and assembly, synaptic transmission, and a number of categories associated with RNA metabolism such as regulation of RNA stability and mRNA processing. In the expression profiling dataset significant categories included regulation of transcription, actin cytoskeleton organisation and biogenesis, and cellular protein catabolism. Consistent with previous studies [20], [21], nervous system development, neurogenesis and multiple G-protein signalling categories — including G-protein coupled receptor signalling (ChIP), and G-protein signalling and Wnt receptor signalling (expression) — were significant in both datasets.

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Figure 2. Gene Ontology categories suggest Foxp2 involvement in known functional pathways.

(A) Gene ontology analysis of Foxp2-ChIP target gene list (see Table S2) and (B) Gene ontology analysis of genes differentially expressed between Foxp2-S321X and wild-type embryos (see Table S4). For each, a selection of biological process ontology categories are shown that were found to be significantly over-represented in the putative target gene lists, including neurogenesis, neuron projection development and axonogenesis (A–B), cell migration (A) and G-protein receptor signalling (B). The number of genes per category and p-values are shown in brackets.

https://doi.org/10.1371/journal.pgen.1002145.g002

We next performed in situ hybridisation on brains from wild-type and Foxp2-S321X E16 embryos, to further assess major targets suggested by the ChIP and expression profiling screens. Consistent with previously published data [16], in addition to the developing striatum, Foxp2 expression at this developmental stage is highest in the diencephalon (developing thalamus), midbrain and cerebellar primordium (Figure 3). The in situ hybridisation data confirmed regulation of Shhrs (also known as Dlx6as1 or Evf1/2), a transcript showing greater than 200-fold increased expression levels in S321X mice. This noncoding RNA is highly specific to the ganglionic eminences in the embryo and is known to play a vital role in the control of the homeodomain transcription factors Dlx5 and Dlx6 [26], [27]. These data illustrate that loss of Foxp2 can influence transcripts central to key neurodevelopmental processes in vivo. We then focused on target genes common to both ChIP and expression profiling datasets, to determine whether expression changes could be observed, not only in the ganglionic eminences, but also elsewhere in the developing brain (Figure 3). Indeed, Nell2, Nrn1 and Cck all demonstrated clear increases in expression in the developing basal ganglia at E16 in the Foxp2 mutant compared to wild-type, in agreement with the array data (Figure 3 and Figure S5), providing further evidence that they are indeed direct targets, repressed by Foxp2. Significantly, Nrn1, a gene important for neuronal outgrowth [28], showed strongly increased expression in mutants in additional regions where Foxp2 is typically expressed, including the developing thalamus and cerebellum (Figure 3). Similarly, Cck shows additional increases in expression in the cerebellum (Figure 3).

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Figure 3. Foxp2 regulates putative target gene expression in the E16 mouse brain.

In situ hybridisation of wild-type (WT) and Foxp2-S321X homozygous mutant brains at E16. Foxp2 is predominantly expressed in the ganglionic eminences (G), diencephalon (D), midbrain (M) and developing cerebellum (C) in WT mice. Figure displays results from selected transcripts that had shown increased levels in expression profiling of Foxp2 mutant mice. Nrn1, Nell2 and Cck were also identified from the ChIP screen. Arrows indicate equivalent regions from brains of both genotypes. In Foxp2 mutants, all target genes show increased expression in the ganglionic eminence, with additional increased expression of Nrn1 in the diencephalon and Nrn1 and Cck in the developing cerebellum. Scale bar is 1 mm. Results are representative of 3 mice of each genotype.

https://doi.org/10.1371/journal.pgen.1002145.g003

Certain putative direct Foxp2 targets with known roles in the CNS, such as Ywhah (tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, eta polypeptide) and Wasf1 (WASP family 1), are ubiquitously expressed in the developing mouse brain [29]. However, other genes are thought to have more localised and/or temporally defined patterns of expression. To ascertain whether these targets are co-expressed with Foxp2, further in situ hybridisation was carried out at E16. The results demonstrate that genes from the ChIP dataset with established CNS functions are found in a range of Foxp2-expressing brain regions (Figure S6).

Foxp2 regulates neurite outgrowth in neurons

The GO analyses of independent Foxp2 target data from genome-wide ChIP and expression profiling uncovered a consistent and statistically significant over-representation of genes involved in neurite development & morphogenesis, axon extension and axon guidance pathways (Table 2). Multiple GO categories associated with such processes were significantly over-represented in at least one of the datasets, and several of these functional classes were significant in both datasets including neurogenesis, neuron projection development and axonogenesis (Figure 2 and Table 2). Furthermore, when we investigated KEGG pathways associated with these datasets, we observed enrichment of genes in a number of pathways (Figure 4A and Table S6), one of the most significant of which was the axon guidance pathway (p = 4.73×10⁻⁸ and p = 3.00×10⁻⁴ in Foxp2-ChIP and expression profiling datasets, respectively). Interestingly, a number of different but interacting genes within this pathway were identified in the two datasets (Figure 4B), suggesting that direct and indirect targets may represent different aspects of the same functional downstream effects of Foxp2-mediated regulation. In sum, our unbiased genomic screens for Foxp2-dependent gene networks predicted that neurite outgrowth and axon guidance should be key biological themes associated with Foxp2 function in the developing brain.

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Figure 4. Neurite outgrowth and axon guidance pathways are major biological themes of Foxp2-dependent networks in embryonic brain.

(A) The Foxp2 target gene datasets obtained via ChIP-chip (see Table S2) and expression profiling (see Table S4) were assessed separately for significantly over-represented KEGG pathways. (B) The axon guidance pathway displayed one of the most highly significant over-representations in the respective datasets (Foxp2-ChIP: p = 4.73×10⁻⁸ and expression profiling: p = 3.00×10⁻⁴). Genes that were present in this pathway from the Foxp2-ChIP target list (window score >0.974) are highlighted on the WebGestalt KEGG pathway in red, with those from expression analysis (p<0.01) highlighted in blue. None of the genes shown were common to both datasets. Expression profiling showed Efnb1 and Plxna4 to be upregulated in vivo in response to Foxp2; all other genes highlighted in blue were downregulated. Figure adapted from the WebGestalt program results (see Materials and Methods).

https://doi.org/10.1371/journal.pgen.1002145.g004

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Table 2. GO categories significantly over-represented in Foxp2-ChIP (Table S2) and expression profiling (Table S4) datasets.

https://doi.org/10.1371/journal.pgen.1002145.t002

We went on to test this prediction through genetic and functional analyses of neuronal cell models and primary neurons. First, we independently assessed whether differences in Foxp2 expression affect expression of putative direct target genes involved in neurite outgrowth, using a well-validated murine cell model. Neuro2a cells are neuron-derived cells that can be differentiated to take on a more neuron-like identity via exposure to retinoic acid. These cells were stably transfected with Foxp2 or with an empty vector control, and then grown in media either with or without the addition of retinoic acid. Cells that were over-expressing Foxp2 demonstrated consistent expression changes for multiple direct target genes that were identified in our E16 ChIP screen and implicated in neurite outgrowth pathways (Figure 5A). Significant repression of target gene expression was observed both pre- and post-differentiation; however most of the neurite-outgrowth genes showed greater fold changes following differentiation. Next, we formally assessed the hypothesis that changes in Foxp2 levels, and the concomitant alterations in expression of neurite-outgrowth genes, yield detectable differences in the growth of neurites in these cells. After 24 and 48 hours of retinoic acid treatment (matching the timepoints for analyses target gene expression) we performed blind scoring of cells and observed striking qualitative differences in neurite outgrowth when cells over-expressed Foxp2, as compared to sham-transfected controls. Cells that had been transfected with Foxp2 prior to differentiation consistently displayed increased neurite length, in a manner that was easy to distinguish from controls (Figure 5B).

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Figure 5. Foxp2 modulates neurite outgrowth in neuronal cell models.

(A) Expression of target genes involved in neurite outgrowth and associated pathways. Quantitative PCR was performed for cDNA reverse-transcribed from stable Neuro2a cell-lines undergoing no differentiation or following 24 or 48 hours differentiation (via the addition of retinoic acid, RA). Expression changes are mean log₂ expression ratios comparing three independent clones stably transfected with Foxp2 and three independent clones stably transfected with an empty vector, normalized for equal expression of the internal control, Gapdh, or U6 (for miRNAs). Error bars indicate the ± SEM, p-values were calculated using a two-tailed unpaired t-test (** = p<0.01, * = p<0.05). (B) Cells expressing Foxp2 demonstrate visible changes in neurite outgrowth. Neuro2a cells stably transfected with Foxp2 or an empty vector control were grown for 24 and 48 hours in differentiation media before images were captured directly under a light microscope. Pictures are representative of >15 images taken for three independent clones of each cell type (Scale bar, 100 µm).

https://doi.org/10.1371/journal.pgen.1002145.g005

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<0.001), median (p = 0.008) and maximum process length (p<0.001); and average number of branches (p = 0.003). Thus, loss of Foxp2 function in striatal neurons that normally express this transcription factor yields significant reductions in multiple indices of neurite outgrowth. When Foxp2-negative cells from the wild-type cultures were compared to equivalent cells from mutants (Figure 6C), it was only the total outgrowth that met significance (p = 0.013).

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Figure 6. Foxp2 regulates neurite outgrowth in primary neurons.

(A) Primary cells were harvested from the ganglionic eminences (developing striatum and pallidum) of wild-type and homozygous Foxp2-R552H E16 littermates and grown in culture for 4 days before fixation and staining for immunofluorescence. Map2 was detected using a TRITC-conjugated (red) antibody and Foxp2 via a FITC-conjugated (green) antibody. The R552H mutation produces a form of Foxp2 protein that appears to be largely non-functional in vitro and in vivo (see references [30] and [9], respectively), but is still detectable via immunofluorescence. DAPI counterstain (blue) indicates the location of nuclei. (Scale bar, 100 µm) (B–C) Quantification of properties of neurite outgrowths in primary culture. Neurites were quantified for all cells in each image and then the data were separated into Foxp2-positive and Foxp2-negative cells. These data represent the mean of 141 Foxp2-positive cells taken from wild-type embryos and 84 Foxp2-positive cells taken from R552H littermates (B) and the mean values of Foxp2-negative cells taken from the same wild-type embryos (142 cells) and the same homozygous R552H littermates (115 cells) (C). Cells were harvested from six wild-type and four Foxp2-R552H embryos, across two litters. All measures of process length as well as the number of branches per cell showed significant differences between wild-type (black bars) and Foxp2-R552H mutant (white bars) mice. However no significant contribution was observed for embryo for any measure except ‘Processes’ (p = 0.018), a measure that was not significant between genotypes. Error bars indicate the ± SEM and p-values were calculated using ANCOVA followed by post-hoc Sidak correction (**** = p<0.001, *** = p = 0.001, ** = p<0.01, * = p<0.05).

https://doi.org/10.1371/journal.pgen.1002145.g006

These findings are strongly in agreement with differences in levels of Foxp2 expression, neurite outgrowth and correlated physiological properties between the two major subpopulations of striatal medium spiny neurons (MSNs) in vivo. While both striatonigral (Drd1a) and striatopallidal (Drd2) MSNs continue to increase their dendritic area well into adulthood, Drd1a MSNs develop significantly more dendrites [31]. This dichotomy in dendritic growth contributes to key physiological differences between both MSN populations, although the underlying mechanisms remain unknown [31]. Furthermore, studies of cultured striatal neurons demonstrate that Drd1a MSNs have larger dendritic trees than Drd2 MSNs, invoking intrinsic mechanisms [31]. To study whether these intrinsic differences in dendritic growth correlate with Foxp2 expression levels, we investigated mice expressing enhanced green fluorescent protein (EGFP) either mainly in Drd1a or Drd2 MSNs [32]. We found that Foxp2 shows consistently high expression in striatonigral Drd1a MSNs and very low expression in Drd2 MSNs throughout the striatum (Figure S7), further supporting roles for Foxp2 in neurite outgrowth.

In vivo chromatin immunoprecipitation

In vivo Foxp2-ChIP in embryonic mouse brain tissue was performed according to the protocol previously described by Vernes and colleagues [21]. Each of the three replicates included whole brain tissue (from the telencephalon to the brain stem at the level of the foramen magnum) at E16 (embryonic day 16), a developmental timepoint of high Foxp2 expression [16], pooled from 5–6 mice of matching genotype. Experiments were carried out either with wild-type embryos, or homozygous Foxp2-S321X mutants as negative controls. S321X mutants carry an early nonsense mutation that disrupts Foxp2; the resulting combination of nonsense-mediated RNA decay and protein instability leads to a complete lack of detectable Foxp2 protein in the brain [9]. The wild-type embryos and mutant controls used in these experiments were all matched littermates, backcrossed for at least ten generations into a C57BL/6J strain, maximizing the homogeneity of the genomic background. Although homozygous mutants display developmental delays and reduced cerebellar growth after birth, they show no gross anomalies in brain anatomy or development during embryogenesis [9]. All animal work was carried out conforming to the regulatory standards of the UK Home Office, under Project Licence 30/2016.

E16 mouse brains were extracted, snap frozen in liquid nitrogen and stored at −80°C until use. Each whole brain was weighed, then chopped finely with a razor on ice. Brains were pooled to achieve a total weight of between 0.3 and 0.5 g of tissue (between 5–6 brains per replicate) and resuspended in 5 ml PBS. A 1/10 volume (500 µl) of cross-linking buffer was added prior to 15 minutes incubation with agitation at room temperature. Formaldehyde was quenched via the addition of 125 mM glycine. Cross-linked tissue was washed in PBS before brief mechanical homogenisation. Pellets were then incubated in two in vivo ChIP lysis buffers at room temperature for ten minutes each: Buffer 1 (50 mM HEPES-KOH pH  = 7.5, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% Triton X-100, protease inhibitors); Buffer 2 (200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 10 mM Tris pH  = 8, protease inhibitors). After collection via centrifugation, nuclei were resuspended in 5 ml sonication buffer (10 mM Tris-HCl pH  = 8, 1 mM EDTA, 0.5 mM EGTA, protease inhibitors). Samples underwent 15 rounds of 20-second sonication pulses at 30% power, with 60 seconds on ice between each round (Branson Digital Sonifier - S450D). Agarose gel electrophoresis was used to confirm that fragment size was 300–1000 bp. Cells were centrifuged at 10,000 g and 4°C for 10 minutes to remove cell debris. 10 µg of polyclonal rabbit anti-Foxp2 antibody (C-terminal antibody, Geschwind Laboratory, UCLA) [20] pre-coupled to 100 µl Dynal M-280 rat anti-rabbit IgG magnetic protein-A beads was added and incubated at 4°C, rotating overnight. Beads were washed five times in RIPA buffer and once in TE buffer. Chromatin was eluted from beads in TE buffer with 1% SDS at 65°C for 10 minutes with agitation. The chromatin was then incubated at 65°C overnight to reverse cross-links. Purified chromatin was amplified via Ligation Mediated PCR (LMPCR) according to published protocols [60]. Size and purity of DNA was assessed via spectrophotometry and gel electrophoresis.

Hybridisation of Foxp2-ChIP products to promoter microarrays

2 µg of amplified immunoprecipitated chromatin, or total input DNA was fluorescently labelled with Cy5 and Cy3 respectively using random primers provided in the BioPrime DNA labelling system (Invitrogen). The labelling reaction was allowed to proceed for 16 hours at 37°C, before purification by sodium acetate precipitation. Hybridisation to mouse promoter arrays (Agilent Technologies, #G4490A) was carried out by the UCLA microarray core facility, according to the manufacturer's instructions. Arrays consisted of 60-mer oligonucleotides spanning ∼8 kb (5.5 kb upstream and 2.5 kb downstream of TSS) at each of ∼17,000 mouse promoter regions. Probes were spaced on average, between 100–300 bp apart, with approximately 25 probes for each promoter region. Three littermate matched sets of pooled wild-type or mutant control chromatin samples were applied to microarrays, each using its respective input DNA sample as the internal reference on the array. Thus, the three wild-type and three mutant control datasets represent signals obtained from a total of 34 individual mouse embryos.

Analysis of promoter microarray data

Array images were scanned using the Axon GenePix 4000B. Data were retrieved and initial quality control carried out using the Axon GenePix 4000B software package. All promoter coordinates and probes were mapped with reference to the NCBI m36 mouse assembly. Microarray data analysis was carried out using the mArray package for R [61]. LOESS normalisation and background correction was performed within each array. Data were normalised between arrays using quantile normalisation, and mean values were calculated from three biological replicates (wild-type or mutant control experiments) for each probe - called ‘probe scores’, such that a score of 1 corresponds to 2-fold enrichment in ChIP versus total input DNA. All negative probe scores were assigned a value of zero. A ‘window-adjusted score’ for each probe was then calculated as the median value of each probe score and its nearest neighbour on either side. Neighbouring probes were only considered if they fell within 500 bp upstream or 500 bp downstream of the central probe. This window size was based on the average size of the labelled DNA fragments, estimated to be approx 1000 bp. Thus, a true binding event would likely be indicated by positive scores of multiple neighbouring probes within a 1000 bp window. In cases where there were less than three probes located within this 1000 bp window showing a signal greater than background then the window-adjusted score was set to zero. This process helps to guard against artificial skewing of enrichment values at edges of promoter regions.

The use of mutant null controls enabled us to robustly assess the empirical significance of wild-type ChIP results. The data from the mutant control experiments were used to estimate a null distribution of window scores; that is, the non-specific signals produced by the Foxp2-ChIP protocol even when there is no Foxp2 protein available for pulldown. (Note that a subset of binding events in mutant null controls could potentially be due to crossreactivity of Foxp2 antibodies with closely related proteins, such as Foxp1 or Foxp4, that may bind to the same promoter. However, prior work with the antibody used here suggests that levels of crossreactivity are extremely low [20].) From this null distribution of window scores we calculated the threshold which excluded 99% of all datapoints in controls. This threshold could then be applied to the wild-type array data.

Semi-quantitative PCR

Chromatin isolated during Foxp2-ChIP in mutant and wild-type mouse brains was amplified using a semi-quantitative PCR technique, as described previously [21], using primers directed towards the peak regions of enrichment (Table 3). The β-actin housekeeping gene promoter was used as a negative control.

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Table 3. Semi-quantitative PCR primers.

https://doi.org/10.1371/journal.pgen.1002145.t003

Expression profiling

The ganglionic eminences, sites of particularly high embryonic Foxp2 expression [16], were dissected from E16 brains of six wild-type mice and six homozygous Foxp2-S321X mutant littermates. For each embryo, the left- and right-hemisphere ganglionic eminences were pooled in TRIzol reagent and RNA was extracted using the QIAGEN RNeasy kit, according to the manufacturer's instructions. RNA yield was measured using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE), and its quality was assessed using RNA6000 Nano Assays on an Agilent Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA).

Gene expression profiling was performed using whole-genome mouse BeadChip arrays from Illumina (San Diego, California, USA), which include 45,281 probes representing 31,492 mouse transcripts. In brief, 500 ng of total RNA was reverse transcribed to synthesize first- and second-strand complementary DNA (cDNA). Following purification on spin columns, in vitro transcription was used to synthesize biotin-labelled complementary RNA (cRNA). 1500 ng of biotin-labelled cRNA was hybridized to Mouse WG-6 V2 Expression BeadChip arrays (Illumina Inc., San Diego, CA) at 55°C for 18 h. The hybridized arrays were washed and labelled with streptavidin-Cy3 according to the manufacturer's protocols before being scanned with the Illumina Bead Array Scanner. Raw data were exported from the Illumina BeadStudio software (v3.4.0) for further processing and analysis using the R statistical software [62] and BioConductor packages [63]. Signal data and detection scores were extracted for each of the 12 samples. Signal data were background corrected by subtracting the average signal from the negative control probes on each array, prior to being transformed and normalised using the ‘VSN’ package [64]. Quality control analyses, including hierarchical clustering and principal component analysis (PCA), identified one outlier sample (from the wild-type group). This sample had very low signal compared to other samples while hybridisation and labelling metrics were normal, suggesting a sample problem rather than a technical issue. It was sufficiently outlying to remove from further analysis and the remaining 11 samples were re-normalised. The dataset was then filtered to remove probes that were not detected (detection score <0.95 in all samples), resulting in a final dataset of 24,479 probes.

Statistical analysis was performed using the ‘Linear Models for Microarray Analysis’ (Limma) package [65]. Differential expression between mutant and wild-type animals was assessed using a linear model that included effects for genotype and litter. Raw p-values were corrected for multiple testing using the false discovery rate (FDR) controlling procedure of Benjamini and Hochberg [66]. Fourteen probes were significant at a FDR of 5%. The larger set of 340 probes significant at p<0.01 was used for further biological investigation. We performed permutation tests on the genotype labels (11 choose 5), taking litter effects into account, and found that ≥340 genes were differentially expressed at p = 0.01 in only 9 out of the possible 108 permutations (∼8%). Gene annotation was added to the final probe list using the relevant annotation file (MouseWG-6_V2_0_R0_11278593_A.txt) from the Illumina website (http://www.illumina.com).

Cell culture and reagents

Neuro2a (murine neuroblastoma) cells were cultured in ‘Growth media’: Modified Eagles Medium (MEM) (Sigma) supplemented with 10% Foetal Calf Serum (Sigma), 2 mM L-glutamine (Sigma) and 2 mM Penicillin/Streptomycin (Sigma). Cells were grown at 37°C in the presence of 5% CO₂. Stable Neuro2a cell-lines overexpressing Foxp2 protein or non-expressing controls, were generated via transfection with pcDNA3.1/Foxp2 (mouse isoform I - untagged) or the empty vector, using Genejuice (Novagen) according to the manufacturers' instructions. Cells were cultured in complete medium supplemented with 500 µg/ml G418 (Calbiochem) as a selective agent. Resistant single colonies were isolated 20 days after transfection, then cultured and expanded independently in the presence of G418 (500 µg/ml). Expression of recombinant Foxp2 was confirmed using qRT-PCR and Western blotting with two polyclonal antibodies recognizing different epitopes of the protein (goat N-terminal antibody, Santa Cruz Biotechnology [30]; rabbit C-terminal antibody, Geschwind Laboratory, UCLA [20]). Three Foxp2-transfected clones with a high and consistent level of expression and three empty vector clones were chosen for use in further experiments. Neuro2a cells were differentiated via the addition of Modified Eagles Medium supplemented with 2% Foetal Calf Serum (Sigma), 2 mM L-glutamine (Sigma), 2 mM Penicillin/Streptomycin (Sigma) and 20 µM all-trans retinoic acid (‘Differentiation media’).

Quantitative RT-PCR

RNA was extracted from three independent clones of Neuro2a cells stably transfected either with murine Foxp2 or the empty control vector following culture in growth media or differentiation media (for 24 or 48 hours). Total RNA was extracted from cells harvested in TRIzol reagent using the RNeasy kit (QIAGEN) according to manufacturers' instructions. Reverse transcription was performed as described previously [21]. Small molecular weight RNA was harvested using the Purelink miRNA isolation kit (Invitrogen) according to manufacturers' instructions. In order to assess miRNA expression levels, the small molecular weight RNA was polyadenylated prior to reverse transcription using the NCode miRNA First-strand cDNA synthesis kit (Invitrogen), as per the manufacturers' protocol.

PCR reactions utilised SYBR Green supermix (BioRad) as described [21]. Primers specific for candidate genes and the control housekeeping genes GAPDH/Gapdh (glyceraldehyde 3-phosphate dehydrogenase) and U6 (small nuclear RNA) were designed using PrimerBank [67] (Table 4). Quantitative PCR reactions were performed on the iQ5 thermal cycler real-time PCR detection system (BioRad) according to manufacturers' instructions. Melting curve analysis was performed to assess the specificity of the amplification. Data analysis was performed using iCycler software (BioRad), and quantification was via the comparative CT method [68]. Fold changes are reported in response to Foxp2 expression compared to cells transfected with an empty vector, following normalisation to an internal control, the GAPDH housekeeping gene (for gene expression) or U6 small nuclear RNA (for miRNA expression). Data are expressed as mean of values from three independent clones ± standard error of the mean (SEM). Statistical significance was assessed using Students t-tests (two-tailed).

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Table 4. Quantitative RT-PCR gene-specific primers.

https://doi.org/10.1371/journal.pgen.1002145.t004

Primary cell culture

Ganglionic eminences from both hemispheres were dissected from wild-type and homozygous Foxp2-R552H E16 littermates [9]. R552H mice carry a missense mutation affecting a conserved arginine residue located in the Foxp2 DNA binding domain, matching an aetiological mutation found in a well-characterised multigenerational family with speech and language disorder (the KE family) [4]. R552H homozygous mice demonstrate comparable phenotypes to homozygous Foxp2 knockouts [9]. This suggests that the introduction of this mutation yields a stable, but non-functional protein product, a conclusion that is supported by in vitro functional studies [30]. Dissections were performed in dissection buffer (15 mM HEPES, 0.01% NaHCO₃, 25 mM glucose in HBSS-CMF) and dissected regions were immediately chopped on ice and pelleted at 800 RPM and 4°C for 5 minutes. The pellet was incubated in papain solution (20 units/ml papain, 1 mM L-cysteine, 0.5 mM EDTA, 100 units/ml DNaseI, in dissection buffer) on ice for 5 minutes then at 37°C for 10 minutes, agitating regularly. The enzymatic reaction was halted by addition of Ovo-BSA solution (10 mg/ml ovomucoid, 10 mg/ml BSA in dissection buffer). Cells were pelleted at 1000 RPM for 5 minutes at 4°C and the pellet was washed then re-suspended in complete medium (neurobasal media (Sigma) supplemented by 2 mM Glutamax (Sigma). 2 mM Penicillin/Streptomycin (Sigma) and 1X B27 supplement). Suspension was triturated using plastic and glass pipettes to dissociate any remaining cell clumps before passing the cell suspension through a 70 µm cell strainer. Single cell suspensions were seeded onto laminin and poly-D-lysine coated coverslips (BD Biosciences) at a density of 6.3×10⁴ cells per well into 24 well plates and grown at 37°C in the presence of 5% CO₂ in complete medium.

After 4 days in culture, cells were fixed using 4% Paraformaldehyde solution for 15 minutes at room temperature and permeablised in wash solution (0.1% Triton X-100 in TBS). Antibodies were diluted in Blocking Solution (1% Fish Gelatine, 0.1% Triton X-100, 5% BSA in PBS). Cells were co-stained at 4°C overnight, using two primary antibodies; an anti-MAP2 rabbit polyclonal antibody (Chemicon) and an anti-Foxp2 mouse monoclonal antibody recognising an epitope near the C-terminal end of the protein (Gift from Prof. A. Banham). Detection was then facilitated via four rounds of antibody incubation, which allowed magnification of the Foxp2 signal. Cells were incubated with anti-rabbit TRITC (Alexa Fluor 568, Molecular Probes) plus anti-mouse biotinylated (BA9200, Vector Labs) secondary antibodies, followed by incubation with anti-rabbit TRITC plus anti-biotin FITC (Alexa Fluor 488, Molecular Probes) antibodies, each for 1 hour, shaking under limited light exposure. This secondary/tertiary antibody incubation was then repeated under the same conditions. Nuclei were visualised using mounting media containing a DAPI counterstain (VectaShield). Cells were viewed on a Nikon Eclipse TE2000U fluorescence inverted microscope. Images were captured using a Hamamatsu black and white C4742-95 Orca hi-sensitivity CCD camera with IPLab imaging software (Scanalytics Inc), and analysed using the neurite outgrowth function of Metamorph Version 7.6 (Molecular Devices). Statistical analyses were carried out using ANCOVA (analysis of covariance) for genotype and embryo, followed by post-hoc Sidak correction. Data are expressed as the mean ± standard error of the mean (SEM).

In situ hybridisation

In situ hybridization was carried out on 10 µM frozen sections of E16.5 embryo heads as previously described [69]. An approximately 500 bp fragment of each target transcript was subcloned into pCR4-TOPO (Invitrogen) for dioxygenin-labelled riboprobe synthesis. Primer sequences for the riboprobes are available on request. Equivalent parasagittal sections were hybridized in parallel from three wild-type and three homozygous Foxp2-S321X mutant embryos and all slides were developed for 16 hours, or 6 hours in the case of Shhrs. In all cases a sense-strand negative control riboprobe gave no specific signal (data not shown).