Protein-Protein Interaction Among the FoxP Family Members and their Regulation of Two Target Genes, VLDLR and CNTNAP2 in the Zebra Finch Song System

The Forkhead transcription factor FOXP2 is implicated in speech perception and production. The avian homolog, FoxP21 contributes to song learning and production in birds. In human cell lines, transcriptional activity of FOXP2 requires homo-dimerization or dimerization with paralogs FOXP1 or FOXP4. Whether FoxP dimerization occurs in the brain is unknown. We recently showed that FoxP1, FoxP2 and FoxP4 (FoxP1/2/4) proteins are co-expressed in neurons of Area X, a song control region in zebra finches. We now report on dimer- and oligomerization of zebra finch FoxPs and how this affects transcription. In cell lines and in the brain we identify homo- and hetero-dimers, and an oligomer composed of FoxP1/2/4. We further show that FoxP1/2 but not FoxP4 bind to the regulatory region of the target gene Contactin-associated protein- like 2 (CNTNAP2). In addition, we demonstrate that FoxP1/4 bind to the regulatory region of very low density lipoprotein receptor (VLDLR), as has been shown for FoxP2 previously. Interestingly, FoxP1/2/4 individually or in combinations regulate the promoters for SV40, zebra finch VLDLR and CNTNAP2 differentially. These data exemplify the potential for complex transcriptional regulation of FoxP1/2/4, highlighting the need for future functional studies dissecting their differential regulation in the brain

“Bird Song Metronomics”: Isochronous Organization of Zebra Finch Song Rhythm

The human capacity for speech and vocal music depends on vocal imitation. Songbirds, in contrast to non-human primates, share this vocal production learning with humans. The process through which birds and humans learn many of their vocalizations as well as the underlying neural system exhibit a number of striking parallels and have been widely researched. In contrast, rhythm, a key feature of language, and music, has received surprisingly little attention in songbirds. Investigating temporal periodicity in bird song has the potential to inform the relationship between neural mechanisms and behavioral output and can also provide insight into the biology and evolution of musicality. Here we present a method to analyze birdsong for an underlying rhythmic regularity. Using the intervals from one note onset to the next as input, we found for each bird an isochronous sequence of time stamps, a “signal-derived pulse,” or pulseS, of which a subset aligned with all note onsets of the bird's song. Fourier analysis corroborated these results. To determine whether this finding was just a byproduct of the duration of notes and intervals typical for zebra finches but not dependent on the individual duration of elements and the sequence in which they are sung, we compared natural songs to models of artificial songs. Note onsets of natural song deviated from the pulseS significantly less than those of artificial songs with randomized note and gap durations. Thus, male zebra finch song has the regularity required for a listener to extract a perceived pulse (pulseP), as yet untested. Strikingly, in our study, pulsesS that best fit note onsets often also coincided with the transitions between sub-note elements within complex notes, corresponding to neuromuscular gestures. Gesture durations often equaled one or more pulseS periods. This suggests that gesture duration constitutes the basic element of the temporal hierarchy of zebra finch song rhythm, an interesting parallel to the hierarchically structured components of regular rhythms in human music

the ‘FOXP2’ gene's journey through time

How did humans evolve language? The fossil record does not yield enough evidence to reconstruct its evolution and animals do not talk. But as the neural and molecular substrates of language are uncovered, their genesis and function can be addressed comparatively in other species. FOXP2 is such a case – a gene with a strong link to language that is also essential for learning in mice, birds and even flies. Comparing the role FOXP2 plays in humans and other animals is starting to reveal common principles that may have provided building blocks for language evolution

Next stop: Language : the ?FOXP2? gene?s journey through time

How did humans evolve language? The fossil record does not yield enough evidence to reconstruct its evolution and animals do not talk. But as the neural and molecular substrates of language are uncovered, their genesis and function can be addressed comparatively in other species. FOXP2 is such a case ? a gene with a strong link to language that is also essential for learning in mice, birds and even flies. Comparing the role FOXP2 plays in humans and other animals is starting to reveal common principles that may have provided building blocks for language evolution

Dynamic FoxP2 levels in male zebra finches are linked to morphology of adult-born Area X medium spiny neurons

The transcription factor FOXP2 is crucial for the formation and function of cortico-striatal circuits. FOXP2 mutations are associated with specific speech and language impairments. In songbirds, experimentally altered FoxP2 expression levels in the striatal song nucleus Area X impair vocal learning and song production. Overall FoxP2 protein levels in Area X are low in adult zebra finches and decrease further with singing. However, some Area X medium spiny neurons (MSNs) express FoxP2 at high levels (FoxP2(high) MSNs) and singing does not change this. Because Area X receives many new neurons throughout adulthood, we hypothesized that the FoxP2(high) MSNs are newly recruited neurons, not yet integrated into the local Area X circuitry and thus not active during singing. Contrary to our expectation, FoxP2 protein levels did not predict whether new MSNs were active during singing, assayed via immediate early gene expression. However, new FoxP2(high) MSNs had more complex dendrites, higher spine density and more mushroom spines than new FoxP2(low) MSNs. In addition, FoxP2 expression levels correlated positively with nucleus size of new MSNs. Together, our data suggest that dynamic FoxP2 levels in new MSNs shape their morphology during maturation and their incorporation into a neural circuit that enables the maintenance and social modulation of adult birdsong

Diminished FoxP2 Levels Affect Dopaminergic Modulation of Corticostriatal Signaling Important to Song Variability

SummaryMutations of the FOXP2 gene impair speech and language development in humans and shRNA-mediated suppression of the avian ortholog FoxP2 disrupts song learning in juvenile zebra finches. How diminished FoxP2 levels affect vocal control and alter the function of neural circuits important to learned vocalizations remains unclear. Here we show that FoxP2 knockdown in the songbird striatum disrupts developmental and social modulation of song variability. Recordings in anesthetized birds show that FoxP2 knockdown interferes with D1R-dependent modulation of activity propagation in a corticostriatal pathway important to song variability, an effect that may be partly attributable to reduced D1R and DARPP-32 protein levels. Furthermore, recordings in singing birds reveal that FoxP2 knockdown prevents social modulation of singing-related activity in this pathway. These findings show that reduced FoxP2 levels interfere with the dopaminergic modulation of vocal variability, which may impede song and speech development by disrupting reinforcement learning mechanisms

Food for Song: Expression of C-Fos and ZENK in the Zebra Finch Song Nuclei during Food Aversion Learning

BACKGROUND: Specialized neural pathways, the song system, are required for acquiring, producing, and perceiving learned avian vocalizations. Birds that do not learn to produce their vocalizations lack telencephalic song system components. It is not known whether the song system forebrain regions are exclusively evolved for song or whether they also process information not related to song that might reflect their 'evolutionary history'. METHODOLOGY/PRINCIPAL FINDINGS: To address this question we monitored the induction of two immediate-early genes (IEGs) c-Fos and ZENK in various regions of the song system in zebra finches (Taeniopygia guttata) in response to an aversive food learning paradigm; this involves the association of a food item with a noxious stimulus that affects the oropharyngeal-esophageal cavity and tongue, causing subsequent avoidance of that food item. The motor response results in beak and head movements but not vocalizations. IEGs have been extensively used to map neuro-molecular correlates of song motor production and auditory processing. As previously reported, neurons in two pallial vocal motor regions, HVC and RA, expressed IEGs after singing. Surprisingly, c-Fos was induced equivalently also after food aversion learning in the absence of singing. The density of c-Fos positive neurons was significantly higher than that of birds in control conditions. This was not the case in two other pallial song nuclei important for vocal plasticity, LMAN and Area X, although singing did induce IEGs in these structures, as reported previously. CONCLUSIONS/SIGNIFICANCE: Our results are consistent with the possibility that some of the song nuclei may participate in non-vocal learning and the populations of neurons involved in the two tasks show partial overlap. These findings underscore the previously advanced notion that the specialized forebrain pre-motor nuclei controlling song evolved from circuits involved in behaviors related to feeding

CNTNAP2 is a direct FoxP2 target in vitro and in vivo in zebra finches: complex regulation by age and activity

Mutations of FOXP2 are associated with altered brain structure, including the striatal part of the basal ganglia, and cause a severe speech and language disorder. Songbirds serve as a tractable neurobiological model for speech and language research. Experimental downregulation of FoxP2 in zebra finch Area X, a nucleus of the striatal song control circuitry, affects synaptic transmission and spine densities. It also renders song learning and production inaccurate and imprecise, similar to the speech impairment of patients carrying FOXP2 mutations. Here we show that experimental downregulation of FoxP2 in Area X using lentiviral vectors leads to reduced expression of CNTNAP2, a FOXP2 target gene in humans. In addition, natural downregulation of FoxP2 by age or by singing also downregulated CNTNAP2 expression. Furthermore, we report that FoxP2 binds to and activates the avian CNTNAP2 promoter in vitro. Taken together these data establish CNTNAP2 as a direct FoxP2 target gene in songbirds, likely affecting synaptic function relevant for song learning and song maintenance