When a cell needs to make a protein, it makes a temporary copy of the corresponding gene so that the genetic code can be carried to its protein-making machinery. When the temporary copy of the code is no longer needed, the cell destroys it. This system is fine-tuned by other small stretches of genetic code called microRNAs, which speed up the destruction and so help to switch genes off faster.

Two genes called FOXP1 and FOXP2 are known to have roles in speech and language development in humans. When these genes do not work properly, people have severe difficulties when speaking and understanding speech. But scientists know little about how the brain controls them. The brains of animals with backbones – like birds and mammals – make a microRNA called miR-9. Scientists thought miR-9 may control how active the FOXP1 and FOXP2 genes are in the brain.

Like humans, zebra finches communicate vocally. Young male birds learn to sing by imitating the song of an adult tutor, usually their father. The process is controlled by a brain region called “Area X”. Now, Shi et al. report on the role of miR-9 in vocal learning and singing in zebra finches.

First, the gene for miR-9 was inserted into a virus-based genetic tool. Shi et al. then injected this virus into Area X of juvenile zebra finches, which delivered the gene to the brain cells and forced them to make excess miR-9. A control group received empty virus with no miR-9 gene for comparison. The juvenile finches then grew up with an adult bird that taught them to sing.

Shi et al. found that the birds that overproduced miR-9 did not learn as well as their normal counterparts. Their songs were shorter, they stuttered, and they missed out syllables, which meant that they simply sounded different to their tutors. These young birds also failed to change their tune in different situations, for example, when they met a female zebra finch. Examination of the birds’ brains four weeks after the viral injection showed that the bird versions of the FOXP1 and FOXP2 genes were less active. There were also changes in other genes involved in brain circuit development.

Humans have a brain area like Area X, called the basal ganglia. The link between miR-9 and vocal learning provides a starting point to understand more about language in general. This could lead to improved understanding of conditions like stuttering, Tourette’s syndrome, dyslexia and autism spectrum disorders.

Like humans developing speech and language, juvenile songbirds learn to sing by imitating the songs of an adult tutor early in life (Doupe and Kuhl, 1999; Immelmann and Hinde, 1969; Marler and Tamura, 1964; Tchernichovski et al., 2001). Juvenile zebra finches (pupils) first hear and memorize a tutor’s song, and then, at about post-hatching day 30 (30 d), the pupils begin to vocalize. Initially, juvenile songs are highly variable. Through thousands of trial-and-error practice sessions guided by auditory feedback, the juvenile song gradually matures into a stereotyped adult song that resembles the tutor’s song (Immelmann and Hinde, 1969; Konishi, 1965; Tchernichovski et al., 2001). The adult song, however, exhibits a residual level of variability. Depending on the social context, adult males sing a courtship song directed toward a female (DS) or an undirected song (UDS) when singing alone (Sossinka and Bohner, 1980). These two types of songs exhibit subtle differences in acoustic features, and the DS is more stereotyped than the UDS (Hessler and Doupe, 1999; Jarvis et al., 1998; Kao and Brainard, 2006; Murugan et al., 2013).

The neural circuit that controls song behavior is organized into two pathways: a motor pathway and an anterior forebrain pathway (AFP) (Figure 1A). The motor pathway controls song production, while the AFP, a cortical-basal ganglia circuit required for song learning and maintenance, guides motor output through a reinforcement mechanism (Bottjer et al., 1984; Brainard and Doupe, 2000; Doupe and Kuhl, 1999; Kao et al., 2005; Nottebohm et al., 1976, 1982; Olveczky et al., 2005; Simpson and Vicario, 1990). Area X is a basal ganglia nucleus within the AFP. Lesions in Area X of juveniles prevent them from crystallizing their song (Scharff and Nottebohm, 1991). Area X receives dopaminergic innervation from the midbrain ventral tegmental area (VTA), and it also modulates the activity of VTA dopaminergic neurons in response to auditory experience, implicating Area X in reinforcement learning (Ding and Perkel, 2002; Gadagkar et al., 2016; Gale and Perkel, 2006, 2010; Lewis et al., 1981).

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MicroRNAs (miRNAs) are small, non-protein-coding RNA molecules that regulate gene expression post-transcriptionally. miR-9 is an evolutionarily conserved miRNA that is highly expressed in vertebrate brains (Landgraf et al., 2007; Luo et al., 2012). In the zebra finch, miR-9 is expressed in Area X. Its expression is regulated during developmental vocal learning and in adult males singing undirected songs (Shi et al., 2013), suggesting that miR-9 plays an active role in these processes. miRNAs regulate gene expression by targeting the 3’ untranslated regions (3’UTRs) of mRNAs, leading to mRNA degradation or suppression of protein synthesis. Many of the genes expressed in the nervous system have long 3’UTRs (Mayr, 2016), highlighting the importance of miRNAs in fine-tuning gene expression in nervous system development and function. FOXP1 and FOXP2 are a pair of paralogous transcription factors that have important roles in nervous system development. Deletions, mutations, and copy number variations of the FOXP1 gene have been implicated in a range of neural developmental disorders, including language delay, intellectual disability, and autism spectrum disorders (ASDs) (Hamdan et al., 2010; Horn et al., 2010; O'Roak et al., 2011). Heterozygous mutations in the FOXP2 gene cause severe speech and language impairments (Lai et al., 2001; Vargha-Khadem et al., 1995), accompanied by structural and functional abnormalities in multiple brain regions. These regions include the basal ganglia (Watkins et al., 2002), which is thought to be a component of the distributed neural circuitry that underlies speech and language (Graham and Fisher, 2013). FOXP1 and FOXP2 regulate the transcription of a large number of downstream genes, many of which are critically involved in neural differentiation, neurite outgrowth, synapse formation, and synaptic transmission (Konopka et al., 2009; Spiteri et al., 2007; Tang et al., 2012; Vernes et al., 2007, 2011). Thus, the functional dosage of FOXP1 and FOXP2 needs to be tightly regulated.

Recent in vitro studies have shown that miR-9 regulates the expression of FOXP2 by targeting specific sequences in its 3’UTR (Fu et al., 2014; Shi et al., 2013). miR-9 also regulates avian FoxP1 in embryonic chicken spinal cord (Otaegi et al., 2011). These findings raise the possibility that miR-9 has a role in language development through regulating FOXP1 and/or FOXP2. Taking advantage of the unique vocal behavior and the underlying neural circuitry of songbirds, we sought to assess the consequences of miR-9 overexpression in Area X of juvenile zebra finches on vocal learning and performance. For these studies, we overexpressed miR-9 using a lentiviral approach. We report here that overexpression of miR-9 in juvenile Area X profoundly impairs basal ganglia-dependent developmental vocal learning in juveniles and impairs song performance in adulthood. We further show that these behavioral deficits are accompanied by downregulation of FoxP1 and FoxP2 expression, disruption of dopamine signaling, and widespread changes in the expression of numerous genes that are important for neural circuit development and function.

We show here that overexpression of miR-9 in the basal ganglia nucleus Area X of juvenile zebra finches impaired developmental vocal learning and adult vocal performance. On the global motif structural level, the most pronounced impairment was that birds with miR-9 overexpression sang shorter song motifs, omitting some of the tutor syllables. This phenomenon in miR-9 pupils (in which FoxP1 and FoxP2 were downregulated) may mirror the limited vocabulary observed in human individuals carrying deletions in the FOXP1 gene, who typically exhibit a working vocabulary of fewer than 100 words at the age of seven (Horn et al., 2010). Songs of birds with miR-9 overexpression in Area X exhibited higher trial-by-trial variability, which was reflected in a more variable sequence of syllable order, truncated motifs, and syllable stuttering. Syntax change has not been reported in prior studies in which FoxP2 was either knocked down or overexpressed in Area X (Haesler et al., 2007; Heston and White, 2015; Murugan et al., 2013). That miR-9 regulates the expression of both FoxP1 and FoxP2 and potentially of other genes in Area X may explain the robust deficits in vocal learning we observed here. Effects of miR-9 on song performance also occurred in a social context-dependent manner. Similar to birds with reduced FoxP2 expression in adult Area X (Murugan et al., 2013), birds with miR-9 overexpression failed to modulate song variability when singing a directed song. Perhaps not coincidentally, miR-9 expression is higher in Area X when adult males naturally sing an undirected song (Shi et al., 2013). It appears that, whether naturally or artificially induced, higher miR-9 levels in Area X render the circuit permissive to a more variable song or interfere with the production of a more stereotyped directed song.

Vocal learning is a developmental process during which a less-structured and highly variable juvenile song gradually transitions to a stereotyped adult song that matches the tutor’s song (Immelmann and Hinde, 1969; Tchernichovski et al., 2001). The effects of miR-9 overexpression on song imitation were apparent at 65 days after hatching, and these birds failed to improve their imitation thereafter (Figure 6A). Acoustic features of syllables of miR-9 pupils also exhibited higher trial-by-trial variability throughout development (Figure 6C–E). It is not clear whether or how higher song variability affects a pupil’s ability to match its song to a tutor’s song. In normal birds, the developmental vocal learning process is accompanied with a gradual increase in the miR-9 level in Area X, reaching its high point as juveniles become adults, and songs become stabilized; meanwhile, throughout the process, the FoxP2 level gradually decreases (Haesler et al., 2004; Shi et al., 2013; Teramitsu et al., 2004). Our current data indicate that an artificially increased miR-9 level (and reduced FoxP2 level) does not result in premature stabilization of a juvenile’s song nor in an increase in similarity to the tutor’s song. Rather, miR-9 overexpression appears to lock Area X in a plasticity-dominant state, preventing further progression toward song stereotypy. Previous lesion studies have shown that lesions in Area X and lMAN, two nuclei in the anterior forebrain pathway, have different consequences on song development. While lesions in lMAN lead to a prematurely stabilized song, birds with Area X lesions never achieve song stability as control birds do (Scharff and Nottebohm, 1991). Although the experimental approaches used in these studies are unrelated (molecular manipulation vs. electrolytic lesion), our observations are more aligned with the effects of lesions in Area X, suggesting that the effect of miR-9 overexpression (gene manipulation) might be restrained by circuit functions, in this case functions of Area X.

According to the reinforcement learning theory, Area X and the AFP guide song motor learning through processes that evaluate the motor output patterns according to auditory feedback, and reinforce favorable motor actions. These processes involve dopamine signaling via D1R and D2R receptors expressed in Area X (Kubikova et al., 2010) and dopaminergic projections from the VTA to Area X (Ding and Perkel, 2002; Doupe et al., 2005; Doya and Tesauro, 1995; Gadagkar et al., 2016; Lewis et al., 1981). Dopamine levels in Area X are higher when birds sing DS (Sasaki et al., 2006). Blocking D1R pharmacologically or reducing D1R expression by knocking down FoxP2 in Area X results in a more variable DS (Leblois et al., 2010; Murugan et al., 2013). Our findings that miR-9 overexpression in Area X selectively downregulated D1R but not D2R provide further evidence of the importance of dopamine signaling in vocal learning and performance. Studies in mammals suggest that the D1R-expressing direct pathway and the D2R-expressing indirect pathway act in an opposing but coordinated manner in the basal ganglia to finely control the timing and synchronization of motor actions; imbalances between the two pathways may lead to movement and cognitive disorders (Cazorla et al., 2015; Gerfen and Surmeier, 2011). The downregulation of D1R but not D2R in Area X, as a consequence of miR-9 overexpression, may have produced a functional imbalance between the two signaling pathways, which may have contributed to impaired song learning and performance. In normal birds, miR-9 expression in Area X is regulated during vocal development, and is upregulated by adult singing UDS (Shi et al., 2013). Thus, the miR-9–FoxP2–dopamine signaling network provides a mechanism that dynamically adjusts the functional balance between the D1R and D2R signaling pathways, allowing birds to adapt to the changing maturation state of song in juveniles and to modulate song stereotypy according to the social context in which a song is produced.

Many of the FOXP1 and FOXP2 downstream transcriptional target genes were identified by genome-wide chromatin immunoprecipitation assays, which depend on the binding of transcription factors FOXP1 or FOXP2 to promoter sequences (Konopka et al., 2009; Spiteri et al., 2007; Vernes et al., 2011). The changes in expression of these downstream genes in Area X when FoxP1 and FoxP2 were downregulated by miR-9 provides further evidence for a regulatory relationship between these genes and FOXP1 or FOXP2 in the basal ganglia circuit critical for vocal communication. Among the genes we tested, many have direct roles in neural circuit development and functions, and their dysregulation has been implicated in various neural developmental disorders. For example, GRIK2, GRIN2B, and GRM8 encode subunits of glutamate receptors. Their altered expression can affect synaptic transmission of Area X neurons. KCNA4 encodes a voltage-gated potassium channel (Ovsepian et al., 2016), and mutations in KCNA4 have been identified in patients exhibiting linguistic disabilities, attention deficit hyperactivity disroder (ADHD), and cognitive impairments (Kaya et al., 2016). MMP2, a member of the matrix metalloproteinase family, plays critical roles in synaptogenesis, dendrite remodeling, and neurogenesis (Fujioka et al., 2012). In songbird brain, MMP2 has been implicated in angiogenesis and neurogenesis in HVC, another song nucleus essential for song learning and production (Kim et al., 2008), suggesting that MMP2 may have a role in Area X circuit development as well. CNTNAP2 encodes a cell surface protein that belongs to the neurexin family, and is important for synaptic formation and clustering of K⁺ channels at synaptic terminals (Poliak et al., 1999). FOXP2 is known to regulate CNTNAP2 expression by binding to a regulatory sequence in its first intron, and CNTNAP2 dysfunctions have been implicated in specific language impairments, intellectual disabilities, and ASDs (Rodenas-Cuadrado et al., 2014; Vernes et al., 2008). DISC1 (Disrupted in schizophrenia 1) plays important roles in cell migration and dendrite development, and it has been linked to schizophrenia, bipolar disorder, depression, and ASDs (Millar et al., 2000; Thomson et al., 2013). The widespread changes in gene expression caused by miR-9 overexpression touch upon an array of cellular functions, including synaptic transmission, dendrite growth, synapse formation, gene regulation, neurotrophin (BDNF) signaling, and protein degradation. We only tested expression changes in a small fraction of FOXP1 and FOXP2 downstream genes; however, given the fact that two-thirds (26 of 37) of these genes changed expression, it is likely that many of the genes we did not test also changed their expression. Thus, a large number of these affected genes may have collectively contributed to the deficits in vocal behavior.

We noted that the magnitudes of changes in gene expression were moderate. Intriguingly, a recent study of a large cohort of schizophrenia patients found moderate changes (ranging from 10% to 40%) in the expression of several hundred genes in the prefrontal cortex (Fromer et al., 2016). Our observations support the emerging view that subtle but broad changes in gene expression might be a molecular signature underlying complex neural developmental and/or neural psychiatric disorders (Fromer et al., 2016; Purcell et al., 2014). Evidence implicating particular genes in language impairments and autism is often established through genome-wide analysis such as screening for mutations and/or copy number variations in human subjects (O'Roak et al., 2012; Sanders et al., 2012; Yuen et al., 2015). Our findings showing that these genes are expressed in the basal ganglia, and that alterations in their expression are accompanied by impairments in vocal communication, provide additional evidence that these genes function in language processes and development. Among the genes we tested, CNTNAP2 and DISC1 have each been implicated in multiple disorders, including language impairments, ASDs, ADHD, intellectual disabilities, and schizophrenia (Fromer et al., 2016; Purcell et al., 2014), suggesting that these neural developmental disorders, cognitive impairments, and psychiatric disorders share common molecular substrates. It is possible that distinct but overlapping phenotypes are manifested depending on where and when these genes are expressed and how they are regulated, emphasizing the need to study these genes in the context of specific functional neural circuits.

miR-9 is expressed in the embryonic human brain and has been shown to regulate human FOXP2 gene expression (Fu et al., 2014). It is likely that miR-9 plays a role in human language development by fine-tuning FOXP1 and FOXP2 expression, thereby coordinating the expression of a large number of genes that are active in neural development and function. Both FOXP1 and FOXP2 mRNAs have long 3’UTRs containing numerous miRNA binding sites, suggesting that these genes can be regulated by many miRNAs in addition to miR-9. Dysregulation of these miRNAs or of miRNA–FOXP1/FOXP2 interactions by genetic, environmental, or physiological factors, thus, may contribute to language impairments and related neurodevelopmental disorders.

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Author details

1. Zhimin Shi

   Neuroscience Center of Excellence, Louisiana State University School of Medicine, New Orleans, United States

   Contribution

   Data curation, Formal analysis, Investigation, Methodology

   Competing interests

   No competing interests declared

2. Zoe Piccus

   Neuroscience Center of Excellence, Louisiana State University School of Medicine, New Orleans, United States

   Contribution

   Data curation, Formal analysis, Methodology

   Competing interests

   No competing interests declared

3. Xiaofang Zhang

   Neuroscience Center of Excellence, Louisiana State University School of Medicine, New Orleans, United States

   Contribution

   Data curation, Formal analysis

   Competing interests

   No competing interests declared

4. Huidi Yang

   Neuroscience Center of Excellence, Louisiana State University School of Medicine, New Orleans, United States

   Contribution

   Data curation, Formal analysis

   Competing interests

   No competing interests declared

5. Hannah Jarrell

   Neuroscience Center of Excellence, Louisiana State University School of Medicine, New Orleans, United States

   Contribution

   Data curation, Formal analysis, Validation

   Competing interests

   No competing interests declared

6. Yan Ding

   Neuroscience Center of Excellence, Louisiana State University School of Medicine, New Orleans, United States

   Contribution

   Data curation, Formal analysis

   Competing interests

   No competing interests declared

7. Zhaoqian Teng

   Neuroscience Center of Excellence, Louisiana State University School of Medicine, New Orleans, United States

   Contribution

   Methodology

   Competing interests

   No competing interests declared

8. Ofer Tchernichovski

   Department of Psychology, Hunter College, New York, United States

   Contribution

   Software, Formal analysis, Methodology, Writing—review and editing

   For correspondence

   tchernichovski@gmail.com

   Competing interests

   No competing interests declared

9. XiaoChing Li

   Neuroscience Center of Excellence, Louisiana State University School of Medicine, New Orleans, United States

   Contribution

   Conceptualization, Formal analysis, Supervision, Funding acquisition, Investigation, Methodology, Writing—original draft, Project administration, Writing—review and editing

   For correspondence

   xli4@lsuhsc.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-7544-494X

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