Learning of most motor skills is constrained in a species-specific manner. However, the proximate mechanisms underlying species-specific learned behaviors remain poorly understood. Songbirds acquire species-specific songs through learning, which is hypothesized to depend on species-specific patterns of gene expression in functionally specialized brain regions for vocal learning and production, called song nuclei. Here, we leveraged two closely related songbird species, zebra finch, owl finch, and their interspecific first-generation (F1) hybrids, to relate transcriptional regulatory divergence between species with the production of species-specific songs. We quantified genome-wide gene expression in both species and compared this with allele-specific expression in F1 hybrids to identify genes whose expression in song nuclei is regulated by species divergence in either cis- or trans-regulation. We found that divergence in transcriptional regulation altered the expression of approximately 10% of total transcribed genes and was linked to differential gene expression between the two species. Furthermore, trans-regulatory changes were more prevalent than cis-regulatory and were associated with synaptic formation and transmission in song nucleus RA, the avian analog of the mammalian laryngeal motor cortex. We identified brain-derived neurotrophic factor (BDNF) as an upstream mediator of trans-regulated genes in RA, with a significant correlation between individual variation in BDNF expression level and species-specific song phenotypes in F1 hybrids. This was supported by the fact that the pharmacological overactivation of BDNF receptors altered the expression of its trans-regulated genes in the RA, thus disrupting the learned song structures of adult zebra finch songs at the acoustic and sequence levels. These results demonstrate functional neurogenetic associations between divergence in region-specific transcriptional regulation and species-specific learned behaviors.

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Learned vocalizations are a crucial acoustic biosignal conveying individual traits in many species. Songbirds learn song patterns by listening to a tutor song and performing vocal practice during a sensitive developmental period. However, when and how individual differences in song patterns develop remain unknown. Here, we report that individual differences in vocal output exist even at the earliest song development stage, called subsong. Experiments involving the manipulation of both breeding pairs and song tutoring conditions revealed that the parental pair combination contributes to generating familial differences in syllable duration and variability in the subsong of offspring. Furthermore, after deafening, juveniles immediately changed their subsong by shortening the syllable durations but maintained the individual variability of their subsong temporal patterns, suggesting both auditory-sensitive modification and independent intrinsic regulation of vocal output. These results indicate that the temporal patterns of subsong are not merely disordered vocalization but are regulated by familial bias with sensitivity to auditory feedback, thus generating individual variability at the initiation of vocal development.Birdsong is a learned behavior that is used as an acoustic biosignal to distinguish between individuals for mating and territorial defense 1 . Among songbirds, the zebra finch acquires its song patterns through two phases: the sensory-learning and sensorimotor-learning phases 2,3 . During the sensory-learning phase, juveniles acquire the sensory memories of songs by listening to the songs of adult birds as a song template to be imitated. The sensorimotor-learning phase starts with the generation of soft and highly variable syllables, in a song-developing state called subsong. Subsongs are generated mainly as vocal output from the neural activity of song nuclei in the basal ganglia-thalamocortical loops in the song system 4,5 . Thereafter, birds start producing what is called plastic song, which is the gradual inclusion of recognizable yet variable syllables. At the end of the learning process, the song is crystallized with acoustically and sequentially stable syllable patterns. During this learning process, auditory feedback is crucial for monitoring vocal outputs to match them with memorized tutor song patterns 6 .However, in the zebra finch (Taeniopygia guttata), even when juveniles listen to the same tutor song during the sensory-learning phase, they generate individually unique song patterns and maintain species-specificity in crystallized song at maturity 7 . Furthermore, during the sensorimotor-learning phase, zebra finch juveniles use multiple learning strategies to develop their song patterns 8 . Given the existing evidence suggesting the developmental origin of individual differences in learned vocalization, we investigated when and how zebra finches initiate the generation of individual variation in song patterns and how auditory feedback contributes to the regulation of vocal output during t...

The emergence of individuality during learned behavior is a general feature of animal species, yet the biological bases of its development remain unknown. Similar to human speech, songbirds develop individually-unique songs with species-specific traits through vocal learning. By taking advantage of songbirds as a model system for studying the neural basis of vocal learning and development, we utilized F1 hybrid songbirds (Taeniopygia guttata cross with T. bichenovii) to examine the developmental and molecular mechanisms underlying individuality in vocal learning. When tutoring with songs from both parental species, F1 pupils showed vast individual differences in their acquired songs. Approximately 30% of F1 hybrids selectively learned either song of the two parental species, whereas others developed merged songs between the parental species. Vocal acoustic biases during vocal babbling were initially observed as individual differences in songs among F1 juveniles, which were maintained through the sensitive period of song vocal learning. These individual differences in vocal acoustic biases appeared independently from the auditory experience of hearing biological farther’s and passive tutored songs. Furthermore, the idiosyncratic traits of F1 hybrids’ songs were not correlated with peripheral vocal organ morphology. However, we identified unique transcriptional signatures from the glutamatergic neurons projecting from the cortical vocal output nucleus to the hypoglossal nuclei associated with individual differences in the acoustic vocal biases, even at the initial stage of vocal learning. These results indicate that a predisposed motor bias influences the individuality observed when learning new motor skills.

The emergence of individuality during learned behavior is a general feature of animal species, yet the biological bases of its development remain unknown. Similar to human speech, songbirds develop individually-unique songs with species-specific traits through vocal learning. By taking advantage of songbirds as a model system for studying the neural basis of vocal learning and development, we utilized F1 hybrid songbirds (Taeniopygia guttata cross with T. bichenovii) to examine the developmental and molecular mechanisms underlying individuality in vocal learning. When tutoring with songs from both parental species, F1 pupils showed vast individual differences in their acquired songs. Approximately 30% of F1 hybrids selectively learned either song of the two parental species, whereas others developed merged songs between the parental species. Vocal acoustic biases during vocal babbling were initially observed as individual differences in songs among F1 juveniles, which were maintained through the sensitive period of song vocal learning. These individual differences in vocal acoustic biases appeared independently from the auditory experience of hearing biological farther’s and passive tutored songs. Furthermore, the idiosyncratic traits of F1 hybrids’ songs were not correlated with peripheral vocal organ morphology. However, we identified unique transcriptional signatures from the glutamatergic neurons projecting from the cortical vocal output nucleus to the hypoglossal nuclei associated with individual differences in the acoustic vocal biases, even at the initial stage of vocal learning. These results indicate that a predisposed motor bias influences the individuality observed when learning new motor skills.