Project description:A male zebra finch begins to learn to sing by memorizing a tutor’s song during a sensitive period in juvenile development. Tutor song memorization requires molecular signaling within the auditory forebrain. Using microarray and in situ hybridizations, we tested whether the auditory forebrain at an age just before tutoring expresses a different set of genes compared with later life after song learning has ceased. Microarray analysis revealed differences in expression of thousands of genes in the male auditory forebrain at posthatch day 20 (P20) compared with adulthood. Furthermore, song playbacks had essentially no impact on gene expression in P20 auditory forebrain, but altered expression of hundreds of genes in adults. Most genes that were song-responsive in adults were expressed at constitutively high levels at P20. Using in situ hybridization with a representative sample of 44 probes, we confirmed these effects and found that birds at P20 and P45 were similar in their gene expression patterns. Additionally, eight of the probes showed male–female differences in expression. We conclude that the developing auditory forebrain is in a very different molecular state from the adult, despite its relatively mature gross morphology and electrophysiological responsiveness to song stimuli. Developmental gene expression changes may contribute to fine-tuning of cellular and molecular properties necessary for song learning.
Project description:A male zebra finch begins to learn to sing by memorizing a tutorM-bM-^@M-^Ys song during a sensitive period in juvenile development. Tutor song memorization requires molecular signaling within the auditory forebrain. Using microarray and in situ hybridizations, we tested whether the auditory forebrain at an age just before tutoring expresses a different set of genes compared with later life after song learning has ceased. Microarray analysis revealed differences in expression of thousands of genes in the male auditory forebrain at posthatch day 20 (P20) compared with adulthood. Furthermore, song playbacks had essentially no impact on gene expression in P20 auditory forebrain, but altered expression of hundreds of genes in adults. Most genes that were song-responsive in adults were expressed at constitutively high levels at P20. Using in situ hybridization with a representative sample of 44 probes, we confirmed these effects and found that birds at P20 and P45 were similar in their gene expression patterns. Additionally, eight of the probes showed maleM-bM-^@M-^Sfemale differences in expression. We conclude that the developing auditory forebrain is in a very different molecular state from the adult, despite its relatively mature gross morphology and electrophysiological responsiveness to song stimuli. Developmental gene expression changes may contribute to fine-tuning of cellular and molecular properties necessary for song learning. Post-hatch day 20 male zebra finches that had been raised in acoustic isolation with a foster female or adult male zebra finches were placed in a song playback chamber. The next day, birds heard either silence (control) or 30 minutes of novel song. All samples were hybridized against the universal SoNG RNA reference pool, 6 biological replicates per group in each of 4 groups.
Project description:We used a combination of ChIPseq, RNAseq, bioinformatic analysis, and molecular biology in the auditory forebrain, a brain area required for tutor song memorization to elucidate the role epigenetics plays in song learning. These analyses revealed thousands of genes were differentially marked by PTMs under these two conditions, indicating greater transcription in the birds with extended learning.
Project description:Like human speech, birdsong is a complex vocal behavior that is acquired by sensorimotor learning based on coordination of auditory input and vocal output to mimic memorized tutor song. Here we investigate neural circuits for vocal learning and production in deafened songbirds to elucidate how sensory-input regulate genetic and epigenetic property of vocal development and its associated gene expression dynamics. Compared with audition-intact birds, in deafened zebra finches, the vocal development is delayed but song crystallization is observed at more than three times later, producing individually different but structured vocal patterns. In contrast to the distinct difference of vocal ontogeny between audition (+) and (-), unexpectedly, developmental regulation of gene expression dynamics is strictly conserved with age-locked trend in vocal motor circuit in both intact and deafened birds, indicating sensory-input independent robustness of developmental gene expression dynamics in the motor circuit for sensorimotor learning. This discrepancy between outward vocal phenotype and inward gene expression dynamics provides new insight into neural regulation at closing of the critical period for vocal learning by two different forms: auditory inputs-dependent ‘active’ crystallization and gene expression dynamics-mediated ‘passive’ crystallization.
Project description:Like human speech, birdsong is a complex vocal behavior that is acquired by sensorimotor learning based on coordination of auditory input and vocal output to mimic memorized tutor song. Here we investigate neural circuits for vocal learning and production in deafened songbirds to elucidate how sensory-input regulate genetic and epigenetic property of vocal development and its associated gene expression dynamics. Compared with audition-intact birds, in deafened zebra finches, the vocal development is delayed but song crystallization is observed at more than three times later, producing individually different but structured vocal patterns. In contrast to the distinct difference of vocal ontogeny between audition (+) and (-), unexpectedly, developmental regulation of gene expression dynamics is strictly conserved with age-locked trend in vocal motor circuit in both intact and deafened birds, indicating sensory-input independent robustness of developmental gene expression dynamics in the motor circuit for sensorimotor learning. This discrepancy between outward vocal phenotype and inward gene expression dynamics provides new insight into neural regulation at closing of the critical period for vocal learning by two different forms: auditory inputs-dependent M-bM-^@M-^XactiveM-bM-^@M-^Y crystallization and gene expression dynamics-mediated M-bM-^@M-^XpassiveM-bM-^@M-^Y crystallization. We collected brain samples from intact and early-deafened birds (deafened at day-post hatch 17-23) under silent and dark condition. Song nuclei in vocal motor circuit, HVC and RA tissue samples (juvenile; n = 3, young; n = 3, old; n = 3 of intact and early-deafened birds for HVC and RA) were laser-microdissected from total 24 birds (intact; n = 12, early-deafened; n = 12). Each sample was hybridized to a single array, totaling 36 arrays. Birds were selected per slide such that early-deafened birds were paired with intact birds. To minimize possible interslide bias or batch effects, intact and early-deafened bird samples matching with brain area and age conditions were hybridized side by side on same array glass.
Project description:Similarities between speech and birdsong make songbirds advantageous for investigating the neurogenetics of learned vocal communication; a complex phenotype likely supported by ensembles of interacting genes in cortico-basal ganglia pathways of both species. To date, only FoxP2 has been identified as critical to both speech and birdsong. We performed weighted gene co-expression network analysis on microarray data from singing zebra finches to discover gene ensembles regulated during vocal behavior. We found ~2,000 singing- regulated genes comprising 3 co-expression groups unique to area X, the basal ganglia subregion dedicated to learned vocal-motor behavior. These contained known targets of human FOXP2 and potential avian targets. We validated novel biological pathways for vocalization. Our findings show that higher-order gene co-expression patterns, rather than expression levels, molecularly distinguish area X from the ventral striato-pallidum during singing. The previously unknown structure of singing-driven networks enables prioritization of molecular interactors that likely bear on human motor disorders, especially those affecting speech. Gene expression was measured in 2 basal ganglia sub-regions (area X & ventral striato-pallidum (VSP)) of 27 adult male zebra finches that sang different amounts of song over a 2hr period in the morning. 18 birds were allowed to sing freely, 9 birds were discouraged from singing by the presence of an investigator and those that sang fewer than 10 song motifs were considered “non-singers”.