Transcriptome profiling of the HVC of the young and adult zebra finch brain by RNA-Seq
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ABSTRACT: Abstract: Juvenile zebra finches learn to sing by imitating songs of adult males early in life. The development of the song control circuit and the song learning and maturation processes are highly intertwined, involving gene expression, neurogenesis, circuit formation, synaptic modification, and sensory-motor learning, which eventually lead to a mature song. To better understand the genetic mechanisms underlying these events, we used RNA-sequencing (RNA-Seq) to profile genome-wide transcriptomes in the HVC, a brain nucleus controlling song behavior, in juvenile and adult zebra finches. We found that gene expression programs related to axon guidance, RNA metabolism, lipid metabolism, and ATP synthesis were enriched in the HVC relative to the rest of the brain in juveniles. As juveniles matured into adults, massive gene expression changes occurred in the HVC. Gene expression shifted from amino acid metabolism, laminin interaction, cell proliferation, and mitochondrial protein translation to synaptic functions; genes associated with G protein coupled-receptors, GTPase signaling, ion channels, and inhibitory transmission increased expression. Unexpectedly, a group of genes known for their functions in the immune system was also developmentally regulated in the HVC. These data will serve as a rich resource for further analysis of development and function of a neural circuit that controls vocal behavior.
Project description:Human speech is one of the few examples of vocal learning among mammals, yet ~half of avian species exhibit this ability. Its genetic basis is unknown beyond a shared requirement for FoxP2 in both humans and zebra finches. Here we manipulated FoxP2 isoforms in Area X during a critical period for song development, delineating, for the first time, unique contributions of each to vocal learning. We used weighted gene coexpression network analysis of RNA-seq data to construct transcriptional profiles and found gene modules correlated to singing, learning, or vocal variability. The juvenile song modules were preserved adults, whereas the learning modules were not. The learning modules were preserved in the striatopallidum adjacent to Area X whereas the song modules were not. The confluence of learning and singing coexpression in juvenile, but not adult, Area X may underscore molecular processes that drive vocal learning in zebra finches and, by analogy, humans.
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:Background: Vocal learning is a rare and complex behavioral trait that serves as a basis for the acquisition of human spoken language. In songbirds, vocal learning and production depend on a set of specialized brain nuclei known as the song system. Methodology/Principal Findings: Using high-throughput functional genomics we have identified, 200 novel molecular markers of adult zebra finch HVC Vocal, a key node of the song system. These markers clearly differentiate HVC from the general pallial region to which HVC belongs, and thus represent molecular specializations of this song nucleus. Bioinformatics analysis reveals that several major neuronal cell functions and specific biochemical pathways are the targets of transcriptional regulation in HVC, including: 1) cell-cell and cell-substrate interactions (e.g., cadherin/catenin-mediated adherens junctions, collagen-mediated focal adhesions, and semaphorin-neuropilin/plexin axon guidance pathways); 2) cell excitability (e.g., potassium channel subfamilies, cholinergic and serotonergic receptors, neuropeptides and neuropeptide receptors); 3) signal transduction (e.g., calcium regulatory proteins, regulators of G-protein-related signaling); 4) cell proliferation/death, migration and differentiation (e.g., TGF-beta/BMP and p53 pathways); and 5) regulation of gene expression (candidate retinoid and steroid targets, modulators of chromatin/nucleolar organization). The overall direction of regulation suggest that processes related to cell stability are enhanced, whereas proliferation, growth and plasticity are largely suppressed in adult HVC, consistent with the observation that song in this songbird species is mostly stable in adulthood. Conclusions/Significance: Our study represents one of the most comprehensive molecular genetic characterizations of a brain nucleus involved in a complex learned behavior in a vertebrate. The data indicate numerous targets for pharmacological and genetic manipulations of the song system, and provide novel insights into mechanisms that might play a role in the regulation of song behavior and/or vocal learning. Comparison of HVC and shelf regions from adult male zebra finches, 6 biological replicates per group. Each sample was hybridized against the SoNG universal reference RNA pool.
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:Background: Vocal learning is a rare and complex behavioral trait that serves as a basis for the acquisition of human spoken language. In songbirds, vocal learning and production depend on a set of specialized brain nuclei known as the song system. Methodology/Principal Findings: Using high-throughput functional genomics we have identified, 200 novel molecular markers of adult zebra finch HVC Vocal, a key node of the song system. These markers clearly differentiate HVC from the general pallial region to which HVC belongs, and thus represent molecular specializations of this song nucleus. Bioinformatics analysis reveals that several major neuronal cell functions and specific biochemical pathways are the targets of transcriptional regulation in HVC, including: 1) cell-cell and cell-substrate interactions (e.g., cadherin/catenin-mediated adherens junctions, collagen-mediated focal adhesions, and semaphorin-neuropilin/plexin axon guidance pathways); 2) cell excitability (e.g., potassium channel subfamilies, cholinergic and serotonergic receptors, neuropeptides and neuropeptide receptors); 3) signal transduction (e.g., calcium regulatory proteins, regulators of G-protein-related signaling); 4) cell proliferation/death, migration and differentiation (e.g., TGF-beta/BMP and p53 pathways); and 5) regulation of gene expression (candidate retinoid and steroid targets, modulators of chromatin/nucleolar organization). The overall direction of regulation suggest that processes related to cell stability are enhanced, whereas proliferation, growth and plasticity are largely suppressed in adult HVC, consistent with the observation that song in this songbird species is mostly stable in adulthood. Conclusions/Significance: Our study represents one of the most comprehensive molecular genetic characterizations of a brain nucleus involved in a complex learned behavior in a vertebrate. The data indicate numerous targets for pharmacological and genetic manipulations of the song system, and provide novel insights into mechanisms that might play a role in the regulation of song behavior and/or vocal 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:Vocal learning and neuronal replacement have been studied extensively in the songbird brain, but until recently, few molecular and genomic tools have been available for this work. Here we describe new molecular/genomic resources for songbird research. We made cDNA libraries from zebra finch (Taeniopygia guttata) brains at different developmental stages. A total of 11000 clones were sequenced from these libraries, representing 5870 unique gene transcripts. A web-based database has been established for sequence analysis and functional annotations. The cDNA libraries were not normalized. Sequence analysis revealed that a cDNA library made from brains at post-hatching day 30-50, when the song system goes through rapid development and birds learn to sing, shows the highest gene discovery rate. We grouped genes into functional categories according to the Gene Ontology classification and found that expression of the functional categories changed as the brain developed. We also identified five microRNAs whose sequences are highly conserved between zebra finch and other species. We printed cDNA microarrays and profiled gene expression in the HVC of both adult male zebra finches and canaries (Serinus canaria). Statistical Analysis of Microarrays (SAM) was used for data analysis. A subset of the differentially regulated genes was validated by in situ hybridization. The bioinformatic tools EASE and Ingenuity Pathway Analysis were used to identify over-represented functional groups and gene networks among the regulated genes. These resources provide songbird biologists with tools for genome annotation, comparative genomics, and microarray gene expression analysis. Keywords: HVC, songbird, cDNA microarray, gene expression
Project description:Song learning in zebra finches is a prototypical example of a complex learned behavior, yet knowledge on the underlying molecular processes is limited. Therefore, we characterized transcriptomic (RNA sequencing) and epigenomic (RRBS, reduced representation bisulfite sequencing; immunofluorescence) dynamics in matched zebra finch telencephalon samples of both sexes from 1 day post hatching (1 dph) to adulthood, spanning the critical period for song learning (20 dph and 65 dph). We identified extensive transcriptional neurodevelopmental changes during postnatal telencephalon development. DNA (hydroxy)methylation was very low, yet increased over time, particularly in song control nuclei. Only a small fraction of the massive differential expression in the developing zebra finch telencephalon could be explained by differential CpG and CpH DNA methylation. However, a strong association between DNA methylation and age dependent gene expression was found for various transcription factors (i.a. OTX2, AR and FOS) involved in neurodevelopment. Additionally, genomic regions featured by age dependent differential methylation in differentially expressed genes were significantly enriched for specific transcription factor binding motifs. Incomplete dosage compensation was found to be largely responsible for sexually dimorphic gene expression, with dosage compensation increasing throughout life. In conclusion, our results indicate that DNA methylation regulates neurodevelopmental gene expression dynamics through steering transcription factor activity, but does not explain sexually dimorphic gene expression patterns in zebra finch telencephalon.
Project description:Song learning in zebra finches is a prototypical example of a complex learned behavior, yet knowledge on the underlying molecular processes is limited. Therefore, we characterized transcriptomic (RNA sequencing) and epigenomic (RRBS, reduced representation bisulfite sequencing; immunofluorescence) dynamics in matched zebra finch telencephalon samples of both sexes from 1 day post hatching (1 dph) to adulthood, spanning the critical period for song learning (20 dph and 65 dph). We identified extensive transcriptional neurodevelopmental changes during postnatal telencephalon development. DNA (hydroxy)methylation was very low, yet increased over time, particularly in song control nuclei. Only a small fraction of the massive differential expression in the developing zebra finch telencephalon could be explained by differential CpG and CpH DNA methylation. However, a strong association between DNA methylation and age dependent gene expression was found for various transcription factors (i.a. OTX2, AR and FOS) involved in neurodevelopment. Additionally, genomic regions featured by age dependent differential methylation in differentially expressed genes were significantly enriched for specific transcription factor binding motifs. Incomplete dosage compensation was found to be largely responsible for sexually dimorphic gene expression, with dosage compensation increasing throughout life. In conclusion, our results indicate that DNA methylation regulates neurodevelopmental gene expression dynamics through steering transcription factor activity, but does not explain sexually dimorphic gene expression patterns in zebra finch telencephalon.
Project description:New experiences can trigger changes in gene expression in the brain. To understand this phenomenon better, we studied zebra finches hearing playbacks of birdsong. Earlier research had shown that initial playbacks of a novel song transiently increase the ZENK (ZIF-268, EGR1, NGFIA, KROX-24) mRNA in the auditory forebrain, but the response selectively habituates after repetition of the stimulus. Here, using DNA microarray analysis, we show that novel song exposure induces rapid changes in thousands of RNAs, with even more RNAs decreasing than increasing. Habituation training leads to the emer- gence of a different gene expression profile a day later, accompanied by loss of essentially all of the rapid "novel" molecular responses. The novel molecular profile is characterized by increases in genes involved in transcription and RNA processing and decreases in ion channels and putative noncoding RNAs. The M-bM-^@M-^XM-bM-^@M-^XhabituatedM-bM-^@M-^YM-bM-^@M-^Y profile is dominated by changes in genes for mitochondrial proteins. A parallel proteomic analysis [2-dimensional difference gel electrophoresis (2D-DIGE) and sequencing by mass spectrometry] also detected changes in mito- chondrial proteins, and direct enzyme assay demonstrated changes in both complexes I and IV in the habituated state. Thus a natural experience, in this case hearing the sound of birdsong, can lead to major shifts in energetics and macromolecular metabolism in higher centers in the brain. Adult male zebra finches were acoustically isolated and exposed to silence, novel song, or familiar song (exposure to testing song for 3 hours on the day prior to testing) on test day. The auditory lobule (AL) was collected 30 minutes after the onset of the testing experience. All samples were hybridized against the universal SoNG reference RNA pool, 6 biological replicates per group in each of 3 groups.