Dynamic transcriptome landscape in the song nucleus HVC between juvenile and adult zebra finches.

Male juvenile zebra finches learn to sing by imitating songs of adult males early in life. The development of the song control circuit and song learning and maturation are highly intertwined processes, involving gene expression, neurogenesis, circuit formation, synaptic modification, and sensory-motor learning. To better understand the genetic and genomic mechanisms underlying these events, we used RNA-Seq to examine genome-wide transcriptomes in the song control nucleus HVC of male juvenile (45 d) and adult (100 d) zebra finches. We report that gene groups related to axon guidance, RNA processing, lipid metabolism, and mitochondrial functions show enriched expression in juvenile HVC compared to the rest of the brain. As juveniles mature into adulthood, massive gene expression changes occur. Expression of genes related to amino acid metabolism, cell cycle, and mitochondrial function is reduced, accompanied by increased and enriched expression of genes with synaptic functions, including genes related to G-protein signaling, neurotransmitter receptors, transport of small molecules, and potassium channels. Unexpectedly, a group of genes with immune system functions is also developmentally regulated, suggesting potential roles in the development and functions of HVC. These data will serve as a rich resource for investigations into the development and function of a neural circuit that controls vocal behavior.

miR-9 regulates basal ganglia-dependent developmental vocal learning and adult vocal performance in songbirds.

miR-9 is an evolutionarily conserved miRNA that is abundantly expressed in Area X, a basal ganglia nucleus required for vocal learning in songbirds. Here, we report that overexpression of miR-9 in Area X of juvenile zebra finches impairs developmental vocal learning, resulting in a song with syllable omission, reduced similarity to the tutor song, and altered acoustic features. miR-9 overexpression in juveniles also leads to more variable song performance in adulthood, and abolishes social context-dependent modulation of song variability. We further show that these behavioral deficits are accompanied by downregulation of FoxP1 and FoxP2, genes that are known to be associated with language impairments, as well as by disruption of dopamine signaling and widespread changes in the expression of genes that are important in circuit development and functions. These findings demonstrate a vital role for miR-9 in basal ganglia function and vocal communication, suggesting that dysregulation of miR-9 in humans may contribute to language impairments and related neurodevelopmental disorders.

Studying the Mechanisms of Developmental Vocal Learning and Adult Vocal Performance in Zebra Finches through Lentiviral Injection.

Here we provide a detailed step-by-step protocol for using lentivirus to manipulate miRNA expression in Area X of juvenile zebra finches and for analyzing the consequences on song learning and song performance. This protocol has four parts: 1) making the lentiviral construct to overexpress miRNA miR-9; 2) packaging the lentiviral vector; 3) stereotaxic injection of the lentivirus into Area X of juvenile zebra finches; 4) analysis of song learning and song performance in juvenile and adult zebra finches. These methods complement the methods employed in recent works that showed changing FoxP2 gene expression in Area X with lentivirus or adeno-associated virus leads to impairments in song behavior.

Multiple microRNAs regulate human FOXP2 gene expression by targeting sequences in its 3' untranslated region.

BACKGROUND: Mutations in the human FOXP2 gene cause speech and language impairments. The FOXP2 protein is a transcription factor that regulates the expression of many downstream genes, which may have important roles in nervous system development and function. An adequate amount of functional FOXP2 protein is thought to be critical for the proper development of the neural circuitry underlying speech and language. However, how FOXP2 gene expression is regulated is not clearly understood. The FOXP2 mRNA has an approximately 4-kb-long 3' untranslated region (3' UTR), twice as long as its protein coding region, indicating that FOXP2 can be regulated by microRNAs (miRNAs). FINDINGS: We identified multiple miRNAs that regulate the expression of the human FOXP2 gene using sequence analysis and in vitro cell systems. Focusing on let-7a, miR-9, and miR-129-5p, three brain-enriched miRNAs, we show that these miRNAs regulate human FOXP2 expression in a dosage-dependent manner and target specific sequences in the FOXP2 3' UTR. We further show that these three miRNAs are expressed in the cerebellum of the human fetal brain, where FOXP2 is known to be expressed. CONCLUSIONS: Our results reveal novel regulatory functions of the human FOXP2 3' UTR sequence and regulatory interactions between multiple miRNAs and the human FOXP2 gene. The expression of let-7a, miR-9, and miR-129-5p in the human fetal cerebellum is consistent with their roles in regulating FOXP2 expression during early cerebellum development. These results suggest that various genetic and environmental factors may contribute to speech and language development and related neural developmental disorders via the miRNA-FOXP2 regulatory network.

miR-9 and miR-140-5p target FoxP2 and are regulated as a function of the social context of singing behavior in zebra finches.

Mutations in the FOXP2 gene cause speech and language impairments, accompanied by structural and functional abnormalities in brain regions underlying speech-related sensory-motor processing, including the striatum and cerebellum. The sequence and expression patterns of FOXP2 are highly conserved among higher vertebrates. In the zebra finch brain, FoxP2 is expressed in Area X, a striatal nucleus required for vocal learning, and reduced FoxP2 expression impairs dendritic development and vocal learning. The FoxP2 gene encodes a transcription factor that controls the expression of many downstream genes. However, how FOXP2 gene expression is regulated is not clearly understood. miRNAs regulate gene expression post-transcriptionally by targeting the 3'-untranslated regions (UTRs) of mRNAs, leading to translational suppression or mRNA degradation. In this study, we identified miR-9 and miR-140-5p as potential regulators of the FoxP2 gene. We show that both miR-9 and miR-140-5p target specific sequences in the FoxP2 3'-UTR and downregulate FoxP2 protein and mRNA expression in vitro. We also show that the expression of miR-9 and miR-140-5p in Area X of the zebra finch brain is regulated during song development in juvenile zebra finches. We further show that in adult zebra finches the expression of miR-9 and miR-140-5p in Area X is regulated as a function of the social context of song behavior in males singing undirected songs. Our findings reveal a post-transcriptional mechanism that regulates FoxP2 expression and suggest that social vocal behavior can influence the basal ganglia circuit controlling vocal learning via a miRNA-FoxP2 gene regulatory network.

Genome-wide annotation and analysis of zebra finch microRNA repertoire reveal sex-biased expression.

BACKGROUND: MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression post-transcriptionally in a wide range of biological processes. The zebra finch (Taeniopygia guttata), an oscine songbird with characteristic learned vocal behavior, provides biologists a unique model system for studying vocal behavior, sexually dimorphic brain development and functions, and comparative genomics. RESULTS: We deep sequenced small RNA libraries made from the brain, heart, liver, and muscle tissues of adult male and female zebra finches. By mapping the sequence reads to the zebra finch genome and to known miRNAs in miRBase, we annotated a total of 193 miRNAs. Among them, 29 (15%) are avian specific, including three novel zebra finch specific miRNAs. Many of the miRNAs exhibit sequence heterogeneity including length variations, untemplated terminal nucleotide additions, and internal substitution events occurring at the uridine nucleotide within a GGU motif. We also identified seven Z chromosome-encoded miRNAs. Among them, miR-2954, an avian specific miRNA, is expressed at significantly higher levels in males than in females in all tissues examined. Target prediction analysis reveals that miR-2954, but not other Z-linked miRNAs, preferentially targets Z chromosome-encoded genes, including several genes known to be expressed in a sexually dimorphic manner in the zebra finch brain. CONCLUSIONS: Our genome-wide systematic analysis of mature sequences, genomic locations, evolutionary sequence conservation, and tissue expression profiles of the zebra finch miRNA repertoire provides a valuable resource to the research community. Our analysis also reveals a miRNA-mediated mechanism that potentially regulates sex-biased gene expression in avian species.

Genomic resources for songbird research and their use in characterizing gene expression during brain development.

Vocal learning and neuronal replacement have been studied extensively in songbirds, but until recently, few molecular and genomic tools for songbird research existed. Here we describe new molecular/genomic resources developed in our laboratory. We made cDNA libraries from zebra finch (Taeniopygia guttata) brains at different developmental stages. A total of 11,000 cDNA clones from these libraries, representing 5,866 unique gene transcripts, were randomly picked and sequenced from the 3' ends. A web-based database was established for clone tracking, sequence analysis, and functional annotations. Our cDNA libraries were not normalized. Sequencing ESTs without normalization produced many developmental stage-specific sequences, yielding insights into patterns of gene expression at different stages of brain development. In particular, the cDNA library made from brains at posthatching day 30-50, corresponding to the period of rapid song system development and song learning, has the most diverse and richest set of genes expressed. 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 high vocal center of both adult male zebra finches and canaries (Serinus canaria). Genes differentially expressed in the high vocal center were identified from the microarray hybridization results. Selected genes were validated by in situ hybridization. Networks among the regulated genes were also identified. These resources provide songbird biologists with tools for genome annotation, comparative genomics, and microarray gene expression analysis.

Expression profiling of intermingled long-range projection neurons harvested by laser capture microdissection.

Gene expression data are most useful if they can be associated with specific cell types. This is particularly so in an organ such as the brain, where many different cell types lie in close proximity to each other. We used zebra finches (Taeniopygia guttata), fluorescent tracers and laser capture microdissection (LCM) to collect projection neurons and their RNAs from two interspersed populations from the same animal. RNA amplified from each cell class was reverse transcribed, fluorescently labeled, and hybridized to cDNA microarrays of genes expressed in the zebra finch brain. We applied strict fold-expression criteria, supplemented by statistical analysis, to single out genes that showed the most extreme and consistent differential expression between the two cell classes. Confirmation of the true expression pattern of these genes was made by in situ hybridization and Taqman quantitative PCR (qPCR). High quality RNA was obtained, too, from backfilled neurons birth-dated with bromodeoxyuridine (BrdU). We also quantified changes in the levels of three genes after singing behavior using qPCR. Thus, we have brought together a combination of techniques allowing for the molecular profiling of intermingled populations of projection neurons of known connectivity, age and experience, which should constitute a powerful tool for CNS research.

Replaceable neurons and neurodegenerative disease share depressed UCHL1 levels.

Might there be systematic differences in gene expression between neurons that undergo spontaneous replacement in the adult brain and those that do not? We first explored this possibility in the high vocal center (HVC) of male zebra finches by using a combination of neuronal tracers, laser capture microdissection, and RNA profiling. HVC has two kinds of projection neurons, one of which continues to be produced and replaced in adulthood. HVC neurons of the replaceable kind showed a consistent and robust underexpression of the deubiquitination gene ubiquitin carboxyl-terminal hydrolase (UCHL1) that is involved with protein degradation. Singing behavior, known to increase the survival of adult-born HVC neurons in birds, significantly up-regulated the levels of UCHL1 in the replaceable neurons but not in their equally active nonreplaceable counterparts. We then looked in the mouse brain and found relatively low UCHL1 expression in granule neurons of the hippocampus and olfactory bulb, two well characterized types of replaceable neurons in mammals. UCHL1 dysfunction has been associated with neurodegeneration in Parkinson's, Alzheimer's, and Huntington's disease patients. In all these instances, reduced UCHL1 function may jeopardize the survival of CNS neurons.