Project description:To investigate the cellular basis of parental species bias at birdsong, we performed single nuclei RNA-seq for six zebra finch and owl finch F1 hybrid juvenile birds.
Project description: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: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:The evolution of the six-layered neocortex is often credited with an increased capacity for complex behaviors and cognition in humans and other mammals. However, birds, despite lacking a laminated neocortex, display complex and non-instinctual behaviors including vocal learning, tool use, and problem-solving1,2. The evolution of brain circuits and cell-types supporting advanced behavioral repertoires remains poorly understood. Here in songbirds, we use single-cell RNA-sequencing to characterize the molecular identities of cells in the song motor pathway, a pallial circuit with function and connectivity that has been likened to the mammalian neocortex1,2. We find that each song region contains different glutamatergic excitatory projection neurons but similar sets of GABAergic inhibitory interneurons, similar to patterns of neuronal diversity in mammals and reptiles3,4. Song motor pathway glutamatergic neurons have gene expression patterns similar to those described in neocortical projection neurons, but at the level of transcription factor expression, display stronger similarity to neurons in the ventral pallium. We observed multiple GABAergic neuron classes that are conserved across amniotes, yet the most abundant class strongly resembles a cell-class that in mammals is not found in the neocortex but is present in non-neocortical pallial regions3,4. Thus, although pallial song-control regions contain neurons with similar molecular profiles to neocortical neurons, these pallial areas have a regional identity distinct from neocortex, indicating that complex behaviors such as vocal learning have evolved through the use of different brain circuits under similar functional constraints.
Project description:To investigate the cellular basis of parental species bias at birdsong, we performed single nuclei RNA-seq for six zebra finch and owl finch F1 hybrid juvenile birds.
Project description:Within the overall project, we performed a set of microarrays to validate RNAseq data (submitted to EBI: PRJEB4463). In this data set, we compare the expression data of song nuclei to the optical tectrum dissected from adult canaries housed at long day cycles to identify nuclei specific genes. 18 total S. canaria samples were analyzed, 6 HVC samples, 5 RA samles and 7 Entopallium samples. The differential expression was analyzed using the group-wise exhaustive analysis with False Discovery Rate set to zero and 10-significant probe minimum coverage, HVc/RA compared to entopallium samples. ChipInspector carries out significance analysis on the single probe level (directly generated from the CEL files). Thus, normalized probe set level data for individual Sample records are not available. Processed data files containing transcripts and the fold changes are available on Series record.
Project description:Photoperiod and hormonal cues drive dramatic seasonal changes in structure and function of the avian song control system. Little is known, however, about the patterns of gene expression associated with seasonal changes. Here we address this issue by exposing Gambel’s white-crowned sparrows to season-appropriate cues and extracting RNA from the telencephalic song control nuclei HVC and RA across multiple time points that capture different stages of growth and regression. We chose HVC and RA because while both nuclei change in volume across seasons, the cellular mechanisms underlying these changes differ. We thus hypothesized that different genes would be expressed between HVC and RA. We tested this by using the extracted RNA to perform a cDNA microarray hybridization developed by the SoNG initiative. We then validated these results using qRT-PCR. Supporting our hypothesis, only 59 of the 363 genes of interest were found to vary by more than |1.5| fold in expression in both nuclei, while 132 gene expression changes were HVC specific and 172 genes were RA specific. We then assigned many of these genes to functional categories relevant to the different mechanisms underlying seasonal change in HVC and RA, including neurogenesis, apoptosis, cell growth, dendrite arborization and axonal growth, angiogenesis, endocrinology, growth factors, and electrophysiology. This revealed categorical differences in the kinds of genes regulated in HVC and RA. These results show that different molecular programs underlie seasonal changes in HVC and RA, and that gene expression is time specific across different reproductive conditions. Our results provide insights into the complex molecular pathways that underlie adult neural plasticity.
Project description:Male zebra finches of a captive population in the University of Sheffield were artificially selected for long or short sperm based on their breeding values for three generations. We used Affymetrix microarrays to examine gene expression differences between testes of selection line male birds.
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:Photoperiod and hormonal cues drive dramatic seasonal changes in structure and function of the avian song control system. Little is known, however, about the patterns of gene expression associated with seasonal changes. Here we address this issue by exposing GambelM-bM-^@M-^Ys white-crowned sparrows to season-appropriate cues and extracting RNA from the telencephalic song control nuclei HVC and RA across multiple time points that capture different stages of growth and regression. We chose HVC and RA because while both nuclei change in volume across seasons, the cellular mechanisms underlying these changes differ. We thus hypothesized that different genes would be expressed between HVC and RA. We tested this by using the extracted RNA to perform a cDNA microarray hybridization developed by the SoNG initiative. We then validated these results using qRT-PCR. Supporting our hypothesis, only 59 of the 363 genes of interest were found to vary by more than |1.5| fold in expression in both nuclei, while 132 gene expression changes were HVC specific and 172 genes were RA specific. We then assigned many of these genes to functional categories relevant to the different mechanisms underlying seasonal change in HVC and RA, including neurogenesis, apoptosis, cell growth, dendrite arborization and axonal growth, angiogenesis, endocrinology, growth factors, and electrophysiology. This revealed categorical differences in the kinds of genes regulated in HVC and RA. These results show that different molecular programs underlie seasonal changes in HVC and RA, and that gene expression is time specific across different reproductive conditions. Our results provide insights into the complex molecular pathways that underlie adult neural plasticity. Experimental groups: long-term Short Day (SD); Long Day (LD) with Testosterone (T) for 3, 7, and 21 days (3LD+T, 7LD+T, 21LD+T respectively); SD and removal of T at 1 and 2 days (1SD-T, 2SD-T); two tissues (HVC, RA) collected separately from each individual animal; one experimental sample and one universal SoNG reference sample per array; dye balanced within groups. Six biological replicates per group, five biological replicates in 3LD+T.HVC group.