Parental developmental experience affects vocal learning in offspring.

Cultural and genetic inheritance combine to enable rapid changes in trait expression, but their relative importance in determining trait expression across generations is not clear. Birdsong is a socially learned cognitive trait that is subject to both cultural and genetic inheritance, as well as being affected by early developmental conditions. We sought to test whether early-life conditions in one generation can affect song acquisition in the next generation. We exposed one generation (F1) of nestlings to elevated corticosterone (CORT) levels, allowed them to breed freely as adults, and quantified their son's (F2) ability to copy the song of their social father. We also quantified the neurogenetic response to song playback through immediate early gene (IEG) expression in the auditory forebrain. F2 males with only one corticosterone-treated parent copied their social father's song less accurately than males with two control parents. Expression of ARC in caudomedial nidopallium (NCM) correlated with father-son song similarity, and patterns of expression levels of several IEGs in caudomedial mesopallium (CMM) in response to father song playback differed between control F2 sons and those with a CORT-treated father only. This is the first study to demonstrate that developmental conditions can affect social learning and neurogenetic responses in a subsequent generation.

Concurrent invasions of European starlings in Australia and North America reveal population-specific differentiation in shared genomic regions.

A species' success during the invasion of new areas hinges on an interplay between the demographic processes common to invasions and the specific ecological context of the novel environment. Evolutionary genetic studies of invasive species can investigate how genetic bottlenecks and ecological conditions shape genetic variation in invasions, and our study pairs two invasive populations that are hypothesized to be from the same source population to compare how each population evolved during and after introduction. Invasive European starlings (Sturnus vulgaris) established populations in both Australia and North America in the 19th century. Here, we compare whole-genome sequences among native and independently introduced European starling populations to determine how demographic processes interact with rapid evolution to generate similar genetic patterns in these recent and replicated invasions. Demographic models indicate that both invasive populations experienced genetic bottlenecks as expected based on invasion history, and we find that specific genomic regions have differentiated even on this short evolutionary timescale. Despite genetic bottlenecks, we suggest that genetic drift alone cannot explain differentiation in at least two of these regions. The demographic boom intrinsic to many invasions as well as potential inversions may have led to high population-specific differentiation, although the patterns of genetic variation are also consistent with the hypothesis that this infamous and highly mobile invader adapted to novel selection (e.g., extrinsic factors). We use targeted sampling of replicated invasions to identify and evaluate support for multiple, interacting evolutionary mechanisms that lead to differentiation during the invasion process.

Transcript- and annotation-guided genome assembly of the European starling.

The European starling, Sturnus vulgaris, is an ecologically significant, globally invasive avian species that is also suffering from a major decline in its native range. Here, we present the genome assembly and long-read transcriptome of an Australian-sourced European starling (S. vulgaris vAU), and a second, North American, short-read genome assembly (S. vulgaris vNA), as complementary reference genomes for population genetic and evolutionary characterization. S. vulgaris vAU combined 10× genomics linked-reads, low-coverage Nanopore sequencing, and PacBio Iso-Seq full-length transcript scaffolding to generate a 1050 Mb assembly on 6222 scaffolds (7.6 Mb scaffold N50, 94.6% busco completeness). Further scaffolding against the high-quality zebra finch (Taeniopygia guttata) genome assigned 98.6% of the assembly to 32 putative nuclear chromosome scaffolds. Species-specific transcript mapping and gene annotation revealed good gene-level assembly and high functional completeness. Using S. vulgaris vAU, we demonstrate how the multifunctional use of PacBio Iso-Seq transcript data and complementary homology-based annotation of sequential assembly steps (assessed using a new tool, saaga) can be used to assess, inform, and validate assembly workflow decisions. We also highlight some counterintuitive behaviour in traditional busco metrics, and present buscomp, a complementary tool for assembly comparison designed to be robust to differences in assembly size and base-calling quality. This work expands our knowledge of avian genomes and the available toolkit for assessing and improving genome quality. The new genomic resources presented will facilitate further global genomic and transcriptomic analysis on this ecologically important species.

Acoustic developmental programming: a mechanistic and evolutionary framework.

Conditions experienced prenatally, by modulating developmental processes, have lifelong effects on individual phenotypes and fitness, ultimately influencing population dynamics. In addition to maternal biochemical cues, prenatal sound is emerging as a potent alternative source of information to direct embryonic development. Recent evidence suggests that prenatal acoustic signals can program individual phenotypes for predicted postnatal environmental conditions, which improves fitness. Across taxonomic groups, embryos have now been shown to have immediate adaptive responses to external sounds and vibrations, and direct developmental effects of sound and noise are increasingly found. Establishing the full developmental, ecological, and evolutionary impact of early soundscapes will reveal how embryos interact with the external world, and potentially transform our understanding of developmental plasticity and adaptation to changing environments.

Acute social isolation alters neurogenomic state in songbird forebrain.

Prolonged social isolation has negative effects on brain and behavior in humans and other social organisms, but neural mechanisms leading to these effects are not understood. Here we tested the hypothesis that even brief periods of social isolation can alter gene expression and DNA methylation in higher cognitive centers of the brain, focusing on the auditory/associative forebrain of the highly social zebra finch. Using RNA sequencing, we first identified genes that individually increase or decrease expression after isolation and observed general repression of gene sets annotated for neurotrophin pathways and axonal guidance functions. We then pursued 4 genes of large effect size: EGR1 and BDNF (decreased by isolation) and FKBP5 and UTS2B (increased). By in situ hybridization, each gene responded in different cell subsets, arguing against a single cellular mechanism. To test whether effects were specific to the social component of the isolation experience, we compared gene expression in birds isolated either alone or with a single familiar partner. Partner inclusion ameliorated the effect of solo isolation on EGR1 and BDNF, but not on FKBP5 and UTS2B nor on circulating corticosterone. By bisulfite sequencing analysis of auditory forebrain DNA, isolation caused changes in methylation of a subset of differentially expressed genes, including BDNF. Thus, social isolation has rapid consequences on gene activity in a higher integrative center of the brain, triggering epigenetic mechanisms that may influence processing of ongoing experience.

The role of the genome in experience-dependent plasticity: Extending the analogy of the genomic action potential.

Our past experiences shape our current and future behavior. These experiences must leave some enduring imprint on our brains, altering neural circuits that mediate behavior and contributing to our individual differences. As a framework for understanding how experiences might produce lasting changes in neural circuits, Clayton [D. F. Clayton, Neurobiol. Learn. Mem. 74, 185-216 (2000)] introduced the concept of the genomic action potential (gAP)-a structured genomic response in the brain to acute experience. Similar to the familiar electrophysiological action potential (eAP), the gAP also provides a means for integrating afferent patterns of activity but on a slower timescale and with longer-lasting effects. We revisit this concept in light of contemporary work on experience-dependent modification of neural circuits. We review the "Immediate Early Gene" (IEG) response, the starting point for understanding the gAP. We discuss evidence for its involvement in the encoding of experience to long-term memory across time and biological levels of organization ranging from individual cells to cell ensembles and whole organisms. We explore distinctions between memory encoding and homeostatic functions and consider the potential for perpetuation of the imprint of experience through epigenetic mechanisms. We describe a specific example of a gAP in humans linked to individual differences in the response to stress. Finally, we identify key objectives and new tools for continuing research in this area.

The variability of song variability in zebra finch (Taeniopygia guttata) populations.

Birdsong is a classic example of a learned social behaviour. Song behaviour is also influenced by genetic factors, and understanding the relative contributions of genetic and environmental influences remains a major goal. In this study, we take advantage of captive zebra finch populations to examine variation in a population-level song trait: song variability. Song variability is of particular interest in the context of individual recognition and in terms of the neuro-developmental mechanisms that generate song novelty. We find that the Australian zebra finch Taeniopygia guttata castanotis (TGC) maintains higher song diversity than the Timor zebra finch T. g. guttata (TGG) even after experimentally controlling for early life song exposure, suggesting a genetic basis to this trait. Although wild-derived TGC were intermediate in song variability between domesticated TGC populations and TGG, the difference between domesticated and wild TGC was not statistically significant. The observed variation in song behaviour among zebra finch populations represents a largely untapped opportunity for exploring the mechanisms of social behaviour.

Urotensin-related gene transcripts mark developmental emergence of the male forebrain vocal control system in songbirds.

Songbirds communicate through learned vocalizations, using a forebrain circuit with convergent similarity to vocal-control circuitry in humans. This circuit is incomplete in female zebra finches, hence only males sing. We show that the UTS2B gene, encoding Urotensin-Related Peptide (URP), is uniquely expressed in a key pre-motor vocal nucleus (HVC), and specifically marks the neurons that form a male-specific projection that encodes timing features of learned song. UTS2B-expressing cells appear early in males, prior to projection formation, but are not observed in the female nucleus. We find no expression evidence for canonical receptors within the vocal circuit, suggesting either signalling to other brain regions via diffusion or transduction through other receptor systems. Urotensins have not previously been implicated in vocal control, but we find an annotation in Allen Human Brain Atlas of increased UTS2B expression within portions of human inferior frontal cortex implicated in human speech and singing. Thus UTS2B (URP) is a novel neural marker that may have conserved functions for vocal communication.

Brain transcriptome sequencing and assembly of three songbird model systems for the study of social behavior.

Emberizid sparrows (emberizidae) have played a prominent role in the study of avian vocal communication and social behavior. We present here brain transcriptomes for three emberizid model systems, song sparrow Melospiza melodia, white-throated sparrow Zonotrichia albicollis, and Gambel's white-crowned sparrow Zonotrichia leucophrys gambelii. Each of the assemblies covered fully or in part, over 89% of the previously annotated protein coding genes in the zebra finch Taeniopygia guttata, with 16,846, 15,805, and 16,646 unique BLAST hits in song, white-throated and white-crowned sparrows, respectively. As in previous studies, we find tissue of origin (auditory forebrain versus hypothalamus and whole brain) as an important determinant of overall expression profile. We also demonstrate the successful isolation of RNA and RNA-sequencing from post-mortem samples from building strikes and suggest that such an approach could be useful when traditional sampling opportunities are limited. These transcriptomes will be an important resource for the study of social behavior in birds and for data driven annotation of forthcoming whole genome sequences for these and other bird species.

Advancing avian behavioral neuroendocrinology through genomics.

Genome technologies are transforming all areas of biology, including the study of hormones, brain and behavior. Annotated reference genome assemblies are rapidly being produced for many avian species. Here we briefly review the basic concepts and tools used in genomics. We then consider how these are informing the study of avian behavioral neuroendocrinology, focusing in particular on lessons from the study of songbirds. We discuss the impact of having a complete "parts list" for an organism; the transformational potential of studying large sets of genes at once instead one gene at a time; the growing recognition that environmental and behavioral signals trigger massive shifts in gene expression in the brain; and the prospects for using comparative genomics to uncover the genetic roots of behavioral variation. Throughout, we identify promising new directions for bolstering the application of genomic information to further advance the study of avian brain and behavior.

Functional genomic analysis and neuroanatomical localization of miR-2954, a song-responsive sex-linked microRNA in the zebra finch.

Natural experience can cause complex changes in gene expression in brain centers for cognition and perception, but the mechanisms that link perceptual experience and neurogenomic regulation are not understood. MicroRNAs (miRNAs or miRs) have the potential to regulate large gene expression networks, and a previous study showed that a natural perceptual stimulus (hearing the sound of birdsong in zebra finches) triggers rapid changes in expression of several miRs in the auditory forebrain. Here we evaluate the functional potential of one of these, miR-2954, which has been found so far only in birds and is encoded on the Z sex chromosome. Using fluorescence in situ hybridization and immunohistochemistry, we show that miR-2954 is present in subsets of cells in the sexually dimorphic brain regions involved in song production and perception, with notable enrichment in cell nuclei. We then probe its regulatory function by inhibiting its expression in a zebra finch cell line (G266) and measuring effects on endogenous gene expression using Illumina RNA sequencing (RNA-seq). Approximately 1000 different mRNAs change in expression by 1.5-fold or more (adjusted p < 0.01), with increases in some but not all of the targets that had been predicted by Targetscan. The population of RNAs that increase after miR-2954 inhibition is notably enriched for ones involved in the MAP Kinase (MAPK) pathway, whereas the decreasing population is dominated by genes involved in ribosomes and mitochondrial function. Since song stimulation itself triggers a decrease in miR-2954 expression followed by a delayed decrease in genes encoding ribosomal and mitochondrial functions, we suggest that miR-2954 may mediate some of the neurogenomic effects of song habituation.

Brain transcriptome of the violet-eared waxbill Uraeginthus granatina and recent evolution in the songbird genome.

Songbirds are important models for the study of social behaviour and communication. To complement the recent genome sequencing of the domesticated zebra finch, we sequenced the brain transcriptome of a closely related songbird species, the violet-eared waxbill (Uraeginthus granatina). Both the zebra finch and violet-eared waxbill are members of the family Estrildidae, but differ markedly in their social behaviour. Using Roche 454 RNA sequencing, we generated an assembly and annotation of 11 084 waxbill orthologues of 17 475 zebra finch genes (64%), with an average transcript length of 1555 bp. We also identified 5985 single nucleotide polymorphisms (SNPs) of potential utility for future population genomic studies. Comparing the two species, we found evidence for rapid protein evolution (ω) and low polymorphism of the avian Z sex chromosome, consistent with prior studies of more divergent avian species. An intriguing outlier was putative chromosome 4A, which showed a high density of SNPs and low evolutionary rate relative to other chromosomes. Genome-wide ω was identical in zebra finch and violet-eared waxbill lineages, suggesting a similar demographic history with efficient purifying natural selection. Further comparisons of these and other estrildid finches may provide insights into the evolutionary neurogenomics of social behaviour.

The genomics of memory and learning in songbirds.

Songbirds have unique value as a model for memory and learning. In their natural social life, they communicate through vocalizations that they must learn to produce and recognize. Song communication elicits abrupt changes in gene expression in regions of the forebrain responsible for song perception and production--what is the functional significance of this genomic response? For 20 years, the focus of research was on just a few genes [primarily ZENK, now known as egr1 (early gene response 1)]. Recently, however, DNA microarrays have been developed and applied to songbird behavioral research, and in 2010 the initial draft assembly of the zebra finch genome was published. Together, these new data reveal that the genomic involvement in song processing is far more complex than anticipated. The concepts of neurogenomic computation and biological embedding are introduced as frameworks for future research.

Noninvasive diffusive optical imaging of the auditory response to birdsong in the zebra finch.

Songbirds communicate by learned vocalizations with concomitant changes in neurophysiological and genomic activities in discrete parts of the brain. Here, we tested a novel implementation of diffusive optical imaging (also known as diffuse optical imaging, DOI) for monitoring brain physiology associated with vocal signal perception. DOI noninvasively measures brain activity using red and near-infrared light delivered through optic fibers (optodes) resting on the scalp. DOI does not harm subjects, so it raises the possibility of repeatedly measuring brain activity and the effects of accumulated experience in the same subject over an entire life span, all while leaving tissue intact for further study. We developed a custom-made apparatus for interfacing optodes to the zebra finch (Taeniopygia guttata) head using 3D modeling software and rapid prototyping technology, and applied it to record responses to presentations of birdsong in isoflurane-anesthetized zebra finches. We discovered a subtle but significant difference between the hemoglobin spectra of zebra finches and mammals which has a major impact in how hemodynamic responses are interpreted in the zebra finch. Our measured responses to birdsong playback were robust, highly repeatable, and readily observed in single trials. Responses were complex in shape and closely paralleled responses described in mammals. They were localized to the caudal medial portion of the brain, consistent with response localization from prior gene expression, electrophysiological, and functional magnetic resonance imaging studies. These results define an approach for collecting neurophysiological data from songbirds that should be applicable to diverse species and adaptable for studies in awake behaving animals.

High throughput analysis reveals dissociable gene expression profiles in two independent neural systems involved in the regulation of social behavior.

BACKGROUND: Production of contextually appropriate social behaviors involves integrated activity across many brain regions. Many songbird species produce complex vocalizations called 'songs' that serve to attract potential mates, defend territories, and/or maintain flock cohesion. There are a series of discrete interconnect brain regions that are essential for the successful production of song. The probability and intensity of singing behavior is influenced by the reproductive state. The objectives of this study were to examine the broad changes in gene expression in brain regions that control song production with a brain region that governs the reproductive state. RESULTS: We show using microarray cDNA analysis that two discrete brain systems that are both involved in governing singing behavior show markedly different gene expression profiles. We found that cortical and basal ganglia-like brain regions that control the socio-motor production of song in birds exhibit a categorical switch in gene expression that was dependent on their reproductive state. This pattern is in stark contrast to the pattern of expression observed in a hypothalamic brain region that governs the neuroendocrine control of reproduction. Subsequent gene ontology analysis revealed marked variation in the functional categories of active genes dependent on reproductive state and anatomical localization. HVC, one cortical-like structure, displayed significant gene expression changes associated with microtubule and neurofilament cytoskeleton organization, MAP kinase activity, and steroid hormone receptor complex activity. The transitions observed in the preoptic area, a nucleus that governs the motivation to engage in singing, exhibited variation in functional categories that included thyroid hormone receptor activity, epigenetic and angiogenetic processes. CONCLUSIONS: These findings highlight the importance of considering the temporal patterns of gene expression across several brain regions when engaging in social behaviors.

Impact of experience-dependent and -independent factors on gene expression in songbird brain.

Songbirds provide rich natural models for studying the relationships between brain anatomy, behavior, environmental signals, and gene expression. Under the Songbird Neurogenomics Initiative, investigators from 11 laboratories collected brain samples from six species of songbird under a range of experimental conditions, and 488 of these samples were analyzed systematically for gene expression by microarray. ANOVA was used to test 32 planned contrasts in the data, revealing the relative impact of different factors. The brain region from which tissue was taken had the greatest influence on gene expression profile, affecting the majority of signals measured by 18,848 cDNA spots on the microarray. Social and environmental manipulations had a highly variable impact, interpreted here as a manifestation of paradoxical "constitutive plasticity" (fewer inducible genes) during periods of enhanced behavioral responsiveness. Several specific genes were identified that may be important in the evolution of linkages between environmental signals and behavior. The data were also analyzed using weighted gene coexpression network analysis, followed by gene ontology analysis. This revealed modules of coexpressed genes that are also enriched for specific functional annotations, such as "ribosome" (expressed more highly in juvenile brain) and "dopamine metabolic process" (expressed more highly in striatal song control nucleus area X). These results underscore the complexity of influences on neural gene expression and provide a resource for studying how these influences are integrated during natural experience.

RNA-seq transcriptome analysis of male and female zebra finch cell lines.

The derivation of stably cultured cell lines has been critical to the advance of molecular biology. We profiled gene expression in the first two generally available cell lines derived from the zebra finch. Using Illumina RNA-seq, we generated ~93 million reads and mapped the majority to the recently assembled zebra finch genome. Expression of most Ensembl-annotated genes was detected, but over half of the mapped reads aligned outside annotated genes. The male-derived G266 line expressed Z-linked genes at a higher level than did the female-derived ZFTMA line, indicating persistence in culture of the distinctive lack of avian sex chromosome dosage compensation. Although these cell lines were not derived from neural tissue, many neurobiologically relevant genes were expressed, although typically at lower levels than in a reference sample from auditory forebrain. These cell lines recapitulate fundamental songbird biology and will be useful for future studies of songbird gene regulation and function.

Seasonal changes in patterns of gene expression in avian song control brain regions.

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 altering the hormonal and photoperiodic conditions in seasonally-breeding Gambel's white-crowned sparrows 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. We found that 363 genes varied by more than 1.5 fold (>log(2) 0.585) in expression in HVC and/or RA. Supporting our hypothesis, only 59 of these 363 genes were found to vary in both nuclei, while 132 gene expression changes were HVC specific and 172 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.

Song exposure regulates known and novel microRNAs in the zebra finch auditory forebrain.

BACKGROUND: In an important model for neuroscience, songbirds learn to discriminate songs they hear during tape-recorded playbacks, as demonstrated by song-specific habituation of both behavioral and neurogenomic responses in the auditory forebrain. We hypothesized that microRNAs (miRNAs or miRs) may participate in the changing pattern of gene expression induced by song exposure. To test this, we used massively parallel Illumina sequencing to analyse small RNAs from auditory forebrain of adult zebra finches exposed to tape-recorded birdsong or silence. RESULTS: In the auditory forebrain, we identified 121 known miRNAs conserved in other vertebrates. We also identified 34 novel miRNAs that do not align to human or chicken genomes. Five conserved miRNAs showed significant and consistent changes in copy number after song exposure across three biological replications of the song-silence comparison, with two increasing (tgu-miR-25, tgu-miR-192) and three decreasing (tgu-miR-92, tgu-miR-124, tgu-miR-129-5p). We also detected a locus on the Z sex chromosome that produces three different novel miRNAs, with supporting evidence from Northern blot and TaqMan qPCR assays for differential expression in males and females and in response to song playbacks. One of these, tgu-miR-2954-3p, is predicted (by TargetScan) to regulate eight song-responsive mRNAs that all have functions in cellular proliferation and neuronal differentiation. CONCLUSIONS: The experience of hearing another bird singing alters the profile of miRNAs in the auditory forebrain of zebra finches. The response involves both known conserved miRNAs and novel miRNAs described so far only in the zebra finch, including a novel sex-linked, song-responsive miRNA. These results indicate that miRNAs are likely to contribute to the unique behavioural biology of learned song communication in songbirds.