The Songbird Neurogenomics (SoNG) Initiative: Community-based tools and strategies for study of brain gene function and evolution

Cell & Developmental Biology, Univ, of Illinois, Urbana, IL, USA.
BMC Genomics (Impact Factor: 3.99). 02/2008; 9(1):131. DOI: 10.1186/1471-2164-9-131
Source: PubMed


Songbirds hold great promise for biomedical, environmental and evolutionary research. A complete draft sequence of the zebra finch genome is imminent, yet a need remains for application of genomic resources within a research community traditionally focused on ethology and neurobiological methods. In response, we developed a core set of genomic tools and a novel collaborative strategy to probe gene expression in diverse songbird species and natural contexts.
We end-sequenced cDNAs from zebra finch brain and incorporated additional sequences from community sources into a database of 86,784 high quality reads. These assembled into 31,658 non-redundant contigs and singletons, which we annotated via BLAST search of chicken and human databases. The results are publicly available in the ESTIMA:Songbird database. We produced a spotted cDNA microarray with 20,160 addresses representing 17,214 non-redundant products of an estimated 11,500-15,000 genes, validating it by analysis of immediate-early gene (zenk) gene activation following song exposure and by demonstrating effective cross hybridization to genomic DNAs of other songbird species in the Passerida Parvorder. Our assembly was also used in the design of the "Lund-zfa" Affymetrix array representing approximately 22,000 non-redundant sequences. When the two arrays were hybridized to cDNAs from the same set of male and female zebra finch brain samples, both arrays detected a common set of regulated transcripts with a Pearson correlation coefficient of 0.895. To stimulate use of these resources by the songbird research community and to maintain consistent technical standards, we devised a "Community Collaboration" mechanism whereby individual birdsong researchers develop experiments and provide tissues, but a single individual in the community is responsible for all RNA extractions, labelling and microarray hybridizations.
Immediately, these results set the foundation for a coordinated set of 25 planned experiments by 16 research groups probing fundamental links between genome, brain, evolution and behavior in songbirds. Energetic application of genomic resources to research using songbirds should help illuminate how complex neural and behavioral traits emerge and evolve.

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Available from: David Clayton
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    • ", a zebra finch Taeniopygia guttata EST microsatellite sequence DV945670 (Replogle et al. 2008) was identified with strong homology to the chicken Gallus gallus Z chromosome. We created a consensus sequence from these homologous sequences using MEGA3 (Kumar et al. 2004) and designed a primer set using PRIMER3 v0.4.0 (Rozen and Skaletsky 2000). "
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    ABSTRACT: A new primer set was developed for sex-typing birds, Z37B. This primer set was designed to amplify al-leles of small size to render it suitable for sex-typing degraded samples, including shed feathers. This marker successfully sex-typed 50 % of the species tested, including passerines, shorebirds, rails, seabirds, eagles and the brown kiwi Apteryx australis (allele size range =81–103 bp), and is therefore expected to be suitable for sex-typing a wide range of species. Z37B sex-typed non-degraded samples (blood), degraded tissue (dead unhatched embryos, dead nestlings and museum specimens) and samples of low quantity DNA (plucked feathers and buccal swabs). The small amplicon sizes in birds suggest that this marker will be of utility for sex-typing feathers, swabs and degraded samples from a wide range of avian species.
    Full-text · Article · Feb 2015 · Conservation Genetics Resources
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    • "Zebra finches are songbirds that communicate using learned vocalizations (Immelmann, 1969; Miller, 1979; Clayton, 1988), and have become important model organisms for studying the neural and genomic mechanisms of social learning, memory, and sex-linked behavior (Replogle et al., 2008; Robinson et al., 2008; Clayton et al., 2009; Clayton, 2013). Both the act of singing, and the experience of hearing other birds sing, can elicit complex changes in gene expression in discrete regions of the higher forebrain (reviewed in Clayton, 2013). "
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    ABSTRACT: 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.
    Full-text · Article · Dec 2014 · Frontiers in Neuroscience
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    • "However, the zebra finch samples clustered closely with the song sparrow samples taken from the same brain region (auditory forebrain), with the white throated sparrow samples from the whole brain clustering together as most divergent, and the Gambel’s white-crowned sparrow samples from hypothalamus in between (Figs. 3C and Fig. 4). This echoes previous findings that brain region is a major determinant of gene expression pattern in songbirds (Replogle et al., 2008; Drnevich et al., 2012). Both euclidean distance-based clustering and PCA also highlight the fact that zebra finches, which were sacrificed in captivity and sequenced in pools of ten, had much reduced variance in expression profile relative to our non-pooled, field-collected white-throated sparrow and song sparrow samples (Fig. 4). "
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    ABSTRACT: 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.
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