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

BACKGROUND: 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. RESULTS: 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 ~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. CONCLUSION: 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

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

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.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

Possible Brucellosis in an Early Hominin Skeleton from Sterkfontein, South Africa

We report on the paleopathological analysis of the partial skeleton of the late Pliocene hominin species Australopithecus africanus Stw 431 from Sterkfontein, South Africa. A previous study noted the presence of lesions on vertebral bodies diagnosed as spondylosis deformans due to trauma. Instead, we suggest that these lesions are pathological changes due to the initial phases of an infectious disease, brucellosis. The macroscopic, microscopic and radiological appearance of the lytic lesions of the lumbar vertebrae is consistent with brucellosis. The hypothesis of brucellosis (most often associated with the consumption of animal proteins) in a 2.4 to 2.8 million year old hominid has a host of important implications for human evolution. The consumption of meat has been regarded an important factor in supporting, directing or altering human evolution. Perhaps the earliest (up to 2.5 million years ago) paleontological evidence for meat eating consists of cut marks on animal remains and stone tools that could have made these marks. Now with the hypothesis of brucellosis in A. africanus, we may have evidence of occasional meat eating directly linked to a fossil hominin

Crystal Structure of the Minimalist Max-E47 Protein Chimera

Max-E47 is a protein chimera generated from the fusion of the DNA-binding basic region of Max and the dimerization region of E47, both members of the basic region/helix-loop-helix (bHLH) superfamily of transcription factors. Like native Max, Max-E47 binds with high affinity and specificity to the E-box site, 5′-CACGTG, both in vivo and in vitro. We have determined the crystal structure of Max-E47 at 1.7 Å resolution, and found that it associates to form a well-structured dimer even in the absence of its cognate DNA. Analytical ultracentrifugation confirms that Max-E47 is dimeric even at low micromolar concentrations, indicating that the Max-E47 dimer is stable in the absence of DNA. Circular dichroism analysis demonstrates that both non-specific DNA and the E-box site induce similar levels of helical secondary structure in Max-E47. These results suggest that Max-E47 may bind to the E-box following the two-step mechanism proposed for other bHLH proteins. In this mechanism, a rapid step where protein binds to DNA without sequence specificity is followed by a slow step where specific protein:DNA interactions are fine-tuned, leading to sequence-specific recognition. Collectively, these results show that the designed Max-E47 protein chimera behaves both structurally and functionally like its native counterparts

Metabolic changes in concussed American football players during the acute and chronic post-injury phases

<p>Abstract</p> <p>Background</p> <p>Despite negative neuroimaging findings many athletes display neurophysiological alterations and post-concussion symptoms that may be attributable to neurometabolic alterations.</p> <p>Methods</p> <p>The present study investigated the effects of sports concussion on brain metabolism using ¹H-MR Spectroscopy by comparing a group of 10 non-concussed athletes with a group of 10 concussed athletes of the same age (mean: 22.5 years) and education (mean: 16 years) within both the acute and chronic post-injury phases. All athletes were scanned 1-6 days post-concussion and again 6-months later in a 3T Siemens MRI.</p> <p>Results</p> <p>Concussed athletes demonstrated neurometabolic impairment in prefrontal and motor (M1) cortices in the acute phase where NAA:Cr levels remained depressed relative to controls. There was some recovery observed in the chronic phase where Glu:Cr levels returned to those of control athletes; however, there was a pathological increase of m-I:Cr levels in M1 that was only present in the chronic phase.</p> <p>Conclusions</p> <p>These results confirm cortical neurometabolic changes in the acute post-concussion phase as well as recovery and continued metabolic abnormalities in the chronic phase. The results indicate that complex pathophysiological processes differ depending on the post-injury phase and the neurometabolite in question.</p

An Alpha-Catulin Homologue Controls Neuromuscular Function through Localization of the Dystrophin Complex and BK Channels in Caenorhabditis elegans

The large conductance, voltage- and calcium-dependent potassium (BK) channel serves as a major negative feedback regulator of calcium-mediated physiological processes and has been implicated in muscle dysfunction and neurological disorders. In addition to membrane depolarization, activation of the BK channel requires a rise in cytosolic calcium. Localization of the BK channel near calcium channels is therefore critical for its function. In a genetic screen designed to isolate novel regulators of the Caenorhabditis elegans BK channel, SLO-1, we identified ctn-1, which encodes an α-catulin homologue with homology to the cytoskeletal proteins α-catenin and vinculin. ctn-1 mutants resemble slo-1 loss-of-function mutants, as well as mutants with a compromised dystrophin complex. We determined that CTN-1 uses two distinct mechanisms to localize SLO-1 in muscles and neurons. In muscles, CTN-1 utilizes the dystrophin complex to localize SLO-1 channels near L-type calcium channels. In neurons, CTN-1 is involved in localizing SLO-1 to a specific domain independent of the dystrophin complex. Our results demonstrate that CTN-1 ensures the localization of SLO-1 within calcium nanodomains, thereby playing a crucial role in muscles and neurons

Genomic and neural analysis of the estradiol-synthetic pathway in the zebra finch

<p>Abstract</p> <p>Background</p> <p>Steroids are small molecule hormones derived from cholesterol. Steroids affect many tissues, including the brain. In the zebra finch, estrogenic steroids are particularly interesting because they masculinize the neural circuit that controls singing and their synthesis in the brain is modulated by experience. Here, we analyzed the zebra finch genome assembly to assess the content, conservation, and organization of genes that code for components of the estrogen-synthetic pathway and steroid nuclear receptors. Based on these analyses, we also investigated neural expression of a cholesterol transport protein gene in the context of song neurobiology.</p> <p>Results</p> <p>We present sequence-based analysis of twenty steroid-related genes using the genome assembly and other resources. Generally, zebra finch genes showed high homology to genes in other species. The diversity of steroidogenic enzymes and receptors may be lower in songbirds than in mammals; we were unable to identify all known mammalian isoforms of the 3β-hydroxysteroid dehydrogenase and 17β-hydroxysteroid dehydrogenase families in the zebra finch genome assembly, and not all splice sites described in mammals were identified in the corresponding zebra finch genes. We did identify two factors, Nobox and NR1H2-RXR, that may be important for coordinated transcription of multiple steroid-related genes. We found very little qualitative overlap in predicted transcription factor binding sites in the genes for two cholesterol transport proteins, the 18 kDa cholesterol transport protein (TSPO) and steroidogenic acute regulatory protein (StAR). We therefore performed in situ hybridization for TSPO and found that its mRNA was not always detected in brain regions where StAR and steroidogenic enzymes were previously shown to be expressed. Also, transcription of TSPO, but not StAR, may be regulated by the experience of hearing song.</p> <p>Conclusions</p> <p>The genes required for estradiol synthesis and action are represented in the zebra finch genome assembly, though the complement of steroidogenic genes may be smaller in birds than in mammals. Coordinated transcription of multiple steroidogenic genes is possible, but results were inconsistent with the hypothesis that StAR and TSPO mRNAs are co-regulated. Integration of genomic and neuroanatomical analyses will continue to provide insights into the evolution and function of steroidogenesis in the songbird brain.</p

Neural expression and post-transcriptional dosage compensation of the steroid metabolic enzyme 17β-HSD type 4

<p>Abstract</p> <p>Background</p> <p>Steroids affect many tissues, including the brain. In the zebra finch, the estrogenic steroid estradiol (E₂) is especially effective at promoting growth of the neural circuit specialized for song. In this species, only the males sing and they have a much larger and more interconnected song circuit than females. Thus, it was surprising that the gene for 17β-hydroxysteroid dehydrogenase type 4 (HSD17B4), an enzyme that converts E₂to a less potent estrogen, had been mapped to the Z sex chromosome. As a consequence, it was likely that HSD17B4 was differentially expressed in males (ZZ) and females (ZW) because dosage compensation of Z chromosome genes is incomplete in birds. If a higher abundance of HSD17B4 mRNA in males than females was translated into functional enzyme in the brain, then contrary to expectation, males could produce less E₂in their brains than females.</p> <p>Results</p> <p>Here, we used molecular and biochemical techniques to confirm the HSD17B4 Z chromosome location in the zebra finch and to determine that HSD17B4 mRNA and activity were detectable in the early developing and adult brain. As expected, HSD17B4 mRNA expression levels were higher in males compared to females. This provides further evidence of the incomplete Z chromosome inactivation mechanisms in birds. We detected HSD17B4 mRNA in regions that suggested a role for this enzyme in the early organization and adult function of song nuclei. We did not, however, detect significant sex differences in HSD17B4 activity levels in the adult brain.</p> <p>Conclusions</p> <p>Our results demonstrate that the HSD17B4 gene is expressed and active in the zebra finch brain as an E₂metabolizing enzyme, but that dosage compensation of this Z-linked gene may occur via post-transcriptional mechanisms.</p