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High-quality and complete reference genome assemblies are fundamental for the application of genomics to biology, disease, and biodiversity conservation. However, such assemblies are available for only a few non-microbial species1–4. To address this issue, the international Genome 10K (G10K) consortium5,6has worked over a five-year period to evaluate and develop cost-effective methods for assembling highly accurate and nearly complete reference genomes. Here we present lessons learned from generating assemblies for 16 species that represent six major vertebrate lineages. We confirm that long-read sequencing technologies are essential for maximizing genome quality, and that unresolved complex repeats and haplotype heterozygosity are major sources of assembly error when not handled correctly. Our assemblies correct substantial errors, add missing sequence in some of the best historical reference genomes, and reveal biological discoveries. These include the identification of many false gene duplications, increases in gene sizes, chromosome rearrangements that are specific to lineages, a repeated independent chromosome breakpoint in bat genomes, and a canonical GC-rich pattern in protein-coding genes and their regulatory regions. Adopting these lessons, we have embarked on the Vertebrate Genomes Project (VGP), an international effort to generate high-quality, complete reference genomes for all of the roughly 70,000 extant vertebrate species and to help to enable a new era of discovery across the life sciences.

Zebra finches respond to certain auditory stimuli with the activation of the immediate early gene zenk . It has been shown that the amount of sound-mediated zenk gene expression varies in the zebra finch caudomedial neostriatum (NCM), apparently correlated with stimulus type (conspecific>heterospecific>noise>tones) and familiarity. Here we tested the impact of two additional factors--song-specific acoustical properties and testing conditions--on the specificity of the sound-mediated zenk response, as assessed by in situ hybridization. A variant of a normal conspecific song was first produced by randomizing the spectral content while retaining the amplitude envelope ('song-enveloped noise'). This stimulus and related controls were presented to birds which were either free in cages or restrained in a stereotaxic instrument, after isolation either overnight or for only 1 h prior to testing. We confirmed prior results that unrestrained birds show a greater zenk response to normal conspecific song than to other acoustic stimuli. However, under restraint, birds showed little or no selectivity for conspecific song compared to matched stimuli lacking a song organization. Thus the specificity of the zenk response to song is not determined simply by the acoustic structure and familiarity of the stimulus. We conclude that the intrinsic selectivity of sensory responses measured in the CNS may be influenced by factors associated with attention, arousal or vigilance, and may be significantly altered by experimental conditions that involve physical restraint.

Detergent-stable multimers of α-synuclein have been found specifically in the brains of patients with Parkinson’s disease and other neurodegenerative diseases. Here we show that recombinant α-synuclein forms multimers in vitroupon exposure to vesicles containing certain polyunsaturated fatty acid (PUFA) acyl groups, including arachidonoyl and docosahexaenoyl. This process occurs at physiological concentrations and much faster than in aqueous solution. PUFA-induced aggregation involves physical association with the vesicle surface via the large apolipoprotein-like lipid-binding domain that constitutes the majority of the protein. β- and γ-synucleins, as well as the Parkinson’s disease-associated α-synuclein variants A30P and A53T, show similar tendencies to multimerize in the presence of PUFAs. Multimerization does not require the presence of any tyrosine residues in the sequence. The membrane-based interaction of the synucleins with specific long chain polyunsaturated phospholipids may be relevant to the protein family’s physiological functions and may also contribute to the aggregation of α-synuclein observed in neurodegenerative disease.

α-Synuclein has been centrally implicated in neurodegenerative disease, and a normal function in developmental synaptic plasticity has been suggested by studies in songbirds. A variety of observations suggest the protein partitions between membrane and cytosol, a behavior apparently conferred by a conserved structural similarity to the exchangeable apolipoproteins. Here we show that the capacity to bind lipids is broadly distributed across exons 3, 4, and 5 (encoding residues 1–102). Binding to phosphatidylserine-containing vesicles requires the presence of all three exons, while binding to phosphatidic acid can be mediated by any one of the three. Consistent with a “class A2” helical binding mechanism, lipid association is disrupted by introduction of charged residues along the hydrophobic face of the predicted α-helix and also by biotinylation of conserved lysines (which line the interfacial region). Circular dichroism spectroscopy reveals a general correlation between the amount of lipid-induced α-helix content and the degree of binding to PS-containing vesicles. Two point mutations associated with Parkinson's disease have little (A30P) or no (A53T) effect on lipid binding or α-helicity. These results are consistent with the hypothesis that α-synuclein's normal functions depend on an ability to undergo a large conformational change in the presence of specific phospholipids.

We show that a single presentation of a zebra finch song, 2 s in duration, will induce an “immediate-early gene” response in the caudomedial neostriatum of zebra finches (Poephila guttata). Repetition of this stimulus 10 times is sufficient to induce a maximal increase in RNA and protein, detected 30 and 90 min later respectively. Thus very brief stimuli can set in motion a slow genomic process in the brain which takes hours to resolve. Immediate-early gene function is often considered in the context of a “feedback” model (i.e., to consolidate memories of the inducing event). However, based on the long lag observed here between initiation and full expression of the molecular response, we suggest an alternative, ethologically based, “feed-forward” model in which exposure to a novel or significant context triggers an increase in the efficiency of memory capture processes for subsequent experiences.

Neurons compute in part by integrating, on a time scale of milliseconds, many synaptic inputs and generating a digital output—the “action potential” of classic electrophysiology. Recent discoveries indicate that neurons also perform a second, much slower, integration operating on a time scale of minutes or even hours. The output of this slower integration involves a pulse of gene expression which may be likened to the electrophysiological action potential. Its function, however, is not directed toward immediate transmission of a synaptic signal but rather toward the experience-dependent modification of the underlying synaptic circuitry. Commonly termed the “immediate early gene” (IEG) response, this phenomenon is often assumed to be a necessary component of a linear, deterministic cascade of memory consolidation. Critical review of the large literature describing the phenomenon, however, leads to an alternative model of IEG function in the brain. In this alternative, IEG activation is not directed at the consolidation of memories of a specific inducing event; instead, it sets the overall gain or efficiency of memory formation and directs it to circuits engaged by behaviorally significant contexts. The net result is a sharpening of the selectivity of memory formation, a recruitment of temporally correlated associations, and an ultimate enhancement of long-term memory retrieval.

The songbird has emerged as an important model for study of brain-behavior relationships by virtue of its rich natural advantages and from the pioneering efforts of explorers using anatomical and behavioral approaches. Now, molecular biology is providing a new and complementary paradigm for discerning songbird brain organization and function. Here, I review the work over the last 10 years that has laid the foundation for approaching songbird biology from the molecular perspective. As a result of this work, specific hypotheses can now be framed and tested regarding the mechanisms behind song circuit formation, behavioral plasticity, and the boundaries of adaptability. Age-related changes in more than 15 molecules have been observed in the song system of juvenile zebra finches, and these changes seem to define specific phases in circuit development. In adult songbirds, ordinary song-related activities such as singing and listening cause dramatic increases in gene expression in brain areas specific to each activity. The sensitivity of gene activation is modulated as a result of experience in adulthood and also changes during juvenile song learning. These studies have provided unexpected insights into the functional organization of the song circuit and the potential role of extrinsic modulatory systems in directing and limiting plastic change in the brain. With this rich base of knowledge, and techniques of gene manipulation on the horizon, answers to old questions seem within our reach: What sets the boundaries of neural plasticity? What limits learning? © 1997 John Wiley & Sons, Inc. J Neurobiol 33: 549–571, 1997

Male zebra finches (Taeniopygia guttata) learn to sing during a critical period in adolescence. We previously described a presynaptic protein, synelfin, whose mRNA is increased early in this critical period in a brain nucleus specifically implicated in song learning, lateral MAN (lMAN). In the current study, in situ hybridization was used to map this change in gene expression to the subregion of lMAN that projects to the robust nucleus of the archistriatum (RA), the principal motor output of the telencephalic circuit that controls song production. Using confocal immunofluorescence microscopy, we detected numerous puncta of synelfin immunoreactivity that apparently represent presynaptic terminals from lMAN in the RA of young males. Synelfin immunoreactivity in RA declined abruptly between 40 and 45 days of age, a time of major synaptic reorganization in RA. This change did not occur until about 10 days after the decline in synelfin mRNA in cell bodies within lMAN, indicating a relatively slow turnover of the protein in presynaptic terminals and suggesting that some of the functional changes that occur during the critical period may arise from regulatory decisions that were initiated a week or more earlier. Depriving birds of tutoring did not halt or delay the decline of synelfin mRNA in lMAN. This change in gene expression must not be a consequence of early song learning, but may reflect an innate or programmed step in song circuit development.

Canaries and other songbirds offer unique advantages for analyzing the relationship between specific gene regulation and neural plasticity. To establish a quantitative profile of the population of RNAs potentially involved in this regulation, we analyzed the solution hybridization kinetics of canary forebrain cytoplasmic polyadenylated RNA. Hybridization of forebrain cDNA to forebrain RNA provides evidence for RNA species at individual concentrations ranging from less than 10−6to about 10−3(by fractional mass). Cross-hybridization to RNA from the rest of the brain, together with other studies, defines a subpopulation of about 5000 rare RNAs that are enriched in the forebrain compared to the rest of the brain. Some of these forebrain-enriched RNAs are likely to play a role in regulating the neural plasticity characteristic of the canary forebrain.

The article talks about research on genetic variation and the holistic study of organisms. In an article in the June 2013 issue of the BioScience journal, the authors propose a renewed emphasis on the study of individual organisms taking into account the environmental exposures they are subjected to on top of their genetic variations. This is in consideration of the gene focused biological research which includes cellular and molecular biology and quantitative genetics.

A novel and highly conserved presynaptic protein has been independently described in rodents (synuclein/SYN-1), songbirds (synelfin), and humans (the precursor protein of the non-Ap component of senile plaques, NACP); a fragment of the latter has been detected in senile plaques in Alzheimer's disease (AD). We characterized the expression of NACP in human AD and non-AD brain. A subcellular fractionation study demonstrated that NACP was mainly localized to cytosolic fractions of human temporal cortex. NACP was also detectable in various membrane and vesicular fractions, suggesting that the protein was associated with membrane structures including synaptic vesicles. Pericellular immunostaining of the neuropil was observed in neocortical and limbic regions, supporting a synaptic localization. Senile plaques in AD brains were not immunoreactive, and confocal microscopy suggested a loss of NACP immunoreactivity in cored plaques. No difference was found in the amount of protein in AD and control frontal cortex, as measured by immunoblotting. PCR analysis showed that the full-length mRNA product was the major splice form in both AD and control human brains. Thus, despite the association of a hydrophobic fragment of NACP with senile plaques, our data suggest that the precursor itself is not a significant component of plaques and NACP synthesis is not substantially altered in AD. Nevertheless, the protein is an abundant component of synaptic regions prone to degeneration in AD, and may have a role in the expression or advancement of the disease.

A mutation in the α-synuclein gene has recently been linked to some cases of familial Parkinson's disease (PD). We characterized the expression of this presynaptic protein in the midbrain, striatum, and temporal cortex of control, PD, and dementia with Lewy bodies (DLB) brain. Control brain showed punctate pericellular immunostaining. PD brain demonstrated α-synuclein immunoreactivity in nigral Lewy bodies, pale bodies and abnormal neurites. Rare neuronal soma in PD brain were immunoreactive for α-synuclein. DLB cases demonstrated these findings as well as α-synuclein immunoreactivity in cortical Lewy bodies and CA2-3 neurites. These results suggest that, even in sporadic cases, there is an early and direct role for α-synuclein in the pathogenesis of PD and the neuropathologically related disorder DLB.