High-quality and complete reference genome assemblies are fundamental for the application of genomics to biology, disease, and biodiversity conservation. However, such assemblies are only available for a few non-microbial species 1-4 . To address this issue, the international Genome 10K (G10K) consortium 5,6 has worked over a five-year period to evaluate and develop cost-effective methods for assembling the most accurate and complete reference genomes to date. Here we summarize these developments, introduce a set of quality standards, and present lessons learned from sequencing and assembling 16 species representing major vertebrate lineages (mammals, birds, reptiles, amphibians, teleost fishes and cartilaginous fishes). 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 error in assemblies. Our new assemblies identify and correct substantial errors in some of the best historical reference genomes. Adopting these lessons, we have embarked on the Vertebrate Genomes Project (VGP), an effort to generate high-quality, complete reference genomes for all ~70,000 extant vertebrate species and help enable a new era of discovery across the life sciences.

The ZENK gene encodes a zinc-finger-containing transcriptional regulator and can be rapidly activated in songbird brain by presentation of birdsong (Mello et al., 1992). Here we map the areas of the songbird forebrain that show this genomic response to birdsong, using in situ hybridization. After 30 min of song presentation ZENK mRNA levels reach a peak in the caudomedial telencephalon, in areas adjacent to or closely related with primary auditory structures. These areas include subfields of field L (L1 and L3), the caudomedial neostriatum (NCM), the caudomedial hyperstriatum ventrale (CMHV) anterior to field L, the caudal paleostriatum, and two field L targets, HVC shelf and RA cup. In contrast, ZENK induction is absent in some areas that show a response to song by other measures and where ZENK induction might have been expected. These include the direct thalamo- recipient field L subfield L2, and the nuclei of the circuit involved in the acquisition and production of learned song. These results demonstrate that ZENK induction following song presentation occurs only in a subset of areas physiologically activated by song, and draw attention to areas previously unsuspected as related to processing of complex auditory stimuli. Based on what is known about ZENK function in mammalian systems (Christy et al., 1989; Cole et al., 1989; Wisden et al., 1990), we speculate that areas revealed by ZENK induction might correspond to sites where critical neuronal modifications occur in response to birdsong presentation, possibly leading to the formation of song-related memories.

Rapid-eye-movement (REM) sleep plays a key role in the consolidation of memories acquired during waking (WK). The search for mechanisms underlying that role has revealed significant correlations in the patterns of neuronal firing, regional blood flow, and expression of the activity-dependent gene zif-268 between WK and subsequent REM sleep. Zif-268 integrates a major calcium signal transduction pathway and is implicated by several lines of evidence in activity-dependent synaptic plasticity. Here we report that the induction of hippocampal long-term potentiation (LTP) during WK in rats leads to an upregulation of zif-268 gene expression in extrahippocampal regions during subsequent REM sleep episodes. This upregulation occurs predominantly in the amygdala, entorhinal, and auditory cerebral cortices during the first REM sleep episodes after LTP induction and reaches somatosensory and motor cerebral cortices as REM sleep recurs. We also show that hippocampal inactivation during REM sleep blocks extrahippocampal zif-268 upregulation, indicating that cortical and amygdalar zif-268 expression during REM sleep is under hippocampal control. Thus, expression of an activity-dependent gene involved in synaptic plasticity propagates gradually from the hippocampus to extrahippocampal regions as REM sleep recurs. These findings suggest that a progressive disengagement of the hippocampus and engagement of the cerebral cortex and amygdala occurs during REM sleep. They are also consistent with the view that REM sleep constitutes a privileged window for hippocampus-driven cortical activation, which may play an instructive role in the communication of memory traces from the hippocampus to the cerebral cortex.

Conspecific song induces a prompt increase in the expression of the zenk gene in the caudo-medial neostriatum (NCM), a part of the auditory telencephalon of songbirds. To test the hypothesis that zenk gene induction in NCM is related to the acquisition of new song-related memories, we presented adult male zebra finches with repeated playbacks of one song. In response, zenk mRNA levels in NCM increased for the first 30 min, but then declined back to baseline levels despite continued stimulation with the same song. When a novel song was then introduced, however, a full zenk response was triggered once again. Even when a full day had passed between the last exposure to a song and the testing period, the song that had been rendered "familiar" by repetition caused no zenk response, whereas a novel song caused a full response. Quantitative analysis indicates that individual cells in NCM must have undergone a selective loss in their zenk responsiveness to the repeated song, while still maintaining their ability to respond to the novel song. These results support the hypothesis that the induction of zenk is related to the formation of long-term memories. Analysis of the stimulus-specific modulation of zenk responses, coupled with appropriate behavioral assays, should provide insight into neural mechanisms responsible for the discrimination and storage of complex perceptual information.

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.