Learning and memory formation are known to require dynamic CpG (de)methylation and gene expression changes. Here, we aimed at establishing a genome-wide DNA methylation map of the zebra finch genome, a model organism in neuroscience, as well as identifying putatively epigenetically regulated genes. RNA- and MethylCap-seq experiments were performed on two zebra finch cell lines in presence or absence of 5-aza-2′-deoxycytidine induced demethylation. First, the MethylCap-seq methodology was validated in zebra finch by comparison with RRBS-generated data. To assess the influence of (variable) methylation on gene expression, RNA-seq experiments were performed as well. Comparison of RNA-seq and MethylCap-seq results showed that at least 357 of the 3,457 AZA-upregulated genes are putatively regulated by methylation in the promoter region, for which a pathway analysis showed remarkable enrichment for neurological networks. A subset of genes was validated using Exon Arrays, quantitative RT-PCR and CpG pyrosequencing on bisulfite-treated samples. To our knowledge, this study provides the first genome-wide DNA methylation map of the zebra finch genome as well as a comprehensive set of genes of which transcription is under putative methylation control.

Birdsong is a precisely timed animal behavior. The connectivity of song premotor neural networks has been proposed to underlie the temporal patterns of neuronal activity that control vo-cal muscle movements during singing. Although the connectivity of premotor nuclei via chemical synapses has been characterized, electrical synapses and their molecular identity remain unex-plored. We show with in situ hybridizations that GJD2 mRNA, coding for the major channel-form-ing electrical synapse protein in mammals, connexin 36, is expressed in the two nuclei that control song production, HVC and RA from canaries and zebra finches. In canaries’ HVC, GJD2 mRNA is extensively expressed in GABAergic and only a fraction of glutamatergic cells. By contrast, in RA, GJD2 mRNA expression is widespread in glutamatergic and GABAergic neurons. Remarkably, GJD2 expression is similar in song nuclei and their respective embedding brain regions, revealing the widespread expression of GJD2 in the avian brain. Inspection of a single-cell sequencing data-base from zebra and Bengalese finches generalizes the distributions of electrical synapses across cell types and song nuclei that we found in HVC and RA from canaries, reveals a differential GJD2 mRNA expression in HVC glutamatergic subtypes and its transient increase along the neurogenic lineage. We propose that songbirds are a suitable model to investigate the contribution of electrical synapses to motor skill learning and production.

Scientific Reports 6: Article number: 20957; published online: 11 February 2016; updated: 17 March 2016 The original version of this Article contained an error in the title of the paper, where the word “epigenetically” was incorrectly given as “eigenetically”. This has now been corrected in the PDF and HTML versions of the Article.

Like other canary reproductive behaviors, song production occurs seasonally and can be triggered by gonadal hormones. Adult female canaries treated with testosterone sing first songs after four days and progressively develop towards typical canary song structure over several weeks, a behavior that females otherwise rarely or never show. We compared gene regulatory networks in the song-controlling brain area HVC after 1 hour (h), 3 h, 8 h, 3 days (d), 7d, and 14d testosterone treatment with placebo-treated control females, paralleling HVC and song development. Rapid onset (1 h or less) of extensive transcriptional changes (2,700 genes) preceded the onset of song production by four days. The highest level of differential gene expression occurred at 14 days when song structure was most elaborate, and song activity was highest. The transcriptomes changed massively several times during the two-week of song production. A total of 9,710 genes were differentially expressed, corresponding to about 60% of the known protein-coding genes of the canary genome. Most (99%) of the differentially expressed genes were regulated only at specific stages. The differentially expressed genes were associated with diverse biological functions, of which cellular level occurring early and nervous system level occurring primarily after prolonged testosterone treatment. Thus, the development of adult songs requires restructuring the entire HVC, including most HVC cell types, rather than altering only neuronal subpopulations or cellular components. Parallel regulation directly by androgen and estrogen receptors and by other hub genes such as the transcription factor SP8, which are under steroidogenic control, lead to massive transcriptomic and neural changes in the specific behavior-controlling brain areas and gradual seasonal occurrence of singing behavior.