Extensive, transient, and long-lasting gene regulation in a song-controlling brain area during testosterone-induced song development in adult female canaries

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, the cellular level occurring early and the nervous system level occurring primarily after prolonged testosterone treatment. Thus, the development of adult songs stimulated by testosterone results in widespread transcriptional changes in the HVC, potentially affecting a majority of neuronal subpopulations rather than altering only specific cellular components. Parallel gene regulation directly by androgen and estrogen receptors, potentially amplified by co-expressed transcription factors that are themselves steroid receptor regulated, leads to substantial transcriptomic and neural changes in specific behavior-controlling brain areas, resulting in the gradual seasonal occurrence of singing behavior. ### Competing Interest Statement The authors have declared no competing interest.