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Molecular profiling of sex-specific development of song and the song control nucleus HVC of songbirds
Molecular profiling of sex-specific development of song and the song control nucleus HVC of songbirds
Singing of songbird species is a behavior that integrates multiple sensory inputs and motor outputs, which primarily rely on interconnected neural circuits in the avian brain, the song control system. The nucleus HVC is of particular interest because of its integrative roles in the song control system. In the majority of Northern temperate songbird species, males sing predominately, and they often use their songs to attract female mates. In contrast, most Northern temperate female songbirds sing either rarely or only in certain contexts. The characteristics and functions of female songs are less clear. In the tropics, many species of females sing regularly and depending on species, their song complexity is less or comparable to that of males. However, the HVC volume is greater in males than females in all songbird species that have been examined. In the first experiment of this thesis (p. 43), I described song features of spontaneously and rarely singing female canaries (Serinus canaria), a Northern temperate songbird species. I observed higher blood testosterone concentrations and greater HVC volume of singing females than that of non-singing females. The results suggest female canary singing is testosterone-dependent. Subcutaneous testosterone implantation induces singing in female canaries. In the second experiment (p. 65), I implanted female canaries with testosterone for six time periods (T1h, T3h, T8h, T3d, T7d, and T14d) and studied changes of gene expression in the HVC. I observed approximately 2,600 genes regulated by testosterone after one hour and the regulation was dynamic throughout the experimental time window. I investigated putative biological functions of testosterone-regulated genes in the six time points by gene ontology (GO)-term enrichment analysis, and showed that the enrichment of angiogenesis began at T1h and the enrichment of neurogenesis began at T3h, with both processes continuing until T14d. Furthermore, genes associated with “GABA” and “spine” were enriched in T3d birds when the birds started singing, while the number of genes associated with “nervous system development” was highest in T14d birds, when the HVC volume was significantly greater than controls. Finally, using approaches integrating gene expression, HVC volume, circulating testosterone levels, and song characteristics, I identified a potential master regulator of testosterone-regulated changes. Male canary songs vary seasonally. Breeding season songs are longer, louder, and more complex than non-breeding season songs. Non-breeding males implanted with testosterone sing songs resembling that of breeding season songs. In the third experiment (p. 81), I studied gene expression in the HVC of seven canary groups, females and males of breeding season and of non-breeding season, non-breeding season females and males treated with testosterone, and spontaneously singing female canaries. Hierarchical clustering and principal component analysis (PCA) showed that circulating testosterone levels and sex were the predominant variables associated with variation in the HVC transcriptomes. Comparison between natural singing canaries with testosterone-induced singing canaries of the same sex revealed large differences in the HVC transcriptomes. Moreover, the intersection of natural and testosterone-induced singing females shared little resemblance with males in terms of genes. GO-term enrichment analysis suggested functional overlap between sex-specific gene networks. However, although strong transcriptional changes in HVC correlate with the transition from non-singing to singing in both sexes, the type of transcriptional changes are sex-specific. In the fourth experiment (p. 89), I studied sex differences in HVC gene expression between three songbird species: the canary, the blue-capped cordon bleus (Uraeginthus cyanocephalus), and the forest weavers (Ploceus bicolor). Cordon bleu females sing regularly with female-specific songs, whereas forest weaver females sing songs identical to males. I found substantial sex differences in HVC gene expression in all three species, and sex-biased genes differed between species. Surprisingly, the majority of sex-biased genes were on autosomes instead the sex chromosome Z. These results provide further evidence for sex differences in brain structure at the molecular and cellular levels in sexually reproductive animals.
Songbirds, canary, blue-capped cordon bleu, forest weaver, HVC, song control system, microarray analysis, WGCNA, song analysis, testosterone, plasticity
Ko, Meng-Ching
2018
English
Universitätsbibliothek der Ludwig-Maximilians-Universität München
Ko, Meng-Ching (2018): Molecular profiling of sex-specific development of song and the song control nucleus HVC of songbirds. Dissertation, LMU München: Faculty of Biology
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Abstract

Singing of songbird species is a behavior that integrates multiple sensory inputs and motor outputs, which primarily rely on interconnected neural circuits in the avian brain, the song control system. The nucleus HVC is of particular interest because of its integrative roles in the song control system. In the majority of Northern temperate songbird species, males sing predominately, and they often use their songs to attract female mates. In contrast, most Northern temperate female songbirds sing either rarely or only in certain contexts. The characteristics and functions of female songs are less clear. In the tropics, many species of females sing regularly and depending on species, their song complexity is less or comparable to that of males. However, the HVC volume is greater in males than females in all songbird species that have been examined. In the first experiment of this thesis (p. 43), I described song features of spontaneously and rarely singing female canaries (Serinus canaria), a Northern temperate songbird species. I observed higher blood testosterone concentrations and greater HVC volume of singing females than that of non-singing females. The results suggest female canary singing is testosterone-dependent. Subcutaneous testosterone implantation induces singing in female canaries. In the second experiment (p. 65), I implanted female canaries with testosterone for six time periods (T1h, T3h, T8h, T3d, T7d, and T14d) and studied changes of gene expression in the HVC. I observed approximately 2,600 genes regulated by testosterone after one hour and the regulation was dynamic throughout the experimental time window. I investigated putative biological functions of testosterone-regulated genes in the six time points by gene ontology (GO)-term enrichment analysis, and showed that the enrichment of angiogenesis began at T1h and the enrichment of neurogenesis began at T3h, with both processes continuing until T14d. Furthermore, genes associated with “GABA” and “spine” were enriched in T3d birds when the birds started singing, while the number of genes associated with “nervous system development” was highest in T14d birds, when the HVC volume was significantly greater than controls. Finally, using approaches integrating gene expression, HVC volume, circulating testosterone levels, and song characteristics, I identified a potential master regulator of testosterone-regulated changes. Male canary songs vary seasonally. Breeding season songs are longer, louder, and more complex than non-breeding season songs. Non-breeding males implanted with testosterone sing songs resembling that of breeding season songs. In the third experiment (p. 81), I studied gene expression in the HVC of seven canary groups, females and males of breeding season and of non-breeding season, non-breeding season females and males treated with testosterone, and spontaneously singing female canaries. Hierarchical clustering and principal component analysis (PCA) showed that circulating testosterone levels and sex were the predominant variables associated with variation in the HVC transcriptomes. Comparison between natural singing canaries with testosterone-induced singing canaries of the same sex revealed large differences in the HVC transcriptomes. Moreover, the intersection of natural and testosterone-induced singing females shared little resemblance with males in terms of genes. GO-term enrichment analysis suggested functional overlap between sex-specific gene networks. However, although strong transcriptional changes in HVC correlate with the transition from non-singing to singing in both sexes, the type of transcriptional changes are sex-specific. In the fourth experiment (p. 89), I studied sex differences in HVC gene expression between three songbird species: the canary, the blue-capped cordon bleus (Uraeginthus cyanocephalus), and the forest weavers (Ploceus bicolor). Cordon bleu females sing regularly with female-specific songs, whereas forest weaver females sing songs identical to males. I found substantial sex differences in HVC gene expression in all three species, and sex-biased genes differed between species. Surprisingly, the majority of sex-biased genes were on autosomes instead the sex chromosome Z. These results provide further evidence for sex differences in brain structure at the molecular and cellular levels in sexually reproductive animals.