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Using the canary genome to decipher the evolution of hormone-sensitive gene regulation in seasonal singing birds.

Frankl-Vilches C, Kuhl H, Werber M, Klages S, Kerick M, Bakker A, de Oliveira EH, Reusch C, Capuano F, Vowinckel J, Leitner S, Ralser M, Timmermann B, Gahr M - Genome Biol. (2015)

Bottom Line: Testosterone-sensitive up-regulated gene networks of HVC of singing males concerned neuronal differentiation.The canary genome sequence and complementary expression analysis reveal intra-regional evolutionary changes in a multi-regional neural circuit controlling seasonal singing behavior and identify gene evolution related to the hormone-sensitivity of this seasonal singing behavior.Such genes that are testosterone- and estrogen-sensitive specifically in the canary and that are involved in rewiring of neurons might be crucial for seasonal re-differentiation of HVC underlying seasonal song patterning.

View Article: PubMed Central - PubMed

Affiliation: Department of Behavioral Neurobiology, Max Planck Institute for Ornithology, 82319, Seewiesen, Germany. frankl@orn.mpg.de.

ABSTRACT

Background: While the song of all songbirds is controlled by the same neural circuit, the hormone dependence of singing behavior varies greatly between species. For this reason, songbirds are ideal organisms to study ultimate and proximate mechanisms of hormone-dependent behavior and neuronal plasticity.

Results: We present the high quality assembly and annotation of a female 1.2-Gbp canary genome. Whole genome alignments between the canary and 13 genomes throughout the bird taxa show a much-conserved synteny, whereas at the single-base resolution there are considerable species differences. These differences impact small sequence motifs like transcription factor binding sites such as estrogen response elements and androgen response elements. To relate these species-specific response elements to the hormone-sensitivity of the canary singing behavior, we identify seasonal testosterone-sensitive transcriptomes of major song-related brain regions, HVC and RA, and find the seasonal gene networks related to neuronal differentiation only in the HVC. Testosterone-sensitive up-regulated gene networks of HVC of singing males concerned neuronal differentiation. Among the testosterone-regulated genes of canary HVC, 20% lack estrogen response elements and 4 to 8% lack androgen response elements in orthologous promoters in the zebra finch.

Conclusions: The canary genome sequence and complementary expression analysis reveal intra-regional evolutionary changes in a multi-regional neural circuit controlling seasonal singing behavior and identify gene evolution related to the hormone-sensitivity of this seasonal singing behavior. Such genes that are testosterone- and estrogen-sensitive specifically in the canary and that are involved in rewiring of neurons might be crucial for seasonal re-differentiation of HVC underlying seasonal song patterning.

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Gene ontology of HVC transcriptomes (A-D) and RA transcriptomes (E) related to season (A,C,E), to testosterone (B), and to the area-specific presence/absence of androgen and estrogen receptors (D) (see Materials and methods sections M10, M11 and M15 for details). Biological processes typical of neuronal differentiation and synaptic transmission are depicted in colour; all others are depicted in grayscale. Due to space limitations, we could not include the names of all significant biological processes in the charts, but we list them in Additional file 5. (A) Of the seasonal biological processes of up-regulated genes of LD HVC versus SD HVC, 34.9% (sum of the coloured segments) relate to synaptic transmission and neuronal differentiation, which includes neurogenesis. (B) Testosterone-induced biological processes of up-regulated genes of HVC of SD + T males versus SD HVC; 36.8% of the biological processes are related to synaptic transmission and neuronal differentiation. (C) None of the seasonal biological processes of down-regulated genes of LD HVC versus SD HVC concern typical neuronal categories. (D) Area-specific biological processes of up-regulated genes of LD HVC versus LD ENT; 30.6% of these HVC-specific processes relate to synaptic transmission and neuronal differentiation. (E) Seasonal biological processes of up-regulated genes of LD RA versus SD RA concern general cell biological processes but not neuronal differentiation. (F) Differentially expressed HVC transcriptomes (genes that are significantly up- or down-regulated in HVC versus ENT) depend on elevated levels of testosterone. The ‘LD male’ curve shows the good agreement between the two techniques for assessing the differential expression of HVC genes (R2 = 0.85) of different groups of LD males, whereas the transcriptomes do not correlate well between LD and SD males (‘SD male’ curve with R2 = 0.46) but do so between LD and SD + T males (‘SD + T male’ curve with R2 = 0.79).
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Fig4: Gene ontology of HVC transcriptomes (A-D) and RA transcriptomes (E) related to season (A,C,E), to testosterone (B), and to the area-specific presence/absence of androgen and estrogen receptors (D) (see Materials and methods sections M10, M11 and M15 for details). Biological processes typical of neuronal differentiation and synaptic transmission are depicted in colour; all others are depicted in grayscale. Due to space limitations, we could not include the names of all significant biological processes in the charts, but we list them in Additional file 5. (A) Of the seasonal biological processes of up-regulated genes of LD HVC versus SD HVC, 34.9% (sum of the coloured segments) relate to synaptic transmission and neuronal differentiation, which includes neurogenesis. (B) Testosterone-induced biological processes of up-regulated genes of HVC of SD + T males versus SD HVC; 36.8% of the biological processes are related to synaptic transmission and neuronal differentiation. (C) None of the seasonal biological processes of down-regulated genes of LD HVC versus SD HVC concern typical neuronal categories. (D) Area-specific biological processes of up-regulated genes of LD HVC versus LD ENT; 30.6% of these HVC-specific processes relate to synaptic transmission and neuronal differentiation. (E) Seasonal biological processes of up-regulated genes of LD RA versus SD RA concern general cell biological processes but not neuronal differentiation. (F) Differentially expressed HVC transcriptomes (genes that are significantly up- or down-regulated in HVC versus ENT) depend on elevated levels of testosterone. The ‘LD male’ curve shows the good agreement between the two techniques for assessing the differential expression of HVC genes (R2 = 0.85) of different groups of LD males, whereas the transcriptomes do not correlate well between LD and SD males (‘SD male’ curve with R2 = 0.46) but do so between LD and SD + T males (‘SD + T male’ curve with R2 = 0.79).

Mentions: Gene Ontology (GO) analysis of the HVC transcriptomes using ClueGo [63] (Materials and methods section M15) showed that, overall, the significant biological processes for LD and SD + T males are rather similar. Of the biological processes of the specifically up-regulated HVC genes of LD males compared with SD males, 34.9% are related to neural and neuronal differentiation (including neurogenesis) and synaptic transmission (Figure 4A; Additional file 5). In the SD + T to SD comparison, the fraction of biological processes associated with these categories is similar (36.8%; Figure 4B; Additional file 5). There are, however, differences among these categories - for example, dendrite development and overall neuron differentiation are more represented in the LD HVC while neuron projection development and synapse organization are more abundant in the SD + T HVC. The most notable differences between LD and SD + T males concern the importance of organelle organization and the regulation of small GTPase-mediated signal transduction pathways in the HVC gene networks of LD males and epithelial morphogenesis (including angiogenesis) and gliogenesis and related networks in the HVC of SD + T males. Since gliogenesis and angiogenesis are induced in the HVC of female canaries following testosterone treatment [30,64] and are likely to occur seasonally, certain testosterone-induced gene networks may be transiently active, resulting in their absence in LD males, or LD males might be physiologically less synchronized than the SD + T males. A similar reasoning might explain the small differences in the neural categories for LD and SD + T males mentioned above. Most significantly, the genes that are more highly expressed in the SD HVC are not associated with typical neuronal processes but are mainly related to the cell cycle process, DNA repair, RNA processing and organelle organization (Figure 4C; Additional file 5). Furthermore and contrary to HVC, seasonal differences in the transcriptomes of RA do not suggest seasonality of neuronal differentiation and synaptic activity (Figure 4E; Additional file 5).Figure 4


Using the canary genome to decipher the evolution of hormone-sensitive gene regulation in seasonal singing birds.

Frankl-Vilches C, Kuhl H, Werber M, Klages S, Kerick M, Bakker A, de Oliveira EH, Reusch C, Capuano F, Vowinckel J, Leitner S, Ralser M, Timmermann B, Gahr M - Genome Biol. (2015)

Gene ontology of HVC transcriptomes (A-D) and RA transcriptomes (E) related to season (A,C,E), to testosterone (B), and to the area-specific presence/absence of androgen and estrogen receptors (D) (see Materials and methods sections M10, M11 and M15 for details). Biological processes typical of neuronal differentiation and synaptic transmission are depicted in colour; all others are depicted in grayscale. Due to space limitations, we could not include the names of all significant biological processes in the charts, but we list them in Additional file 5. (A) Of the seasonal biological processes of up-regulated genes of LD HVC versus SD HVC, 34.9% (sum of the coloured segments) relate to synaptic transmission and neuronal differentiation, which includes neurogenesis. (B) Testosterone-induced biological processes of up-regulated genes of HVC of SD + T males versus SD HVC; 36.8% of the biological processes are related to synaptic transmission and neuronal differentiation. (C) None of the seasonal biological processes of down-regulated genes of LD HVC versus SD HVC concern typical neuronal categories. (D) Area-specific biological processes of up-regulated genes of LD HVC versus LD ENT; 30.6% of these HVC-specific processes relate to synaptic transmission and neuronal differentiation. (E) Seasonal biological processes of up-regulated genes of LD RA versus SD RA concern general cell biological processes but not neuronal differentiation. (F) Differentially expressed HVC transcriptomes (genes that are significantly up- or down-regulated in HVC versus ENT) depend on elevated levels of testosterone. The ‘LD male’ curve shows the good agreement between the two techniques for assessing the differential expression of HVC genes (R2 = 0.85) of different groups of LD males, whereas the transcriptomes do not correlate well between LD and SD males (‘SD male’ curve with R2 = 0.46) but do so between LD and SD + T males (‘SD + T male’ curve with R2 = 0.79).
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Related In: Results  -  Collection

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Fig4: Gene ontology of HVC transcriptomes (A-D) and RA transcriptomes (E) related to season (A,C,E), to testosterone (B), and to the area-specific presence/absence of androgen and estrogen receptors (D) (see Materials and methods sections M10, M11 and M15 for details). Biological processes typical of neuronal differentiation and synaptic transmission are depicted in colour; all others are depicted in grayscale. Due to space limitations, we could not include the names of all significant biological processes in the charts, but we list them in Additional file 5. (A) Of the seasonal biological processes of up-regulated genes of LD HVC versus SD HVC, 34.9% (sum of the coloured segments) relate to synaptic transmission and neuronal differentiation, which includes neurogenesis. (B) Testosterone-induced biological processes of up-regulated genes of HVC of SD + T males versus SD HVC; 36.8% of the biological processes are related to synaptic transmission and neuronal differentiation. (C) None of the seasonal biological processes of down-regulated genes of LD HVC versus SD HVC concern typical neuronal categories. (D) Area-specific biological processes of up-regulated genes of LD HVC versus LD ENT; 30.6% of these HVC-specific processes relate to synaptic transmission and neuronal differentiation. (E) Seasonal biological processes of up-regulated genes of LD RA versus SD RA concern general cell biological processes but not neuronal differentiation. (F) Differentially expressed HVC transcriptomes (genes that are significantly up- or down-regulated in HVC versus ENT) depend on elevated levels of testosterone. The ‘LD male’ curve shows the good agreement between the two techniques for assessing the differential expression of HVC genes (R2 = 0.85) of different groups of LD males, whereas the transcriptomes do not correlate well between LD and SD males (‘SD male’ curve with R2 = 0.46) but do so between LD and SD + T males (‘SD + T male’ curve with R2 = 0.79).
Mentions: Gene Ontology (GO) analysis of the HVC transcriptomes using ClueGo [63] (Materials and methods section M15) showed that, overall, the significant biological processes for LD and SD + T males are rather similar. Of the biological processes of the specifically up-regulated HVC genes of LD males compared with SD males, 34.9% are related to neural and neuronal differentiation (including neurogenesis) and synaptic transmission (Figure 4A; Additional file 5). In the SD + T to SD comparison, the fraction of biological processes associated with these categories is similar (36.8%; Figure 4B; Additional file 5). There are, however, differences among these categories - for example, dendrite development and overall neuron differentiation are more represented in the LD HVC while neuron projection development and synapse organization are more abundant in the SD + T HVC. The most notable differences between LD and SD + T males concern the importance of organelle organization and the regulation of small GTPase-mediated signal transduction pathways in the HVC gene networks of LD males and epithelial morphogenesis (including angiogenesis) and gliogenesis and related networks in the HVC of SD + T males. Since gliogenesis and angiogenesis are induced in the HVC of female canaries following testosterone treatment [30,64] and are likely to occur seasonally, certain testosterone-induced gene networks may be transiently active, resulting in their absence in LD males, or LD males might be physiologically less synchronized than the SD + T males. A similar reasoning might explain the small differences in the neural categories for LD and SD + T males mentioned above. Most significantly, the genes that are more highly expressed in the SD HVC are not associated with typical neuronal processes but are mainly related to the cell cycle process, DNA repair, RNA processing and organelle organization (Figure 4C; Additional file 5). Furthermore and contrary to HVC, seasonal differences in the transcriptomes of RA do not suggest seasonality of neuronal differentiation and synaptic activity (Figure 4E; Additional file 5).Figure 4

Bottom Line: Testosterone-sensitive up-regulated gene networks of HVC of singing males concerned neuronal differentiation.The canary genome sequence and complementary expression analysis reveal intra-regional evolutionary changes in a multi-regional neural circuit controlling seasonal singing behavior and identify gene evolution related to the hormone-sensitivity of this seasonal singing behavior.Such genes that are testosterone- and estrogen-sensitive specifically in the canary and that are involved in rewiring of neurons might be crucial for seasonal re-differentiation of HVC underlying seasonal song patterning.

View Article: PubMed Central - PubMed

Affiliation: Department of Behavioral Neurobiology, Max Planck Institute for Ornithology, 82319, Seewiesen, Germany. frankl@orn.mpg.de.

ABSTRACT

Background: While the song of all songbirds is controlled by the same neural circuit, the hormone dependence of singing behavior varies greatly between species. For this reason, songbirds are ideal organisms to study ultimate and proximate mechanisms of hormone-dependent behavior and neuronal plasticity.

Results: We present the high quality assembly and annotation of a female 1.2-Gbp canary genome. Whole genome alignments between the canary and 13 genomes throughout the bird taxa show a much-conserved synteny, whereas at the single-base resolution there are considerable species differences. These differences impact small sequence motifs like transcription factor binding sites such as estrogen response elements and androgen response elements. To relate these species-specific response elements to the hormone-sensitivity of the canary singing behavior, we identify seasonal testosterone-sensitive transcriptomes of major song-related brain regions, HVC and RA, and find the seasonal gene networks related to neuronal differentiation only in the HVC. Testosterone-sensitive up-regulated gene networks of HVC of singing males concerned neuronal differentiation. Among the testosterone-regulated genes of canary HVC, 20% lack estrogen response elements and 4 to 8% lack androgen response elements in orthologous promoters in the zebra finch.

Conclusions: The canary genome sequence and complementary expression analysis reveal intra-regional evolutionary changes in a multi-regional neural circuit controlling seasonal singing behavior and identify gene evolution related to the hormone-sensitivity of this seasonal singing behavior. Such genes that are testosterone- and estrogen-sensitive specifically in the canary and that are involved in rewiring of neurons might be crucial for seasonal re-differentiation of HVC underlying seasonal song patterning.

Show MeSH
Related in: MedlinePlus