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Using the Canary Genome to Decipher the Evolution of Hormone-Sensitive Gene Regulation in Seasonal Singing Birds

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Using the Canary Genome to Decipher the Evolution of Hormone-Sensitive Gene Regulation in Seasonal Singing Birds

Carolina Frankl-Vilches et al. Genome Biol.

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.

Figures

Figure 1
Figure 1
Schematic of the song control system of songbirds and seasonal features of the canary song. (A) HVC and RA are essential nuclei of the descending motor pathway of the song system that controls song production. All interconnected areas are parts of loops that feed back to the song motor pathway. The androgen receptors (red dots) are expressed in both the HVC and RA of canaries, while only HVC contains estrogen receptors (green dots) among song areas. Both areas express the 5α-reductase gene (blue squares) but only HVC expresses aromatase (yellow squares). The entopallium (ENT; the bird visual cortex) expresses neither androgen or estrogen receptors nor androgen- or estrogen-producing enzymes and was used as a contrasting brain area [20-23] (this study). (B) Song features such as song length, the number of non-repeated syllables, the number of sexy syllables and the maximal repetition rate of syllables change seasonally while the syllable repertoire of canaries does not change between their breeding and non-breeding season. The abundance of song segments with high repetition rates is a sexually attractive song pattern in canaries. Plotted are the percent differences between the breeding males (control) producing high testosterone levels and the non-breeding males producing low testosterone levels (redrawn after [16]).
Figure 2
Figure 2
Collinearity increases the superscaffold sizes of the canary genome significantly (yellow field) so that the assembly quality approaches that of the chicken, turkey and zebra finch, all based on additional sequencing information such as genetic linkage maps (blue field). Note that the values are given as log bp. Without collinearity, the superscaffold length of the canary genome is among the highest among genomes sequenced with next generation technologies (ngs) only (green field). Note that details of the assembly procedures for the budgerigar, ground finch, emperor penguin, Adelie penguin, and rock dove have not been published. The N50 contig length is in red, and the N50 superscaffold length is in blue. For scientific species names and sources of genome assemblies see Materials and methods section M5.
Figure 3
Figure 3
Whole genome alignments of the canary genome and 13 other publicly available bird genome assemblies using the zebra finch genome as a reference underline the high long-range continuity of the canary genome assembly and highly conserved collinearity and synteny of genomes throughout the bird taxa. Scaffold colours were chosen in a random fashion to visualize the assembly N50 length of the top level sequences (chromosomes, superscaffolds or scaffolds, depending on genome project), resulting in highly heterogeneous coloured plots for low quality genome assemblies (outside rings) and homogeneous coloured plots for high quality genome assemblies (inside rings). Black arcs depict putative intra-chromosomal rearrangements of the genome assemblies compared with zebra finch, many of which are found in different bird taxa and thus likely trace back to zebra finch-specific rearrangements or mis-assemblies in the zebra finch assembly. For the canary genome we also show five putative inter-chromosomal rearrangements (red arcs). For scientific species names and sources of genome assemblies see Materials and methods section M5.
Figure 4
Figure 4
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).
Figure 5
Figure 5
Hormone sensitivity of seasonal transcriptomes. (A,B) Venn diagrams of up-regulated transcriptomes (A) and down-regulated transcriptomes (B) related to season (HVC LD versus HVC SD), testosterone (HVC SD versus HVC SD + T), and area-specificity (HVC LD versus ENT LD) (Figure 4). (C-E) From the various resulting transcriptomes of these comparisons, we calculated the frequency of genes with an androgen response element (ARE) or estrogen response element (ERE) in their promoters. Numbers below the headers of (C-G) relate to the differential transcriptomes of (A) and (B). Many more genes are down-regulated (B) compared with up-regulated (A) in each of the comparisons: 833 genes are up-regulated seasonally in HVC, among which 208 are testosterone-inducible; 2,410 are down-regulated seasonally in HVC, among which 1,695 are testosterone-inducible. Genes containing ERE are enriched particularly among the testosterone-sensitive seasonal gene pool (C) compared with seasonal but not testosterone-sensitive genes (E) or random (not shown) gene pools. Among the down-regulated transcriptomes, AREs are particularly enriched among the testosterone-sensitive seasonal genes (D). The frequency of AREs and EREs in (D,E) is depicted as the percentage enrichment compared with ARE and ERE abundance in genes not expressed in HVC. (F-H) The frequency of canary-specific AREs (CAN-ARE) and of canary-specific EREs (CAN-ERE) among the various gene pools. Canary-specific means the AREs and EREs are absent in orthologous genes of the zebra finch genome. Percentage is based on all genes of a gene pool (here 208, 1,695 and 1,000; see Additional file 6 for gene lists). Note that AREs of testosterone-sensitive seasonally expressed genes (F,G) are conserved, that is, the frequency of CAN-AREs is much lower compared with genes not expressed in HVC (H). This is not the case for CAN-EREs.
Figure 6
Figure 6
In situ hybridization for mRNA of genes differentially expressed in HVC, RA or entopallium. Expression, either up- or down-regulated in HVC and RA compared with the ENT, was as expected from the RNA-seq for all six depicted genes. Next to the high expression in HVC (small arrows for ALDH1A2, CNTFR, GPR83, GLI2, RASGRP1) or RA (large arrow for GPR83) of LD canaries, the selected genes are expressed in other, distinct brain regions, such as the medial striatum (large arrow for RASGRP1), the arcopallium (large arrow for CNTFR) and the mesopallium (large arrow for ALDH1A2). KCNQ5 is down-regulated in the HVC of the depicted SD canary but year-round highly expressed in the ENT (large arrow). Abbreviations: ALDH1A2, aldehyde dehydrogenase 1 family, member A2; CNTFR, ciliary neurotrophic factor receptor; GLI2, GLI family zinc finger 2; GPR83, G protein-coupled receptor 83; KCNH5, potassium voltage-gated channel, subfamily H (eag-related), member 5; RASGRP1, RAS guanyl releasing protein 1 (calcium and DAG-regulated). Shown are photomicrographs of autoradiograms of sagittal sections of the canary brain. Labelled areas appear darkened. The scale bar equals 5 mm.
Figure 7
Figure 7
RNA-seq-based HVC gene expression correlated with HVC protein SWATH-MS results. The data were normalized to the expression levels of the ENT; positive values indicate higher expression in HVC. For 10 of the 13 investigated proteins, the correlation between protein and mRNA abundance is strong (r = 0.76). Nonetheless, we also found examples where the post-translational control of gene expression appears to be crucial: the endophilin SH3GL2 had a substantially higher protein expression level (as predicted from its RNA level), whereas levels of the calcium-transporting ATPase ATP2B1were lower than its mRNA expression would indicate. The insert shows an exemplary peptide chromatogram from SWATH-MS [77] for the peptide ASDPAAPPEEAK, which is specific for the protein MBP and is less expressed in the ENT compared with the HVC. Y-axis: intensity cps × 103.
Figure 8
Figure 8
Canary-specific estrogen response elements (see inserts) occur in the orthologous promoters of canary and zebra finch genes. (A) Genes with orthologous promoters but that contain nucleotide sequences in zebra finch deviating from known ERE sequences (see insert in (A)). (B) Genes with EREs in the promoters of canaries for which we did not find orthologous promoters in zebra finch. In these cases, sequence analysis of 1 kb of the putative promoter region of the zebra finch did not identify EREs. The 15 genes analysed in detail were randomly selected from the list of genes with canary-specific EREs. Thus, of the about 550 seasonally up-regulated genes that contain canary-specific EREs, about one-half are classified as type ‘A’ and the other half as type ‘B’. Some genes such as SRGAP3 are in both categories. Orthologous promoters are in yellow, those containing EREs are in orange, and heterologous promoters are in red.

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