DOI: 10.5061/dryad.5hqbzkh8c
title: Extensive, transient, and long-lasting gene regulation in a song-controlling brain area during testosterone-induced song development in adult female canaries

description: Seasonal song production in canaries, influenced by gonadal hormones, is a
 well-documented phenomenon. We explored testosterone-induced song
 development in adult female canaries—a behavior rarely exhibited
 naturally. Gene regulatory networks in the song-controlling brain area HVC
 were compared at multiple time points (1 hour to 14 days) post-treatment
 with those of placebo-treated controls, paralleling HVC and song
 development. Females began vocalizing within four days of testosterone
 treatment, with song complexity and HVC volume increasing progressively
 over two weeks. Rapid transcriptional changes involving 2,739 genes
 preceded song initiation. Over two weeks, 9,913 genes—approximately 64% of
 the canary's protein-coding genome—were differentially expressed,
 with 98% being transiently regulated. These genes are linked to various
 biological functions, with early changes at the cellular level and later
 changes affecting the nervous system level after prolonged hormone
 exposure. Our findings suggest that testosterone-induced song development
 is accompanied by extensive and dynamic transcriptional changes in the
 HVC, implicating widespread neuronal involvement. The data reveal
 extensive transcriptomic changes, including alterations in steroid
 receptor expression and numerous transcription factors, coinciding with
 significant neural transformations. These changes underpin the gradual
 emergence of singing behavior, providing insights into the neural basis of
 seasonal behavioral patterns.
description: We compared the expression data of song nuclei HVC dissected from
 adult female canaries (<em>Serinus canaria</em>) implanted
 with 7-mm SilasticTM tubes filled with testosterone for 6 time durations
 (1h, 3h, 8h, 3d, 7d, and 14d) with control birds (implanted with empty
 tube) to identify testosterone effects on gene expression.
 We performed microarray analysis using the Custom Affymetrix Gene
 Chip® MPIO-ZF1s520811 Exon Array. The CEL files were generated using
 Affymetrix GeneChip Command Console® Software (AGCC). Differential
 expression analyses were performed using the R package limma, following
 the workflow of Klaus and Reisenauer (<em>F1000Research
 2016</em>).​ The HVC volumes, plasma androgen
 levels, and physiological measurements of testosterone-treated and control
 birds were also deposited.  The song parameters of
 birds in T7d and T14d Multi_Channel_Analyser (MCA), a custom program with
 a graphical user interface written in MATLAB (version R2016b, Mathworks),
 as described previously (Ko et al., 2020) and available to the public
 (https://doi.org/10.5281/zenodo.1489098).
description: # Extensive, transient, and long-lasting gene regulation in a
 song-controlling brain area during testosterone-induced song development
 in adult female canaries Provenance for this README This README.md was
 updated on 30-November-2024 by Meng-Ching Ko. This repository contains all
 datasets required to reproduce the figures, supplementary files, and
 supplementary figures in "From silence to song: Testosterone triggers
 extensive transcriptional changes in the female canary HVC,"
 published in the *Journal of Neuroendocrinology*. doi: 10.1111/jne.13476
 Prior to publication, preprint versions of this manuscript were available
 on bioRxiv:
 [https://doi.org/10.1101/2022.06.13.495861](https://doi.org/10.1101/2022.06.13.495861) The scripts for reproducing figures, supplementary files, and supplementary figures were deposited on GitHub ([https://github.com/maggieMCKO/TimeLapseTestoFemaleCanary](https://github.com/maggieMCKO/TimeLapseTestoFemaleCanary)). * AUTHOR INFORMATION * Ko, Meng-Ching, Department of Behavioural Neurobiology, Max Planck Institute for Biological Intelligence * orcid: [https://orcid.org/0000-0002-2234-9380](https://orcid.org/0000-0002-2234-9380) * email: [mengching.ko@bi.mpg.de](mailto:mengching.ko@bi.mpg.de) * alternate email: [mengchingko@gmail.com](mailto:mengchingko@gmail.com) * Frankl-Vilches, Carolina, Department of Behavioural Neurobiology, Max Planck Institute for Biological Intelligence * Bakker, Antje, Department of Behavioural Neurobiology, Max Planck Institute for Biological Intelligence * Sohnius-Wilhelmi, Nina, Department of Behavioural Neurobiology, Max Planck Institute for Biological Intelligence * Alcami, Pepe, Department of Behavioural Neurobiology, Max Planck Institute for Biological Intelligence; Division of Neurobiology, Faculty of Biology, Ludwig-Maximilians-University Munich * Gahr, Manfred, Department of Behavioural Neurobiology, Max Planck Institute for Biological Intelligence ## FILE OVERVIEW ### Table of Contents # Dataset Overview This repository contains the following files that are critical for reproducing the data analysis and results. Each file is briefly described to help you understand its purpose and content. ## Table of Contents ### Primary Data Files * **DiffExpression.tsv** * The output of the differential analysis is stored in this file. * **HVC_volume.tsv** * This file includes data on HVC volume and normalized HVC volume. * **Physiological_measurements.tsv** * This file contains measurements of brain weight, body weight, and oviduct weight. * **PlasmaAndrogenLv.tsv** * This file contains data on plasma androgen levels. * **RNAscope_NormalizedStainedArea.tsv** * This file includes the quantification of RNAscope staining. * **Expression_est_perBird.tsv** * output of RMA (Robust Multichip Analysis). ### Song analysis-related files * **DayLv_data.tsv** * This file contains daily singing rates. * **SongLv_data.tsv** * Song parameters such as song length, syllable repetition rate, and the number of syllables per song are stored in this file. ### File/Folder Details #### Details for DiffExpression.tsv * Description: This tab-delimited file contains the filtered output of the differential expression analysis performed using the R package limma. It includes only genes with human orthologous annotation meeting the significance criteria (adjusted P-value, fold change, and power thresholds) and showing the consistent direction of regulation, as described in the 'Analysis of differentially expressed genes' section of the associated manuscript. The file contains normalized expression values, log fold changes, adjusted p-values, and power values for these differentially expressed genes across different time points. * Format(s): .tsv * Variables: * GeneSymbol: Differentially expressed genes * logFC: Log2 fold change of the gene * AveExpr: Average expression of the gene across all samples * t: t-statistic * P.Value: Raw p-value * adj.P.Val: Adjusted p-value (Benjamini-Hochberg method) * B: B-statistic (log-odds that the gene is differentially expressed) * contrast: The group in which the gene was identified as differentially expressed (relative to the control group) * direction: Direction of regulation (-1 for down-regulated, 1 for up-regulated) * logFC_ave: The average log2 fold change across all probes for each differentially expressed gene * power: Power value for the gene (≥ 0.8) * TF: Indicates whether the gene is a transcription factor (1) or not (0) * Missing data codes: Cells with missing data are coded as NA ("NA") #### Details for: HVC\_volume.tsv * Description: This tab-delimited file provides the HVC volume data for the testosterone-treated group. Both raw and body weight-normalized HVC volumes are included. * Format(s): .tsv * Variables: * Group: Testosterone-treated group * ProcessNum: Bird (process) ID * HVC Volume (mm3): HVC volume * HVC_nor: HVC volume normalized by body weight #### Details for: Physiological\_measurements.tsv * Description: This tab-delimited file contains measurements of brain weight, body weight, and oviduct weight. * Format(s): .tsv * Variables: * Group: Testosterone-treated group * ProcessNum: Bird (process) ID * Brain (mg): Brain weight * Body Weight (g): Body weight * Oviduct (mg): Oviduct weight * Missing data codes: Cells with missing data are coded as NA ("NA") #### Details for: PlasmaAndrogenLv.tsv * Description: This tab-delimited file contains data on plasma androgen levels. * Format(s): .tsv * Variables: * Group: Testosterone-treated group * PrePost: Blood sampled before or after testosterone implantation * ProcessNum: Bird (process) ID * ngml: Plasma androgen levels * date: Date of blood sampling (yyyy/mm/dd) #### Details for: RNAscope\_NormalizedStainedArea.tsv * Description: This tab-delimited file contains the quantification of RNAscope staining. Please see the methods section "RNAScope® in situ hybridization assay" in the manuscript for further details. * Format(s): .tsv * Variables: * Probe: Gene examined using RNAscope * Group: Testosterone-treated group * Individual: Bird ID * Slide: Slide ID * sum_slide: The sum of the area of accumulated chromogenic particles in the stained HVC sections * Area: The HVC volume of the section * n_particle: The number of accumulated chromogenic particles in the stained HVC sections * Density: The density of accumulated chromogenic particles within the HVC (n_particle/Area) * percent_area_slide: The area of accumulated chromogenic particles normalized by HVC volume * Missing data codes: Cells with missing data are coded as NA ("NA") #### Details for: Expression\_est\_perBird.tsv * Description: This tab-delimited file contains the output of the Robust Multichip Analysis (RMA). Each column from the second to the 12360st represents the average expression of a specific gene. * Format(s): .tsv * Variables: * Sample: Sample (Bird) ID * Column 2 to 12360: Average expression of each gene (each column corresponds to a specific gene) #### Song analysis-related files #### Details for: DayLv\_data.tsv * Description: This tab-delimited file contains daily singing rates. * Format(s): .tsv * Variables: * Ind: Bird ID * Individual: Bird ID as integer * Group: Testosterone-treated group * n_Group: Testosterone-treated group as integer * n_Day: days after testosterone implantation * Daily_SongRate: daily song rate #### Details for: SongLv\_data.tsv * Description: This tab-delimited file contains song parameters such as song length, syllable repetition rate, the number of syllables per song, and the slope coefficient (α). * Format(s): .tsv * Variables: * Ind: Bird ID * Individual: Bird ID as integer * Group: Testosterone-treated group * n_Group: Testosterone-treated group as integer * FisrtDate: Testosterone implantation date * n_Day: days after testosterone implantation * StartTime: The recording date * Daily_SongRate: daily song rate * SongLen: Song length * SylperSong: number of syllables in a song * RepetitionRate: syllable repetition rate * slope: the slope coefficient (α) ### Version changes 30-November-2024:  * DiffExpression.tsv: * As per reviewer's request ([https://www.biorxiv.org/content/10.1101/2022.06.13.495861v3#review](https://www.biorxiv.org/content/10.1101/2022.06.13.495861v3#review)), we have removed the results generated using ChipInspector software. In the current version, differential expression analysis was performed using the R package limma. The following columns from the May 19, 2023 version have been removed: 'Group', 'Coverage', 'LFC.ave', 'regulatory.function', 'matrix.family', 'Chromosome', 'description', and 'power &gt;= 0.8'. * Columns 'logFC', 'AveExpr', 't', 'P.Value', 'adj.P.Val', 'B', 'contrast', 'direction', 'logFC_ave', 'power', 'TF' were added. * Expression_est_perBird.tsv * The duplicated column 'PGM2' was removed. * Removed the following files as they were not included in the published manuscript: * CellType_GeneList.tsv * ClueGO_full_results_KEGG.csv * ClueGO_full_results.csv * ClueGO_Top10.csv" * WGCNA folder