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Results for “Molecular Biology”

Search indexed bioRxiv preprints in genomics, neuroscience, cell biology and bioinformatics. Read source abstracts and check manuscript versions; preprints are not peer reviewed.

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bioRxiv2022

Synergistic Combined-proteomics Guided Mapping strategy identifies mTOR mediated phosphorylation of LARP1 in nutrient responsiveness and dilated cardiomyopathy

Increased activity of the mammalian target of rapamycin (mTOR) signalling pathway, a crucial nutrient sensor, exacerbates ageing and ageing-related diseases, including cancer and heart failure. To further elucidate the physiological role of the serine/threonine kinase mTOR, we devised a novel tractable proteomics strategy that combines interaction proteomics, proximity-based proteomics and quantitative phosphoproteomics to identify interactors with and potential substrates of mTOR. We identified 58 candidate mTOR substrates, several of which were further validated. Interestingly, several of these candidate mTOR substrates are involved in various aspects of RNA biology, including regulating stability and processing. We characterized in-depth one of the validated mTOR substrates, LARP1, an RNA binding protein. mTOR-dependent phosphorylation of LARP1 is nutrient-sensitive and controls the RNA-binding ability of LARP1. We show that mTOR activity and LARP1 and LARP1 phosphorylation levels are increased in a congenital mouse model of dilated cardiomyopathy (DCM) caused by a mutation in the Lamin A gene. This implicates LARP1 in the development of DCM.

molecular biology↗

bioRxiv2023

Analysis of ageing-dependent thiol oxidation reveals early oxidation of proteins involved in core proteostasis functions

Oxidants have a profound impact on biological systems in physiology and under pathological conditions. Oxidative post-translational modifications of protein thiols are well-recognized as a readily occurring alteration of proteins. Changes in protein thiol redox state can modify the function of proteins and thus can control cellular processes. However, chronic oxidative stress causes oxidative damage to proteins with detrimental consequences for cellular function and organismal health. The development of techniques enabling the site-specific and quantitative assessment of protein thiol oxidation on a proteome-wide scale significantly expanded the number of known oxidation-sensitive protein thiols. However, lacking behind are large-scale data on the redox state of proteins during ageing, a physiological process accompanied by increased levels of endogenous oxidants. Here, we present the landscape of protein thiol oxidation in chronologically aged wild-type Saccharomyces cerevisiae in a time-dependent manner. Our data determine early oxidation targets in key biological processes governing the de novo production of proteins, folding, and protein degradation. Comparison to existing datasets reveals evolutionary conservation of early oxidation targets. To facilitate accessibility and cross-species comparison of the experimental data obtained, we created the OxiAge Database, a free online tool for the research community that integrates current datasets on thiol redoxomes in aged yeast, nematode Caenorhabditis elegans, fruit fly Drosophila melanogaster, and mouse Mus musculus. The database can be accessed through an interactive web application at http://oxiage.ibb.waw.pl.

molecular biology↗

bioRxiv2025

Genetic response to light and carbon source variations in Trichoderma harzianum: The key regulatory roles of env1, cre1, and blr2

In filamentous fungi, light plays a key role in regulating physiological processes such as growth, conidiation, secondary metabolism, and the expression of hydrolytic enzymes. The processes that depend on light are controlled by photoreceptors, including BLR1, BLR2, and ENV1, as well as by signaling pathways involving heterotrimeric G-proteins and cyclic adenosine monophosphate (cAMP). Trichoderma harzianum is a promising candidate for biotechnological use and is able to promote hydrolytic reactions under biomass degradation conditions. However, the genetic mechanisms underlying its response to light remain poorly understood, especially under degradative conditions. This study aimed to assess the expression of carbohydrate-active enzymes (CAZymes), transcription factors (TFs), and signaling pathway proteins under different light conditions and carbon sources. The results revealed distinct patterns of relative gene expression influenced by these environmental factors, highlighting the complex regulatory mechanisms at play in T. harzianum. Moreover, our results suggested that env1, cre1, and blr2 are critical for adjusting to different light conditions and carbon sources. This highlights the importance of both factors in regulating gene expression and supporting metabolic adaptation in T. harzianum. To our knowledge, such findings have not been previously reported in the context of cellulose degradation for this species. Overall, these results offer valuable insights into how T. harzianum responds to environmental changes, revealing a complex regulatory network that is not only crucial for optimizing fungal growth in industrial applications but also deepens our understanding of its biology and ecological interactions. Highlights{blacksquare} Trichoderma harzianum is a key candidate for biotechnology applications. {blacksquare}Light and carbon control CAZymes, photoreceptors, key regulators, and signaling. {blacksquare}T. harzianum has distinct gene expression patterns under different light conditions and carbon sources. {blacksquare}env1, cre1, and blr2 regulate metabolic adaptation in T. harzianum. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=185 SRC="FIGDIR/small/657067v1_ufig1.gif" ALT="Figure 1"> View larger version (72K): org.highwire.dtl.DTLVardef@8167c7org.highwire.dtl.DTLVardef@1c46f6borg.highwire.dtl.DTLVardef@16eddc8org.highwire.dtl.DTLVardef@1a2afa6_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2017

An open-source cryo-storage solution

The field of cryo-electron microscopy is a rapidly growing method in structural biology. With this development, access to cryo-EM facilities becomes a bottleneck that results in long wait times between sample preparation and data acquisition. To improve sample storage, we developed a cryo-storage system with a more efficient and larger storage capacity that enables cryo-sample storage in a highly organized manner. This system is simple to use, cost-effective and easily adaptable for any type of grid box and storage dewar and any size cryo-EM laboratory.

molecular biology↗

bioRxiv2024

Comprehensive Phenotyping of Extracellular Vesicles in Blood of Healthy Humans - Insights into Cellular Origin and Biological Variability

Despite immense interest in biomarker applications of extracellular vesicles (EVs) from blood, our understanding of their physiological population in healthy humans remains limited. Using imaging and multiplex bead-based flow cytometry, we comprehensively quantified circulating EVs with respect to their cellular origin in a large cohort of healthy blood donors. We assessed coefficients of variations to characterise their biological variability and explored demographic, clinical, and lifestyle factors contributing to this variability. Cell-specific circulating EV subsets show a wide range of concentrations, which do not directly reflect concentrations of blood cells, indicating diverse patterns of EV subset release and/or uptake, even for EVs originating from the same cell type. Interestingly, tetraspanin+ circulating EVs largely originate from platelets and to a lesser extent from lymphocytes. PCA and association analyses demonstrate high biological inter-individual variability in circulating EVs across healthy humans, which can be only partly explained by the influence of sex, menopausal status, age and smoking on specific circulating EV and/or tetraspanin+ circulating EV subsets. No global influence of the explored subjects factors on circulating EVs was detected. Our findings provide the first comprehensive, quantitative data towards the cell-origin atlas of blood EVs, with important implications in the clinical use of EVs as biomarkers of disease.

molecular biology↗

bioRxiv2026

Cross-talk among miRNAs, lncRNAs, and DNA methylation in three coral species reveal conserved epigenetic regulatory architecture

Epigenetic mechanisms support phenotypic plasticity across metazoans, enabling dynamic response to environmental change. DNA methylation and non-coding RNAs, including microRNAs (miRNAs) and long non-coding RNAs (lncRNAs), regulate gene expression through distinct but interconnected mechanisms. In vertebrate systems, these layers form integrated networks in which specific miRNAs directly target the protein machinery of other epigenetic processes ("epi-miRNAs") and specialized lncRNAs act as competing endogenous RNAs (ceRNAs), sequestering miRNAs from their mRNA targets. Whether equivalent cross-layer regulatory architectures exist in cnidarians, whose methylomes are invertebrate-characteristic and whose miRNAs function mechanistically like those of plants, is unknown. Here we integrate matched RNA-seq, small RNA-seq, and whole-genome bisulfite sequencing across three species of reef-building coral (Acropora pulchra, Porites evermanni, and Pocillopora tuahiniensis) to characterize the landscape and regulatory interactions of microRNAs (miRNAs), long non-coding RNAs (lncRNAs) and DNA methylation, including the first description of epi-miRNAs and ceRNA networks in cnidarian taxa. Across the study species, miRNAs putatively targeted transcripts encoding a suite of epigenetic processes, including DNA methylation regulators (TET3, MBD, PRDM14), ubiquitin-signaling and histone-modifying machinery, and components of the miRNA pathway itself (e.g., AGO, TNRC6). The conserved miRNA miR-100 also exhibited species-divergent target coexpression, suggesting lineage-specific regulatory roles for deeply conserved miRNAs. Candidate ceRNA networks were also recovered, including predicted derepression of epimachinery transcripts, indicating that lncRNA-mediated buffering operates alongside direct miRNA control. Recovery of these regulatory interactions across three evolutionarily divergent species, despite few orthologous miRNA or lncRNA, suggests that multi-layered epigenetic regulation is a conserved feature of cnidarian biology. These results establish direct miRNA and lncRNA control of epigenetic machinery as an active component of coral gene regulation, and provide foundational resources for studying how multilayered epigenetic interactions contribute to coral resilience to environmental change. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=140 SRC="FIGDIR/small/739451v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@7e07dforg.highwire.dtl.DTLVardef@36cf0forg.highwire.dtl.DTLVardef@5406d3org.highwire.dtl.DTLVardef@8c09e3_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2020

Quantification of extracellular vesicles with unaltered surface membranes using an internalized oligonucleotide tracer and droplet digital PCR applied to modeling of in vivo kinetics

Extracellular vesicles (EVs) continue to attract interest for their potential role in targeted therapeutics and as biomarkers of disease and drug response. In order to achieve clinical utility, it is important to determine the pharmacokinetic parameters of candidate EVs during preclinical development using in vivo animal models. To date, no methods exist for studying EV kinetics without modification to surface ligands that may affect normal behavior. Here we introduce an accessible method for labeling and quantifying EVs administered to conscious animals, without disrupting endogenous ligands. Our method relies upon established laboratory techniques and can be tailored to a variety of biological questions. Digital PCR is leveraged to detect a non-homologous oligonucleotide tracer introduced into the vesicles, allowing for quantification over a wide dynamic range. Using an application of this method, we found differences in the in vivo kinetics of EVs from three different cell types using non-linear mixed effects modeling. We propose that this method will provide a complementary approach for the of study EV ligand-receptor interactions in the context of EV uptake and targeted therapeutics.

molecular biology↗

bioRxiv2022

SARS-CoV2 associated secretion of nanoLuciferase reports on virus and Virus-Like Particle production

SARS-CoV2 is a positive-strand RNA virus in the Coronaviridae family that has caused world-wide morbidity and mortality. While much progress has been made we still need expanded rapid anti-virals. The top advanced antiviral candidates all target stages of RNA replication, leaving virus assembly an unexplored avenue of antiviral research. To address this gap, and explore the biochemical and cell biological features of viral assembly, we have employed an improved virus-like particle (VLP) system. We exploited the small nanoLuciferase protein for enhanced signal and surprisingly found that the protein itself appears to be packaged into both SARS CoV2 VLPs and virions and secreted from cells. Interestingly, nLuc is not co-secreted with dengue or Zika infection, suggesting the large virion of Coronavirus can encaspidate and secrete a cellularly expressed reporter protein. Our findings open the way for powerful new approaches to measure viral particle production, egress and viral entry mechanisms.

molecular biology↗

bioRxiv2021

In silico analysis for potential proteins and microRNAs in Glioblastoma and Parkinsonism.

In todays world, neurodegenerative diseases such as Alzheimers disease, Parkinsons Disease, Huntingtons Disease as well as brain cancers such as astrocytomas, ependymomas, glioblastomas have become a great threat to us. In this study, we are trying to find a probable molecular connection associated with two very much different diseases, Glioblastoma, also known as Glioblastoma Multiforme (cancers of microglial cells of our brain) and Parkinsons disease. We at first downloaded the microarray datasets of these two diseases from Gene Expression Omnibus (GEO) and then analyzed them by the GEO2R tool. After analysis, we found 249 common upregulated differential expressed genes and 135 common downregulated differential expressed genes of these two diseases. Therefore the common differentially expressed genes, both upregulated and downregulated, were imported into STRING online tool to find out the protein-protein interactions. Now, this whole network was subjected to Cytoscape and the top ten hub genes were found by Cyto-Hubba plug-in. The top then hub genes are EGFR, CCNB1, CDK1, CCNA2, CHEK1, RAD51, MAD2L1, KIF20A, BUB1, and CCNB2. These all genes are upregulated in both diseases. To find out the biological processes, molecular functions, cellular components, and pathways associated with these hub genes Enrichr online software was used. We used miRNet software to determine the interactions of hub genes with microRNAs. This study will be useful in the future for drug targets discovery for these diseases.

neuroscience↗

bioRxiv2020

Long-read Individual-molecule Sequencing Reveals CRISPR-induced Genetic Heterogeneity in Human ESCs

Accurately quantifying the genetic heterogeneity of a cell population is essential to understanding of biological systems. We develop a universal method to label individual DNA molecules for analyzing diverse types of rare genetic variants, with frequency as low as 4x10-5, using short- or long-read sequencing. It enables base-resolution haplotype-resolved quantitative characterization of rare variants. It provides the first quantitative evidence of persistent nonrandom large deletions and insertions following DNA repair of double-strand breaks induced by CRISPR-Cas9 in human pluripotent stem cells.

molecular biology↗

bioRxiv2020

Bioinformatic analysis based genome-wide identification, characterization, diversification and regulatory transcription components of RNA silencing machinery genes in wheat (Triticum aestivum L.)

Dicer-Like (DCL), Argonaute (AGO), and RNA-dependent RNA polymerase (RDR) gene families are known as RNA silencing machinery genes or RNAi genes. They have important activities at post-transcriptional and chromatin modification levels. They regulate gene expression relating to different stresses, growth, and development in eukaryotes. A complete cycle of gene silencing is occurred by the collaboration of these three families. However, these gene families are not yet rigorously studied in the economically important wheat genome. Our bioinformatic analysis based genome-wide identification, characterization, diversification and regulatory components of these gene families identified 7 TaDCL, 39 TaAGO and 16 TaRDR genes from wheat genome against RNAi genes of Arabidopsis thaliana. Phylogenetic analysis of wheat genome with Arabidopsis and rice RNAi genes showed that TaDCL, TaAGO and TaRDR proteins are clustered into four, eight and four subgroups respectively. Domain, motif and exon-intron structure analyses showed that the TaDCL, TaAGO and TaRDR proteins conserve identical characteristics within groups while retain diverse differences between groups. GO annotations implied that a number of biological and molecular pathways are linked to RNAi mechanism in wheat. Gene networking between transcription factors and RNAi proteins indicates that ERF is the leading family linked to maximum RNAi genes followed by MIKC-MADS, C2H2, BBR-BPC, MYB, and Dof. Cis-regulatory elements associated to RNAi genes are predicted to act as regulatory components against various environmental conditions. Expressed sequence tag analysis showed that larger numbers of RNAi genes are expressed in different tissues and organs predicted to play roles for healthy plants and grains. Expression analysis of 7 TaDCL genes using qRT-PCR showed that only TaDCL3a and TaDCL3b had root specific significant expression (p-value<0.05) with no expression in leaf validated EST results. Besides, TaDCL3b and TaDCL4 significantly prompted in drought condition indicating their potential role in drought stress tolerance. Overall results would however help researchers for in-depth biological investigation of these RNAi genes in wheat crop improvement.

genomics↗

bioRxiv2023

Using RNASeq to investigate the involvement of the Ophiocordyceps clock in ant host infection and behavioral manipulation

IntroductionParasites can modify host behavior to ensure their own growth and transmission. Multiple species of the fungi Ophiocordyceps infect ants, but in a species-specific manner; one fungal species co-evolved to successfully modify the behavior of one ant species. However, several characteristics of the behavioral modification seem to be similar across different Ophiocordyceps-ant systems, including a preference for the time of the day for manipulating host behavior. In this study, we explored the various mechanisms via which the circadian clock of Ophiocordyceps might be playing a role in modifying host behavior. We studied O. camponoti-floridani that modifies the behavior of its ant host Camponotus floridanus. To separate the role of the clock in behavior manipulation, from its role in growth and survival, we compared the daily gene expression profile of O. camponoti-floridani to a generalist, non-manipulating fungal parasite, Beauveria bassiana, which also successfully infects the same ant host. ResultsMajority of the 24h rhythmic O. camponoti-floridani genes show peak expression before or at the transitions between light and dark. Rhythmic genes in O. camponoti-floridani, for which B. bassiana lacks an ortholog, were overrepresented for enterotoxin genes. Around half of all genes that show 24h rhythms in either O. camponoti-floridani or B. bassiana showed a consistent difference in their temporal pattern of daily expression. At the halfway mark in O. camponoti-floridani infections, when diseased ants show a loss of 24h rhythms in daily foraging, several fungal clock genes, including Frequency, showed differential expression. Network analyses revealed a single gene cluster, containing White Collar 1 and 2, that showed overrepresentation for genes oscillating every 24h in liquid culture as well as genes differentially expressed while growing inside the ant head. ConclusionThis study identifies several sets of putatively clock-controlled genes and biological processes in O. camponoti-floridani that likely plays a role in modifying the behavior of its ant host. Differential expression of O. camponoti-floridani clock genes or 24h-rhythmic genes during infection is suggestive of either a loss of daily rhythm or a change in the amplitude of rhythmic gene expression. Both possibilities would suggest that a disease-associated change occurs to the functioning of the O. camponoti-floridani clock, and its output, while the fungi grows inside the ant head.

molecular biology↗

bioRxiv2025

AXL-GAS6/PROS1 Interaction: A Critical Switch Between Aberrant- and Healthy Repair Following Alveolar Lung Injury

RationaleIdiopathic pulmonary fibrosis (IPF) is a progressive lung disease characterized by aberrant alveolar repair and excessive fibrosis. The TAM-family receptor tyrosine kinase AXL, activated by GAS6 and PROS1, is implicated in tissue remodeling, but ligand-specific AXL signaling during alveolar repair remains poorly defined. ObjectivesTo investigate ligand specific AXL signaling in IPF and how it impacts epithelial proliferation and repair after alveolar injury in-vivo and in-vitro. MethodsSingle cell RNA sequencing was utilized to understand cell specific expression patterns in IPF patients, followed by functional studies in primary human cell culture and functional spatial digital profiling (FuncOmap) analysis in patient tissue. Longitudinal assessment of repair process after alveolar-specific injury in-vivo was used to complement the in-vitro approach. ResultsAXL expression showed enrichment in basal and aberrant basaloid cells of IPF patients. In-vitro GAS6 increased proliferation of basal cells, while PROS1 counteracted this effect. FuncOmap analysis demonstrates direct in-situ interactions between AXL and both ligands, providing evidence for biological relevance. Investigating longitudinal repair processes in-vivo revealed dynamic regulation of AXL ligands as well as AXL. ConclusionsThese findings highlight the importance of ligand-specific AXL signaling in lung repair and suggest that it dysregulation may contribute to IPF pathogenesis, offering potential therapeutic targets for restoring normal repair processes.

molecular biology↗

bioRxiv2025

Plasmodium Repetome: A mysterious space with a wealth of information

Eukaryotic proteomes harbour repetitive stretches of amino acids that may play critical roles in the biology of that organism. While several tandem repeats (TR) are known to contribute to protein structure and function, information about the vast majority of repeat regions remains obscure. In this article, we have analysed the repeat content of different Plasmodium species and found the leading human malaria-causing P. falciparum (Pf) and P. vivax to be exceptionally rich in TR regions (>40% TR containing proteins). Detailed analysis of Pf repetome showed this intracellular parasite to carry longer TRs, several of which were present in exported proteins important for parasite survival and immune evasion. The repeat regions of Pf were enriched in acidic amino acids and asparagine (Asn), where Asn was more abundant in short and intermediate TRs, suggesting an evolutionary bias influenced by replication slippage and positive selection. Gene ontology analysis of TR containing Pf proteins helped us to understand their cellular localization along with the molecular and biological processes they are involved in. The Pf variable surface antigen families with roles in important pathogenic processes like cytoadherence, immune evasion etc. had low repeat content present within seroreactive peptides. Three-dimensional structure predictions of TR regions revealed several repeats to adopt ordered super-secondary conformations that are known to facilitate intermolecular interactions. Overall, this is a comprehensive study attempting to gain insights on the importance of TRs in malaria parasite biology and suggests a novel route to understanding protein function through the characterization of repeat content.

bioinformatics↗

bioRxiv2022

A quantitative and site-specific atlas of the in vivo citrullinome reveals widespread existence of citrullination

Citrullination is the conversion of peptidyl-arginine into the non-coded amino acid citrulline. Despite its importance in physiology and disease, global identification of citrullinated proteins and precise modification sites has remained challenging. Here, we employed quantitative mass spectrometry-based proteomics to generate a comprehensive atlas of citrullination sites in a physiologically relevant cell type. Collectively, we identified 14.056 citrullination sites within 4.008 proteins and quantified their regulation upon inhibition of the citrullinating enzyme PADI4. Using this rich dataset, we uncover general mechanistic and cell biological principles of citrullination function, while providing site-specific and quantitative information on thousands of PAD4 substrates within cells. Our findings include signature histone marks and numerous modifications on transcriptional regulators and chromatin-related signaling effectors. Additionally, we identify precise citrullination sites on an extensive list of known autoantigens. Collectively, we describe systems attributes of the human citrullinome and provide a resource framework for understanding citrullinaiton at the mechanistic level.

molecular biology↗

bioRxiv2023

Improved detection and quantitation of RNA-interactomes using DIA SILAC

The RNA-interacting proteome is commonly characterized by UV-crosslinking followed by RNA purification, with protein recovery quantified using SILAC labeling followed by data-dependent acquisition (DDA) of proteomic data. However, the low efficiency of UV-crosslinking, combined with limited sensitivity of the DDA approach often restricts detection to relatively abundant proteins, necessitating multiple mass spec injections of fractionated peptides for each biological sample. Here we report an application of data-independent acquisition (DIA) with SILAC in a total RNA-associated protein purification (TRAPP) UV-crosslinking experiment. This gave 15% greater protein detection and lower inter-replicate variation relative to the same biological materials analyzed using DDA, while allowing single-shot analysis of the sample. As proof of concept, we determined the effects of arsenite treatment on the RNA-bound proteome of HEK293T cells. The DIA dataset yielded similar GO term enrichment for RNA-binding proteins involved in cellular stress responses to the DDA dataset while detecting extra proteins unseen by DDA. Overall, the DIA SILAC approach improved detection of proteins over conventional DDA SILAC for generating RNA-interactome datasets, at a lower cost due to reduced machine time.

molecular biology↗

bioRxiv2020

A single N6-methyladenosine site in lncRNA HOTAIR regulates its function in breast cancer cells

N6-methyladenosine (m6A) modification of RNA plays important roles in normal and cancer biology, but knowledge of its function on long noncoding RNAs (lncRNAs) remains limited. Here, we investigate whether m6A regulates the function of the human HOTAIR lncRNA, which contributes to multiple pro-tumor phenotypes in triple-negative breast cancer (TNBC) cells. We identify at least 8 individual m6A sites within HOTAIR, with a single site (A783) consistently methylated. Mutation of A783 impairs cellular proliferation and invasion in HOTAIR-overexpressing TNBC cells. m6A at A783 regulates HOTAIRs ability to localize to chromatin and induce gene pathways that affect tumor progression. In contrast, A783U mutant HOTAIR demonstrates loss-of-function and antimorph behaviors by impairing gene expression changes induced by WT HOTAIR and, in some cases, inducing opposite changes in gene expression. HOTAIR interacts with nuclear m6A reader YTHDC1 and high HOTAIR is significantly associated with shorter overall patient survival, particularly in the context of high YTHDC1. At the molecular level, YTHDC1-HOTAIR interactions are required for chromatin localization and regulation of gene repression. Our work demonstrates how modification of one base in a lncRNA can elicit a distinct gene regulation mechanism and drive disease-associated phenotypic changes such as proliferation and invasion.

molecular biology↗

bioRxiv2022

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

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 canarys 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.

molecular biology↗