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Project Number16-14-10216

Project titleIntegrated Analysis of Gene Expression in the Brain of the Tame Foxes: A Study on a Unique Selection Model of Domestication

Project LeadTrut Lyudmila

AffiliationInstitute of Cytology and Genetics, Siberian Branch of Russian Academy of Sciences,

Implementation period2016 - 2018

Research area 04 - BIOLOGY AND LIFE SCIENCES, 04-104 - General genetics

KeywordsAnimal domestication, social behavior, tolerance, aggression, regulatory systems, gene expression, epigenetics, DNA methylation, miRNA, high throughput sequencing, transcriptomics



A fundamental aim of this research project is to explore molecular-genetic mechanisms underlying the fast and complex transformations of animals occurring in some evolutionary situations. The most illustrative example is the process of historical domestication of animals during which the whole biological organization of their wild ancestors, in particular their behavior, underwent transformation. The original idea behind the project objectives comes to the fact that a principal molecular-genetic mechanism underlying the fast transformation of wild species into domestic forms is the regulatory changes of genetic expression. Testing this idea by comparing the levels of gene expression in contemporary domestic animals which have been exposed to multi-pathway selection pressure during several thousand years with those in their contemporary ancestor forms is uninformative and not convincing. The project will utilize a unique experimental model of domestication developed at the Institute of Cytology and Genetics. This model is based on long-term systematic selection of silver foxes (Vulpes vulpes) for responses to humans: first for the elimination of aggression-avoidance reactions and then for positive and even friendly responses to humans. Thus, the world’s only population of domesticated foxes has been developed. Our unique selection model has the topicality and importance that provide an opportunity to perform comparative analysis of model animal stocks consisting of the animals of the original stocks living at the same time under similar conditions, which has not been made before. To evaluate the differences in gene expression in the brain structures of different fox populations several approaches will be used. In the studies of animal domestication for the first time under comparison will be the level of gene expression and epigenetic modifications involved in the regulation of gene expression, namely microRNA and DNA methylation. For this purpose the next generation high performance sequencing methods will be used. The results obtained will be verified by experimental analysis of expression and methylation of separate genes and also by miRNA. In addition, variations in the content of proteins encoded by differentially expressed genes will be analyzed. Therefore, the cohort of anticipated results will enable us to identify not only the differences in expression of genes in the brain structures of domesticated and not domesticated foxes but also the mechanism of such differences for a number of genes and will bring us closer to understanding of the molecular-genetic mechanisms playing a key role not only in domestication but also in other evolutionary situations, including human evolution.

Expected results
Previously it was demonstrated that domestication results in an altered expression balance of many genes. This could be due to both correlated genetic alterations in the regulation of expression of such genes and the general character of epigenetic modification of their activity. Both mechanisms will be investigated during the project. This study will facilitate the identification of the genes with a differential level of expression in the brain depending on aggressive or domestic behavior as well as the elucidation of the molecular-genetic mechanisms underlying the regulation of expression of these genes. The data will be obtained in a complex study of regulation of gene expression – the level of expression and epigenetic modifications, namely miRNA and DNA methylation. No similar data have so far been presented by any other research teams so particular importance could be attached to such data. Although the anticipated results of the project will be obtained using the model of experimental domestication, such data could provide a unique opportunity for answering some fundamental questions not only on the mechanisms of evolutionary transformations under animal domestication but also on the mechanism of the general biological evolution. The study gains in significance because of its implications for the mechanisms of evolutionary making of human-specific psycho-physiological and behavioral traits such as social relationships, aggression and amicability. An important role in their making was obviously played by selection for such behavioral characteristics, which in the modern literature is referred to as “self-domestication”.



Annotation of the results obtained in 2018
The project “Integrated Analysis of Gene Expression in the Brain of the Tame Foxes: A Study on a Unique Selection Model of Domestication” was carried out by scientists from Novosibirsk Institute of Cytology and Genetics (SB RAS). For three years, geneticists, physiologists, molecular biologists, animal behavior and bioinformatics experts were involved in the study focused on domestication. How did emerge the human-friendly behavior which had never occurred in the wild nature? How did change brain function of a wild animal that became domestic? These questions remain in focus of specialists’ attention since Charles Darwin’s times. The main difficulty of using modern domestic animals for studying domestication is the fact that they have gone through a long history of adaptation and selection for economically useful traits. Therefore, our model for the early stages of the domestication of a silver fox created in the Institute of Cytology and Genetics is particularly valuable. These animals were selected exclusively for friendly behavior towards humans. Over many decades, their friendly behavior became inherited – now the offspring is born already tame. The Institute has also a population of aggressive foxes. Like tame ones, these foxes were also selected generation by generation, but for aggressive behavior towards humans. It is believed that at the initial stage of domestication, aggressive reaction disappeared and animals no longer experienced stress when a human appeared. Instead, they probably showed emotionally positive reactions: they supposedly approached humans, perhaps they wagged their tails and even touched humans. Therefore, the project participants studied mainly those structures of the brain that are associated with the response to stress, learning, memorizing living and nonliving objects. One of the key structures in these processes is hippocampus. In order to understand how its activity changed during the domestication, a series of experiments was performed on foxes. Populations of aggressive and domesticated foxes were tested to reveal the most prominent individuals from which the biomaterial was taken. Further processing of this biomaterial required many steps: from molecular to bioinformatic. Using the sequenced fox genome, scientists have identified 52 genes which expressed differently in the hippocampus of tame and aggressive animals. In addition, about 100 selective sweep regions were found in the genome, which were subjected to positive selection pressure on behavior. 4 from these 52 genes were located in these regions. The most interesting is CYP26B1 gene, which is involved in the regulation of neurogenesis, learning and memory processes through the degradation of all-trans retinoic acid (atRA), a derivative of vitamin A. Besides CYP26B1, 2 genes responsible for the synthesis of atRA were found in hippocampus. Their expression differs in tame and aggressive animals, but they were not located in selective sweep. On the other hand, another 4 genes from the atRA metabolic network were found in “selective regions”, while having almost no expression in hippocampus. They probably are involved in the cellular differentiation of other brain structures, or maybe they are activated at another stage of ontogenesis. To understand the mechanisms of gene expression changes in the brain of tame and aggressive foxes, the scientists decided to investigate the genome methylation profile in hippocampus. They found 40 differentially methylated CpG-islands and about 1.5 thousand individual differentially methylated loci. Thus, selection of experimental foxes by behavior, apparently, affected the epigenetic mechanisms of gene expression regulation. In particular, a locus was found in the regulatory region of the CYP26B1 gene, with methylation level in the hippocampus differing by more than 85% in tame and aggressive foxes. In tame foxes, this site may bind with a transcription factor which facilitates the synthesis of mRNA. The results of our work allow us to conclude that there is a marked increase in neurogenesis in the hippocampus of tame foxes compared to aggressive ones. In the network of atRA metabolism, some genes were detected as possible mechanisms of this effect. We also revealed differential methylation of a key gene - CYP26B1 gene, which have probably affected selection by behavior. Apparently, one of the reasons for the difference in behavior between tame and aggressive foxes lies in the change in atRA metabolism.



1. Ванг Х., Пайпс Л., Трут Л.Н., Гербек Ю., Владимирова А.В., Гулевич Р.Г., Харламова А.В., Джонсон Й.Л., Ацланд Дж.М., Кукекова А.В., Кларк А.Г. Genomic responses to selection for tame/aggressive behaviors in the silver fox (Vulpes vulpes) Proceedings of the National Academy of Sciences of the United States of America, 115 (41), pp. 10398-10403 (year - 2018).

2. Кукекова А, Джонсон Дж, Хианг Х, Фенг С, Лиу С, Рандо Х, Харламова А, Гербек Ю, Сердюкова Н, Хионг З, Беклемишева В, Кэпфли К, Гулевич Р, Владимирова А, Хекман Дж, Перельман П, Графодатский А, О Брейн С, Ванг Х, Кларк А, Окланд Г, Трут Л, Жанг Г Red fox genome assembly identifies genomic regions associated with tame and aggressive behaviours Nature Ecology and Evolution, 2, pages1479–1491 (year - 2018).

3. Хекман Дж.П., Джонсон Дж.Л., Эдвардс В., Владимирова А.В., Гулевич Р.Г., Форд А.Л., Харламова А.В., Гербек Ю., Окланд Г.М., Рецман Л.Т., Трут Л.Н., Кукекова А.В Anterior pituitary transcriptome suggests differences in ACTH release in tame and aggressive foxes G3: Genes, Genomes, Genetics, 8(3), с. 859-873 (year - 2018).

4. Гербек Ю.Э., Гулевич Р.Г. Neuropeptides as facilitators of domestication Cell and Tissue Research, - (year - 2018).



7. Дугаткин Л., Трут Л.Н. How to Tame a Fox and Build a Dog University of Chicago Press, Chicago, U.S.A., 240с (year - 2017).

8. - Российские и зарубежные исследователи секвенировали геном одомашненных лисиц Наука в Сибири, 07 августа 2018 (year - ).

Annotation of the results obtained in 2016
The Project is directed to clarification of molecular genetic mechanisms of rapid complex animal transformations occurred in the process of historical domestication. A fundamental change in the behavior of domestic animals toward humans as compared with their wild relatives occurred in the evolutionary short period (several thousand years) and was accompanied by morphophysiological changes (the appearance of facial skeleton and limb anomalies, floppy ears, curled tail, piebald, change in the reproduction periods, etc.) (Price, 2002). In the present work, silver foxes obtained in the Institute of Cytology and Genetics (Siberian Branch, Russian Academy of Sciences) by long-term selection on emotionally positive reactions to humans (a unique experimental domestication model) was used (Trut et al., 2009). Based on this model, the same behavioral transformation as during the historical domestication, as well as the appearance of morphophysiological changes in the population similar to those in domestic animals, was demonstrated. The population with genetically increased aggression to humans was obtained by the fox selection in the opposite direction. Thus, the model represents a population with the complete absence of aggression (and moreover friendly attitude towards humans) and a population with increased aggression, as well as the control population; this allows to model early stages of domestication associated exactly with a change in the animal behavior, but not with their economically useful traits (Trut et al., 2009). The goal of this work is to clarify the mechanisms of the gene expression regulation in dorsal hippocampus of domesticated (“tame”) and undomesticated foxes, the brain structure playing a key role in social behavior and stress reactivity, as well as in cognitive abilities and training (Weaver et al., 2004; Doherty et al., 2016; Pollano et al., 2016). The solution of this problem can provide not only new data on molecular genetic mechanisms of experimental domestication, but also help to understand the transformation mechanisms of all biological organization of domestic animals, which can be associated with a cascade character of the gene expression changes and their wide pleiotropic effects. The foxes with the largest expression of tame or aggressive behavior were selected for the experiment. High-quality RNA was isolated from the dorsal hippocampus samples and cDNA libraries were obtained; they were sequenced on the Illumina HiSeq platform calculated 75 nt direct reads and at least 40 million reads (more than 400 million reads for 8 libraries). The filtered reads were mapped on the dog genome by means of the STAR program and processed by the Cufflinks program line. The non-metric multidimensional scaling method allocated the clusters that consist only of the tame fox samples and only of the aggressive fox samples. The search for differentially expressing genes was conducted by means of the Cuffdiff program; 496 genes were detected. The analysis of data obtained by means of the KEGG database demonstrated that a significant amount of detected differentially expressing genes refer to the functional groups associated with metabolic pathways and cellular processes. This is probably explained by the adaptation significance of these genes that rapidly react to any habitat changes and the processes of species divergence, but not only by their role in the domestication processes. At the same time, functional groups of the genes associated with the nervous system were detected: calcium signaling pathway, long-term potentiation, glutamatergic synapse, GABAergic synapse; they are associated with the regulation of different forms of behavior, such as anxiety, fear, social recognition, learning and memory, as well as with neuronal plasticity and stress. In addition, the functional “axon guidance” group (associated with the neuron proliferation and axon growth) was allocated. The allocation of this group is probably associated with increased neurogenesis in adult hippocampus in less aggressive foxes as it was previously demonstrated (Huang et al., 2015). Thus, a change in the expression of the genes of key neurohumoral brain systems (that probably play a leading role in the change of the fox behavior during the selection on domestication) was found. The analysis of data obtained also demonstrated a change in the expression of endocrine system genes, namely estrogen signaling pathway and functional groups of the genes associated with thyroid hormones. These changes can be associated with a decrease in the interrelation between the reproductive rate and environmental photoperiod during the experimental fox domestication (that is more pronounced in almost all domestic animals). • Doherty TS, Forster A., Roth TL. Global and gene-specific DNA methylation alterations in the adolescent amygdala and hippocampus in an animal model of caregiver maltreatment // Behav Brain Res. 2016. 298(Pt A):55-61. doi: 10.1016/j.bbr.2015.05.028. • Huang S., Slomianka L., Farmer A.J., Kharlamova A.V., Gulevich R.G., Herbeck Y.E., Trut L.N., Wolfer D.P., Amrein I. Selection for tameness, a key behavioral trait of domestication, increases adult hippocampal neurogenesis in foxes.// Hippocampus. 2015 Aug;25(8):963-75. doi: 10.1002/hipo.22420. • Pollano A, Zalosnik MI, Durando PE, Suárez MM. Differential effects of tianeptine on the dorsal hippocampal volume of rats submitted to maternal separation followed by chronic unpredictable stress in adulthood // Stress. 2016. 19(6): 599-608. • Price, E.O. Animal Domestication and Behaviour. CAB International. 2002. Wallingford. UK. • Trut L., Oskina I., Kharlamova A. Animal evolution during domestication: the domesticated fox as a model // BioEssays. 2009. 31: 349–360. • Weaver I. C., Cervoni N., Champagne F. A., D'Alessio A. C., Sharma S., Seckl J. R., ... Meaney M. J. Epigenetic programming by maternal behavior // Nature Neurosci. 2004. 7(8): 847-854.



1. Гербек Ю.Э., Хантемирова А.Р., Антонов Е.В., Гончарова Н.И., Гулевич Р. Г., Шепелева Д. В., Трут Л. Н. Expression of the genes for DNA methyltransferases in silver foxes experimentally selected for domestication Russian Journal of Genetics, - (year - 2017).

Annotation of the results obtained in 2017
The project “Integrated Analysis of Gene Expression in the Brain of the Tame Foxes: A Study on a Unique Selection Model of Domestication” is performed by scientists of the Institute of Cytology and Genetics, Novosibirsk, Russia. Geneticists, physiologists, molecular biologists, animal behaviorists, and computational biology experts have worked for it for two years. How did the friendly attitude to humans, never found in wild nature, arise? How did the work of the brain change in a wild animal as it became domestic? These questions have attracted scientists' attention since Charles Darwin's time. The main difficulty in studying domestication in present-day domestic animals is their long history of adaptation and selection for commercial traits. In this respect, the silver fox model of early domestication stages developed at the Institute of Cytology and Genetics is of special value. These animals were obtained solely by selection for attitude to humans. Their friendly behavior was inherited for several decades, and their current generations are born tame. The Institute also supports a population of aggressive foxes. They have been selected in the opposite direction, for aggressive response to humans. It is believed that early stages of domestication resulted in nonaggressive response and absence of stress in animals when contacting humans. Therefore, the investigators trace changes in the operation of the brain divisions involved in stress regulation rather than changes in higher nervous activity, occurring at later domestication stages. The hippocampus is the key brain division regulating stress. To reveal changes in its operation occurring in domestication, experiments were performed with foxes. Populations of aggressive and domesticated foxes were screened to detect the most prominent individuals, and biologic samples were taken from them. The samples were analyzed by many methods, from molecular to computational. Different expression levels in the hippocampi of domesticated and aggressive animals were found in 500 genes. Tens of them formed 10 functional groups reproducibly differing between domesticated and aggressive foxes. Presumably, they make the largest contribution to the formation of differences in hippocampus operation. These groups are associated with behavior formation, stress, and neurogenesis in adult animals. These processes form a triangle whose apexes influence each other. For instance, stress decrease reduces aggression and increases neurogenesis. Elevated neurogenesis mitigates the adverse effects of stress. Finally, social behavior reduces stress and increases neurogenesis. It is conceivable that the recognized functional groups of genes constitute the molecular base of these interactions. An interesting commonly recognized effect of domestication is the emergence of traits not directly related to behavior. They make up the so-called ‘domestication syndrome’. It includes morphological skull changes, the emergence of white patches on the forehead, deviation from the seasonal reproduction cycle, etc. It was believed for long that these traits were sort of markers resulting from the selection for friendly behavior towards humans. The experiments conducted in our project yielded an unexpected result. The mentioned traits, thought to be typical solely of domesticated animals, appeared in the aggressive fox population. It is likely that they are associated with aberrations in regulatory systems, similar to those occurring in domestication and bringing about various forms. Now it is clear that the cause was not the selection for friendly behavior but the selection for a social behavior shift in any direction, including aggression. In particular, it was found that the expression of the TSHR gene, essential for the seasonal reproduction habit and called the domestication gene, changed in both aggressive and domesticated foxes in comparison to unselected ones. These results may demand that the concept of the domestication syndrome be revised. An important problem is the molecular base of the detected changes in gene expression in the hippocampus. One of the main factors whose role in stress and behavior regulation is increasingly recognized is the degradation of mRNA by microRNAs. Nevertheless, it follows from our studies that this important mechanism of gene regulation plays a minor role in the difference between the hippocampi of domesticated and aggressive foxes. Therefore, our future work will be aimed at search for chemical modifications (methylation) of DNA in genes differentially expressed in domesticated and aggressive foxes. Other scientists report that DNA methylation is essential in the regulation of both behavior and stress; thus, the expression of genes studied in the project may be regulated by these chemical modifications of the genome.



1. Трут Л.Н., Харламова А.В., Владимирова А.В., Гербек Ю.Э. On selection of foxes for enhanced aggressiveness and its correlated implications Вавиловский журнал генетики и селекции., 21(4):392-401. (year - 2017).

2. Овчинников В.Ю., Антонов Е.В. , Васильев Г.В., Шихевич С.Г., Шепелева Д.В., Гербек Ю.Э. Hippocampal glucocorticoid receptor and microRNA gene expression and serum cortisol concentration in foxes selected for behavior toward humans Вавиловский журнал генетики и селекции, - (year - 2017).

3. Орлов Ю.Л., Баранова А.В., Гербек Ю.Э. Evolutionary Biology at Belyaev Conference – 2017: Introductory Note BMC Evolutionary Biology, - (year - 2017).

4. Науменко Ф.М., Абнизова И.И., Бека Н., Генаев М.А., Орлов Ю.Л. Novel read density distribution score shows possible aligner artefacts, when mapping a single chromosome BMC Genomics, - (year - 2017).

5. - Агрессивная среда Наука 2.0, декабрь 2017 (year - ).