Genome sequencing reveals loci under artificial selection that underlie disease phenotypes in the laboratory rat.

Large numbers of inbred laboratory rat strains have been developed for a range of complex disease phenotypes. To gain insights into the evolutionary pressures underlying selection for these phenotypes, we sequenced the genomes of 27 rat strains, including 11 models of hypertension, diabetes, and insulin resistance, along with their respective control strains. Altogether, we identified more than 13 million single-nucleotide variants, indels, and structural variants across these rat strains. Analysis of strain-specific selective sweeps and gene clusters implicated genes and pathways involved in cation transport, angiotensin production, and regulators of oxidative stress in the development of cardiovascular disease phenotypes in rats. Many of the rat loci that we identified overlap with previously mapped loci for related traits in humans, indicating the presence of shared pathways underlying these phenotypes in rats and humans. These data represent a step change in resources available for evolutionary analysis of complex traits in disease models.

Cooperativity and rapid evolution of cobound transcription factors in closely related mammals.

To mechanistically characterize the microevolutionary processes active in altering transcription factor (TF) binding among closely related mammals, we compared the genome-wide binding of three tissue-specific TFs that control liver gene expression in six rodents. Despite an overall fast turnover of TF binding locations between species, we identified thousands of TF regions of highly constrained TF binding intensity. Although individual mutations in bound sequence motifs can influence TF binding, most binding differences occur in the absence of nearby sequence variations. Instead, combinatorial binding was found to be significant for genetic and evolutionary stability; cobound TFs tend to disappear in concert and were sensitive to genetic knockout of partner TFs. The large, qualitative differences in genomic regions bound between closely related mammals, when contrasted with the smaller, quantitative TF binding differences among Drosophila species, illustrate how genome structure and population genetics together shape regulatory evolution.

Region-Specific Genetic Diversity of Black Rats (Rattus rattus Complex) in Southeast and East Asia Shaped by Rapid Population Expansion Events.

Among the six mitochondrial DNA lineages of the black rat (Rattus rattus Complex; RrC), lineages II and IV are widespread in Southeast and East Asia. This study explored their demographic history using 17 new sequences from the Miyako Islands in the Ryukyu archipelago, together with 178 publicly available cytochrome b sequences. We defined six and two haplotype groups showing rapid population expansion signals in Lineages II and IV, respectively. The six haplotype groups of Lineage II were represented by haplotypes from 1) Myanmar/Bangladesh/Northeast India, 2) Laos, 3) Thailand, 4) Indonesia/Philippines, 5) Vietnam/southern China, and 6) the Ryukyu archipelago. These expansion times were estimated using time-dependent evolutionary rates to be 115,300 years ago (ya), 128,500 ya, 9600 ya, 10,600 ya, 7200 ya, and 1400 ya, respectively, although all had large confidence intervals. The two groups of Lineage IV were recovered from the mainland and islands of Southeast Asia with predicted expansion times of 197,000 ya and 5800 ya, respectively. These results suggest that climatic fluctuations during the last 200,000 years of the Quaternary, affected the population dynamics in subtropical areas at different times. Furthermore, the results of the younger rapid expansion events of RrC suggest the possibility of agricultural advancement and dispersal of Neolithic farmers to different areas within the mainland and islands of Southeast Asia during the Holocene. A subset of rats from the Miyako Islands were found to have the same lineage IV haplotypes as those in Southeast Asia, suggesting a recent introduction of these new lineages.

Brown rat demography reveals pre-commensal structure in eastern Asia before expansion into Southeast Asia.

Fossil evidence indicates that the globally distributed brown rat (Rattus norvegicus) originated in northern China and Mongolia. Historical records report the human-mediated invasion of rats into Europe in the 1500s, followed by global spread because of European imperialist activity during the 1600s-1800s. We analyzed 14 genomes representing seven previously identified evolutionary clusters, and tested alternative demographic models to infer patterns of range expansion, divergence times, and changes in effective population (N e) size for this globally important pest species. We observed three range expansions from the ancestral population that produced the Pacific (diverged ∼16.1 kya), eastern China (∼17.5 kya), and Southeast (SE) Asia (∼0.86 kya) lineages. Our model shows a rapid range expansion from SE Asia into the Middle East and then continued expansion into central Europe 788 yr ago (1227 AD). We observed declining N e within all brown rat lineages from 150-1 kya, reflecting population contractions during glacial cycles. N e increased since 1 kya in Asian and European, but not in Pacific, evolutionary clusters. Our results support the hypothesis that northern Asia was the ancestral range for brown rats. We suggest that southward human migration across China between the 800s-1550s AD resulted in the introduction of rats to SE Asia, from which they rapidly expanded via existing maritime trade routes. Finally, we discovered that North America was colonized separately on both the Atlantic and Pacific seaboards, by evolutionary clusters of vastly different ages and genomic diversity levels. Our results should stimulate discussions among historians and zooarcheologists regarding the relationship between humans and rats.

The Year of the Rat: The Rat Genome Database at 20: a multi-species knowledgebase and analysis platform.

Formed in late 1999, the Rat Genome Database (RGD, https://rgd.mcw.edu) will be 20 in 2020, the Year of the Rat. Because the laboratory rat, Rattus norvegicus, has been used as a model for complex human diseases such as cardiovascular disease, diabetes, cancer, neurological disorders and arthritis, among others, for >150 years, RGD has always been disease-focused and committed to providing data and tools for researchers doing comparative genomics and translational studies. At its inception, before the sequencing of the rat genome, RGD started with only a few data types localized on genetic and radiation hybrid (RH) maps and offered only a few tools for querying and consolidating that data. Since that time, RGD has expanded to include a wealth of structured and standardized genetic, genomic, phenotypic, and disease-related data for eight species, and a suite of innovative tools for querying, analyzing and visualizing this data. This article provides an overview of recent substantial additions and improvements to RGD's data and tools that can assist researchers in finding and utilizing the data they need, whether their goal is to develop new precision models of disease or to more fully explore emerging details within a system or across multiple systems.

Disruption of insulin receptor substrate-2 impairs growth but not insulin function in rats.

Insulin receptor substrate (IRS)-2, along with IRS-1, is a key signaling molecule that mediates the action of insulin and insulin-like growth factor (IGF)-I. The activated insulin and IGF-I receptors phosphorylate IRSs on tyrosine residues, leading to the activation of downstream signaling pathways and the induction of various physiological functions of insulin and IGF-I. Studies using IRS-2 knockout (KO) mice showed that the deletion of IRS-2 causes type 2 diabetes due to peripheral insulin resistance and impaired ß-cell function. However, little is known about the roles of IRS-2 in other animal models. Here, we created IRS-2 KO rats to elucidate the physiological functions of IRS-2 in rats. The body weights of IRS-2 KO rats at birth were lower compared with those of their WT littermates. The postnatal growth of both male and female IRS-2 KO rats was also suppressed. Compared with male WT rats, the glucose and insulin tolerance of male IRS-2 KO rats were slightly enhanced, whereas a similar difference was not observed between female WT and IRS-2 KO rats. Besides the modestly increased insulin sensitivity, male IRS-2 KO rats displayed the enhanced insulin-induced activation of the mTOR complex 1 pathway in the liver compared with WT rats. Taken together, these results indicate that in rats, IRS-2 plays important roles in the regulation of growth but is not essential for the glucose-lowering effects of insulin.

Zp4 is completely dispensable for fertility in female rats†.

Zona pellucida (ZP), which is composed of at most four extracellular glycoproteins (ZP1, ZP2, ZP3, and ZP4) in mammals, shelters the oocytes and is vital in female fertility. Several studies have identified the indispensable roles of ZP1-3 in maintaining normal female fertility. However, the understanding of ZP4 is still very poor because only one study on ZP4-associated infertility performed in rabbits has been reported up to date. Here we investigated the function of mammalian Zp4 by creating a knockout (KO) rat strain (Zp4-/- rat) using CRISPR-Cas9-mediated DNA-editing method. The influence of Zp4 KO on ZP morphology and some pivotal processes of reproduction, including oogenesis, ovulation, fertilization, and pup production, were studied using periodic acid-Schiff's staining, superovulation, in vitro fertilization, and natural mating. The ZP morphology in Zp4-/- rats was normal, and none of these pivotal processes was affected. This study renewed the knowledge of mammalian Zp4 by suggesting that Zp4 was completely dispensable for female fertility.

Genomic analyses reveal three independent introductions of the invasive brown rat (Rattus norvegicus) to the Faroe Islands.

Population genomics offers innovative approaches to test hypotheses related to the source and timing of introduction of invasive species. These approaches are particularly appropriate to study colonization of island ecosystems. The brown rat is a cold-hardy global invasive that has reached most of the world's island ecosystems, including even highly isolated archipelagoes such as the Faroe Islands in the North Atlantic Ocean. Historic records tell of rats rafting to the southern island of Suðuroy in 1768 following a shipwreck off the coast of Scotland, then expanding across the archipelago. We investigated the demographic history of brown rats in the Faroes using 50,174 SNPs. We inferred three independent introductions of rats, including to Suðuroy, the islands of Borðoy and Viðoy, and onto Streymoy from which they expanded to Eysturoy and Vágar. All Faroese populations showed signs of strong bottlenecks and declining effective population size. We inferred that these founder events removed low frequency alleles, the exact data needed to estimate recent demographic histories. Therefore, we were unable to accurately estimate the timing of each invasion. The difficulties with demographic inference may be applicable to other invasive species, particularly those with extreme and recent bottlenecks. We identified three invasions of brown rats to the Faroe Islands that resulted in highly differentiated populations that will be useful for future studies of life history variation and genomic adaptation.

Pleiotropic Impacts of Macrophage and Microglial Deficiency on Development in Rats with Targeted Mutation of the Csf1r Locus.

We have produced Csf1r-deficient rats by homologous recombination in embryonic stem cells. Consistent with the role of Csf1r in macrophage differentiation, there was a loss of peripheral blood monocytes, microglia in the brain, epidermal Langerhans cells, splenic marginal zone macrophages, bone-associated macrophages and osteoclasts, and peritoneal macrophages. Macrophages of splenic red pulp, liver, lung, and gut were less affected. The pleiotropic impacts of the loss of macrophages on development of multiple organ systems in rats were distinct from those reported in mice. Csf1r-/- rats survived well into adulthood with postnatal growth retardation, distinct skeletal and bone marrow abnormalities, infertility, and loss of visceral adipose tissue. Gene expression analysis in spleen revealed selective loss of transcripts associated with the marginal zone and, in brain regions, the loss of known and candidate novel microglia-associated transcripts. Despite the complete absence of microglia, there was little overt phenotype in brain, aside from reduced myelination and increased expression of dopamine receptor-associated transcripts in striatum. The results highlight the redundant and nonredundant functions of CSF1R signaling and of macrophages in development, organogenesis, and homeostasis.

Mitogenomes Reveal Multiple Colonization of Mountains by Rattus in Sundaland.

Tropical mountains are cradles of biodiversity and endemism. Sundaland, tropical Southeast Asia, hosts 3 species of Rattus endemic to elevations above 2000 m with an apparent convergence in external morphology: Rattus korinchi and R. hoogerwerfi from Sumatra, and R. baluensis from Borneo. A fourth one, R. tiomanicus, is restricted to lowland elevations across the whole region. The origins of these endemics are little known due to the absence of a robust phylogenetic framework. We use complete mitochondrial genomes from the 3 high altitude Rattus, and several related species to determine their relationships, date divergences, reconstruct their history of colonization, and test for selection on the mitochondrial DNA. We show that mountain colonization happened independently in Borneo (<390 Kya) and Sumatra (~1.38 Mya), likely from lowland lineages. The origin of the Bornean endemic R. baluensis is very recent and its genetic diversity is nested within the diversity of R. tiomanicus. We found weak evidence of positive selection in the high-elevation lineages and attributed the greater nonsynonymous mutations on these branches (specially R. baluensis) to lesser purifying selection having acted on the terminal branches in the phylogeny.

Out of Southern East Asia of the Brown Rat Revealed by Large-Scale Genome Sequencing.

The geographic origin and migration of the brown rat (Rattus norvegicus) remain subjects of considerable debate. In this study, we sequenced whole genomes of 110 wild brown rats with a diverse world-wide representation. We reveal that brown rats migrated out of southern East Asia, rather than northern Asia as formerly suggested, into the Middle East and then to Europe and Africa, thousands of years ago. Comparison of genomes from different geographical populations reveals that many genes involved in the immune system experienced positive selection in the wild brown rat.

The rat as a model for studying noise injury and otoprotection.

A major challenge for those studying noise-induced injury pre-clinically is the selection of an animal model. Noise injury models are particularly relevant in an age when people are constantly bombarded by loud noise due to occupation and/or recreation. The rat has been widely used for noise-related morphological, physiological, biochemical, and molecular assessment. Noise exposure resulting in a temporary (TTS) or permanent threshold shift (PTS) yields trauma in peripheral and central auditory related pathways. While the precise nature of noise-related injuries continues to be delineated, both PTS and TTS (with or without hidden hearing loss) result in homeostatic changes implicated in conditions such as tinnitus and hyperacusis. Compared to mice, rats generally tolerate exposure to loud sounds reasonably well, often without exhibiting other physical non-inner ear related symptoms such as death, loss of consciousness, or seizures [Skradski, Clark, Jiang, White, Fu, and Ptacek (2001). Neuron 31, 537-544; Faingold (2002). Hear. Res. 168, 223-237; Firstova, Abaimov, Surina, Poletaeva, Fedotova, and Kovalev (2012). Bull Exp. Biol. Med. 154, 196-198; De Sarro, Russo, Citraro, and Meldrum (2017). Epilepsy Behav. 71, 165-173]. This ability of the rat to thrive following noise exposure permits study of long-term effects. Like the mouse, the rat also offers a well-characterized genome allowing genetic manipulations (i.e., knock-out, viral-based gene expression modulation, and optogenetics). Rat models of noise-related injury also provide valuable information for understanding mechanistic changes to identify therapeutic targets for treatment. This article provides a framework for selection of the rat as a model for noise injury studies.

l-Methionine activates Nrf2-ARE pathway to induce endogenous antioxidant activity for depressing ROS-derived oxidative stress in growing rats.

BACKGROUND: Methionine is an essential sulfur-containing amino acid. To elucidate the influence of l-methionine on activation of the nuclear factor erythroid 2-related factor 2-antioxidant responsive element (Nrf2-ARE) antioxidant pathway to stimulate the endogenous antioxidant activity for depressing reactive oxygen species (ROS)-derived oxidative stress, male Wistar rats were orally administered l-methionine daily for 14 days. RESULTS: With the intake of l-methionine, Nrf2 was activated by l-methionine through depressing Keap1 and Cul3, resulting in upregulation of ARE-driven antioxidant expression (glutamate cysteine ligase catalytic subunit, glutamate cysteine ligase modulatory subunit, glutathione synthase (GS), catalase (CAT), superoxide dismutase (SOD), heme oxygenase 1, NAD(P)H:quinone oxidoreductase 1, glutathione reductase (GR), glutathione S-transferase (GST), glutathione peroxidase (GPx)) with increasing l-methionine availability. Upon activation of Nrf2, glutathione synthesis was increased through upregulated expression of methionine adenosyltransferase, S-adenosylhomocysteine hydrolase, cystathionine ß-synthase, cystathionine γ-lyse, glutamate cysteine ligase (GCL) and GS, while hepatic expressions of methionine sulfoxide reductases (MsrA, MsrB2, MsrB3) and hepatic enzyme activities (CAT, SOD, GCL, GR, GST, GPx) were uniformly stimulated with increasing consumption of l-methionine. As a result, hepatic content of ROS and MDA were effectively reduced by l-methionine intake. CONCLUSION: The present study demonstrates that methionine availability plays a critical role in activation of the Nrf2-ARE pathway to induce an endogenous antioxidant response for depressing ROS-derived oxidative stress, which is primarily attributed to the stimulation of methionine sulfoxide reductase expression and glutathione synthesis. © 2019 Society of Chemical Industry.

Population Genomics Reveals Speciation and Introgression between Brown Norway Rats and Their Sibling Species.

Murine rodents are excellent models for study of adaptive radiations and speciation. Brown Norway rats (Rattus norvegicus) are successful global colonizers and the contributions of their domesticated laboratory strains to biomedical research are well established. To identify nucleotide-based speciation timing of the rat and genomic information contributing to its colonization capabilities, we analyzed 51 whole-genome sequences of wild-derived Brown Norway rats and their sibling species, R. nitidus, and identified over 20 million genetic variants in the wild Brown Norway rats that were absent in the laboratory strains, which substantially expand the reservoir of rat genetic diversity. We showed that divergence of the rat and its siblings coincided with drastic climatic changes that occurred during the Middle Pleistocene. Further, we revealed that there was a geographically widespread influx of genes between Brown Norway rats and the sibling species following the divergence, resulting in numerous introgressed regions in the genomes of admixed Brown Norway rats. Intriguing, genes related to chemical communications among these introgressed regions appeared to contribute to the population-specific adaptations of the admixed Brown Norway rats. Our data reveals evolutionary history of the Brown Norway rat, and offers new insights into the role of climatic changes in speciation of animals and the effect of interspecies introgression on animal adaptation.

Rapid Evolution of Genes Involved in Learning and Energy Metabolism for Domestication of the Laboratory Rat.

The laboratory rat, widely used in biomedical research, is domesticated from wild brown rat. The origin and genetic mechanism underlying domestication of the laboratory rat remain largely elusive. In the present study, large scale genomes supported a single origin for the laboratory rat, possibly from a sister group to wild rats from Europe/Africa/Middle East. Genomic and transcriptomic analyses uncovered many artificially selected genes (e.g., FOXP2, B3GAT1, and CLOCK) involved in the nervous system. These genes associate with learning ability and regulation of circadian rhythm, which likely enabled the successful domestication of the laboratory rat. Particularly, many genes, including mitochondrial genes responsible for energy metabolism, displayed a substantially increased expression in the brain of laboratory rats compared with wild rats. Our findings demystify the origin and evolution of this model animal, and provide insight into the process of its domestication.

Relatedness decreases and reciprocity increases cooperation in Norway rats.

Kin selection and reciprocity are two mechanisms underlying the evolution of cooperation, but the relative importance of kinship and reciprocity for decisions to cooperate are yet unclear for most cases of cooperation. Here, we experimentally tested the relative importance of relatedness and received cooperation for decisions to help a conspecific in wild-type Norway rats (Rattus norvegicus). Test rats provided more food to non-kin than to siblings, and they generally donated more food to previously helpful social partners than to those that had refused help. The rats thus applied reciprocal cooperation rules irrespective of relatedness, highlighting the importance of reciprocal help for cooperative interactions among both related and unrelated conspecifics.

Collagen-induced arthritis in Dark Agouti rats as a model for study of immunological sexual dimorphisms in the human disease.

Collagen-induced arthritis (CIA) is a frequently used animal model of rheumatoid arthritis, human autoimmune disease that exhibits clear sex bias in incidence and clinical course. Female Dark Agouti rats immunized for CIA showed also greater incidence and higher arthritic score than their male counterparts. The study investigated sex differences in mechanisms controlling the primary immune responses in draining lymph nodes (dLNs), as a factor contributing to this dimorphism. The higher frequencies of CD4 + CD25 + Foxp3- cells, presumably activated effector T (Teff) cells, and IL-17+, IFN-γ + and IL-17 + IFN-γ + T cells were found in female compared with male rat dLNs. However, the frequency of CD4 + CD25 + Foxp3+ T regulatory cells (Treg) did not differ between sexes. Thus, CD4+ Teff cells/Treg ratio, and IL-17+ T cells/Treg and IFN-γ + T cells/Treg ratios were higher in female than in male rats, and among them was found lower frequency of PD-1+ cells. This suggested less efficient control of (auto)immune Th1/Th17 cell responses in female rat dLNs. On the contrary, the frequency of IL-4+ T cells was lower in female than in male rat dLNs. Consistently, the ratio of serum levels of collagen-specific IgG2a (IFN-γ-dependent, with an important pathogenic role in CIA) and IgG1 (IL-4-dependent) was shifted towards IgG2a in female compared with male rats. As a whole, the study suggests that sexual dimorphism in the control of T cell activation/polarization could contribute to sex bias in the susceptibility to CIA. Moreover, the study advises the use of animals of both sexes in the preclinical testing of new drugs for rheumatoid arthritis.

Metals Differentially Activate Ovarian Cancer G Protein-Coupled Receptor 1 in Various Species.

Human, mouse, and zebrafish ovarian cancer G protein-coupled receptors (OGR1s) are activated by both metals and extracellular protons. In the present study, we examined whether pig, rat, chicken, and Xenopus OGR1 homologs could sense and be activated by protons and metals. We found that all homologs stimulated serum response element (SRE)-driven promoter activities when they are stimulated by protons. On the other hand, metals differentially activated the homologs. The results using chimeric receptors of human and zebrafish OGR1s indicate that the specificity of the metal-induced activation lies in the extracellular region. These results suggest that protons are an evolutionally conserved agonist of OGR1. However, the types of metals that activated the receptor differed among the homologs.

Avian and murine neurosecretory protein GL participates in the regulation of feeding and energy metabolism.

Probing previously unknown neuropeptides and/or peptide hormones is essential for our understanding of the regulation of energy homeostasis in the brain. We recently performed a cDNA subtractive screening of the chicken hypothalamus, which contained one of the feeding and energy metabolic centers. We found a gene encoding a novel protein of 182 amino acid residues, including one putative small secretory protein of 80 amino acid residues. The C-terminal amino acids of the small protein were Gly-Leu-NH2, and as a result, the small protein was termed neurosecretory protein GL (NPGL). Subcutaneous and intracerebroventricular infusions of NPGL increased body mass gain in chicks, suggesting a central role for this protein in regulating growth and energy homeostasis. A database search revealed that the Npgl gene is conserved in vertebrates, including mice and rats. This review summarizes the advances in the characterization, localization, and biological action of NPGL, in birds and rodents.

Sexual Dimorphism of NADPH Oxidase/H2O2 System in Rat Thyroid Cells; Effect of Exogenous 17ß-Estradiol.

It has long been observed that females are more susceptible to thyroid diseases than males. Epidemiological and experimental data show that actions of hormonal factors-especially estrogens-may explain such disparity. However, the exact cause and mechanisms of this sexual dimorphism remain so far unknown. Therefore, we aimed at evaluating the effect of 17ß-estradiol on the redox balance in thyroids of male and female rats. Expression of nicotinamide adenine dinucleotide phosphate (NADPH) oxidases, i.e., dual oxidase 1 (DUOX1), dual oxidase 2 (DUOX2) and NADPH oxidase 4 (NOX4), and hydrogen peroxide (H2O2) levels were evaluated in the primary cell cultures derived from thyroid glands of adult male or female Wistar rats. The measurement was made before and after treatment with 17ß-estradiol alone or with addition of one of its receptor antagonists. We found that under basal conditions female thyroid cells are exposed to higher concentrations of H2O2, most likely due to NOX/DUOX enzymes activity. Additionally, exogenous 17ß-estradiol stimulated NOX/DUOX expression as well as H2O2 production, and this effect was mainly mediated through ERα. In conclusion, oxidative processes may constitute mechanisms responsible for sexual dimorphism of thyroid diseases. Exogenous 17ß-estradiol may play a crucial pathogenic role in thyroid diseases via oxidative mechanisms, however without any gender differences.