Unraveling patterns of disrupted gene expression across a complex tissue

Whole tissue RNASeq is the standard approach for studying gene expression divergence in evolutionary biology and provides a snapshot of the comprehensive transcriptome for a given tissue. However, whole tissues consist of diverse cell types differing in expression profiles, and the cellular composition of these tissues can evolve across species. Here, we investigate the effects of different cellular composition on whole tissue expression profiles. We compared gene expression from whole testes and enriched spermatogenesis populations in two species of house mice, Mus musculus musculus and M. m. domesticus, and their sterile and fertile F1 hybrids, which differ in both cellular composition and regulatory dynamics. We found that cellular composition differences skewed expression profiles and differential gene expression in whole testes samples. Importantly, both approaches were able to detect large-scale patterns such as disrupted X chromosome expression although whole testes sampling resulted in decreased power to detect differentially expressed genes. We encourage researchers to account for histology in RNASeq and consider methods that reduce sample complexity whenever feasible. Ultimately, we show that differences in cellular composition between tissues can modify expression profiles, potentially altering inferred gene ontological processes, insights into gene network evolution, and processes governing gene expression evolution.

Homogeneously Staining Regions (HSR) in Chromosome 1 of the House Mouse: Synapsis and Recombination at Meiosis

Amplified sequences constitute a large part of mammalian genomes. A chromosome 1 containing 2 large (up to 50 Mb) homogeneously staining regions (HSRs) separated by a small inverted euchromatic region is present in many natural populations of the house mouse (Mus musculus musculus). The HSRs are composed of a long-range repeat cluster, Sp100-rs, with a repeat length of 100 kb. In order to understand the organization and function of HSRs in meiotic chromosomes, we examined synapsis and recombination in male mice hetero- and homozygous for the HSR-carrying chromosome using FISH with an HSR-specific DNA probe and immunolocalization of the key meiotic proteins. In all homozygous and heterozygous pachytene nuclei, we observed fully synapsed linear homomorphic bivalents 1 marked by the HSR FISH probe. The synaptic adjustment in the heterozygotes was bilateral: the HSR-carrying homolog was shortened and the wild-type homolog was elongated. The adjustment was reversible: desynapsis at diplotene was accompanied by elongation of the HSRs. Immunolocalization of H3K9me2/3 indicated that the HSRs in the meiotic chromosome retained the epigenetic modification typical for C-heterochromatin in somatic cells. MLH1 foci, marking mature recombination nodules, were detected in the proximal HSR band in heterozygotes and in both HSR bands of homozygotes. Unequal crossing over within the long-range repeat cluster can cause variation in size of the HSRs, which has been detected in the natural populations of the house mouse.

Primed to vocalize: Wild-derived male house mice increase vocalization rate and diversity after a previous encounter with a female

Males in a wide variety of taxa, including insects, birds and mammals, produce vocalizations to attract females. Male house mice emit ultrasonic vocalizations (USVs), especially during courtship and mating, which are surprising complex. It is often suggested that male mice vocalize at higher rates after interacting with a female, but the evidence is mixed depending upon the strain of mice. We conducted a study with wild-derived house mice (Mus musculus musculus) to test whether male courtship vocalizations (i.e., vocalizations emitted in a sexual context) are influenced by a prior direct interaction with a female, and if so, determine how long the effect lasts. We allowed sexually naïve males to directly interact with a female for five minutes (sexual priming), and then we recorded males’vocalizations either 1, 10, 20, or 30 days later when presented with an unfamiliar female (separated by a perforated partition) and female scent. We automatically detected USVs and processed recordings using the Automatic Mouse Ultrasound Detector (A-MUD version 3.2), and we describe our improved version of this tool and tests of its performance. We measured vocalization rate and spectro-temporal features and we manually classified USVs into 15 types to investigate priming effects on vocal repertoire diversity and composition. After sexual priming, males emitted nearly three times as many USVs, they had a larger repertoire diversity, and their vocalizations had different spectro-temporal features (USV length, slope and variability in USV frequency) compared to unprimed controls. Unprimed control males had the most distinctive repertoire composition compared to the primed groups. Most of the effects were found when comparing unprimed to all primed males (treatment models), irrespective of the time since priming. Timepoint models showed that USV length increased 1 day after priming, that repertoire diversity increased 1 and 20 days after priming, and that the variability of USV frequencies was lower 20 and 30 days after priming. Our results show that wild-derived male mice increased the number and diversity of courtship vocalizations if they previously interacted with a female. Thus, the USVs of house mice are not only context-dependent, they depend upon previous social experience and perhaps the contexts of these experiences. The effect of sexual priming on male courtship vocalizations is likely mediated by neuro-endocrine-mechanisms, which may function to advertise males’ sexual arousal and facilitate social recognition.

Sex-specific variation in the genome-wide recombination rate

In most species that reproduce sexually, successful gametogenesis requires recombination during meiosis. The number and placement of crossovers (COs) vary among individuals, with females and males often presenting the most striking contrasts. Despite the recognition that the sexes recombine at different rates (heterochiasmy), existing data fail to answer the question of whether patterns of genetic variation in recombination rate are similar in the two sexes. To fill this gap, we measured the genome-wide recombination rate in both sexes from a panel of wild-derived inbred strains from multiple subspecies of house mice (Mus musculus) and from a few additional species of Mus. To directly compare recombination rates in females and males from the same genetic backgrounds, we applied established methods based on immunolocalization of recombination proteins to inbred strains. Our results reveal discordant patterns of genetic variation in the two sexes. Whereas male genome-wide recombination rates vary substantially among strains, female recombination rates measured in the same strains are more static. The direction of heterochiasmy varies within two subspecies, Mus musculus molossinus and Mus musculus musculus. The direction of sex differences in the length of the synaptonemal complex and CO positions is consistent across strains and does not track sex differences in genome-wide recombination rate. In males, contrasts between strains with high recombination rate and strains with low recombination rate suggest more recombination is associated with stronger CO interference and more double-strand breaks. The sex-specific patterns of genetic variation we report underscore the importance of incorporating sex differences into recombination research.

Chromosome-wide characterization of meiotic noncrossovers (gene conversions) in mouse hybrids

During meiosis, the recombination-initiating DNA double-strand breaks (DSBs) are repaired by crossovers or noncrossovers (gene conversions). While crossovers are easily detectable, noncrossover identification is hampered by the small size of their converted tracts and the necessity of sequence polymorphism. We report identification and characterization of a mouse chromosome-wide set of noncrossovers by next-generation sequencing of 10 mouse intersubspecific chromosome substitution strains. Based on 94 identified noncrossovers, we determined the mean length of a conversion tract to be 32 bp. The spatial chromosome-wide distribution of noncrossovers and crossovers significantly differed, although both sets overlapped the known hotspots of PRDM9-directed histone methylation and DNA DSBs, thus supporting their origin in the standard DSB repair pathway. A significant deficit of noncrossovers descending from asymmetric DSBs proved their proposed adverse effect on meiotic recombination and pointed to sister chromatids as an alternative template for their repair. The finding has implications for the molecular mechanism of hybrid sterility in mice from crosses between closely related Mus musculus musculus and Mus musculus domesticus subspecies.

Further resolution of the house mouse (Mus musculus) phylogeny by integration over isolation-with-migration histories

Background The three main subspecies of house mice, Mus musculus castaneus, Mus musculus domesticus, and Mus musculus musculus, are estimated to have diverged ~ 350-500KYA. Resolution of the details of their evolutionary history is complicated by their relatively recent divergence, ongoing gene flow among the subspecies, and complex demographic histories. Previous studies have been limited to some extent by the number of loci surveyed and/or by the scope of the method used. Here, we apply a method (IMa3) that provides an estimate of a population phylogeny while allowing for complex histories of gene exchange. Results Results strongly support a topology with M. m. domesticus as sister to M. m. castaneus and M. m. musculus. In addition, we find evidence of gene flow between all pairs of subspecies, but that gene flow is most restricted from M. m. musculus into M. m. domesticus. Estimates of other key parameters are dependent on assumptions regarding generation time and mutation rate in house mice. Nevertheless, our results support previous findings that the effective population size, Ne, of M. m. castaneus is larger than that of the other two subspecies, that the three subspecies began diverging ~ 130 - 420KYA, and that the time between divergence events was short. Conclusions Joint demographic and phylogenetic analyses of genomic data provide a clearer picture of the history of divergence in house mice.

Morphofunctional characteristics of mouse (Mus musculus musculus) liver on the application of various doses of nanostructural sapropel

The liver is considered to be the main organ in the processes of regulating metabolism, neutralizing toxins and maintaining the constancy of the internal environment of the body. The goal of the research was to study the morphofunctional state of the liver under the effect of different concentrations of nanostructured sapropel. The experiments were carried out on non-linear (outbred) white mice weighing 24.9 ± 1.8 g. Twelve mature males were allotted to four groups. Mice of the experimental groups I, II and III intragastrically through the atraumatic flexible probe were once injected with nanostructured sapropel (particle size of 45.0–180.0 nm) in the following doses: lethal – 3.0 g/kg of the body weight; toxic – 1.8 g/kg of the body weight and safe – 0.6 g/kg of the body weight. Mice of group IV served as a control one and received deionized water in the same way. The choice of liver as the organ for analyzing is justified by the fact that the liver did not have direct contact with sapropel nanoparticles in the process of its intragastric administration into the body of white mice. Four hours after the introduction of nanostructured sapropel, three mice from each group were killed by cervical dislocation. After preparation and staining with hematoxylin and eosin, identical pieces of the liver were evaluated using light microscopy. Histological studies have established that the introduction of a lethal dose of nanostructured sapropel caused hemodynamic vascular disorders; focal necrosis and necrobiosis of hepatocytes were also observed in the research. Furthermore, the research noted a migration of reticuloendotheliocytes to the centrolobular regions of the lobules and enhancement of their activity. The microstructure of the liver when introducing a toxic dose of nanostructured sapropel was characterized by moderate plethora of sinusoidal capillaries, deformation of hepatocytes, focal destruction with the development of karyopiknosis, karyorhexis and karyolysis. The study revealed the activation of reticuloendothelial cells. Liver histology when introducing a safe dose of nanostructured sapropel was characterized by the preservation of the integrity of the structural elements, polyploid (two- and multi-core) hepatocytes were identified in the periportal part of the lobes. The changes in the structural and functional state of the mice liver were found to be depending on the dose of the nanostructured sapropel.

Widespread introgression of the Mus musculus musculus Y chromosome in Central Europe

According to Haldane’s rule, sex chromosomes should harbour more incompatibilities than autosomes. As a consequence, transmission of sex-linked genes across a genetic barrier is expected to be hampered. A remarkable example of a contradiction of this assumption was reported from the hybrid zone between two house mouse subspecies in western Czechia and south-eastern Germany where unidirectional east→west Y chromosome introgression was observed. Since the phenomenon was coupled with differences in sex ratio, this was hypothesised to be caused by a genetic conflict between sex-specific genes on sex chromosomes or elsewhere in the genome. Here we capitalise on a large material consisting of almost 7500 mice collected across a vast area from the Baltic Sea to the Alps embracing a ~900 km long portion of the zone with the aim to (i) detect its exact course and (ii) reveal the extent and pattern of the Y chromosome introgression in Central Europe. We show that the path of the zone is quite tortuous even at the global scale and the introgression is rather a rule than an exception. We also show that although sex ratio perturbations described in our previous study appear also in other introgression areas, they may not be ubiquitous. Finally, we reveal that although not all Y chromosome types are associated with the introgression, it is not restricted to a single ‘winning’ haplotype.

Instability of the pseudoautosomal boundary in house mice

Faithful segregation of homologous chromosomes at meiosis requires pairing and recombination. In taxa with dimorphic sex chromosomes, pairing between them in the heterogametic sex is limited to a narrow interval of residual sequence homology known as the pseudoautosomal region (PAR). Failure to form the obligate crossover in the PAR is associated with male infertility in house mice (Mus musculus) and humans. Yet despite this apparent functional constraint, the boundary and organization of the PAR is highly variable in mammals, and even between subspecies of mice. Here we estimate the genetic map in a previously-documented expansion of the PAR in the Mus musculus castaneus subspecies and show that the local recombination rate is 100-fold higher than the autosomal background. We identify an independent shift in the PAR boundary in the Mus musculus musculus subspecies and show that it involves a complex rearrangement but still recombines in heterozygous males. Finally, we demonstrate pervasive copy-number variation at the PAR boundary in wild populations of M. m. domesticus, M. m. musculus and M. m. castaneus. Our results suggest that the intensity of recombination activity in the PAR, coupled with relatively weak constraints on its sequence, permit the generation and maintenance in the population of unusual levels of polymorphism of unknown functional significance.

Automated Nuclear Cartography Reveals Conserved Sperm Chromosome Territory Localization across 2 Million Years of Mouse Evolution

Measurements of nuclear organization in asymmetric nuclei in 2D images have traditionally been manual. This is exemplified by attempts to measure chromosome position in sperm samples, typically by dividing the nucleus into zones, and manually scoring which zone a fluorescence in-situ hybridisation (FISH) signal lies in. This is time consuming, limiting the number of nuclei that can be analyzed, and prone to subjectivity. We have developed a new approach for automated mapping of FISH signals in asymmetric nuclei, integrated into an existing image analysis tool for nuclear morphology. Automatic landmark detection defines equivalent structural regions in each nucleus, then dynamic warping of the FISH images to a common shape allows us to generate a composite of the signal within the entire cell population. Using this approach, we mapped the positions of the sex chromosomes and two autosomes in three mouse lineages (Mus musculus domesticus, Mus musculus musculus and Mus spretus). We found that in all three, chromosomes 11 and 19 tend to interact with each other, but are shielded from interactions with the sex chromosomes. This organization is conserved across 2 million years of mouse evolution.