Selection shapes the landscape of functional variation in wild house mice

Background Through human-aided dispersal over the last ~ 10,000 years, house mice (Mus musculus) have recently colonized diverse habitats across the globe, promoting the emergence of new traits that confer adaptive advantages in distinct environments. Despite their status as the premier mammalian model system, the impact of this demographic and selective history on the global patterning of disease-relevant trait variation in wild mouse populations is poorly understood. Results Here, we leveraged 154 whole-genome sequences from diverse wild house mouse populations to survey the geographic organization of functional variation and systematically identify signals of positive selection. We show that a significant proportion of wild mouse variation is private to single populations, including numerous predicted functional alleles. In addition, we report strong signals of positive selection at many genes associated with both complex and Mendelian diseases in humans. Notably, we detect a significant excess of selection signals at disease-associated genes relative to null expectations, pointing to the important role of adaptation in shaping the landscape of functional variation in wild mouse populations. We also uncover strong signals of selection at multiple genes involved in starch digestion, including Mgam and Amy1. We speculate that the successful emergence of the human-mouse commensalism may have been facilitated, in part, by dietary adaptations at these loci. Finally, our work uncovers multiple cryptic structural variants that manifest as putative signals of positive selection, highlighting an important and under-appreciated source of false-positive signals in genome-wide selection scans. Conclusions Overall, our findings highlight the role of adaptation in shaping wild mouse genetic variation at human disease-associated genes. Our work also highlights the biomedical relevance of wild mouse genetic diversity and underscores the potential for targeted sampling of mice from specific populations as a strategy for developing effective new mouse models of both rare and common human diseases.

Efficient Targeted Transgenesis of Large Donor DNA Into Multiple Mouse Genetic Backgrounds Using Bacteriophage Bxb1 Integrase

The development of mouse models of human disease and synthetic biology research by targeted transgenesis of large DNA constructs represent a significant genetic engineering hurdle. We developed an efficient, precise, single-copy integration of large transgenes directly into zygotes using multiple mouse genetic backgrounds. We used in vivo Bxb1 mediated recombinase-mediated cassette exchange (RMCE) with a transgene “landing pad” composed of dual heterologous Bxb1 attachment (att) sites in cis, within the Gt(ROSA)26Sor safe harbor locus. RMCE of donor was achieved by microinjection of vector DNA carrying cognate attachment sites flanking the donor transgene with Bxb1-integrase mRNA. This approach achieves perfect vector-free integration of donor constructs at efficiencies >40% with up to ~43kb transgenes. Coupled with a nanopore-based Cas9-targeted sequencing (nCATS), complete verification of precise insertion sequence was achieved. As a proof-of-concept we describe the development of C57BL/6J and NSG Krt18-ACE2 models for SARS-CoV2 research with verified heterozygous N1 animals within ~4 months. Additionally, we created a series of mice with diverse backgrounds carrying a single att site including FVB/NJ, PWK/PhJ, NOD/ShiLtJ, CAST/EiJ and DBA/2J allowing for rapid transgene insertion. Combined, this system enables predictable, rapid development combined with simplified characterization of precisely targeted transgenic animals across multiple genetic backgrounds.

Towards mouse genetic-specific RNA-sequencing read mapping

Genetic variations affect behavior and cause disease but understanding how these variants drive complex traits is still an open question. A common approach is to link the genetic variants to intermediate molecular phenotypes such as the transcriptome using RNA-sequencing (RNA-seq). Paradoxically, these variants between the samples are usually ignored at the beginning of RNA-seq analyses of many model organisms. This can skew the transcriptome estimates that are used later for downstream analyses, such as expression quantitative trait locus (eQTL) detection. Here, we assessed the impact of reference-based analysis on the transcriptome and eQTLs in a widely-used mouse genetic population: the BXD panel of recombinant inbred lines. We highlight existing reference bias in the transcriptome data analysis and propose practical solutions which combine available genetic variants, genotypes, and genome reference sequence. The use of custom BXD line references improved downstream analysis compared to classical genome reference. These insights would likely benefit genetic studies with a transcriptomic component and demonstrate that genome references might need to be reassessed and improved.

Efficient targeted transgenesis of large donor DNA into multiple mouse genetic backgrounds using bacteriophage Bxb1 integrase

Efficient, targeted integration of large DNA constructs represent a significant hurdle in genetic engineering for the development of mouse models of human disease and synthetic biology research. To address this, we developed a system for efficient and precise, targeted single-copy integration of large transgenes directly into the zygote using multiple mouse genetic backgrounds. Conventional approaches, such as random transgenesis, CRISPR/Cas9-mediated homology-directed repair (HDR), lentivirus-based insertion, or DNA transposases all have significant limitations. Our strategy uses in vivo Bxb1 mediated recombinase-mediated cassette exchange (RMCE) to efficiently generate precise single-copy integrations of transgenes. This is achieved using a transgene landing pad composed of dual heterologous Bxb1 attachment (att) sites in cis, pre-positioned in the Gt(ROSA)26Sor safe harbor locus. Successful RMCE is achieved in att carrier zygotes using donor DNA carrying cognate attachment sites flanking the desired donor transgene microinjected along with Bxb1-integrase mRNA. This approach routinely achieves perfect vector-free integration of donor constructs at efficiencies as high as 43% and has generated transgenic animals containing inserts up to ~43kb. Furthermore, when coupled with a nanopore-based Cas9-targeted sequencing (nCATS) approach, complete verification of the precise insertion sequence can be achieved. As a proof-of-concept we describe the creation and characterization of C57BL/6J and NSG Krt18-ACE2 transgenic mouse models for SARS-CoV2 research with verified heterozygous N1 animals available for experimental use in ~4 months. In addition, we created a diverse series of mouse backgrounds carrying a single att site version of the landing pad allele in C57BL/6J, NSG, B6(Cg)-Tyrc-2J/J, FVB/NJ, PWK/PhJ, 129S1/SvImJ, A/J, NOD/ShiLtJ, NZO/HILtJ, CAST/EiJ, and DBA/2J for rapid transgene insertion. Combined, this system enables predictable, rapid creation of precisely targeted transgenic animals across multiple genetic backgrounds, simplifying characterization, speeding expansion and use.

Ferroptotic stress promotes the accumulation of pro-inflammatory proximal tubular cells in maladaptive renal repair

Overwhelming lipid peroxidation induces ferroptotic stress and ferroptosis, a non-apoptotic form of regulated cell death that has been implicated in maladaptive renal repair in mice and humans. Using single-cell transcriptomic and mouse genetic approaches, we show that proximal tubular (PT) cells develop a molecularly distinct, pro-inflammatory state following injury. While these inflammatory PT cells transiently appear after mild injury and return to their original state without inducing fibrosis, after severe injury they accumulate and contribute to persistent inflammation. This transient inflammatory PT state significantly downregulates glutathione metabolism genes, making the cells vulnerable to ferroptotic stress. Genetic induction of high ferroptotic stress in these cells after mild injury leads to the accumulation of the inflammatory PT cells, enhancing inflammation and fibrosis. Our study broadens the roles of ferroptotic stress from being a trigger of regulated cell death to include the promotion and accumulation of proinflammatory cells that underlie maladaptive repair.

Oligodendrocyte precursor cells guide the migration of cortical interneurons by unidirectional contact repulsion

The cerebral cortex is built by neural cells that migrate away from their birthplace. In the forebrain, ventrally-derived oligodendrocyte precursor cells (vOPCs) travel tangentially together with cortical interneurons (cINs) to reach the cortex. After birth, vOPCs form transient synapses with cINs before engaging later into myelination. Here we tested whether these populations interact during embryogenesis while migrating. By coupling histological analysis of mouse genetic models with live imaging, we showed that, while responding to the chemokine Cxcl12, vOPCs and cINs occupy mutually-exclusive forebrain territories. Moreover, vOPCs depletion selectively disrupts the migration and distribution of cINs. At the cellular level, we found that by promoting unidirectional contact-repulsion (UCoRe) of cINs, vOPCs steer their migration away from blood vessels and contribute to their allocation to proper migratory streams. UCoRe is thus an efficient strategy to spatially control the competition for a shared chemoattractant, thereby allowing cINs to reach proper cortical territories.

Selection shapes the landscape of functional variation in wild house mice

Background Through human-aided dispersal, house mice have recently colonized new and diverse habitats across the globe, promoting the emergence of new traits that confer adaptive advantages in distinct environments. Despite their status as the premiere mammalian model system, the impact of this demographic and selective history on the global patterning of disease-relevant trait variation in wild mouse populations is poorly understood. Results Here, we leveraged 154 whole-genome sequences from diverse wild house mouse populations, subspecies, and species to survey the geographic organization of functional variation and systematically identify signals of positive selection. We show that a significant proportion of wild mouse variation is private to single populations, including numerous predicted functional alleles. In addition, we report strong signals of positive selection at numerous genes associated with both complex and Mendelian diseases in humans. Notably, we detect a significant excess of selection signals at disease-associated genes relative to null expectations, pointing to the important role of adaptation in shaping the landscape of functional variation in wild mouse populations. We also uncover strong signals of selection at multiple genes involved in starch digestion, including Mgam and Amy1. We speculate that the successful emergence of the human-mouse commensalism may have been facilitated, in part, by dietary adaptations at these loci. Finally, our work uncovers multiple cryptic structural variants that manifest as putative signals of positive selection, highlighting an important and under-appreciated source of false-positive signals in genome-wide selection scans. Conclusions Overall, our findings underscore the role of adaptation in shaping wild mouse genetic variation at human disease-associated genes. Our work highlights the biomedical relevance of wild mouse genetic diversity and underscores the potential for targeted sampling of mice from specific populations as a strategy for developing effective new mouse models of both rare and common human diseases.

Maternal vitamin B12 in mice positively regulates bone, but not muscle mass and strength in post-weaning and mature offspring

Vitamin B12 deficiency has been shown to affect bone mass in rodents and negatively impact bone formation in humans. In this study using mouse models we define the effect of B12 supplementation in the wild-type mother and B12 deficiency in a mouse genetic model (Gif-/- mice) during gestation on the bone and muscle architecture, and mechanical properties in the offspring. Analysis of bones from 4 weeks-old offspring of the wild-type mother following vehicle or B12 supplementation during gestation (From embryonic day 0.5-20.5) showed an increase in bone mass caused by an isolated increase in bone formation in the B12 supplemented group compared to vehicle controls. Analysis of effect of B12 deficiency in the mother in a mouse genetic model (Gif-/- mice) on long bone architecture of the offspring showed a compromised cortical and trabecular bone mass, which was completely prevented by a single injection of B12 in the B12-deficient Gif-/- mothers.Biomechanical analysis of long bones of the offspring born from B12 supplemented wild-type mothers showed an increase in bone strength, and conversely offspring born from B12-deficient Gif-/- mothers revealed a compromised bone strength, which could be rescued by a single injection of B12 in the B12-deficient Gif-/- mother. Muscle structure and function analysis however revealed no significant effect on muscle mass, structure and grip strength of B12 deficiency or supplementation in Gif-/- mice compared to littermate controls. Together, these results demonstrate the beneficial effect of maternally-derived B12 in the regulation of bone structure and function in the offspring.