Disrupted Gene Networks in Subfertile Hybrid House Mice.

The Dobzhansky-Muller (DM) model provides a widely accepted mechanism for the evolution of reproductive isolation: incompatible substitutions disrupt interactions between genes. To date, few candidate incompatibility genes have been identified, leaving the genes driving speciation mostly uncharacterized. The importance of interactions in the DM model suggests that gene coexpression networks provide a powerful framework to understand disrupted pathways associated with postzygotic isolation. Here, we perform weighted gene coexpression network analysis to infer gene interactions in hybrids of two recently diverged European house mouse subspecies, Mus mus domesticus and M. m. musculus, which commonly show hybrid male sterility or subfertility. We use genome-wide testis expression data from 467 hybrid mice from two mapping populations: F2s from a laboratory cross between wild-derived pure subspecies strains and offspring of natural hybrids captured in the Central Europe hybrid zone. This large data set enabled us to build a robust consensus network using hybrid males with fertile phenotypes. We identify several expression modules, or groups of coexpressed genes, that are disrupted in subfertile hybrids, including modules functionally enriched for spermatogenesis, cilium and sperm flagellum organization, chromosome organization, and DNA repair, and including genes expressed in spermatogonia, spermatocytes, and spermatids. Our network-based approach enabled us to hone in on specific hub genes likely to be influencing module-wide gene expression and hence potentially driving large-effect DM incompatibilities. A disproportionate number of hub genes lie within sterility loci identified previously in the hybrid zone mapping population and represent promising candidate barrier genes and targets for future functional analysis.

Genomic networks of hybrid sterility.

Hybrid dysfunction, a common feature of reproductive barriers between species, is often caused by negative epistasis between loci ("Dobzhansky-Muller incompatibilities"). The nature and complexity of hybrid incompatibilities remain poorly understood because identifying interacting loci that affect complex phenotypes is difficult. With subspecies in the early stages of speciation, an array of genetic tools, and detailed knowledge of reproductive biology, house mice (Mus musculus) provide a model system for dissecting hybrid incompatibilities. Male hybrids between M. musculus subspecies often show reduced fertility. Previous studies identified loci and several X chromosome-autosome interactions that contribute to sterility. To characterize the genetic basis of hybrid sterility in detail, we used a systems genetics approach, integrating mapping of gene expression traits with sterility phenotypes and QTL. We measured genome-wide testis expression in 305 male F2s from a cross between wild-derived inbred strains of M. musculus musculus and M. m. domesticus. We identified several thousand cis- and trans-acting QTL contributing to expression variation (eQTL). Many trans eQTL cluster into eleven 'hotspots,' seven of which co-localize with QTL for sterility phenotypes identified in the cross. The number and clustering of trans eQTL-but not cis eQTL-were substantially lower when mapping was restricted to a 'fertile' subset of mice, providing evidence that trans eQTL hotspots are related to sterility. Functional annotation of transcripts with eQTL provides insights into the biological processes disrupted by sterility loci and guides prioritization of candidate genes. Using a conditional mapping approach, we identified eQTL dependent on interactions between loci, revealing a complex system of epistasis. Our results illuminate established patterns, including the role of the X chromosome in hybrid sterility. The integrated mapping approach we employed is applicable in a broad range of organisms and we advocate for widespread adoption of a network-centered approach in speciation genetics.

Craniofacial shape transition across the house mouse hybrid zone: implications for the genetic architecture and evolution of between-species differences.

Craniofacial shape differences between taxa have often been linked to environmental adaptation, e.g., new food sources, or have been studied in the context of domestication. Evidence for the genetic basis of such phenotypic differences to date suggests that between-species as well as between-population variation has an oligogenic basis, i.e., few loci of large effect explain most of the variation. In mice, it has been shown that within-population craniofacial variation has a highly polygenic basis, but there are no data regarding the genetic basis of between-species differences in natural populations. Here, we address this question using a phenotype-focused approach. Using 3D geometric morphometrics, we phenotyped a panel of mice derived from a natural hybrid zone between Mus musculus domesticus and Mus mus musculus and quantify the transition of craniofacial shape along the hybridization gradient. We find a continuous shape transition along the hybridization gradient and unaltered developmental stability associated with hybridization. This suggests that the morphospace between the two subspecies is continuous despite reproductive isolation and strong barriers to gene flow. We show that quantitative changes in overall genome composition generate quantitative changes in craniofacial shape; this supports a highly polygenic basis for between-species craniofacial differences in the house mouse. We discuss our findings in the context of oligogenic versus polygenic models of the genetic architecture of morphological traits.

Use of a natural hybrid zone for genomewide association mapping of craniofacial traits in the house mouse.

The identification of the genes involved in morphological variation in nature is still a major challenge. Here, we explore a new approach: we combine 178 samples from a natural hybrid zone between two subspecies of the house mouse (Mus musculus domesticus and Mus musculus musculus), and high coverage of the genome (~ 145K SNPs) to identify loci underlying craniofacial shape variation. Due to the long history of recombination in the hybrid zone, high mapping resolution is anticipated. The combination of genomes from subspecies allows the mapping of both, variation within subspecies and inter-subspecific differences, thereby increasing the overall amount of causal genetic variation that can be detected. Skull and mandible shape were measured using 3D landmarks and geometric morphometrics. Using principal component axes as phenotypes, and a linear mixed model accounting for genetic relatedness in the mapping populations, we identified nine genomic regions associated with skull shape and 10 with mandible shape. High mapping resolution (median size of significant regions = 148 kb) enabled identification of single or few candidate genes in most cases. Some of the genes act as regulators or modifiers of signalling pathways relevant for morphological development and bone formation, including several with known craniofacial phenotypes in mice and humans. The significant associations combined explain 13% and 7% of the skull and mandible shape variation, respectively. In addition, a positive correlation was found between chromosomal length and proportion of variation explained. Our results suggest a complex genetic architecture for shape traits and support a polygenic model.

Gene regulation and speciation in a migratory divide between songbirds.

Behavioral variation abounds in nature. This variation is important for adaptation and speciation, but its molecular basis remains elusive. Here, we use a hybrid zone between two subspecies of songbirds that differ in migration - an ecologically important and taxonomically widespread behavior---to gain insight into this topic. We measure gene expression in five brain regions. Differential expression between migratory states was dominated by circadian genes in all brain regions. The remaining patterns were largely brain-region specific. For example, expression differences between the subspecies that interact with migratory state likely help maintain reproductive isolation in this system and were documented in only three brain regions. Contrary to existing work on regulatory mechanisms underlying species-specific traits, two lines of evidence suggest that trans- (vs. cis) regulatory changes underlie these patterns - no evidence for allele-specific expression in hybrids and minimal associations between genomic differentiation and expression differences. Additional work with hybrids shows expression levels were often distinct (transgressive) from parental forms. Behavioral contrasts and functional enrichment analyses allowed us to connect these patterns to mitonuclear incompatibilities and compensatory responses to stress that could exacerbate selection on hybrids and contribute to speciation.

Key features of the genetic architecture and evolution of host-microbe interactions revealed by high-resolution genetic mapping of the mucosa-associated gut microbiome in hybrid mice.

Determining the forces that shape diversity in host-associated bacterial communities is critical to understanding the evolution and maintenance of metaorganisms. To gain deeper understanding of the role of host genetics in shaping gut microbial traits, we employed a powerful genetic mapping approach using inbred lines derived from the hybrid zone of two incipient house mouse species. Furthermore, we uniquely performed our analysis on microbial traits measured at the gut mucosal interface, which is in more direct contact with host cells and the immune system. Several mucosa-associated bacterial taxa have high heritability estimates, and interestingly, 16S rRNA transcript-based heritability estimates are positively correlated with cospeciation rate estimates. Genome-wide association mapping identifies 428 loci influencing 120 taxa, with narrow genomic intervals pinpointing promising candidate genes and pathways. Importantly, we identified an enrichment of candidate genes associated with several human diseases, including inflammatory bowel disease, and functional categories including innate immunity and G-protein-coupled receptors. These results highlight key features of the genetic architecture of mammalian host-microbe interactions and how they diverge as new species form.

The digestive system, particularly the large intestine, hosts many types of bacteria which together form the gut microbiome. The exact makeup of different bacterial species is specific to an individual, but microbiomes are often more similar between related individuals, and more generally, across related species. Whether this is because individuals share similar environments or similar genetic backgrounds remains unclear. These two factors can be disentangled by breeding different animal lineages ­ which have different genetic backgrounds while belonging to the same species ­ and then raising the progeny in the same environment. To investigate this question, Doms et al. studied the genes and microbiomes of mice resulting from breeding strains from multiple locations in a natural hybrid zone between different subspecies. The experiments showed that 428 genetic regions affected the makeup of the microbiome, many of which were known to be associated with human diseases. Further analysis revealed 79 genes that were particularly interesting, as they were involved in recognition and communication with bacteria. These results show how the influence of the host genome on microbiome composition becomes more specialized as animals evolve. Overall, the work by Doms et al. helps to pinpoint the genes that impact the microbiome; this knowledge could be helpful to examine how these interactions contribute to the emergence of conditions such as diabetes or inflammatory bowel disease, which are linked to perturbations in gut bacteria.

Monogamy evolves through multiple mechanisms: evidence from V1aR in deer mice.

Genetic variation in Avpr1a, the locus encoding the arginine vasopressin receptor 1A (V1aR), has been implicated in pair-bonding behavior in voles (genus Microtus) and humans, raising the possibility that this gene may contribute commonly to mating-system variation in mammals. In voles, differential expression of V1aR in the brain is associated with male partner-preference behavior in a comparison of a monogamous (Microtus ochrogaster) and promiscuous (Microtus montanus) species. This expression difference is correlated, in turn, with a difference in length of a 5' regulatory microsatellite in Avpr1a. Here, we use a combination of comparative sequencing of coding and regulatory regions, analysis of neural expression patterns, and signaling assays to test for differences in V1aR expression and function among eight species of deer mice (genus Peromyscus). Despite well-documented variation in Peromyscus social behavior, we find no association between mating system and length variation in the microsatellite locus linked to V1aR expression in voles. Further, there are no consistent differences in V1aR expression pattern between monogamous and promiscuous species in regions of the brain known to influence mating behavior. We do find statistical evidence for positive selection on the V1aR coding sequence including several derived amino acid substitutions in a monogamous Peromyscus lineage, yet these substitutions have no measurable effect on V1aR signaling activity. Together, these results suggest that mating-system variation in rodents is mediated by multiple genetic mechanisms.

Genome-wide analysis of alternative splicing evolution among Mus subspecies.

Alternative splicing, the combination of different exons to produce a variety of transcripts from a single gene, contributes enormously to transcriptome diversity in mammals, and the majority of genes encode alternatively spliced products. Previous research comparing mouse, rat and human has shown that a significant proportion of splice forms are not conserved across species, suggesting that alternative transcripts are an important source of evolutionary novelty. Here, we studied the evolution of alternative splicing in the early stages of species divergence in the house mouse. We sequenced the testis transcriptomes of three Mus musculus subspecies and Mus spretus using Illumina technology. On the basis of a genome-wide analysis of read coverage differences among subspecies, we identified several hundred candidate alternatively spliced regions. We conservatively estimate that 6.5% of testis-expressed genes show alternative splice differences between at least one pair of M. musculus subspecies, a proportion slightly higher than the proportion of genes differentially expressed among subspecies. These results suggest that differences in both the structure and abundance of transcripts contribute to early transcriptome divergence.

Spondyloepiphyseal dysplasia congenita.

We report a case of spondyloepiphyseal dysplasia congenita (SED congenita), diagnosed at autopsy of a term infant. Prenatal ultrasound at 20 weeks of gestation had shown shortening of all the fetal long bones, with bowing of the femora and humeri, clubfeet, and small thoracic cage. We discuss the diagnostic features of SED and the main differential diagnoses.

Comparative analysis of testis protein evolution in rodents.

Genes expressed in testes are critical to male reproductive success, affecting spermatogenesis, sperm competition, and sperm-egg interaction. Comparing the evolution of testis proteins at different taxonomic levels can reveal which genes and functional classes are targets of natural and sexual selection and whether the same genes are targets among taxa. Here we examine the evolution of testis-expressed proteins at different levels of divergence among three rodents, mouse (Mus musculus), rat (Rattus norvegicus), and deer mouse (Peromyscus maniculatus), to identify rapidly evolving genes. Comparison of expressed sequence tags (ESTs) from testes suggests that proteins with testis-specific expression evolve more rapidly on average than proteins with maximal expression in other tissues. Genes with the highest rates of evolution have a variety of functional roles including signal transduction, DNA binding, and egg-sperm interaction. Most of these rapidly evolving genes have not been identified previously as targets of selection in comparisons among more divergent mammals. To determine if these genes are evolving rapidly among closely related species, we sequenced 11 of these genes in six Peromyscus species and found evidence for positive selection in five of them. Together, these results demonstrate rapid evolution of functionally diverse testis-expressed proteins in rodents, including the identification of amino acids under lineage-specific selection in Peromyscus. Evidence for positive selection among closely related species suggests that changes in these proteins may have consequences for reproductive isolation.

Causes and consequences of the evolution of reproductive proteins.

Proteins involved in reproduction often evolve rapidly, raising the possibility that changes in these proteins contribute to reproductive isolation between species. We review the evidence for rapid and adaptive change in reproductive proteins in animals, focusing on studies in recently diverged vertebrates. We identify common patterns and point out promising directions for future research. In particular, we highlight the ways that integrating the different but complementary approaches of evolutionary and developmental biology will provide new insights into fertilization processes.

Reproductive protein evolution within and between species: maintenance of divergent ZP3 alleles in Peromyscus.

In a variety of animal taxa, proteins involved in reproduction evolve more rapidly than nonreproductive proteins. Most studies of reproductive protein evolution, however, focus on divergence between species, and little is known about differentiation among populations within a species. Here we investigate the molecular population genetics of the protein ZP3 within two Peromyscus species. ZP3 is an egg coat protein involved in primary binding of egg and sperm and is essential for fertilization. We find that amino acid polymorphism in the sperm-combining region of ZP3 is high relative to silent polymorphism in both species of Peromyscus. In addition, while there is geographical structure at a mitochondrial gene (Cytb), a nuclear gene (Lcat) and eight microsatellite loci, we find no evidence for geographical structure at Zp3 in Peromyscus truei. These patterns are consistent with the maintenance of ZP3 alleles by balancing selection, possibly due to sexual conflict or pathogen resistance. However, we do not find evidence that reinforcement promotes ZP3 diversification; allelic variation in P. truei is similar among populations, including populations allopatric and sympatric with sibling species. In fact, most alleles are present in all populations sampled across P. truei's range. While additional data are needed to identify the precise evolutionary forces responsible for sequence variation in ZP3, our results suggest that in Peromyscus, selection to maintain divergent alleles within species contributes to the pattern of rapid amino acid substitution observed among species.

Using the Mus musculus hybrid zone to assess covariation and genetic architecture of limb bone lengths.

Two subspecies of the house mouse, Mus musculus domesticus and Mus musculus musculus, meet in a narrow contact zone across Europe. Mice in the hybrid zone are highly admixed, representing the full range of mixed ancestry from the two subspecies. Given the distinct morphologies of these subspecies, these natural hybrids can be used for genomewide association mapping at sufficiently high resolution to directly infer candidate genes. We focus here on limb bone length differences, which is of special interest for understanding the evolution of developmentally correlated traits. We used 172 first-generation descendants of wild-caught mice from the hybrid zone to measure the length of stylopod (humerus/femur), zeugopod (ulna/tibia) and autopod (metacarpal/metatarsal) elements in skeletal CT scans. We find phenotypic covariation between limb elements in the hybrids similar to patterns previously described in Mus musculus domesticus inbred strains, suggesting that the hybrid genotypes do not influence the covariation pattern in a major way. Mapping was performed using 143,592 SNPs and identified several genomic regions associated with length differences in each bone. Bone length was found to be highly polygenic. None of the candidate regions include the canonical genes known to control embryonic limb development. Instead, we are able to identify candidate genes with known roles in osteoblast differentiation and bone structure determination, as well as recently evolved genes of, as yet, unknown function.

Cryptic "chromo-fibroma".

Chromoblastomycosis is an uncommon chronic fungal infection capable of presenting in a variety of clinical guises. Herein, we present the histopathological features of an unusual dermal response engendered by this organism, consisting of dermal effacement by a spindle cell proliferation arranged in sweeping fascicles.

Genomic regions controlling shape variation in the first upper molar of the house mouse.

Numerous loci of large effect have been shown to underlie phenotypic variation between species. However, loci with subtle effects are presumably more frequently involved in microevolutionary processes but have rarely been discovered. We explore the genetic basis of shape variation in the first upper molar of hybrid mice between Mus musculus musculus and M. m. domesticus. We performed the first genome-wide association study for molar shape and used 3D surface morphometrics to quantify subtle variation between individuals. We show that many loci of small effect underlie phenotypic variation, and identify five genomic regions associated with tooth shape; one region contained the gene microphthalmia-associated transcription factor Mitf that has previously been associated with tooth malformations. Using a panel of five mutant laboratory strains, we show the effect of the Mitf gene on tooth shape. This is the first report of a gene causing subtle but consistent variation in tooth shape resembling variation in nature.

BRAF inhibition for advanced locoregional BRAF V600E mutant melanoma: a potential neoadjuvant strategy.

Selective BRAF inhibitors (BRAFi) yield objective responses in 50% of patients with metastatic BRAF V600E mutant melanoma. Adding an MEK inhibitor increases this response rate to 70%. Limited data are available on the outcomes of unresectable stage III patients, and it remains unclear whether BRAF-targeted therapy can be utilized as a neoadjuvant strategy. Data on patients with advanced locoregional BRAF V600E mutant melanoma treated with BRAF-targeted therapy at Moffitt Cancer Center were analyzed to determine response rates, subsequent resection rates after tumor downsizing, pathologic responses, and patient survival. Fifteen patients with locoregional disease treated with BRAF-targeted therapy, either BRAFi alone (vemurafenib; 11 patients) or a combination of BRAFi and an MEK inhibitor (dabrafenib plus trametinib or placebo; four patients), were identified. The median age was 50 years; the median follow-up was 25.4 months. The median BRAF-targeted therapy treatment duration was 6.0 months (range 1.2-29.4 months). Response Evaluation Criteria In Solid Tumors-based evaluation demonstrated objective response in 11 patients (73.3%). Six patients underwent resection of the remaining disease after therapy. Pathological analysis showed complete pathologic response (n=2), partial pathologic response (n=2), or no pathologic response (n=2). Four of six patients undergoing surgery have been alive for more than 2 years, including three patients currently free from active disease. No complications attributable to BRAF-targeted therapy were observed in the perioperative period. Dose reduction or discontinuation because of toxicities occurred in 10/15 patients. Neoadjuvant BRAF-targeted therapy may be effective in advanced locoregional BRAF V600E mutant melanoma patients in increasing resectability, yielding pathological responses, and achieving prolonged survival.

Serum gamma-glutamyltransferase as a prognostic indicator of neonatal viability in nondomestic ruminants.

Rapid assessment of immune status in neonatal ruminants of endangered species facilitates early intervention in cases of inadequate passive transfer of maternal immunoglobulins. Serum gamma-glutamyltransferase (GGT) was used to evaluate suspected passive transfer status in 25 North Indian muntjac (Muntiacus muntjak vaginalis), 45 Cretan goats (Capra algagrus cretica), 20 white-lipped deer (Cervus albirostris), 25 Mhorr gazelles (Gazella dama mhorr), and 31 Soemmerring's gazelles (Gazella soemmerringi soemmerringi). Serum GGT, measured within 48 hr of birth, was compared with clinical condition at 5 days of age. Neonatal Soemmerring's and Mhorr gazelles with GGT > 600 U/L were likely to survive without medical intervention, whereas GGT < 400 U/L was a good indicator that the gazelle neonate would need medical intervention. Neonatal muntjac with GGT > 200 U/L were also likely to survive without medical intervention. Because there is no gold standard for evaluating passive transfer status in neonatal nondomestic ruminants, it is recommended to evaluate the results of more than one diagnostic test, as well as clinical condition, in considering health status and disposition of neonatal ruminants of endangered species.

Analysis of intestinal microbiota in hybrid house mice reveals evolutionary divergence in a vertebrate hologenome.

Recent evidence suggests that natural selection operating on hosts to maintain their microbiome contributes to the emergence of new species, that is, the 'hologenomic basis of speciation'. Here we analyse the gut microbiota of two house mice subspecies, Mus musculus musculus and M. m. domesticus, across their Central European hybrid zone, in addition to hybrids generated in the lab. Hybrid mice display widespread transgressive phenotypes (that is, exceed or fall short of parental values) in a variety of measures of bacterial community structure, which reveals the importance of stabilizing selection operating on the intestinal microbiome within species. Further genetic and immunological analyses reveal genetic incompatibilities, aberrant immune gene expression and increased intestinal pathology associated with altered community structure among hybrids. These results provide unique insight into the consequences of evolutionary divergence in a vertebrate 'hologenome', which may be an unrecognized contributing factor to reproductive isolation in this taxonomic group.

Genome-wide mapping in a house mouse hybrid zone reveals hybrid sterility loci and Dobzhansky-Muller interactions.

Mapping hybrid defects in contact zones between incipient species can identify genomic regions contributing to reproductive isolation and reveal genetic mechanisms of speciation. The house mouse features a rare combination of sophisticated genetic tools and natural hybrid zones between subspecies. Male hybrids often show reduced fertility, a common reproductive barrier between incipient species. Laboratory crosses have identified sterility loci, but each encompasses hundreds of genes. We map genetic determinants of testis weight and testis gene expression using offspring of mice captured in a hybrid zone between M. musculus musculus and M. m. domesticus. Many generations of admixture enables high-resolution mapping of loci contributing to these sterility-related phenotypes. We identify complex interactions among sterility loci, suggesting multiple, non-independent genetic incompatibilities contribute to barriers to gene flow in the hybrid zone.

Reduced male fertility is common but highly variable in form and severity in a natural house mouse hybrid zone.

Barriers to gene flow between naturally hybridizing taxa reveal the initial stages of speciation. Reduced hybrid fertility is a common feature of reproductive barriers separating recently diverged species. In house mice (Mus musculus), hybrid male sterility has been studied extensively using experimental crosses between subspecies. Here, we present the first detailed picture of hybrid male fertility in the European M. m. domesticus-M. m. musculus hybrid zone. Complete sterility appears rare or absent in natural hybrids but a large proportion of males (~30%) have sperm count or relative testis weight below the range in pure subspecies, and likely suffer reduced fertility. Comparison of a suite of traits related to fertility among subfertile males indicates reduced hybrid fertility in the contact zone is highly variable among individuals and ancestry groups in the type, number, and severity of spermatogenesis defects present. Taken together, these results suggest multiple underlying genetic incompatibilities are segregating in the hybrid zone, which likely contribute to reproductive isolation between subspecies.