Pawel Michalak

Insights into the Evolution of Longevity from the Bowhead Whale Genome

Summary The bowhead whale (Balaena mysticetus) is estimated to live over 200 years and is possibly the longest-living mammal. These animals should possess protective molecular adaptations relevant to age-related diseases, particularly cancer. Here, we report the sequencing and comparative analysis of the bowhead whale genome and two transcriptomes from different populations. Our analysis identifies genes under positive selection and bowhead-specific mutations in genes linked to cancer and aging. In addition, we identify gene gain and loss involving genes associated with DNA repair, cell-cycle regulation, cancer, and aging. Our results expand our understanding of the evolution of mammalian longevity and suggest possible players involved in adaptive genetic changes conferring cancer resistance. We also found potentially relevant changes in genes related to additional processes, including thermoregulation, sensory perception, dietary adaptations, and immune response. Our data are made available online (http://www.bowhead-whale.org) to facilitate research in this long-lived species.

Sterility and Gene Expression in Hybrid Males of Xenopus laevis and X. muelleri

Background Reproductive isolation is a defining characteristic of populations that represent unique biological species, yet we know very little about the gene expression basis for reproductive isolation. The advent of powerful molecular biology tools provides the ability to identify genes involved in reproductive isolation and focuses attention on the molecular mechanisms that separate biological species. Herein we quantify the sterility pattern of hybrid males in African Clawed Frogs (Xenopus) and apply microarray analysis of the expression pattern found in testes to identify genes that are misexpressed in hybrid males relative to their two parental species (Xenopus laevis and X. muelleri). Methodology/Principal Findings Phenotypic characteristics of spermatogenesis in sterile male hybrids (X. laevis x X. muelleri) were examined using a novel sperm assay that allowed quantification of live, dead, and undifferentiated sperm cells, the number of motile vs. immotile sperm, and sperm morphology. Hybrids exhibited a dramatically lower abundance of mature sperm relative to the parental species. Hybrid spermatozoa were larger in size and accompanied by numerous undifferentiated sperm cells. Microarray analysis of gene expression in testes was combined with a correction for sequence divergence derived from genomic hybridizations to identify candidate genes involved in the sterility phenotype. Analysis of the transcriptome revealed a striking asymmetric pattern of misexpression. There were only about 140 genes misexpressed in hybrids compared to X. laevis but nearly 4,000 genes misexpressed in hybrids compared to X. muelleri. Conclusions/Significance Our results provide an important correlation between phenotypic characteristics of sperm and gene expression in sterile hybrid males. The broad pattern of gene misexpression suggests intriguing mechanisms creating the dominance pattern of the X. laevis genome in hybrids. These findings significantly contribute to growing evidence for allelic dominance in hybrids and have implications for the mechanism of species differentiation at the transcriptome level.

Adaptive differentiation of thermotolerance in Drosophila along a microclimatic gradient

We examined whether a remarkable occurrence – the physiological evolution of two Drosophila melanogaster populations, despite a spatial separation of only 100–400 m, was idiosyncratic and temporary, or persisted over multiple years. We ascertained the high-temperature tolerance of Drosophila descended from populations on the north-facing slope (NFS) and south-facing slope (SFS) of ‘Evolution Canyon’ (Lower Nahal Oren, Mt Carmel, Israel), which were collected in 1997, 1999, and 2000. Results for these Drosophila uniformly resembled other studies in many respects: an inverse relationship between survival and heat-shock temperature, male–female differences in thermotolerance, and inducible thermotolerance. Importantly, for all years of collection, SFS flies consistently exceeded NFS flies in basal and inducible thermotolerance after diverse heat shocks, with and without thermal pretreatment, and whether isofemale lines, synthetic populations, or inbred lines were compared. Inbred lines, however, had lower thermotolerance than outbred lines. Several nonexclusive processes may explain the evolution of such physiological differentiation.

Physiological sex predicts hybrid sterility regardless of genotype.

Reproductive isolation between biological species is characterized by Haldanes rule, which states that the heterogametic sex (XY or ZW) suffers the most dysfunctional effects of hybridization. It has been hypothesized that, in addition to X-linked recessive genes, Haldanes rule also reflects the faster evolution of genes related to male reproduction (faster-male evolution). We used sex-reversed hybrid Xenopus to test faster-male evolution by examining the fertility of sex-reversed individuals with the genotype of the inverse phenotypic sex. Hybrid males are sterile and hybrid females are fertile irrespective of their genotypic sex. Gene expression profiles match these adult phenotypes, and our results demonstrate that faster-male evolution is the most likely mechanism producing hybrid male sterility.

Genome differentiation of Drosophila melanogaster from a microclimate contrast in Evolution Canyon, Israel

Significance The microclimatic contrast between opposing slopes of “Evolution Canyon” (Mount Carmel, Israel) provides a natural laboratory for testing the effects of abiotic factors on biodiversity and population genetic differentiation in a geographical microscale. Drosophila melanogaster fruitflies originating from the opposite canyon slopes are subject to divergent selection leading to slope-specific adaptations, accompanied by incipient mating isolation, all in the face of pervasive demographic processes, including ongoing genetic exchange. We demonstrate that interslope genetic changes in this species accumulate in a number of chromosomal differentiation “islands” and that gene networks related to adaptive responses and reproductive processes are thus significantly affected. The opposite slopes of “Evolution Canyon” in Israel have served as a natural model system of adaptation to a microclimate contrast. Long-term studies of Drosophila melanogaster populations inhabiting the canyon have exhibited significant interslope divergence in thermal and drought stress resistance, candidate genes, mobile elements, habitat choice, mating discrimination, and wing-shape variation, all despite close physical proximity of the contrasting habitats, as well as substantial interslope migration. To examine patterns of genetic differentiation at the genome-wide level, we used high coverage sequencing of the flies’ genomes. A total of 572 genes were significantly different in allele frequency between the slopes, 106 out of which were associated with 74 significantly overrepresented gene ontology (GO) terms, particularly so with response to stimulus and developmental and reproductive processes, thus corroborating previous observations of interslope divergence in stress response, life history, and mating functions. There were at least 37 chromosomal “islands” of interslope divergence and low sequence polymorphism, plausible signatures of selective sweeps, more abundant in flies derived from one (north-facing) of the slopes. Positive correlation between local recombination rate and the level of nucleotide polymorphism was also found.

Sex-biased gene expression in a ZW sex determination system.

Studies of the transcriptome have shown that a substantial fraction of interspecific differences in gene expression is the result of sex-biased gene expression. These results suggest that sex-dependent selection may be an important force in generating differences between species but to date all studies have focused on Drosophila. We examined a sample of the transcriptome in the gonads of two species of Xenopus to provide an additional test of how sex-biased gene expression may contribute to differences in gene expression between species. In contrast to Drosophila, Xenopus provides an example of a ZW system with morphologically indistinguishable sex chromosomes. About 81% of the transcriptome was differentially expressed between X. laevis and X. muelleri and there were more genes that were male-biased compared to the number of genes that were female-biased or non-sex-biased. While there were more genes up-regulated in males of Xenopus, the largest magnitude difference in expression between species occurred in female-biased genes, and male-biased genes were proportionally more abundant for the smallest changes in expression between species. Our results suggest that more genes have a role in the function of the testis compared to the ovary and female-biased genes play a principle role in expression divergence between species. These results differ from those in the Drosophila XY system in that more female-biased genes had >4-fold difference of expression between species than male-biased genes, suggesting that ZW sex chromosomes may facilitate enhanced gene expression divergence between species.

Divergence of Drosophila melanogaster repeatomes in response to a sharp microclimate contrast in Evolution Canyon, Israel

Significance Repeatome, or the ensemble of all repeat sequences, with its enormous variability and internal epigenetic dynamics, emerges as a critical source of potentially adaptive changes and evolutionary novelties. This conclusion is exemplified here by the cosmopolitan Drosophila melanogaster from a sharp ecological contrast in North Israel. Flies derived from the opposing sides of this long-studied microsite exhibit a significant difference in the contents and distribution of mobile elements, as well as microsatellite allele frequencies, corresponding well with earlier reported phenotypic patterns of stress resistance and assortative mating in the system. Repeat sequences, especially mobile elements, make up large portions of most eukaryotic genomes and provide enormous, albeit commonly underappreciated, evolutionary potential. We analyzed repeatomes of Drosophila melanogaster that have been diverging in response to a microclimate contrast in Evolution Canyon (Mount Carmel, Israel), a natural evolutionary laboratory with two abutting slopes at an average distance of only 200 m, which pose a constant ecological challenge to their local biotas. Flies inhabiting the colder and more humid north-facing slope carried about 6% more transposable elements than those from the hot and dry south-facing slope, in parallel to a suite of other genetic and phenotypic differences between the two populations. Nearly 50% of all mobile element insertions were slope unique, with many of them disrupting coding sequences of genes critical for cognition, olfaction, and thermotolerance, consistent with the observed patterns of thermotolerance differences and assortative mating.

Modeling the Role of Peroxisome Proliferator-Activated Receptor γ and MicroRNA-146 in Mucosal Immune Responses to Clostridium difficile

Clostridium difficile is an anaerobic bacterium that has re-emerged as a facultative pathogen and can cause nosocomial diarrhea, colitis or even death. Peroxisome proliferator-activated receptor (PPAR) γ has been implicated in the prevention of inflammation in autoimmune and infectious diseases; however, its role in the immunoregulatory mechanisms modulating host responses to C. difficile and its toxins remains largely unknown. To characterize the role of PPARγ in C. difficile-associated disease (CDAD), immunity and gut pathology, we used a mouse model of C. difficile infection in wild-type and T cell-specific PPARγ null mice. The loss of PPARγ in T cells increased disease activity and colonic inflammatory lesions following C. difficile infection. Colonic expression of IL-17 was upregulated and IL-10 downregulated in colons of T cell-specific PPARγ null mice. Also, both the loss of PPARγ in T cells and C. difficile infection favored Th17 responses in spleen and colonic lamina propria of mice with CDAD. MicroRNA (miRNA)-sequencing analysis and RT-PCR validation indicated that miR-146b was significantly overexpressed and nuclear receptor co-activator 4 (NCOA4) suppressed in colons of C. difficile-infected mice. We next developed a computational model that predicts the upregulation of miR-146b, downregulation of the PPARγ co-activator NCOA4, and PPARγ, leading to upregulation of IL-17. Oral treatment of C. difficile-infected mice with the PPARγ agonist pioglitazone ameliorated colitis and suppressed pro-inflammatory gene expression. In conclusion, our data indicates that miRNA-146b and PPARγ activation may be implicated in the regulation of Th17 responses and colitis in C. difficile-infected mice.

Genetic Divergence between Freshwater and Marine Morphs of Alewife (Alosa pseudoharengus): A ‘Next-Generation’ Sequencing Analysis

Alewife Alosa pseudoharengus, a small clupeid fish native to Atlantic Ocean, has recently (∼150 years ago) invaded the North American Great Lakes and despite challenges of freshwater environment its populations exploded and disrupted local food web structures. This range expansion has been accompanied by dramatic changes at all levels of organization. Growth rates, size at maturation, or fecundity are only a few of the most distinct morphological and life history traits that contrast the two alewife morphs. A question arises to what extent these rapidly evolving differences between marine and freshwater varieties result from regulatory (including phenotypic plasticity) or structural mutations. To gain insights into expression changes and sequence divergence between marine and freshwater alewives, we sequenced transcriptomes of individuals from Lake Michigan and Atlantic Ocean. Population specific single nucleotide polymorphisms were rare but interestingly occurred in sequences of genes that also tended to show large differences in expression. Our results show that the striking phenotypic divergence between anadromous and lake alewives can be attributed to massive regulatory modifications rather than coding changes.

Gene expression polymorphism in Drosophila populations.

Although changes in gene expression have long been recognized as critical to evolutionary processes, the extent of natural polymorphism in gene expression has yet to be assessed, thus opening a new area of active research. We present microarray and quantitative real‐time polymerase chain reaction (RT‐PCR) data from Cosmopolitan and Zimbabwe morphs of Drosophila melanogaster. These morphs provide a useful model for investigations into the incipient stages of speciation because Zimbabwe females tend to preferentially mate with their own males and discriminate against Cosmopolitan males, while Cosmopolitan females mate indiscriminately. We analysed expression profiles from heads of mated and nonmated females and identified 45 candidate genes whose expression levels were associated with the behavioural morphs and were modified by mating. Genes with altered transcription levels were randomly distributed across the genome and fell into diverse categories of biological activities. Several candidate genes, such as desaturase2 and Odorant receptor 63a, were additionally subjected to quantitative RT‐PCR analysis. Notably, desaturase2, which has been invoked to play a role in sexual isolation between Cosmopolitan and Zimbabwe D. melanogaster/races/strains and predicted to be translational‐inactive in Cosmopolitan due to a major deletion, was found to be up‐regulated in Zimbabwe and down‐regulated, but still expressed, in Cosmopolitan.