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A central goal of modern genetics is to understand how and why organisms in the wild differ in phenotype. To date, the field has advanced largely on the strength of linkage and association mapping methods, which trace the relationship between DNA sequence variants and phenotype across recombinant progeny from matings between individuals of a species. These approaches, although powerful, are not well suited to trait differences between reproductively isolated species. Here we describe a new method for genome-wide dissection of natural trait variation that can be readily applied to incompatible species. Our strategy, RH-seq, is a genome-wide implementation of the reciprocal hemizygote test. We harnessed it to identify the genes responsible for the striking high temperature growth of the yeast Saccharomyces cerevisiae relative to its sister species S. paradoxus. RH-seq utilizes transposon mutagenesis to create a pool of reciprocal hemizygotes, which are then tracked through a high-temperature competition via high-throughput sequencing. Our RH-seq workflow as laid out here provides a rigorous, unbiased way to dissect ancient, complex traits in the budding yeast clade, with the caveat that resource-intensive deep sequencing is needed to ensure genomic coverage for genetic mapping. As sequencing costs drop, this approach holds great promise for future use across eukaryotes.

Alzheimer's disease (AD), the most common form of dementia, is characterized by the abnormal accumulation of amyloid plaques and hyperphosphorylated tau aggregates, as well as microgliosis. Hemizygous missense variants in Triggering Receptor Expressed on Myeloid Cells 2 (TREM2) are associated with elevated risk for developing late-onset AD. These variants are hypothesized to result in loss of function, mimicking TREM2 haploinsufficiency. However, the consequences of TREM2 haploinsufficiency on tau pathology and microglial function remain unknown. We report the effects of partial and complete loss of TREM2 on microglial function and tau-associated deficits. In vivo imaging revealed that microglia from aged TREM2-haploinsufficient mice show a greater impairment in their injury response compared with microglia from aged TREM2-KO mice. In transgenic mice expressing mutant human tau, TREM2 haploinsufficiency, but not complete loss of TREM2, increased tau pathology. In addition, whereas complete TREM2 deficiency protected against tau-mediated microglial activation and atrophy, TREM2 haploinsufficiency elevated expression of proinflammatory markers and exacerbated atrophy at a late stage of disease. The differential effects of partial and complete loss of TREM2 on microglial function and tau pathology provide important insights into the critical role of TREM2 in AD pathogenesis.

Alzheimer's disease (AD), the most common form of dementia, is characterized by the abnormal accumulation of amyloid plaques and hyperphosphorylated tau aggregates, as well as microgliosis. Hemizygous missense variants in Triggering Receptor Expressed on Myeloid Cells 2 (TREM2) are associated with elevated risk for developing late-onset AD. These variants are hypothesized to result in loss of function, mimicking TREM2 haploinsufficiency. However, the consequences of TREM2 haploinsufficiency on tau pathology and microglial function remain unknown. We report the effects of partial and complete loss of TREM2 on microglial function and tau-associated deficits. In vivo imaging revealed that microglia from aged TREM2-haploinsufficient mice show a greater impairment in their injury response compared with microglia from aged TREM2-KO mice. In transgenic mice expressing mutant human tau, TREM2 haploinsufficiency, but not complete loss of TREM2, increased tau pathology. In addition, whereas complete TREM2 deficiency protected against tau-mediated microglial activation and atrophy, TREM2 haploinsufficiency elevated expression of proinflammatory markers and exacerbated atrophy at a late stage of disease. The differential effects of partial and complete loss of TREM2 on microglial function and tau pathology provide important insights into the critical role of TREM2 in AD pathogenesis.

Importance:Accumulation of the protein tau is a defining characteristic of several neurodegenerative diseases. Thorough assessment of transgenic (Tg) mouse lines that replicate this process is critical for establishing the models used for testing anti-tau therapeutics in vivo. Objective:To define a consistent mouse model of disease for use in future compound efficacy studies. Design, Setting, and Participants:In this time course study, cohorts of Tg and control mice were euthanized at defined intervals. Collected brains were bisected down the midline. One half was frozen and used to measure the tau prion content, while the other half was fixed for immunostaining with anti-tau antibodies. All mice were maintained at the Hunters Point Animal Facility at the University of California, San Francisco, and all experiments were performed at the Mission Bay Campus of the University of California, San Francisco. Study animals were PS19, homozygous and hemizygous Tg(MAPT*P301S), and B6/J mice. The study dates were August 9, 2010, to October 3, 2016. Main Outcomes and Measures:Tau prions were measured using a cell-based assay. Neuropathology was measured by determining the percentage area positive for immunostaining in defined brain regions. A separate cohort of mice was aged until each mouse developed neurological signs as determined by trained animal technicians to assess mortality. Results:A total of 1035 mice were used in this time course study. These included PS19 mice (51.2% [126 of 246] male and 48.8% [120 of 246] female), Tg(MAPT*P301S+/+) mice (52.3% [216 of 413] male, 43.8% [181 of 413] female, and 3.9% [16 of 413] undetermined), Tg(MAPT*P301S+/-) mice (51.8% [101 of 195] male and 48.2% [94 of 195] female), and B6/J mice (49.7% [90 of 181] male and 50.3% [91 of 181] female). While considerable interanimal variability in neuropathology, disease onset, and tau prion formation in the PS19 mice was observed, all 3 measures of disease were more uniform in the Tg(MAPT*P301S+/+) mice. Comparing tau prion formation in Tg(MAPT*P301S+/+) mice with B6/J controls, the 95% CIs for the 2 mouse lines diverged before age 5 weeks, and significant (P

Importance:Accumulation of the protein tau is a defining characteristic of several neurodegenerative diseases. Thorough assessment of transgenic (Tg) mouse lines that replicate this process is critical for establishing the models used for testing anti-tau therapeutics in vivo. Objective:To define a consistent mouse model of disease for use in future compound efficacy studies. Design, Setting, and Participants:In this time course study, cohorts of Tg and control mice were euthanized at defined intervals. Collected brains were bisected down the midline. One half was frozen and used to measure the tau prion content, while the other half was fixed for immunostaining with anti-tau antibodies. All mice were maintained at the Hunters Point Animal Facility at the University of California, San Francisco, and all experiments were performed at the Mission Bay Campus of the University of California, San Francisco. Study animals were PS19, homozygous and hemizygous Tg(MAPT*P301S), and B6/J mice. The study dates were August 9, 2010, to October 3, 2016. Main Outcomes and Measures:Tau prions were measured using a cell-based assay. Neuropathology was measured by determining the percentage area positive for immunostaining in defined brain regions. A separate cohort of mice was aged until each mouse developed neurological signs as determined by trained animal technicians to assess mortality. Results:A total of 1035 mice were used in this time course study. These included PS19 mice (51.2% [126 of 246] male and 48.8% [120 of 246] female), Tg(MAPT*P301S+/+) mice (52.3% [216 of 413] male, 43.8% [181 of 413] female, and 3.9% [16 of 413] undetermined), Tg(MAPT*P301S+/-) mice (51.8% [101 of 195] male and 48.2% [94 of 195] female), and B6/J mice (49.7% [90 of 181] male and 50.3% [91 of 181] female). While considerable interanimal variability in neuropathology, disease onset, and tau prion formation in the PS19 mice was observed, all 3 measures of disease were more uniform in the Tg(MAPT*P301S+/+) mice. Comparing tau prion formation in Tg(MAPT*P301S+/+) mice with B6/J controls, the 95% CIs for the 2 mouse lines diverged before age 5 weeks, and significant (P

Meiotic recombination establishes connections between homologous chromosomes to promote segregation. Hemizygous regions of sex chromosomes have no homologous chromosome to recombine with, yet must be transmitted through meiosis. An extreme case of hemizygosity exists in the genus Caenorhabditis, where males have a single X chromosome that completely lacks a homologous partner. To determine whether similar strategies have evolved to accommodate hemizygosity of the X during male meiosis in Caenorhabditis with distinct modes of sexual reproduction, we examined induction and processing of meiotic double strand breaks (DSBs) in androdioecious (hermaphrodite/male) Caenorhabditis elegans and C. briggsae, and gonochoristic (female/male) C. remanei and C. brenneri Analysis of the recombinase RAD-51 suggests more meiotic DSBs are induced in gonochoristic vs. androdioecious species. However, in late prophase in all species, chromosome pairs are restructured into bivalents around a single axis, suggesting that the holocentric nature of Caenorhabditis chromosomes dictates a single crossover per bivalent regardless of the number of DSBs induced. Interestingly, RAD-51 foci were readily observed on the X chromosome of androdioecious male germ cells, while very few were detected in gonochoristic male germ cells. As in C. elegans, the X chromosome in C. briggsae male germ cells undergoes transient pseudosynapsis and flexibility in DSB repair pathway choice. In contrast, in C. remanei and C. brenneri male germ cells, the X chromosome does not undergo pseudosynapsis and appears refractory to SPO-11-induced breaks. Together our results suggest that distinct strategies have evolved to accommodate sex chromosome hemizygosity during meiosis in closely related Caenorhabditis species.

Meiotic recombination establishes connections between homologous chromosomes to promote segregation. Hemizygous regions of sex chromosomes have no homologous chromosome to recombine with, yet must be transmitted through meiosis. An extreme case of hemizygosity exists in the genus Caenorhabditis, where males have a single X chromosome that completely lacks a homologous partner. To determine whether similar strategies have evolved to accommodate hemizygosity of the X during male meiosis in Caenorhabditis with distinct modes of sexual reproduction, we examined induction and processing of meiotic double strand breaks (DSBs) in androdioecious (hermaphrodite/male) Caenorhabditis elegans and C. briggsae, and gonochoristic (female/male) C. remanei and C. brenneri Analysis of the recombinase RAD-51 suggests more meiotic DSBs are induced in gonochoristic vs. androdioecious species. However, in late prophase in all species, chromosome pairs are restructured into bivalents around a single axis, suggesting that the holocentric nature of Caenorhabditis chromosomes dictates a single crossover per bivalent regardless of the number of DSBs induced. Interestingly, RAD-51 foci were readily observed on the X chromosome of androdioecious male germ cells, while very few were detected in gonochoristic male germ cells. As in C. elegans, the X chromosome in C. briggsae male germ cells undergoes transient pseudosynapsis and flexibility in DSB repair pathway choice. In contrast, in C. remanei and C. brenneri male germ cells, the X chromosome does not undergo pseudosynapsis and appears refractory to SPO-11-induced breaks. Together our results suggest that distinct strategies have evolved to accommodate sex chromosome hemizygosity during meiosis in closely related Caenorhabditis species.

Charcot-Marie-Tooth disease type 2A (CMT2A), the most common axonal form of hereditary sensory motor neuropathy, is caused by mutations of mitofusin-2 (MFN2). Mitofusin-2 is a GTPase required for fusion of mitochondrial outer membranes, repair of damaged mitochondria, efficient mitochondrial energetics, regulation of mitochondrial-endoplasmic reticulum calcium coupling and axonal transport of mitochondria. We knocked T105M MFN2 preceded by a loxP-flanked STOP sequence into the mouse Rosa26 locus to permit cell type-specific expression of this pathogenic allele. Crossing these mice with nestin-Cre transgenic mice elicited T105M MFN2 expression in neuroectoderm, and resulted in diminished numbers of mitochondria in peripheral nerve axons, an alteration in skeletal muscle fiber type distribution, and a gait abnormality.

Charcot-Marie-Tooth disease type 2A (CMT2A), the most common axonal form of hereditary sensory motor neuropathy, is caused by mutations of mitofusin-2 (MFN2). Mitofusin-2 is a GTPase required for fusion of mitochondrial outer membranes, repair of damaged mitochondria, efficient mitochondrial energetics, regulation of mitochondrial-endoplasmic reticulum calcium coupling and axonal transport of mitochondria. We knocked T105M MFN2 preceded by a loxP-flanked STOP sequence into the mouse Rosa26 locus to permit cell type-specific expression of this pathogenic allele. Crossing these mice with nestin-Cre transgenic mice elicited T105M MFN2 expression in neuroectoderm, and resulted in diminished numbers of mitochondria in peripheral nerve axons, an alteration in skeletal muscle fiber type distribution, and a gait abnormality.

The long noncoding X-inactivation-specific transcript (Xist gene) is responsible for mammalian X-chromosome dosage compensation between the sexes, the process by which one of the two X chromosomes is inactivated in the female soma. Xist is essential for both the random and imprinted forms of X-chromosome inactivation. In the imprinted form, Xist is paternally marked to be expressed in female embryos. To investigate the mechanism of Xist imprinting, we introduce Xist transgenes (Tg) into the male germ line. Although ectopic high-level Xist expression on autosomes can be compatible with viability, transgenic animals demonstrate reduced fitness, subfertility, defective meiotic pairing, and other germ-cell abnormalities. In the progeny, paternal-specific expression is recapitulated by the 200-kb Xist Tg. However, Xist imprinting occurs efficiently only when it is in an unpaired or unpartnered state during male meiosis. When transmitted from a hemizygous father (+/Tg), the Xist Tg demonstrates paternal-specific expression in the early embryo. When transmitted by a homozygous father (Tg/Tg), the Tg fails to show imprinted expression. Thus, Xist imprinting is directed by sequences within a 200-kb X-linked region, and the hemizygous (unpaired) state of the Xist region promotes its imprinting in the male germ line.

BackgroundThe genetic basis of nonobstructive azoospermia is unknown in the majority of infertile men.MethodsWe performed array comparative genomic hybridization testing in blood samples obtained from 15 patients with azoospermia, and we performed mutation screening by means of direct Sanger sequencing of the testis-expressed 11 gene (TEX11) open reading frame in blood and semen samples obtained from 289 patients with azoospermia and 384 controls.ResultsWe identified a 99-kb hemizygous loss on chromosome Xq13.2 that involved three TEX11 exons. This loss, which was identical in 2 patients with azoospermia, predicts a deletion of 79 amino acids within the meiosis-specific sporulation domain SPO22. Our subsequent mutation screening showed five novel TEX11 mutations: three splicing mutations and two missense mutations. These mutations, which occurred in 7 of 289 men with azoospermia (2.4%), were absent in 384 controls with normal sperm concentrations (P=0.003). Notably, five of those TEX11 mutations were detected in 33 patients (15%) with azoospermia who received a diagnosis of azoospermia with meiotic arrest. Meiotic arrest in these patients resembled the phenotype of Tex11-deficient male mice. Immunohistochemical analysis showed specific cytoplasmic TEX11 expression in late spermatocytes, as well as in round and elongated spermatids, in normal human testes. In contrast, testes of patients who had azoospermia with TEX11 mutations had meiotic arrest and lacked TEX11 expression.ConclusionsIn our study, hemizygous TEX11 mutations were a common cause of meiotic arrest and azoospermia in infertile men. (Funded by the National Institutes of Health and others.).

The Adaptor Protein (AP)-3 complex is an evolutionary conserved, molecular sorting device that mediates the intracellular trafficking of proteins to lysosomes and related organelles. Genetic defects in AP-3 subunits lead to impaired biogenesis of lysosome-related organelles (LROs) such as mammalian melanosomes and insect eye pigment granules. In this work, we have performed a forward screening for genetic modifiers of AP-3 function in the fruit fly, Drosophila melanogaster. Specifically, we have tested collections of large multi-gene deletions--which together covered most of the autosomal chromosomes-to identify chromosomal regions that, when deleted in single copy, enhanced or ameliorated the eye pigmentation phenotype of two independent AP-3 subunit mutants. Fine-mapping led us to define two non-overlapping, relatively small critical regions within fly chromosome 3. The first critical region included the Atg2 gene, which encodes a conserved protein involved in autophagy. Loss of one functional copy of Atg2 ameliorated the pigmentation defects of mutants in AP-3 subunits as well as in two other genes previously implicated in LRO biogenesis, namely Blos1 and lightoid, and even increased the eye pigment content of wild-type flies. The second critical region included the ArfGAP1 gene, which encodes a conserved GTPase-activating protein with specificity towards GTPases of the Arf family. Loss of a single functional copy of the ArfGAP1 gene ameliorated the pigmentation phenotype of AP-3 mutants but did not to modify the eye pigmentation of wild-type flies or mutants in Blos1 or lightoid. Strikingly, loss of the second functional copy of the gene did not modify the phenotype of AP-3 mutants any further but elicited early lethality in males and abnormal eye morphology when combined with mutations in Blos1 and lightoid, respectively. These results provide genetic evidence for new functional links connecting the machinery for biogenesis of LROs with molecules implicated in autophagy and small GTPase regulation.

The Adaptor Protein (AP)-3 complex is an evolutionary conserved, molecular sorting device that mediates the intracellular trafficking of proteins to lysosomes and related organelles. Genetic defects in AP-3 subunits lead to impaired biogenesis of lysosome-related organelles (LROs) such as mammalian melanosomes and insect eye pigment granules. In this work, we have performed a forward screening for genetic modifiers of AP-3 function in the fruit fly, Drosophila melanogaster. Specifically, we have tested collections of large multi-gene deletions--which together covered most of the autosomal chromosomes-to identify chromosomal regions that, when deleted in single copy, enhanced or ameliorated the eye pigmentation phenotype of two independent AP-3 subunit mutants. Fine-mapping led us to define two non-overlapping, relatively small critical regions within fly chromosome 3. The first critical region included the Atg2 gene, which encodes a conserved protein involved in autophagy. Loss of one functional copy of Atg2 ameliorated the pigmentation defects of mutants in AP-3 subunits as well as in two other genes previously implicated in LRO biogenesis, namely Blos1 and lightoid, and even increased the eye pigment content of wild-type flies. The second critical region included the ArfGAP1 gene, which encodes a conserved GTPase-activating protein with specificity towards GTPases of the Arf family. Loss of a single functional copy of the ArfGAP1 gene ameliorated the pigmentation phenotype of AP-3 mutants but did not to modify the eye pigmentation of wild-type flies or mutants in Blos1 or lightoid. Strikingly, loss of the second functional copy of the gene did not modify the phenotype of AP-3 mutants any further but elicited early lethality in males and abnormal eye morphology when combined with mutations in Blos1 and lightoid, respectively. These results provide genetic evidence for new functional links connecting the machinery for biogenesis of LROs with molecules implicated in autophagy and small GTPase regulation.

De novo phosphatase and tensin homolog on chromosome ten (PTEN) mutations are a cause of sporadic autism. How single-copy loss of PTEN alters neural function is not understood. Here we report that Pten haploinsufficiency increases the expression of small-conductance calcium-activated potassium channels. The resultant augmentation of this conductance increases the amplitude of the afterspike hyperpolarization, causing a decrease in intrinsic excitability. In vivo, this change in intrinsic excitability reduces evoked firing rates of cortical pyramidal neurons but does not alter receptive field tuning. The decreased in vivo firing rate is not associated with deficits in the dendritic integration of synaptic input or with changes in dendritic complexity. These findings identify calcium-activated potassium channelopathy as a cause of cortical dysfunction in the PTEN model of autism and provide potential molecular therapeutic targets.

Studies of striatal physiology and motor control have increasingly relied on the use of bacterial artificial chromosome (BAC) transgenic mice expressing fluorophores or other genes under the control of genetic regulatory elements for the dopamine D1 receptor (D1R) or dopamine D2 receptor (D2R). Three recent studies have compared wild-type, D1R, and D2R BAC transgenic mice, and found significant differences in physiology and behavior, calling into question the use of these mice in studies of normal circuit function. We repeated the behavioral portions of these studies in wild-type C57BL/6 mice and hemizygous Drd1a-td Tomato (D1-Tmt), Drd1a-eGFP (D1-GFP), and Drd2-eGFP (D2-GFP) mice backcrossed into the C57BL/6 background. Our three laboratories independently found that open-field locomotion, acute locomotor responses to cocaine (20 mg/kg), locomotor sensitization to 5 d of daily injections of cocaine (15 mg/kg) or amphetamine (3 mg/kg), cocaine (20 mg/kg) conditioned place preference, and active avoidance learning to paired light and footshock were indistinguishable in these four mouse lines. These results suggest that while it is crucial to screen new transgenic mouse lines for abnormal behavior and physiology, these BAC transgenic mouse lines remain extremely valuable tools for evaluating the cellular, synaptic, and circuit basis of striatal motor control and associative learning.