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CVN424 is a novel small molecule and first-in-class candidate therapeutic to selectively modulate GPR6, an orphan G-protein coupled receptor. Expression of GPR6 is largely confined to the subset of striatal projection neurons that give rise to the indirect (striatopallidal) pathway, important in the control of movement. CVN424 improves motor function in preclinical animal models of Parkinson's disease. Here, we report results of a phase 1, first-in-human study investigating the safety, tolerability, and pharmacokinetics of CVN424 in healthy volunteers. The study (NCT03657030) was randomized, double-blind, and placebo controlled. CVN424 was orally administered in ascending doses to successive cohorts as inpatients in a clinical research unit. Single doses ranged from 1 mg to 225 mg, and repeated (7 day) daily doses were 25, 75, or 150 mg. CVN424 peak plasma concentrations were reached within 2 h post-dose in the fasted state and increased with increasing dose. Dosing after a standardized high-fat meal reduced and delayed the peak plasma concentration, but total plasma exposure was similar. Mean terminal half-life ranged from 30 to 41 h. CVN424 was generally well tolerated: no serious or severe adverse effects were observed, and there were no clinically significant changes in vital signs or laboratory parameters. We conclude that CVN424, a nondopaminergic compound that modulates a novel therapeutic target, was safe and well tolerated. A phase 2 study in patients with Parkinson's disease is underway. SIGNIFICANCE STATEMENT: This is the first-in-human clinical study of a first-in-class candidate therapeutic. CVN424 modulates a novel drug target, GPR6, which is selectively expressed in a pathway in the brain that has been implicated in the motor dysfunction of patients with Parkinson's disease. This study paves the way for investigating this novel mechanism of action in patients with Parkinson's disease.

Irregular N-methyl-D-aspartate receptor (NMDAR) function is one of the main hypotheses employed to facilitate understanding of the underlying disease state of schizophrenia. Although direct agonism of the NMDAR has not yielded promising therapeutics, advances have been made by modulating the NMDAR co-agonist site which is activated by glycine and D-serine. One approach to activate the co-agonist site is to increase synaptic D-serine levels through inhibition of D-amino acid oxidase (DAO), the major catabolic clearance pathway for this and other D-amino acids. A number of DAO inhibitors have been developed but most have not entered clinical trials. One exception to this is sodium benzoate which has demonstrated efficacy in small trials of schizophrenia and Alzheimer's disease. Herein we provide data on the effect of sodium benzoate and an optimised Takeda compound, PGM030756 on ex vivo DAO enzyme occupancy and cerebellar D-serine levels in mice. Both compounds achieve high levels of enzyme occupancy; although lower doses of PGM030756 (1, 3 and 10 mg/kg) were required to achieve this compared to sodium benzoate (300, 1000 mg/kg). Cerebellar D-serine levels were increased by both agents with a delay of approximately 6 h after dosing before the peak effect was achieved. Our data and methods may be useful in understanding the effects of sodium benzoate that have been seen in clinical trials of schizophrenia and Alzheimer's disease and to support the potential clinical assessment of other DAO inhibitors, such as PGM030756, which demonstrate good enzyme occupancy and D-serine increases following administration of low oral doses.

Background Negative symptoms in schizophrenia (SCZ) are poorly treated and interfere with the function of patients. Studies focusing on structural and functional imaging and non-invasive electrophysiology implicate perturbations of the frontocortico-temporal circuits and disruption of cortico-striatal loops to negative symptoms in SCZ. GPR139 is an orphan GPCR that is specifically expressed in the CNS and enriched in the habenula, a brain structure involved in reward and motivation. Structural and functional alterations of the habenula have been found in SCZ patients, who show deficits in feedback processing and lack habenula activation in response to negative outcomes. Rodent experiments demonstrate the involvement of direct projection from the cortex to the habenula controlling social behavior. Alteration in habenula activity have been correlated with depression, and normalization of aberrant habenula activity has been proposed as therapeutic strategy to reverse anhedonia. Building on data previously reported at SIRS2018, we further explored modulation of GPR139 receptors and habenula circuitry in vivo as a novel mechanism to treat negative symptoms in SCZ. Methods Animals were submitted to an unpredictable chronic mild stress (uCMS) protocol, an animal model of depression that produces deficits in several behavioral domains affected in depressed patients. The three-chamber (Crawley’s) paradigm has been used to study social interaction in rodents. Induction of the immediate early gene c-fos expression was studied as marker for habenula neuron activation in vivo. Dopamine release in the nucleus accumbens (NAc) was studied by standard in vivo microdialysis in freely moving rats. Results The selective GPR139 agonist TAK-041 increased cFOS expression in the habenula in wild type mice, but not in GPR139 knock out mice. No desensitization of cFOS in the habenula was observed after chronic dosing of TAK-041. Microdialysis studies showed that TAK-041 reduced amphetamine- and nicotine-induced dopamine release in the NAc in rats. TAK-041 dosed acutely and chronically reversed anhedonia caused by uCMS in rats. TAK-041 also reversed anxiety related behavior assessed in the novelty suppressed feeding test and depressive-like behavior in the forced swim test in the uCMS model. uCMS-exposed animals develop a disrupted circadian regulation of corticosterone secretion, which was also normalized by TAK-041 treatment. Furthermore, TAK-041 reversed the uCMS-induced atrophy in the basal dendrites of pyramidal neurons in the hippocampus and the uCMS-induced hypertrophy of medium-spiny neurons in the NAc. These results suggest a potential benefit of TAK-041 in the treatment of anhedonia and possibly depression. TAK-041 also completely reversed social interaction (SI) deficits in the maternal immune activation poly-I:C model of SCZ, the subchronic PCP-SI model and in Balb/C and BTBR mice. Discussion The GPR139 agonist TAK-041 is proposed as a modulator of habenula circuitry to treat negative symptoms in SCZ based on efficacy in reversing anhedonia and social interaction deficits in multiple rodent models related to negative symptoms in SCZ. *Employed by Takeda when the study was done.

Background Individuals with schizophrenia fail to appropriately use negative feedback to guide learning. These learning deficits are thought to arise from abnormalities in midbrain dopamine activity. The habenula is a well conserved paired structure that sits in the midline, adjacent to the third ventricle, and dorsal and posterior to the thalamus. Classic studies have shown that it is part of the reward pathway and functions with dopamine neurons in the ventral tegmental area (VTA) to mediate reward related signals, specifically aversive and negative stimulus (Hikosaka, 2010). Several studies point to pathology in the habenula as contributing to schizophrenia (Sandyk, 1992; Caputo et al., 1998; Shepard et al., 2006). Despite the many studies on the habenula, the precise function of the habenula remains unclear. Here we describe functional consequences of regulation of GPR139 an orphan GPCR that is specifically expressed in the CNS and enriched in the habenula (Matsuo et al., 2005) in mouse models of domain of schizophrenia. Methods Specific expression of mouse GPR139 in the habenula was evaluated using bacterial artificial chromosome translating ribosome affinity purification (bacTRAP) and confirmed by immunohistochemistry. GPR139-/- mice were generated by removal of a 736bp region encoding the seven transmembrane domain (7TM) domain. Knockout animals were maintained inbred on a 129/SvEv background and evaluated in behavioral models that reflect aspects of negative symptoms. High throughput screen (HTS) for small molecules was performed. We expressed full length GPR139 into CHO cells and screened a 600K library using a calcium assay to identify small molecule agonist. Hits were identified and physical properties optimized to produce a molecule suitable for in vivo evaluation. GPR139 agonist was testedin vivo in social interaction in Poly(I:C) and cognitive test in subchronic PCP, models of schizophrenia. Results Using bacTRAP we observed enriched expression of GPR139 in substance P positive- cells of the medial habenula. This expression was confirmed with immunohistochemistry. To determine if GPR139 regulates the known role of the habenula in learning, motivation, and social behaviour, GPR139 -/- animals were generated and phenotyped. These animals appeared normal and performed comparably to wild-type animals in a range of standard tasks. However, they were significantly impaired in models that reflect aspects of negative symptoms such as progressive ratio (Killeen et al., 2009; Heath et al., 2016), a measure of motivation and nest-building (Pedersen et al., 2014; Nakajima et al., 2013), a model of self-neglect. Furthermore, these animals showed deficits in novel object recognition model of working memory (Antunes and Biala, 2012). The small molecule agonist was observed to reverse deficits in models of schizophrenia including cognition in a subchronic PCP induced attentional set-shifting paradigm (Birrell and Brown, 2000) and social interaction in the Poly(I:C) maternal model (Meyer et al., 2008; Bitanihirwe et al., 2010). Discussion These findings identify an orphan receptor that plays an important role in habenular function. Modulation of this receptor may provide an opportunity to address an important unmet need in schizophrenia.

MLL5 is a divergent member of the Drosophila Trithorax-related (SET) domain and plant homeodomain (PHD) domain-containing chromatin regulators that are involved in the regulation of transcriptional “memory” during differentiation. Human MLL5 is located on chromosome 7q22, which frequently is deleted in myeloid leukemias, suggesting a possible role in hemopoiesis. To address this question, we generated a loss-of-function allele (Mll5tm1Apa) in the murine Mll5 locus. Unlike other Mll genes, Mll5tm1Apa homozygous mice are viable but display defects in immunity and hematopoiesis. First, Mll5tm1Apa homozygous mice show increased susceptibility to spontaneous eye infections, associated with a cell-autonomous impairment of neutrophil function. Second, Mll5tm1Apa/tm1Apa mice exhibit a mild impairment of erythropoiesis. Third, Mll5tm1Apa/tm1Apa hematopoietic stem cells (HSCs) have impaired competitive repopulating capacity both under normal conditions and when subjected to self-renewal stimulation by NUP98-HOXA10. Fourth, Mll5tm1Apa homozygous HSCs show a dramatic sensitivity to DNA demethylation–induced differentiation (5-azadeoxycytidine). Taken together, our data show that MLL5 is involved in terminal myeloid differentiation and the regulation of HSC self-renewal by a mechanism that involves DNA methylation. These data warrant investigation of MLL5 expression levels as a predictive marker of demethylating-agent response in patients with myelodysplastic syndromes and leukemias and identify MLL5 as a key regulator of normal hematopoiesis.

The G protein-coupled receptor GPR54, and its peptide ligand kisspeptin (Kp), are crucial for the induction and maintenance of mammalian reproductive function. GPR54 is expressed by GnRH neurons and is directly activated by Kp to stimulate GnRH release. We hypothesized that Kp may be able to act at the GnRH nerve terminals located in the mediobasal hypothalamus (MBH) region. To test this hypothesis, we used organotypic culture of MBH explants challenged with Kp, followed by RIA to detect GnRH released into the cultured medium. Kp stimulation for 1 h induced GnRH release from wild-type male MBH in a dose-dependent manner, whereas this did not occur in MBH explants isolated from Gpr54 null mice. Continuous Kp stimulation caused a sustained GnRH release for 4 h, followed by a decrease of GnRH release, suggesting a desensitization of GPR54 activity. Tetrodotoxin did not alter the Kp-induced GnRH release, indicating that Kp can act directly at the GnRH nerve terminals. To localize Gpr54 expression within the MBH, we used transgenic mice, in which Gpr54 expression is tagged with an IRES-LacZ reporter gene and can be visualized by β-galactosidase staining. Gpr54 expression was detected outside of the median eminence, in the pars tuberalis. In conclusion, our results provide evidence for a potent stimulating effect of Kp at GnRH nerve terminals in the MBH of the mouse. This study suggests a new point at which Kp can act on GnRH neurons.

The nuclear receptor peroxisome proliferator-activated receptor-gamma (PPARgamma) is critically required for adipogenesis. PPARgamma exists as two isoforms, gamma1 and gamma2. PPARgamma2 is the more potent adipogenic isoform in vitro and is normally restricted to adipose tissues, where it is regulated more by nutritional state than PPARgamma1. To elucidate the relevance of the PPARgamma2 in vivo, we generated a mouse model in which the PPARgamma2 isoform was specifically disrupted. Despite similar weight, body composition, food intake, energy expenditure, and adipose tissue morphology, male mice lacking the gamma2 isoform were more insulin resistant than wild-type animals when fed a regular diet. These results indicate that insulin resistance associated with ablation of PPARgamma2 is not the result of lipodystrophy and suggests a specific role for PPARgamma2 in maintaining insulin sensitivity independently of its effects on adipogenesis. Furthermore, PPARgamma2 knockout mice fed a high-fat diet did not become more insulin resistant than those on a normal diet, despite a marked increase in their mean adipocyte cell size. These findings suggest that PPARgamma2 is required for the maintenance of normal insulin sensitivity in mice but also raises the intriguing notion that PPARgamma2 may be necessary for the adverse effects of a high-fat diet on carbohydrate metabolism.

Significance Hormonal factors from specialized enteroendocrine cells in the gut epithelium link the availability of dietary nutrients to energy utilization and storage. Many gut hormones also affect behaviors such as appetite and foraging, conveying for example the satiating effects of food consumption. Here we identify insulin-like peptide 5 (Insl5) as a product of colonic endocrine L-cells, and show that levels were elevated in calorie-restricted mice and reduced after feeding. Consistent with this profile Insl5 administration stimulated food intake in mice, indicating it should join ghrelin as only the second identified gut hormone that enhances appetite. Modulating the Insl5 axis presents a new strategy for the treatment of metabolic disease and obesity.

Spermatogenesis is a complex developmental pro-cess involving cell division and differentiation. Approximately half of all sterile males have defects in spermatogenesis or sperm function. An insight into the molecular control points regulating this process might help in treating male infertility. Gene trapping in embryonic stem cells and the generation of transgenic mice represents one route to identify genes expressed during spermatogenesis. The trapped gene is tagged with a lacZ reporter gene so that the expression pattern of the gene can be visualized by staining for beta-galactosidase activity. We have screened transgenic mouse lines for expression of trapped genes in the gonads. One such trap event was shown to be in the replacement histone 3.3A gene ( H3.3A ). This gene was expressed ubiquitously during embryonic development until 13.5 days post-coitum and in the adult heart, kidney, brain, testes and ovaries. This mutation resulted in postnatal death of 50% of homozygous mutants. Surviving mutants displayed reduced growth rates when competing with wild-type siblings for food. Mutant mice also had a neuro-muscular deficit and males displayed reduced copulatory activity. When copulations did occur, these resulted in very few pregnancies, suggesting that mutations in the H3.3A gene may contribute to some cases of impaired fertility in man.

Gene trapping with the retrovi-ral ROSA b geo vector was used to generate linesof mice carrying disrupted genes. Both cDNAandgenomic flanks have been cloned from a numberof these lines. One mutation has been shown todisrupt the a -enolase gene by insertion of thesplice-trap vector into the first intron. In adultmice, lacZ expression was detected only in testes.Embryonic expression was detected from 10.5-day postcoitum embryos and was seen as a dif-fuse staining pattern over much of the embryo,consistent with the housekeeping gene functionof a -enolase. This mutation results in an earlyrecessive embryonic lethality. Mice heterozygousfor the mutation have no obvious phenotype.Mutations of this gene in humans are reported tobeassociatedwithrareautosomal-dominant,non-spherocytic haemolytic anaemia. This phenotypeis not reproduced in mice heterozygous for thismutation. Dev. Dyn. 1998;212:284–292. r 1998 Wiley-Liss, Inc. Key words: a -enolase; embryonic gene trap; EScells; embryonic lethalINTRODUCTION

Mature osteoclasts specifically express the purple, band 5 isozyme (Acp 5) of tartrate-resistant acid phosphatase, a binuclear metalloenzyme that can generate reactive oxygen species. The function of Acp 5 was investigated by targeted disruption of the gene in mice. Animals homozygous for the null Acp 5 allele had progressive foreshortening and deformity of the long bones and axial skeleton but apparently normal tooth eruption and skull plate development, indicating a rôle for Acp 5 in endochondral ossification. Histomorphometry and mineralization density analysis of backscattered electron imaging revealed widened and dis-organized epiphyseal growth plates with delayed mineralization of cartilage in 6- to 8-week-old mutant mice. The membrane bones of the skull showed increased density at all ages examined, indicating defective osteoclastic bone turnover. Increased mineralization density was observed in the long bones of older animals which showed modelling deformities at their extremities: heterozygotes and homozygous Acp 5 mutant mice had tissue that was more mineralized and occupied a greater proportion of the bone in all regions. Thus the findings reflect a mild osteopetrosis due to an intrinsic defect of osteoclastic modelling activity that was confirmed in the resorption pit assay in vitro. We conclude that this bifunctional metalloprotein of the osteoclast is required for normal mineralization of cartilage in developing bones; it also maintains integrity and turnover of the adult skeleton by a critical contribution to bone matrix resorption.

Background: Adhesion G protein-coupled receptors (aGPCR) constitute a structurally and functionally diverse class of seven-transmembrane receptor proteins. Although for some of the members important roles in immunology, neurology, as well as developmental biology have been suggested, most receptors have been poorly characterized. Results: We have studied evolution, expression, and function of an entire receptor group containing four uncharacterized aGPCR: Gpr110, Gpr111, Gpr115 ,a ndGpr116 .W e show that the genomic loci of these four receptors are clustered tightly together in mouse and human genomes and that this cluster likely derives from a single common ancestor gene. Using transcriptional profiling on wild-type and knockout/LacZ reporter knockin mice strains, we have obtained detailed expression maps that show ubiquitous expression of Gpr116, co-expression of Gpr111 and Gpr115 in developing skin, and expression of Gpr110 in adult kidney. Loss of Gpr110, Gpr111 ,o rGpr115 function did not result in detectable defects, indicating that genes of this aGPCR group might function redundantly. Conclusions: The aGPCR cluster Gpr110, Gpr111, Gpr115 ,a ndGpr116 developed from one common ancestor in vertebrates. Expression suggests a role in epithelia, and one can speculate about a possible redundant function of GPR111 and GPR115. Developmental Dynamics 241:1591–1602, 2012.

The LIM domain protein rbtn2 is associated with T cell acute leukemias. We demonstrate that rbtn2 is a nuclear protein expressed in the erythroid lineage in vivo, and using homologous recombination, we show that it is essential for erythroid development in mice. The homozygous rbtn2 null mutation leads to failure of yolk sac erythropoiesis and embryonic lethality around E10.5. Moreover, in vitro differentiation of yolk sac tissue from homozygous mutant mice and sequentially targeted double-mutant ES cells demonstrates a block to erythroid development. This shows a pivotal role for a LIM domain protein in lineage specification during mammalian development and suggests that RBTN2 and GATA-1 are critical at similar stages of erythroid differentiation.

Adhesion-GPCRs provide essential cell-cell and cell-matrix interactions in development, and have been implicated in inherited human diseases like Usher Syndrome and bilateral frontoparietal polymicrogyria. They are the second largest subfamily of seven-transmembrane spanning proteins in vertebrates, but the function of most of these receptors is still not understood. The orphan Adhesion-GPCR GPR126 has recently been shown to play an essential role in the myelination of peripheral nerves in zebrafish. In parallel, whole-genome association studies have implicated variation at the GPR126 locus as a determinant of body height in the human population. The physiological function of GPR126 in mammals is still unknown. We describe a targeted mutation of GPR126 in the mouse, and show that GPR126 is required for embryonic viability and cardiovascular development.

The G protein-coupled receptor GPR54 (AXOR12, OT7T175) is central to acquisition of reproductive competency in mammals. Peptide ligands (kisspeptins) for this receptor are encoded by the Kiss1 gene, and administration of exogenous kisspeptins stimulates hypothalamic gonadotropin-releasing hormone (GnRH) release in several species, including humans. To establish that kisspeptins are the authentic agonists of GPR54 in vivo and to determine whether these ligands have additional physiological functions we have generated mice with a targeted disruption of the Kiss1 gene. Kiss1 -null mice are viable and healthy with no apparent abnormalities but fail to undergo sexual maturation. Mutant female mice do not progress through the estrous cycle, have thread-like uteri and small ovaries, and do not produce mature Graffian follicles. Mutant males have small testes, and spermatogenesis arrests mainly at the early haploid spermatid stage. Both sexes have low circulating gonadotropin (luteinizing hormone and follicle-stimulating hormone) and sex steroid (β-estradiol or testosterone) hormone levels. Migration of GnRH neurons into the hypothalamus appears normal with appropriate axonal connections to the median eminence and total GnRH content. The hypothalamic–pituitary axis is functional in these mice as shown by robust luteinizing hormone secretion after peripheral administration of kisspeptin. The virtually identical phenotype of Gpr54 - and Kiss1 -null mice provides direct proof that kisspeptins are the true physiological ligand for the GPR54 receptor in vivo. Kiss1 also does not seem to play a vital role in any other physiological processes other than activation of the hypothalamic–pituitary–gonadal axis, and loss of Kiss1 cannot be overcome by compensatory mechanisms.

We reconstruct a phylogeny of the African and Asian Psittacula parakeets using approximately 800 bp of mitochondrial cytochrome b sequence to examine their evolutionary relationships in reference to their head plumage and major morphological tail innovations. Our phylogeny identifies three groups, whose distinctiveness is also apparent from their possession of three different head plumage characters: a neck ring, a distinctive colouration of the head, and a 'moustache'-shaped pattern that extends from the chin to the cheek. We examine the extent of sexual dimorphism in tail length across the phylogeny and reveal large differences between closely related forms. We apply a range of published avian cytochrome b substitution rates to our data, as an alternative to internal calibration of a molecular clock arising from incomplete paleontological information. An ancestral Psittacula form appears to have evolved during the late Miocene-early Pliocene (3.4-9.7 MYA), a time when regional geological processes on the Asian continent may have promoted subsequent diversity at the species level, and many forms diverged relatively early on in the evolutionary history of Psittacula (between 2.5 and 7.7 MYA). However, others, such as the derbyan and moustached parakeets, diverged as recently as 0.2 MYA. Our phylogeny also suggests that the echo parakeet from Mauritius diverged from the Indian ringneck parakeet as opposed to the African ringneck, and may have done so relatively recently. The molecular results indicate support for a southwards radiation from India across the Indian Ocean to Mauritius, where the arrival-date of the echo parakeet appears consistent with the island's volcanic formation. (C) 2003 Elsevier Inc. All rights reserved.

The earliest cell fate decision in the mammalian embryo separates the extra-embryonic trophoblast lineage, which forms the fetal portion of the placenta, from the embryonic cell lineages. The body plan of the embryo proper is established only later at gastrulation, when the pluripotent epiblast gives rise to the germ layers ectoderm, mesoderm and endoderm. Here we show that the T-box gene Eomesodermin performs essential functions in both trophoblast development and gastrulation. Mouse embryos lacking Eomesodermin arrest at the blastocyst stage. Mutant trophoectoderm does not differentiate into trophoblast, indicating that Eomesodermin may be required for the development of trophoblast stem cells. In the embryo proper, Eomesodermin is essential for mesoderm formation. Although the specification of the anterior-posterior axis and the initial response to mesoderm-inducing signals is intact in mutant epiblasts, the prospective mesodermal cells are not recruited into the primitive streak. Our results indicate that Eomesodermin defines a conserved molecular pathway controlling the morphogenetic movements of germ layer formation and has acquired a new function in mammals in the differentiation of trophoblast.

SPARC (secreted protein acidic and rich in cysteine, also known as osteonectin/BM40) is a secreted Ca2+-binding glycoprotein that interacts with a range of extracellular matrix molecules, including collagen IV. It is widely expressed during embryogenesis, and in vitro studies have suggested roles in the regulation of cell adhesion and proliferation, and in the modulation of cytokine activity. In order to analyse the function of this protein in vivo, the endogenous Sparc locus was disrupted by homologous recombination in murine embryonic stem cells. SPARC-deficient mice (Sparctm1Cam) appear normal and fertile until around 6 months of age, when they develop severe eye pathology characterized by cataract formation and rupture of the lens capsule. The first sign of lens pathology occurs in the equatorial bow region where vacuoles gradually form within differentiating epithelial cells and fibre cells. The lens capsule, however, shows no qualitative changes in the major basal lamina proteins laminin, collagen IV, perlecan or entactin. These mice are an excellent resource for further studies on how SPARC affects cell behaviour in vivo.

During evolution, up to 10% of the mammalian genome may have arisen by rare retroposition events. This process involves reverse transcription of RNA intermediates that originate from retroviral and retroviral-like sequences, highly and middle repetitive DNA elements, and processed pseudogenes. The mechanism, and contemporary nature, for retrotransposition of the viral family and long interspersed elements has been well studied; however, it has proven difficult to demonstrate that the process by which pseudogenes retropose is continuing. In this report a mutation in the murine hypoxanthine-guanosine phosphoribosyl transferase (hprt) gene, which was previously isolated following retroviral infection of ES cells, is shown to result from a de novo retroposition of an alpha-tubulin pseudogene. Repair of this insertion by homologous recombination restores the activity of the hprt locus, thus confirming the site of mutation. This retroposon bears all the hallmarks of a naturally processed pseudogene [intron loss, presence of a poly(A) tail, and target site duplication] while the retroposition event took place at a known time in well-defined conditions, during retroviral infection of ES cells. The study of this mutation demonstrates that under appropriate conditions pseudogenes of protein-coding genes can still retropose in the mammalian genome. The coincidence of this mutagenic event with retroviral infection suggests that in this situation the reverse transcriptase may have had a retroviral origin, which would implicate a retroviral role in facilitating pseudogene formation.

THE c-mos proto-oncogene encodes a 37-39K cytoplasmic serine/ threonine kinase1 implicated in the meiotic maturation events during murine spermatogenesis2 and oogenesis3–6. In Xenopus, ectopic expression of pp39mos can promote both the meiotic matur-ation of oocytes7–9 and also arrest the cleavage of blastomeres10. To elucidate the role of pp39mos we have generated homozygous mutant mice by gene targeting in embryonic stem cells11. These mice are viable and mutant males are fertile, demonstrating that pp39mos is not essential for spermatogenesis. In contrast, mutant females, have a reduced fertility because of the failure of mature eggs to arrest during meiosis. c-mos-/- oocytes undergo germinal vesicle breakdown and extrusion of both polar bodies followed in some cases by progression into cleavage. Mutant females also develop ovarian cysts. These results demonstrate that a major role for pp39mos is to prevent the spontaneous parthenogenetic activation of unfertilized eggs.

Classical genetics depends upon investigation of function by random destruction with little information on structure. Modern mapping using random polymorphisms, cloning and sequencing investigates structure without function. The genome projects with their rapid gene discovery are, however, redefining classical genetic approaches. The efficient translation of this wealth of new information into insights in biological function at molecular, cellular and organismal levels requires large-scale approaches to the generation of mutants. Gene trapping in embryonic stem (ES) cells allows an efficient approach to the functional analysis of the murine genome. The usually separate processes of gene discovery, mapping, the observation of the expression pattern and the mutant phenotype in vivo, can be integrated by the use of an indexed library of insertionally mutated ES cell clones. It should be possible to generate mutants for a large proportion of the genes of the mammalian genome.