A missense mutation in Katnal1 underlies behavioural, neurological and ciliary anomalies.

Microtubule severing enzymes implement a diverse range of tissue-specific molecular functions throughout development and into adulthood. Although microtubule severing is fundamental to many dynamic neural processes, little is known regarding the role of the family member Katanin p60 subunit A-like 1, KATNAL1, in central nervous system (CNS) function. Recent studies reporting that microdeletions incorporating the KATNAL1 locus in humans result in intellectual disability and microcephaly suggest that KATNAL1 may play a prominent role in the CNS; however, such associations lack the functional data required to highlight potential mechanisms which link the gene to disease symptoms. Here we identify and characterise a mouse line carrying a loss of function allele in Katnal1. We show that mutants express behavioural deficits including in circadian rhythms, sleep, anxiety and learning/memory. Furthermore, in the brains of Katnal1 mutant mice we reveal numerous morphological abnormalities and defects in neuronal migration and morphology. Furthermore we demonstrate defects in the motile cilia of the ventricular ependymal cells of mutants, suggesting a role for Katnal1 in the development of ciliary function. We believe the data we present here are the first to associate KATNAL1 with such phenotypes, demonstrating that the protein plays keys roles in a number of processes integral to the development of neuronal function and behaviour.

Sleep-like behavior and 24-h rhythm disruption in the Tc1 mouse model of Down syndrome.

Down syndrome is a common disorder associated with intellectual disability in humans. Among a variety of severe health problems, patients with Down syndrome exhibit disrupted sleep and abnormal 24-h rest/activity patterns. The transchromosomic mouse model of Down syndrome, Tc1, is a trans-species mouse model for Down syndrome, carrying most of human chromosome 21 in addition to the normal complement of mouse chromosomes and expresses many of the phenotypes characteristic of Down syndrome. To date, however, sleep and circadian rhythms have not been characterized in Tc1 mice. Using both circadian wheel-running analysis and video-based sleep scoring, we showed that these mice exhibited fragmented patterns of sleep-like behaviour during the light phase of a 12:12-h light/dark (LD) cycle with an extended period of continuous wakefulness at the beginning of the dark phase. Moreover, an acute light pulse during night-time was less effective in inducing sleep-like behaviour in Tc1 animals than in wild-type controls. In wheel-running analysis, free running in constant light (LL) or constant darkness (DD) showed no changes in the circadian period of Tc1 animals although they did express subtle behavioural differences including a reduction in total distance travelled on the wheel and differences in the acrophase of activity in LD and in DD. Our data confirm that Tc1 mice express sleep-related phenotypes that are comparable with those seen in Down syndrome patients with moderate disruptions in rest/activity patterns and hyperactive episodes, while circadian period under constant lighting conditions is essentially unaffected.

Social jetlag, obesity and metabolic disorder: investigation in a cohort study.

BACKGROUND: Obesity is one of the leading causes of preventable death worldwide. Circadian rhythms are known to control both sleep timing and energy homeostasis, and disruptions in circadian rhythms have been linked with metabolic dysfunction and obesity-associated disease. In previous research, social jetlag, a measure of chronic circadian disruption caused by the discrepancy between our internal versus social clocks, was associated with elevated self-reported body mass index, possibly indicative of a more generalized association with obesity and metabolic dysfunction. METHODS: We studied participants from the population-representative Dunedin Longitudinal Study (N=1037) to determine whether social jetlag was associated with clinically assessed measurements of metabolic phenotypes and disease indicators for obesity-related disease, specifically, indicators of inflammation and diabetes. RESULTS: Our analysis was restricted to N=815 non-shift workers in our cohort. Among these participants, we found that social jetlag was associated with numerous clinically assessed measures of metabolic dysfunction and obesity. We distinguished between obese individuals who were metabolically healthy versus unhealthy, and found higher social jetlag levels in metabolically unhealthy obese individuals. Among metabolically unhealthy obese individuals, social jetlag was additionally associated with elevated glycated hemoglobin and an indicator of inflammation. CONCLUSIONS: The findings are consistent with the possibility that 'living against our internal clock' may contribute to metabolic dysfunction and its consequences. Further research aimed at understanding that the physiology and social features of social jetlag may inform obesity prevention and have ramifications for policies and practices that contribute to increased social jetlag, such as work schedules and daylight savings time.

Clusterin regulates ß-amyloid toxicity via Dickkopf-1-driven induction of the wnt-PCP-JNK pathway.

Although the mechanism of Aß action in the pathogenesis of Alzheimer's disease (AD) has remained elusive, it is known to increase the expression of the antagonist of canonical wnt signalling, Dickkopf-1 (Dkk1), whereas the silencing of Dkk1 blocks Aß neurotoxicity. We asked if clusterin, known to be regulated by wnt, is part of an Aß/Dkk1 neurotoxic pathway. Knockdown of clusterin in primary neurons reduced Aß toxicity and DKK1 upregulation and, conversely, Aß increased intracellular clusterin and decreased clusterin protein secretion, resulting in the p53-dependent induction of DKK1. To further elucidate how the clusterin-dependent induction of Dkk1 by Aß mediates neurotoxicity, we measured the effects of Aß and Dkk1 protein on whole-genome expression in primary neurons, finding a common pathway suggestive of activation of wnt-planar cell polarity (PCP)-c-Jun N-terminal kinase (JNK) signalling leading to the induction of genes including EGR1 (early growth response-1), NAB2 (Ngfi-A-binding protein-2) and KLF10 (Krüppel-like factor-10) that, when individually silenced, protected against Aß neurotoxicity and/or tau phosphorylation. Neuronal overexpression of Dkk1 in transgenic mice mimicked this Aß-induced pathway and resulted in age-dependent increases in tau phosphorylation in hippocampus and cognitive impairment. Furthermore, we show that this Dkk1/wnt-PCP-JNK pathway is active in an Aß-based mouse model of AD and in AD brain, but not in a tau-based mouse model or in frontotemporal dementia brain. Thus, we have identified a pathway whereby Aß induces a clusterin/p53/Dkk1/wnt-PCP-JNK pathway, which drives the upregulation of several genes that mediate the development of AD-like neuropathologies, thereby providing new mechanistic insights into the action of Aß in neurodegenerative diseases.

Reaching and grasping phenotypes in the mouse (Mus musculus): a characterization of inbred strains and mutant lines.

Skilled movements, such as reaching and grasping, have classically been considered as originating in the primate lineage. For this reason, the use of rodents to investigate the genetic and molecular machinery of reaching and grasping has been limited in research. A few studies in rodents have now shown that these movements are not exclusive to primates. Here we present a new test, the Mouse Reaching and Grasping (MoRaG) performance scale, intended to help researchers in the characterization of these motor behaviors in the mouse. Within the MoRaG test battery we identified early phenotypes for the characterization of motor neurone (Tg[SOD1-G93A](dl)1Gur mice) and neurodegenerative (TgN(HD82Gln)81Dbo transgenic mice) disease models in addition to specific motor deficits associated with aging (C3H/HeH inbred strain). We conclude that the MoRaG test can be used to further investigate complex neuromuscular, neurological, neurodegenerative and behavioral disorders. Moreover, our study supports the validity of the mouse as a model for reaching and grasping studies.

Genetic and molecular analysis of the central and peripheral circadian clockwork of mice.

A hierarchy of interacting, tissue-based clocks controls circadian physiology and behavior in mammals. Preeminent are the suprachiasmatic nuclei (SCN): central hypothalamic pacemakers synchronized to solar time via retinal afferents and in turn responsible for internal synchronization of other clocks present in major organ systems. The SCN and peripheral clocks share essentially the same cellular timing mechanism. This consists of autoregulatory transcriptional/posttranslational feedback loops in which the Period (Per) and Cryptochrome (Cry) "clock" genes are negatively regulated by their protein products. Here, we review recent studies directed at understanding the molecular and cellular bases to the mammalian clock. At the cellular level, we demonstrate the role of F-box protein Fbxl3 (characterized by the afterhours mutation) in directing the proteasomal degradation of Cry and thereby controlling negative feedback and circadian period of the molecular loops. Within SCN neural circuitry, we describe how neuropeptidergic signaling by VIP synchronizes and sustains the cellular clocks. At the hypothalamic level, signaling via a different SCN neuropeptide, prokineticin, is not required for pacemaking but is necessary for control of circadian behavior. Finally, we consider how metabolic pathways are coordinated in time, focusing on liver function and the role of glucocorticoid signals in driving the circadian transcriptome and proteome.

Screening for novel ENU-induced rhythm, entrainment and activity mutants.

Chemical mutagenesis has provided an opportunity to develop and expand the repertoire of behavioural mutants for gene function studies. With this in mind, we have established a screen in mice for mutations affecting circadian rhythms, entrainment to light and other wheel-running parameters. The screen consists of an assessment of mouse wheel-running activity in a 12:12 h light/dark cycle for 7-10 days followed by assessment in constant darkness for up to 20 days. Responses to light are assessed using two protocols; a 15 minute light pulse given at circadian time 16 on the tenth day in constant darkness and an additional 12 h of light upon transition from light/dark conditions to constant darkness. To date, approximately 1300 progeny of chemically mutagenised mice have been screened. Computer-aided assessment of wheel-running parameters has helped in identifying abnormal phenotypes in approximately 5% of all animals screened. Inheritance testing of mice with abnormal phenotypes has confirmed the number of robustly inherited mutant phenotypes to be 1% of the total screened. Confirmed mutants including those affecting free-running period, light-responsiveness and wheel-running endurance have been identified. Thus far, low-resolution map positions have been established for four mutants by completing genome scans in backcross progeny. Mutant loci do not correspond with those previously associated with wheel-running behaviour. This result confirms that phenotype-driven approaches such as this should continue to provide material for mammalian gene function studies.

Characterization of mycoplasma gallisepticum infection in captive house finches (Carpodacus mexicanus) in 1998.

Since 1995, the epidemic of mycoplasmal conjunctivitis in eastern house finches has affected the Auburn, AL, house finch population. To better characterize the current status of this host-parasite interaction, we established a captive flock of 38 seronegative, healthy finches in fall 1998. After a minimum quarantine period of 4 wk, two Mycoplasma gallisepticum (MG)-infected house finches were introduced into this flock. Over a 12-wk period, the flock was captured every 2 wk and each bird was observed for conjunctivitis. Blood and choanal swabs were collected from each bird for serologic analysis and for the detection of MG by polymerase chain reaction. The infection spread rapidly through the flock just as it had in a similar study performed in 1996 at the height of the epidemic. Unlike the earlier study in which birds remained chronically infected, most of the birds in our study recovered rapidly, and only three of the birds died during the study. Two patterns of host response to infection with MG were observed. Twenty-seven birds (73%) experienced an acute conjunctivitis that resolved, and the birds appeared to clear the infection. Ten birds (27%) suffered prolonged clinical disease, and MG could be detected in these birds intermittently throughout the experiment. These results, in conjunction with our surveys of MG in the wild population, suggest an evolving host-parasite interaction.

Characterization of the mycoplasmal conjunctivitis epizootic in a house finch population in the southeastern USA.

An epidemiological study of the prevalence of mycoplasmal conjunctivitis in the house finch (Carpodacus mexicanus) was conducted in Auburn (Alabama, USA) between March 1998 and February 1999. Clinical disease was observed in 4% of the 1,214 finches trapped and examined. This rate is comparable to the average annual prevalence observed in this population since 1996, although the prevalence of clinical disease observed in the peak months of September through November was lower than in previous years. Clinically ill birds were observed in all months of the study. To estimate the prevalence of recovering and asymptomatic, infected birds, we tested a subset of 334 house finches serologically for exposure to Mycoplasma gallisepticum (MG) using the serum plate agglutination (SPA) assay. The prevalence of clinical disease in this subsample was slightly higher (7%) than in the entire sample, reflecting the fact that the serological survey was initiated in the late summer when the prevalence of MG infection peaks in our study population and a sampling bias for symptomatic birds. The serological survey indicated that 13% of this subpopulation had been exposed to MG. We also tested 46 of 334 finches by polymerase chain reaction (PCR) to detect MG in seropositive, asymptomatic birds. Use of the PCR in conjunction with the SPA detected six asymptomatic, infected birds that may represent potential carriers or birds in the early stages of infection. The decreasing prevalence of clinical disease observed during the peak months suggests a changing host-parasite relationship. Continued surveillance of this population, employing both clinical observation and serological analysis will be useful in characterizing these changes over time.

A systematic, genome-wide, phenotype-driven mutagenesis programme for gene function studies in the mouse.

As the human genome project approaches completion, the challenge for mammalian geneticists is to develop approaches for the systematic determination of mammalian gene function. Mouse mutagenesis will be a key element of studies of gene function. Phenotype-driven approaches using the chemical mutagen ethylnitrosourea (ENU) represent a potentially efficient route for the generation of large numbers of mutant mice that can be screened for novel phenotypes. The advantage of this approach is that, in assessing gene function, no a priori assumptions are made about the genes involved in any pathway. Phenotype-driven mutagenesis is thus an effective method for the identification of novel genes and pathways. We have undertaken a genome-wide, phenotype-driven screen for dominant mutations in the mouse. We generated and screened over 26,000 mice, and recovered some 500 new mouse mutants. Our work, along with the programme reported in the accompanying paper, has led to a substantial increase in the mouse mutant resource and represents a first step towards systematic studies of gene function in mammalian genetics.

Identification of two new Pmp22 mouse mutants using large-scale mutagenesis and a novel rapid mapping strategy.

Mouse mutants have a key role in discerning mammalian gene function and modelling human disease; however, at present mutants exist for only 1-2% of all mouse genes. In order to address this phenotype gap, we have embarked on a genome-wide, phenotype-driven, large-scale N-ethyl-N--nitrosourea (ENU) mutagenesis screen for dominant mutations of clinical and pharmacological interest in the mouse. Here we describe the identification of two similar neurological phenotypes and determination of the underlying mutations using a novel rapid mapping strategy incorporating speed back-crosses and high throughput genotyping. Two mutant mice were identified with marked resting tremor and further characterized using the SHIRPA behavioural and functional assessment protocol. Back-cross animals were generated using in vitro fertilization and genome scans performed utilizing DNA pools derived from multiple mutant mice. Both mutants were mapped to a region on chromosome 11 containing the peripheral myelin protein 22 gene (Pmp22). Sequence analysis revealed novel point mutations in Pmp22 in both lines. The first mutation, H12R, alters the same amino acid as in the severe human peripheral neuropathy Dejerine Sottas syndrome and Y153TER in the other mutant truncates the Pmp22 protein by seven amino acids. Histological analysis of both lines revealed hypo-myelination of peripheral nerves. This is the first report of the generation of a clinically relevant neurological mutant and its rapid genetic characterization from a large-scale mutagenesis screen for dominant phenotypes in the mouse, and validates the use of large-scale screens to generate desired clinical phenotypes in mice.

Implementation of a large-scale ENU mutagenesis program: towards increasing the mouse mutant resource.

Systematic approaches to mouse mutagenesis will be vital for future studies of gene function. We have begun a major ENU mutagenesis program incorporating a large genome-wide screen for dominant mutations. Progeny of ENU-mutagenized mice are screened for visible defects at birth and weaning, and at 5 weeks of age by using a systematic and semi-quantitative screening protocol-SHIRPA. Following this, mice are screened for abnormal locomotor activity and for deficits in prepulse inhibition of the acoustic startle response. Moreover, in the primary screen, blood is collected from mice and subjected to a comprehensive clinical biochemical analysis. Subsequently, secondary and tertiary screens of increasing complexity can be used on animals demonstrating deficits in the primary screen. Frozen sperm is archived from all the male mice passing through the screen. In addition, tail tips are stored for DNA. Overall, the program will provide an extensive new resource of mutant and phenotype data to the mouse and human genetics communities at large. The challenge now is to employ the expanding mouse mutant resource to improve the mutant map of the mouse. An improved mutant map of the mouse will be an important asset in exploiting the growing gene map of the mouse and assisting with the identification of genes underlying novel mutations-with consequent benefits for the analysis of gene function and the identification of novel pathways.

Informatics for mutagenesis: the design of mutabase--a distributed data recording system for animal husbandry, mutagenesis, and phenotypic analysis.

The increasing use of high-throughput methods for the production of biologically important information and the increasing diversity of that information pose considerable bioinformatics challenges. These challenges will be met by implementing electronic data management systems not only to capture the data, but increasingly to provide a platform for data integration and mining as we enter the post-genomic era. We discuss the design and implementation of such a data capture system, 'Mutabase', as a model of how such electronic systems might be designed and implemented. Mutabase was created in support of a large-scale, phenotype-driven mouse mutagenesis program at MRC Mammalian Genetics Unit, Harwell, in collaboration with SmithKline Beecham Pharmaceuticals, Queen Mary and Westfield College, London, and Imperial College of Science, Technology and Medicine, London. The aim of this mutagenesis project is to make a significant contribution to the existing mouse mutant resource, closing the phenotype gap and providing many more models for fundamental research and disease modeling. Mutabase records experimental details at the 'point of generation' and provides a number of dissemination and analysis tools for the experimental data, as well as providing a means of assessing various aspects of progress of the program. Mutabase uses a hypertext-based interface to provide interaction between a number of intranet-based client workstations and a central industrial strength database. Mutabase utilizes a variety of techniques in order to implement the user interface system including Perl/CGI, Java Servlets, and an experimental CORBA server. We discuss the relative merits of these methods in the context of the need to provide sound informatics approaches for the support of systematic mutagenesis programs.

Towards new models of disease and physiology in the neurosciences: the role of induced and naturally occurring mutations.

There is a dearth of good mouse models for central nervous system (CNS) disorders. However, the development of gene-targeted technology and the recognition of the importance of the mouse as a model organism have led to the development of a range of behavioural tests for mice. Spontaneous mutations in mice have already provided important information about the role of novel gene products in disorders such as epilepsy and deafness. This has provided the impetus to the establishment of large-scale mutagenesis programmes to generate new mutations. Tests of sensory and motor function have previously been most frequently used as these are simple to perform and the phenotypes are relatively obvious. Subtle phenotypes, of relevance to pyschiatric disorders such as anxiety and schizophrenia, can be detected using more complex tests. Screens such as prepulse inhibition and startle have been adapted for mice and these can be run with relatively high throughput using fully automated equipment. Other behaviours such as sleep and circadian rhythms, learning and memory and nociception can also be assessed. New technological advances in non-invasive imaging and neurochemical analyses have meant that these techniques can be readily applied to mouse phenotyping. The use of these screens together with mutagenesis is already beginning to increase the numbers of mouse models of potential relevance to CNS diseases.

Generation of mouse mutants as a tool for functional genomics.

As sequence information becomes available from the Human Genome Project, key developments include systematic methods for assigning function to each of the 100,000 or so genes. Strategies for coping with this sequence information, including microarray analysis and proteomics, will further our understanding of how genes function and interact. Ultimately, however, the simplest way to understand how a gene works is to examine the consequences of interference with its function: mutational analysis. The mouse represents the model organism of choice in the analysis of gene function; close enough to human to represent a satisfactory model organism, yet relatively easy to manipulate at a genetic level. Two complementary approaches, genotype- and phenotype-based, have been established in the mouse genetics and genomics communities to systematically generate new mouse mutations. Genotype-based approaches are advantageous in that molecular analysis of mutations is facilitated. Phenotypic analysis, however, is often assumed based on gene expression patterns, often leading to unexpected results. Phenotype-based approaches do not make prior assumptions about gene function. Often, however, it may be difficult to define the underlying genetic lesion. Progress in each of these approaches will be considered and situations in which they might be mutually beneficial will be investigated.

The evolution of sexual size dimorphism in the house finch. II. Population divergence in relation to local selection.

Recent colonization of ecologically distinct areas in North America by the house finch (Carpodacus mexicanus) was accompanied by strong population divergence in sexual size dimorphism. Here we examined whether this divergence was produced by population differences in local selection pressures acting on each sex. In a long-term study of recently established populations in Alabama, Michigan, and Montana, we examined three selection episodes for each sex: selection for pairing success, overwinter survival, and within-season fecundity. Populations varied in intensity of these selection episodes, the contribution of each episode to the net selection, and in the targets of selection. Direction and intensity of selection strongly differed between sexes, and different selection episodes often favored opposite changes in morphological traits. In each population, current net selection for sexual dimorphism was highly concordant with observed sexual dimorphism--in each population, selection for dimorphism was the strongest on the most dimorphic traits. Strong directional selection on sexually dimorphic traits, and similar intensities of selection in both sexes, suggest that in each of the recently established populations, both males and females are far from their local fitness optimum, and that sexual dimorphism has arisen from adaptive responses in both sexes. Population differences in patterns of selection on dimorphism, combined with both low levels of ontogenetic integration in heritable sexually dimorphic traits and sexual dimorphism in growth patterns, may account for the close correspondence between dimorphism in selection and observed dimorphism in morphology across house finch populations.

Maintenance of a captive flock of house finches free of infection by Mycoplasma gallisepticum.

Since the beginning of an epidemic of conjunctivitis in wild house finches caused by Mycoplasma gallisepticum (MG), all captive colonies established by capturing free-ranging house finches from the eastern population have also either been infected at the time of capture or developed infection shortly after capture. In an attempt to avoid this infection in captive flocks being maintained for studies of the finches' behavior and ecology, we compared two different flock management strategies and were able to prevent the development of mycoplasmal conjunctivitis with one of the strategies. Single-sex flocks were built by introducing only seronegative wild-caught birds showing no clinical signs of conjunctivitis and covering their outdoor flight cages with netting to prevent interaction with other wild birds although only the female flocks were initially treated with a 6-wk course of tylosin tartrate (0.3 mg/ml). The female flocks never developed conjunctivitis although the disease did develop in the male flocks. Furthermore, serologic assessments of the healthy flock by serum plate agglutination assays for MG indicated that the females remained free of MG infection in the final 7 wk of the study, during which they were unmedicated. We conclude that any low-level MG infection not diagnosed by the initial test for seroconversion was cleared by the prolonged drug treatment.

A retroviral gene trap insertion into the histone 3.3A gene causes partial neonatal lethality, stunted growth, neuromuscular deficits and male sub-fertility in transgenic mice.

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.

Mouse mutagenesis-systematic studies of mammalian gene function.

The mouse will play a pivotal role in mammalian gene function studies as we enter the post-genomics era. The challenge is to develop systematic, genome-wide mutagenesis approaches to the study of gene function. The current mouse mutant resource has been an important source of human genetic disease models. However, despite an apparently large catalogue of mouse mutations, we have access to mutations at only a small fraction of the likely total number of mammalian genes-there is a phenotype gap that needs to be filled by the establishment of new mutagenesis programmes. Two routes, genotype- and phenotype-driven, can be used for the recovery of novel mouse mutations. For the former, gene trap embryonic stem cell libraries appear set to deliver a large number of mutations around the mouse genome. The advantage of genotype-driven approaches is the ease of identification of the mutated locus; the disadvantage that a priori assumptions have to be made concerning the function and likely phenotype of the mutated gene. In contrast, phenotype-driven mutagenesis emphasizes the recovery of novel phenotypes. One phenotype-driven approach that will play an important role in expanding the mouse mutant resource employs the mutagen N-ethyl-N-nitrosourea (ENU). The phenotype-driven route makes no assumptions about the underlying genes involved, and ENU mutagenesis programmes can be expected to play a significant role in uncovering novel pathways and genes; the disadvantage is that the identification of the mutant gene is still not trivial. Together, the complementary routes of genotype- and phenotype-driven mutagenesis will provide a much enlarged catalogue of mouse mutations and phenotypes for future gene function studies.