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1.
Dis Model Mech ; 2021 Sep 03.
Artigo em Inglês | MEDLINE | ID: mdl-34477842

RESUMO

Down syndrome (DS), trisomy 21, results in many complex phenotypes including cognitive deficits, heart defects and craniofacial alterations. Phenotypes arise from an extra copy of human chromosome 21 (Hsa21) genes. However, these dosage-sensitive causative genes remain unknown. Animal models enable identification of genes and pathological mechanisms. The Dp1Tyb mouse model of DS has an extra copy of 63% of Hsa21-orthologous mouse genes. In order to establish if this model recapitulates DS phenotypes, we comprehensively phenotyped Dp1Tyb mice using 28 tests of different physiological systems and found that 468 out of 1800 parameters were significantly altered. We show that Dp1Tyb mice have wide-ranging DS-like phenotypes including aberrant erythropoiesis and megakaryopoiesis, reduced bone density, craniofacial changes, altered cardiac function, a pre-diabetic state and deficits in memory, locomotion, hearing and sleep. Thus, Dp1Tyb mice are an excellent model for investigating complex DS phenotype-genotype relationships for this common disorder.

2.
FASEB J ; 35(9): e21802, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34383984

RESUMO

Mutations in transcription factors often exhibit pleiotropic effects related to their complex expression patterns and multiple regulatory targets. One such mutation in the zinc finger homeobox 3 (ZFHX3) transcription factor, short circuit (Sci, Zfhx3Sci/+ ), is associated with significant circadian deficits in mice. However, given evidence of its retinal expression, we set out to establish the effects of the mutation on retinal function using molecular, cellular, behavioral and electrophysiological measures. Immunohistochemistry confirms the expression of ZFHX3 in multiple retinal cell types, including GABAergic amacrine cells and retinal ganglion cells including intrinsically photosensitive retinal ganglion cells (ipRGCs). Zfhx3Sci/+ mutants display reduced light responsiveness in locomotor activity and circadian entrainment, relatively normal electroretinogram and optomotor responses but exhibit an unexpected pupillary reflex phenotype with markedly increased sensitivity. Furthermore, multiple electrode array recordings of Zfhx3Sci/+ retina show an increased sensitivity of ipRGC light responses.


Assuntos
Ritmo Circadiano/fisiologia , Proteínas de Homeodomínio/metabolismo , Retina/metabolismo , Células Amácrinas/metabolismo , Animais , Luz , Locomoção/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Estimulação Luminosa/métodos , Células Ganglionares da Retina/metabolismo , Visão Ocular/fisiologia
3.
Curr Protoc Mouse Biol ; 10(3): e81, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32865891

RESUMO

The 24-hr cycle of activity and sleep provides perhaps the most familiar example of circadian rhythms. In mammals, circadian activity rhythms are generated by a master biological clock located in the hypothalamic suprachiasmatic nuclei (SCN). This clock is synchronized (entrained) to the external light environment via light input from retinal photoreceptors. However, sleep is not a simple circadian output and also is regulated by a homeostatic process whereby extended wakefulness increases the need for subsequent sleep. As such, the amount and distribution of sleep depends upon the interaction between both circadian and homeostatic processes. Moreover, the study of circadian activity and sleep is not confined only to these specialized fields. Sleep and circadian rhythm disruption is common in many conditions, ranging from neurological and metabolic disorders to aging. Such disruption is associated with a range of negative consequences including cognitive impairment and mood disorders, as well as immune and metabolic dysfunction. As circadian activity and sleep are hallmarks of normal healthy physiology, they also provide valuable welfare indicators. However, traditional methods for the monitoring of circadian rhythms and sleep in mice can require separate specialized resources as well as significant expertise. Here, we outline a low-cost, non-invasive, and open-source method for the simultaneous assessment of circadian activity and sleep in mice. This protocol describes both the assembly of the hardware used and the capture and analysis of data without the need for expertise in electronics or data processing. © 2020 Wiley Periodicals LLC. Basic Protocol: Assembly of a PIR system for basic activity and sleep recordings Alternate Protocol: Data collection using Raspberry Pi Support Protocol: Circadian analysis using PIR sensors.

4.
Curr Protoc Mouse Biol ; 10(3): e80, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32813317

RESUMO

Over the last century, the study of mouse behavior has uncovered insights into brain molecular mechanisms while revealing potential causes of many neurological disorders. To this end, researchers have widely exploited the use of mutant strains, including those generated in mutagenesis screens and those produced using increasingly sophisticated genome engineering technologies. It is now relatively easy to access mouse models carrying alleles that faithfully recapitulate changes found in human patients or bearing variants of genes that provide data on those genes' functions. Concurrent with these developments has been an appreciation of the limitations of some current testing platforms, especially those monitoring complex behaviors. Out-of-cage observational testing is useful in describing overt persistent phenotypes but risks missing sporadic or intermittent events. Furthermore, measuring the progression of a phenotype, potentially over many months, can be difficult while relying on assays that may be susceptible to changes in the testing environment. In recent years, there has also been increasing awareness that measurement of behaviors in isolation can be limiting, given that mice attempt to hide behavioral cues of vulnerability. To overcome these limitations, laboratory animal science is capitalizing on progress in data capture and processing expertise. Moreover, as additional recording modes become commonplace, ultrasonic vocalization recording is an appealing focus, as mice use vocalizations in various social contexts. Using video and audio technologies, we record the voluntary, unprovoked behaviors and vocalizations of mice in social groups. Adoption of these approaches is undoubtedly set to increase, as they capture the round-the-clock behavior of mouse strains. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Continuous recording of home cage activity using the Home Cage Analyzer (HCA) system Support Protocol: Subcutaneous insertion of a radio frequency identification microchip in the inguinal area Basic Protocol 2: Continuous recording of mouse ultrasonic vocalizations in the home cage.

5.
Sci Adv ; 6(33): eabb3567, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32851175

RESUMO

Switches between global sleep and wakefulness states are believed to be dictated by top-down influences arising from subcortical nuclei. Using forward genetics and in vivo electrophysiology, we identified a recessive mouse mutant line characterized by a substantially reduced propensity to transition between wake and sleep states with an especially pronounced deficit in initiating rapid eye movement (REM) sleep episodes. The causative mutation, an Ile102Asn substitution in the synaptic vesicular protein, VAMP2, was associated with morphological synaptic changes and specific behavioral deficits, while in vitro electrophysiological investigations with fluorescence imaging revealed a markedly diminished probability of vesicular release in mutants. Our data show that global shifts in the synaptic efficiency across brain-wide networks leads to an altered probability of vigilance state transitions, possibly as a result of an altered excitability balance within local circuits controlling sleep-wake architecture.

7.
PLoS Biol ; 17(9): e3000414, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31479441

RESUMO

Bardet-Biedl syndrome (BBS), a ciliopathy, is a rare genetic condition characterised by retinal degeneration, obesity, kidney failure, and cognitive impairment. In spite of progress made in our general understanding of BBS aetiology, the molecular and cellular mechanisms underlying cognitive impairment in BBS remain elusive. Here, we report that the loss of BBS proteins causes synaptic dysfunction in principal neurons, providing a possible explanation for the cognitive impairment phenotype observed in BBS patients. Using synaptosomal proteomics and immunocytochemistry, we demonstrate the presence of Bbs proteins in the postsynaptic density (PSD) of hippocampal neurons. Loss of Bbs results in a significant reduction of dendritic spines in principal neurons of Bbs mouse models. Furthermore, we show that spine deficiency correlates with events that destabilise spine architecture, such as impaired spine membrane receptor signalling, known to be involved in the maintenance of dendritic spines. Our findings suggest a role for BBS proteins in dendritic spine homeostasis that may be linked to the cognitive phenotype observed in BBS.


Assuntos
Síndrome de Bardet-Biedl/patologia , Proteínas do Citoesqueleto/metabolismo , Espinhas Dendríticas/patologia , Animais , Ansiedade , Síndrome de Bardet-Biedl/metabolismo , Síndrome de Bardet-Biedl/fisiopatologia , Síndrome de Bardet-Biedl/psicologia , Giro Denteado/fisiopatologia , Modelos Animais de Doenças , Potenciais Pós-Sinápticos Excitadores , Feminino , Masculino , Memória , Camundongos , Receptor IGF Tipo 1/metabolismo , Sinaptossomos/metabolismo
8.
Mamm Genome ; 30(3-4): 54-62, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-31020388

RESUMO

Spermatogenesis-associated protein 13 (Spata13) is a guanine nucleotide exchange factor (GEF) enriched in discrete brain regions in the adult, with pronounced expression in the extended central amygdala (CeA). Loss of Spata13, also known as the adenomatous polyposis coli exchange factor Asef2, has no identifiable phenotype although it has been shown to reduce the number and size of intestinal tumours in Apc (Min/+) mice. Nevertheless, its brain-related functions have not been investigated. To pursue this, we have generated a Spata13 knockout mouse line using CRISPR-mediated deletion of an exon containing the GTPase domain that is common to multiple isoforms. Homozygous mutants were viable and appeared normal. We subjected both male and female cohorts to a comprehensive battery of behavioural tests designed to investigate particular CeA-related functions. Here, we show that Spata13 modulates social behaviour with homozygous mutants being subordinate to wildtype controls. Furthermore, female homozygotes show increased activity in home cages during the dark phase of the light-dark cycle. In summary, Spata13 modulates social hierarchy in both male and female mice in addition to affecting voluntary activity in females.


Assuntos
Ritmo Circadiano/efeitos da radiação , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Comportamento Social , Animais , Comportamento Animal/efeitos da radiação , Feminino , Fatores de Troca do Nucleotídeo Guanina/genética , Hierarquia Social , Humanos , Masculino , Camundongos , Camundongos Knockout , Fotoperíodo , Predomínio Social
9.
Dis Model Mech ; 12(2)2019 02 22.
Artigo em Inglês | MEDLINE | ID: mdl-30692144

RESUMO

Loss-of-function mutations in a human AMPA receptor-associated protein, ferric chelate reductase 1-like (FRRS1L), are associated with a devastating neurological condition incorporating choreoathetosis, cognitive deficits and epileptic encephalopathies. Furthermore, evidence from overexpression and ex vivo studies has implicated FRRS1L in AMPA receptor biogenesis, suggesting that changes in glutamatergic signalling might underlie the disorder. Here, we investigated the neurological and neurobehavioural correlates of the disorder using a mouse Frrs1l null mutant. The study revealed several neurological defects that mirrored those seen in human patients. We established that mice lacking Frrs1l suffered from a broad spectrum of early-onset motor deficits with no progressive, age-related deterioration. Moreover, Frrs1l-/- mice were hyperactive, irrespective of test environment, exhibited working memory deficits and displayed significant sleep fragmentation. Longitudinal electroencephalographic (EEG) recordings also revealed abnormal EEG results in Frrs1l-/- mice. Parallel investigations into disease aetiology identified a specific deficiency in AMPA receptor levels in the brain of Frrs1l-/- mice, while the general levels of several other synaptic components remained unchanged, with no obvious alterations in the number of synapses. Furthermore, we established that Frrsl1 deletion results in an increased proportion of immature AMPA receptors, indicated by incomplete glycosylation of GLUA2 (also known as GRIA2) and GLUA4 (also known as GRIA4) AMPA receptor proteins. This incomplete maturation leads to cytoplasmic retention and a reduction of those specific AMPA receptor levels in the postsynaptic membrane. Overall, this study determines, for the first time in vivo, how loss of FRRS1L function can affect glutamatergic signalling, and provides mechanistic insight into the development and progression of a human hyperkinetic disorder.This article has an associated First Person interview with the first author of the paper.


Assuntos
Cognição , Fenômenos Eletrofisiológicos , Proteínas de Membrana/metabolismo , Atividade Motora , Proteínas do Tecido Nervoso/metabolismo , Sistema Nervoso/crescimento & desenvolvimento , Sistema Nervoso/patologia , Receptores de AMPA/metabolismo , Sinapses/metabolismo , Animais , Animais Recém-Nascidos , Tamanho Corporal , Encéfalo/metabolismo , Encéfalo/patologia , Transtornos Cognitivos/patologia , Citoplasma/metabolismo , Glicosilação , Proteínas de Membrana/genética , Camundongos Endogâmicos C57BL , Proteínas do Tecido Nervoso/genética , Sistema Nervoso/fisiopatologia , Sono , Análise de Sobrevida
10.
FASEB J ; 32(8): 4302-4314, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29561690

RESUMO

Cryptochromes 1 and 2 (CRY1/2) are key components of the negative limb of the mammalian circadian clock. Like many peripheral tissues, Cry1 and -2 are expressed in the retina, where they are thought to play a role in regulating rhythmic physiology. However, studies differ in consensus as to their localization and function, and CRY1 immunostaining has not been convincingly demonstrated in the retina. Here we describe the expression and function of CRY1 and -2 in the mouse retina in both sexes. Unexpectedly, we show that CRY1 is expressed throughout all retinal layers, whereas CRY2 is restricted to the photoreceptor layer. Retinal period 2::luciferase recordings from CRY1-deficient mice show reduced clock robustness and stability, while those from CRY2-deficient mice show normal, albeit long-period, rhythms. In functional studies, we then investigated well-defined rhythms in retinal physiology. Rhythms in the photopic electroretinogram, contrast sensitivity, and pupillary light response were all severely attenuated or abolished in CRY1-deficient mice. In contrast, these physiological rhythms are largely unaffected in mice lacking CRY2, and only photopic electroretinogram rhythms are affected. Together, our data suggest that CRY1 is an essential component of the mammalian retinal clock, whereas CRY2 has a more limited role.-Wong, J. C. Y., Smyllie, N. J., Banks, G. T., Pothecary, C. A., Barnard, A. R., Maywood, E. S., Jagannath, A., Hughes, S., van der Horst, G. T. J., MacLaren, R. E., Hankins, M. W., Hastings, M. H., Nolan, P. M., Foster, R. G., Peirson, S. N. Differential roles for cryptochromes in the mammalian retinal clock.


Assuntos
Criptocromos/metabolismo , Mamíferos/metabolismo , Mamíferos/fisiologia , Retina/metabolismo , Retina/fisiologia , Animais , Relógios Circadianos/fisiologia , Ritmo Circadiano/fisiologia , Eletrorretinografia/métodos , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Células Fotorreceptoras/metabolismo , Células Fotorreceptoras/fisiologia
11.
J Neurosci Methods ; 300: 37-47, 2018 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-28456660

RESUMO

An important factor in reducing variability in mouse test outcomes has been to develop assays that can be used for continuous automated home cage assessment. Our experience has shown that this has been most evidenced in long-term assessment of wheel-running activity in mice. Historically, wheel-running in mice and other rodents have been used as a robust assay to determine, with precision, the inherent period of circadian rhythms in mice. Furthermore, this assay has been instrumental in dissecting the molecular genetic basis of mammalian circadian rhythms. In teasing out the elements of this test that have determined its robustness - automated assessment of an unforced behaviour in the home cage over long time intervals - we and others have been investigating whether similar test apparatus could be used to accurately discriminate differences in distinct behavioural parameters in mice. Firstly, using these systems, we explored behaviours in a number of mouse inbred strains to determine whether we could extract biologically meaningful differences. Secondly, we tested a number of relevant mutant lines to determine how discriminative these parameters were. Our findings show that, when compared to conventional out-of-cage phenotyping, a far deeper understanding of mouse mutant phenotype can be established by monitoring behaviour in the home cage over one or more light:dark cycles.


Assuntos
Comportamento Animal/fisiologia , Pesquisa Comportamental/métodos , Ritmo Circadiano/fisiologia , Abrigo para Animais , Fotoperíodo , Corrida/fisiologia , Bem-Estar do Animal , Animais , Pesquisa Comportamental/instrumentação , Camundongos
12.
Sci Rep ; 7(1): 17765, 2017 12 19.
Artigo em Inglês | MEDLINE | ID: mdl-29259298

RESUMO

Circadian clock is known to adapt to environmental changes and can significantly influence cognitive and physiological functions. In this work, we report specific behavioral, cognitive, and sleep homeostatic defects in the after hours (Afh) circadian mouse mutant, which is characterized by lengthened circadian period. We found that the circadian timing irregularities in Afh mice resulted in higher interval timing uncertainty and suboptimal decisions due to incapability of processing probabilities. Our phenotypic observations further suggested that Afh mutants failed to exhibit the necessary phenotypic plasticity for adapting to temporal changes at multiple time scales (seconds-to-minutes to circadian). These behavioral effects of Afh mutation were complemented by the specific disruption of the Per/Cry circadian regulatory complex in brain regions that govern food anticipatory behaviors, sleep, and timing. We derive statistical predictions, which indicate that circadian clock and sleep are complementary processes in controlling behavioral/cognitive performance during 24 hrs. The results of this study have pivotal implications for understanding how the circadian clock modulates sleep and behavior.


Assuntos
Adaptação Fisiológica/fisiologia , Comportamento Animal/fisiologia , Relógios Circadianos/fisiologia , Ritmo Circadiano/fisiologia , Homeostase/fisiologia , Sono/fisiologia , Adaptação Fisiológica/genética , Animais , Encéfalo/fisiologia , Relógios Circadianos/genética , Ritmo Circadiano/genética , Feminino , Homeostase/genética , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Mutação/genética , Sono/genética
13.
Mol Metab ; 6(11): 1419-1428, 2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-29107289

RESUMO

OBJECTIVE: Genetic studies in obese rodents and humans can provide novel insights into the mechanisms involved in energy homeostasis. METHODS: In this study, we genetically mapped the chromosomal region underlying the development of severe obesity in a mouse line identified as part of a dominant N-ethyl-N-nitrosourea (ENU) mutagenesis screen. We characterized the metabolic and behavioral phenotype of obese mutant mice and examined changes in hypothalamic gene expression. In humans, we examined genetic data from people with severe early onset obesity. RESULTS: We identified an obese mouse heterozygous for a missense mutation (pR108W) in orthopedia homeobox (Otp), a homeodomain containing transcription factor required for the development of neuroendocrine cell lineages in the hypothalamus, a region of the brain important in the regulation of energy homeostasis. OtpR108W/+ mice exhibit increased food intake, weight gain, and anxiety when in novel environments or singly housed, phenotypes that may be partially explained by reduced hypothalamic expression of oxytocin and arginine vasopressin. R108W affects the highly conserved homeodomain, impairs DNA binding, and alters transcriptional activity in cells. We sequenced OTP in 2548 people with severe early-onset obesity and found a rare heterozygous loss of function variant in the homeodomain (Q153R) in a patient who also had features of attention deficit disorder. CONCLUSIONS: OTP is involved in mammalian energy homeostasis and behavior and appears to be necessary for the development of hypothalamic neural circuits. Further studies will be needed to investigate the contribution of rare variants in OTP to human energy homeostasis.


Assuntos
Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Sequência de Aminoácidos , Animais , Ansiedade/metabolismo , Sequência de Bases , Encéfalo/metabolismo , Mapeamento Cromossômico , Bases de Dados Genéticas , Feminino , Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento/genética , Genes Homeobox , Proteínas de Homeodomínio/fisiologia , Humanos , Hipotálamo/metabolismo , Masculino , Camundongos , Proteínas do Tecido Nervoso/fisiologia , Sistemas Neurossecretores/metabolismo , Obesidade/metabolismo , Fatores de Transcrição/genética , Transcriptoma/genética
14.
Nucleic Acids Res ; 45(17): 9860-9873, 2017 Sep 29.
Artigo em Inglês | MEDLINE | ID: mdl-28973476

RESUMO

The master circadian pacemaker in mammals is located in the suprachiasmatic nuclei (SCN) which regulate physiology and behaviour, as well as coordinating peripheral clocks throughout the body. Investigating the function of the SCN has often focused on the identification of rhythmically expressed genes. However, not all genes critical for SCN function are rhythmically expressed. An alternative strategy is to characterize those genes that are selectively enriched in the SCN. Here, we examined the transcriptome of the SCN and whole brain (WB) of mice using meta-analysis of publicly deposited data across a range of microarray platforms and RNA-Seq data. A total of 79 microarrays were used (24 SCN and 55 WB samples, 4 different microarray platforms), alongside 17 RNA-Seq data files (7 SCN and 10 WB). 31 684 MGI gene symbols had data for at least one platform. Meta-analysis using a random effects model for weighting individual effect sizes (derived from differential expression between relevant SCN and WB samples) reliably detected known SCN markers. SCN-enriched transcripts identified in this study provide novel insights into SCN function, including identifying genes which may play key roles in SCN physiology or provide SCN-specific drivers.


Assuntos
Relógios Circadianos/genética , Ritmo Circadiano/genética , Redes Reguladoras de Genes , Núcleo Supraquiasmático/fisiologia , Transcriptoma , Animais , Química Encefálica , Mineração de Dados , Conjuntos de Dados como Assunto , Ontologia Genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Anotação de Sequência Molecular , Análise de Sequência com Séries de Oligonucleotídeos , Análise de Sequência de RNA
15.
J Biol Rhythms ; 32(5): 433-443, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28816086

RESUMO

The transcription factor zinc finger homeobox 3 (ZFHX3) plays a key role in coupling intracellular transcriptional-translational oscillations with intercellular synchrony in mouse suprachiasmatic nucleus (SCN). However, like many key players in central nervous system function, ZFHX3 serves an important role in neurulation and neuronal terminal differentiation while retaining discrete additional functions in the adult SCN. Recently, using a dominant missense mutation in mouse Zfhx3, we established that this gene can modify circadian period and sleep in adult animals. Nevertheless, we were still concerned that the neurodevelopmental consequences of ZFHX3 dysfunction in this mutant may interfere with, or confound, its critical adult-specific roles in SCN circadian function. To circumvent the developmental consequences of Zfhx3 deletion, we crossed a conditional null Zfhx3 mutant to an inducible, ubiquitously expressed Cre line (B6.Cg-Tg(UBC-cre/ERT2)1Ejb/J). This enabled us to assess circadian behavior in the same adult animals both before and after Cre-mediated excision of the critical Zfhx3 exons using tamoxifen treatment. Remarkably, we found a strong and significant alteration in circadian behavior in tamoxifen-treated homozygous animals with no phenotypic changes in heterozygous or control animals. Cre-mediated excision of Zfhx3 critical exons in adult animals resulted in shortening of the period of wheel-running in constant darkness by more than 1 h in the majority of homozygotes while, in 30% of animals, excision resulted in complete behavioral arrhythmicity. In addition, we found that homozygous animals reentrain almost immediately to 6-h phase advances in the light-dark cycle. No additional overt phenotypic changes were evident in treated homozygous animals. These findings confirm a sustained and significant role for ZFHX3 in maintaining rhythmicity in the adult mammalian circadian system.


Assuntos
Relógios Circadianos , Ritmo Circadiano , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/fisiologia , Fatores Etários , Animais , Relógios Circadianos/efeitos dos fármacos , Relógios Circadianos/genética , Ritmo Circadiano/genética , Regulação da Expressão Gênica , Camundongos , Camundongos Knockout , Mutação , Proteínas Circadianas Period/genética , Proteínas Circadianas Period/metabolismo , Fotoperíodo , Núcleo Supraquiasmático/fisiologia , Tamoxifeno/farmacologia
16.
Mamm Genome ; 28(7-8): 377-382, 2017 08.
Artigo em Inglês | MEDLINE | ID: mdl-28756587

RESUMO

The unprecedented efficiency of the CRISPR/Cas9 system in genome engineering has opened the prospect of employing mutant founders for phenotyping cohorts, thus accelerating research projects by circumventing the requirement to generate cohorts using conventional two- or three-generation crosses. However, these first-generation mutants are often genetic mosaics, with a complex and difficult to define genetic make-up. Here, we discuss the potential benefits, challenges and scientific validity of such models.


Assuntos
Edição de Genes , Genoma , Mutação , Fenótipo , Animais , Sistemas CRISPR-Cas , Quimerismo , Edição de Genes/métodos , Edição de Genes/normas , Estudos de Associação Genética/métodos , Estudos de Associação Genética/normas , Engenharia Genética/métodos , Engenharia Genética/normas , Mosaicismo
17.
Nat Commun ; 7: 12444, 2016 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-27534441

RESUMO

Determining the genetic bases of age-related disease remains a major challenge requiring a spectrum of approaches from human and clinical genetics to the utilization of model organism studies. Here we report a large-scale genetic screen in mice employing a phenotype-driven discovery platform to identify mutations resulting in age-related disease, both late-onset and progressive. We have utilized N-ethyl-N-nitrosourea mutagenesis to generate pedigrees of mutagenized mice that were subject to recurrent screens for mutant phenotypes as the mice aged. In total, we identify 105 distinct mutant lines from 157 pedigrees analysed, out of which 27 are late-onset phenotypes across a range of physiological systems. Using whole-genome sequencing we uncover the underlying genes for 44 of these mutant phenotypes, including 12 late-onset phenotypes. These genes reveal a number of novel pathways involved with age-related disease. We illustrate our findings by the recovery and characterization of a novel mouse model of age-related hearing loss.


Assuntos
Envelhecimento/genética , Testes Genéticos , Mutagênese/genética , Animais , Cóclea/metabolismo , Modelos Animais de Doenças , Epitélio/ultraestrutura , Potenciais Evocados Auditivos do Tronco Encefálico/fisiologia , Feminino , Audição/genética , Masculino , Camundongos Endogâmicos C57BL , Mutação/genética , Linhagem , Fenótipo
18.
Cell Rep ; 16(3): 615-21, 2016 07 19.
Artigo em Inglês | MEDLINE | ID: mdl-27373158

RESUMO

An AT motif-dependent axis, modulated by the transcription factor Zfhx3, influences the circadian clock in mice. In particular, gain of function of Zfhx3 significantly shortens circadian rhythms and alters the transcriptional activity of an important class of neuropeptides that controls intercellular signaling in the suprachiasmatic nucleus (SCN) of the hypothalamus. The ZFHX3/AT axis revealed an important, largely cell-nonautonomous control of the circadian clock. Here, by studying the recently identified circadian mouse mutant Zfhx3(Sci/+), we identify significant effects on sleep homeostasis, a phenomenon that is outside the canonical circadian clock system and that is modulated by the activity of those neuropeptides at a circuit level. We show that the Zfhx3(Sci/+) mutation accelerates the circadian clock at both the hourly scale (i.e., advancing circadian rhythms) and the seconds-to-minutes scale (i.e., anticipating behavioral responses) in mice. The in vivo results are accompanied by a significant presence of sleep targets among protein-protein interactions of the Zfhx3(Sci/+)-dependent network.


Assuntos
Relógios Circadianos/fisiologia , Ritmo Circadiano/fisiologia , Proteínas de Homeodomínio/metabolismo , Sono/fisiologia , Animais , Homeostase/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neuropeptídeos/metabolismo , Mapas de Interação de Proteínas/fisiologia , Núcleo Supraquiasmático/metabolismo , Núcleo Supraquiasmático/fisiologia
19.
Front Behav Neurosci ; 10: 106, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27375446

RESUMO

Central nervous system disorders such as autism as well as the range of neurodegenerative diseases such as Huntington's disease are commonly investigated using genetically altered mouse models. The current system for characterizing these mice usually involves removing the animals from their home-cage environment and placing them into novel environments where they undergo a battery of tests measuring a range of behavioral and physical phenotypes. These tests are often only conducted for short periods of times in social isolation. However, human manifestations of such disorders are often characterized by multiple phenotypes, presented over long periods of time and leading to significant social impacts. Here, we have developed a system which will allow the automated monitoring of individual mice housed socially in the cage they are reared and housed in, within established social groups and over long periods of time. We demonstrate that the system accurately reports individual locomotor behavior within the group and that the measurements taken can provide unique insights into the effects of genetic background on individual and group behavior not previously recognized.

20.
PLoS Biol ; 14(6): e1002482, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-27276063

RESUMO

Light plays a critical role in the regulation of numerous aspects of physiology and behaviour, including the entrainment of circadian rhythms and the regulation of sleep. These responses involve melanopsin (OPN4)-expressing photosensitive retinal ganglion cells (pRGCs) in addition to rods and cones. Nocturnal light exposure in rodents has been shown to result in rapid sleep induction, in which melanopsin plays a key role. However, studies have also shown that light exposure can result in elevated corticosterone, a response that is not compatible with sleep. To investigate these contradictory findings and to dissect the relative contribution of pRGCs and rods/cones, we assessed the effects of light of different wavelengths on behaviourally defined sleep. Here, we show that blue light (470 nm) causes behavioural arousal, elevating corticosterone and delaying sleep onset. By contrast, green light (530 nm) produces rapid sleep induction. Compared to wildtype mice, these responses are altered in melanopsin-deficient mice (Opn4-/-), resulting in enhanced sleep in response to blue light but delayed sleep induction in response to green or white light. We go on to show that blue light evokes higher Fos induction in the SCN compared to the sleep-promoting ventrolateral preoptic area (VLPO), whereas green light produced greater responses in the VLPO. Collectively, our data demonstrates that nocturnal light exposure can have either an arousal- or sleep-promoting effect, and that these responses are melanopsin-mediated via different neural pathways with different spectral sensitivities. These findings raise important questions relating to how artificial light may alter behaviour in both the work and domestic setting.


Assuntos
Nível de Alerta/efeitos da radiação , Luz , Opsinas de Bastonetes/metabolismo , Sono/efeitos da radiação , Animais , Nível de Alerta/fisiologia , Corticosterona/sangue , Corticosterona/metabolismo , Expressão Gênica/efeitos da radiação , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Biológicos , Proteínas Circadianas Period/genética , Células Fotorreceptoras de Vertebrados/metabolismo , Células Fotorreceptoras de Vertebrados/efeitos da radiação , Área Pré-Óptica/metabolismo , Área Pré-Óptica/efeitos da radiação , Proteínas Proto-Oncogênicas c-fos/genética , Células Ganglionares da Retina/metabolismo , Células Ganglionares da Retina/efeitos da radiação , Opsinas de Bastonetes/genética , Sono/fisiologia , Núcleo Supraquiasmático/metabolismo , Núcleo Supraquiasmático/efeitos da radiação , Fatores de Tempo
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