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1.
BMC Biol ; 20(1): 67, 2022 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-35296311

RESUMO

BACKGROUND: Mice carrying targeted mutations are important for investigating gene function and the role of genes in disease, but off-target mutagenic effects associated with the processes of generating targeted alleles, for instance using Crispr, and culturing embryonic stem cells, offer opportunities for spontaneous mutations to arise. Identifying spontaneous mutations relies on the detection of phenotypes segregating independently of targeted alleles, and having a broad estimate of the level of mutations generated by intensive breeding programmes is difficult given that many phenotypes are easy to miss if not specifically looked for. Here we present data from a large, targeted knockout programme in which mice were analysed through a phenotyping pipeline. Such spontaneous mutations segregating within mutant lines may confound phenotypic analyses, highlighting the importance of record-keeping and maintaining correct pedigrees. RESULTS: Twenty-five lines out of 1311 displayed different deafness phenotypes that did not segregate with the targeted allele. We observed a variety of phenotypes by Auditory Brainstem Response (ABR) and behavioural assessment and isolated eight lines showing early-onset severe progressive hearing loss, later-onset progressive hearing loss, low frequency hearing loss, or complete deafness, with vestibular dysfunction. The causative mutations identified include deletions, insertions, and point mutations, some of which involve new genes not previously associated with deafness while others are new alleles of genes known to underlie hearing loss. Two of the latter show a phenotype much reduced in severity compared to other mutant alleles of the same gene. We investigated the ES cells from which these lines were derived and determined that only one of the 8 mutations could have arisen in the ES cell, and in that case, only after targeting. Instead, most of the non-segregating mutations appear to have occurred during breeding of mutant mice. In one case, the mutation arose within the wildtype colony used for expanding mutant lines. CONCLUSIONS: Our data show that spontaneous mutations with observable effects on phenotype are a common side effect of intensive breeding programmes, including those underlying targeted mutation programmes. Such spontaneous mutations segregating within mutant lines may confound phenotypic analyses, highlighting the importance of record-keeping and maintaining correct pedigrees.


Assuntos
Surdez , Perda Auditiva , Alelos , Animais , Surdez/genética , Perda Auditiva/genética , Camundongos , Mutagênese , Mutação
2.
PLoS Biol ; 17(4): e3000194, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30973865

RESUMO

Adult-onset hearing loss is very common, but we know little about the underlying molecular pathogenesis impeding the development of therapies. We took a genetic approach to identify new molecules involved in hearing loss by screening a large cohort of newly generated mouse mutants using a sensitive electrophysiological test, the auditory brainstem response (ABR). We review here the findings from this screen. Thirty-eight unexpected genes associated with raised thresholds were detected from our unbiased sample of 1,211 genes tested, suggesting extreme genetic heterogeneity. A wide range of auditory pathophysiologies was found, and some mutant lines showed normal development followed by deterioration of responses, revealing new molecular pathways involved in progressive hearing loss. Several of the genes were associated with the range of hearing thresholds in the human population and one, SPNS2, was involved in childhood deafness. The new pathways required for maintenance of hearing discovered by this screen present new therapeutic opportunities.


Assuntos
Percepção Auditiva/genética , Potenciais Evocados Auditivos do Tronco Encefálico/genética , Perda Auditiva/genética , Estimulação Acústica/métodos , Adulto , Animais , Proteínas de Transporte de Ânions/genética , Criança , Fenômenos Eletrofisiológicos/genética , Potenciais Evocados Auditivos do Tronco Encefálico/fisiologia , Feminino , Estudos de Associação Genética , Audição/genética , Perda Auditiva/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL
3.
J Neurosci ; 38(44): 9539-9550, 2018 10 31.
Artigo em Inglês | MEDLINE | ID: mdl-30242051

RESUMO

We identify Sox14 as an exclusive marker of inhibitory projection neurons in the lateral and interposed, but not the medial, cerebellar nuclei. Sox14+ neurons make up ∼80% of Gad1+ neurons in these nuclei and are indistinguishable by soma size from other inhibitory neurons. All Sox14+ neurons of the lateral and interposed cerebellar nuclei are generated at approximately E10/10.5 and extend long-range, predominantly contralateral projections to the inferior olive. A small Sox14+ population in the adjacent vestibular nucleus "Y" sends an ipsilateral projection to the oculomotor nucleus. Cerebellar Sox14+ and glutamatergic projection neurons assemble in non-overlapping populations at the nuclear transition zone, and their integration into a coherent nucleus depends on Sox14 function. Targeted ablation of Sox14+ cells by conditional viral expression of diphtheria toxin leads to significantly impaired motor learning. Contrary to expectations, associative learning is unaffected by unilateral Sox14+ neuron elimination in the interposed and lateral nuclei.SIGNIFICANCE STATEMENT The cerebellar nuclei are central to cerebellar function, yet how they modulate and process cerebellar inputs and outputs is still primarily unknown. Our study gives a direct insight into how nucleo-olivary projection neurons are generated, their projections, and their function in an intact behaving mouse. These neurons play a critical conceptual role in all models of cerebellar function, and this study represents the first specific analysis of their molecular identity and function and offers a powerful model for future investigation of cerebellar function in motor control and learning.


Assuntos
Aprendizagem por Associação/fisiologia , Núcleos Cerebelares/metabolismo , Núcleo Olivar/metabolismo , Fatores de Transcrição SOXB2/deficiência , Animais , Células Cultivadas , Núcleos Cerebelares/química , Cerebelo/química , Cerebelo/metabolismo , Feminino , Locomoção/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Vias Neurais/química , Vias Neurais/metabolismo , Núcleo Olivar/química , Fatores de Transcrição SOXB2/genética
4.
Science ; 381(6659): eadd7564, 2023 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-37590359

RESUMO

The extraembryonic yolk sac (YS) ensures delivery of nutritional support and oxygen to the developing embryo but remains ill-defined in humans. We therefore assembled a comprehensive multiomic reference of the human YS from 3 to 8 postconception weeks by integrating single-cell protein and gene expression data. Beyond its recognized role as a site of hematopoiesis, we highlight roles in metabolism, coagulation, vascular development, and hematopoietic regulation. We reconstructed the emergence and decline of YS hematopoietic stem and progenitor cells from hemogenic endothelium and revealed a YS-specific accelerated route to macrophage production that seeds developing organs. The multiorgan functions of the YS are superseded as intraembryonic organs develop, effecting a multifaceted relay of vital functions as pregnancy proceeds.


Assuntos
Desenvolvimento Embrionário , Saco Vitelino , Feminino , Humanos , Gravidez , Coagulação Sanguínea/genética , Macrófagos , Saco Vitelino/citologia , Saco Vitelino/metabolismo , Desenvolvimento Embrionário/genética , Atlas como Assunto , Expressão Gênica , Perfilação da Expressão Gênica , Hematopoese/genética , Fígado/embriologia
5.
Hear Res ; 387: 107879, 2020 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-31927188

RESUMO

The underlying causes of age-related hearing loss (ARHL) are not well understood, but it is clear from heritability estimates that genetics plays a role in addition to environmental factors. Genome-wide association studies (GWAS) in human populations can point to candidate genes that may be involved in ARHL, but follow-up analysis is needed to assess the role of these genes in the disease process. Some genetic variants may contribute a small amount to a disease, while other variants may have a large effect size, but the genetic architecture of ARHL is not yet well-defined. In this study, we asked if a set of 17 candidate genes highlighted by early GWAS reports of ARHL have detectable effects on hearing by knocking down expression levels of each gene in the mouse and analysing auditory function. We found two of the genes have an impact on hearing. Mutation of Dclk1 led to late-onset progressive increase in ABR thresholds and the A430005L14Rik (C1orf174) mutants showed worse recovery from noise-induced damage than controls. We did not detect any abnormal responses in the remaining 15 mutant lines either in thresholds or from our battery of suprathreshold ABR tests, and we discuss the possible reasons for this.


Assuntos
Percepção Auditiva/genética , Variação Genética , Perda Auditiva Provocada por Ruído/genética , Audição/genética , Presbiacusia/genética , Fatores Etários , Animais , Quinases Semelhantes a Duplacortina , Potenciais Evocados Auditivos do Tronco Encefálico/genética , Feminino , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Perda Auditiva Provocada por Ruído/fisiopatologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Masculino , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Camundongos Transgênicos , Fenótipo , Presbiacusia/fisiopatologia , Proteínas Serina-Treonina Quinases/genética , Medição de Risco , Fatores de Risco
6.
Sci Rep ; 9(1): 15218, 2019 10 23.
Artigo em Inglês | MEDLINE | ID: mdl-31645601

RESUMO

The developing cerebellum of amniotes is characterised by a unique, transient, secondary proliferation zone: the external germinal layer (EGL). The EGL is comprised solely of granule cell precursors, whose progeny migrate inwardly to form the internal granule cell layer. While a range of cell morphologies in the EGL has long been known, how they reflect the cells' differentiation status has previously only been inferred. Observations have suggested a deterministic maturation from outer to inner EGL that we wished to test experimentally. To do this, we electroporated granule cell precursors in chick with plasmids encoding fluorescent proteins and probed labelled cells with markers of both proliferation (phosphohistone H3) and differentiation (Axonin1/TAG1 and NeuroD1). We show that granule cell precursors can display a range of complex forms throughout the EGL while mitotically active. Overexpression of full length NeuroD1 within granule cell precursors does not abolish proliferation, but biases granule cells towards precocious differentiation, alters their migration path and results in a smaller and less foliated cerebellum. Our results show that granule cells show a greater flexibility in differentiation than previously assumed. We speculate that this allows the EGL to regulate its proliferative activity in response to overall patterns of cerebellar growth.


Assuntos
Cerebelo/embriologia , Embrião de Galinha/embriologia , Células-Tronco Neurais/citologia , Animais , Proteínas Aviárias/análise , Fatores de Transcrição Hélice-Alça-Hélice Básicos/análise , Movimento Celular , Proliferação de Células , Cerebelo/citologia , Galinhas , Mitose , Proteínas do Tecido Nervoso/análise , Neurogênese
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