RESUMEN
The directed differentiation of pluripotent stem cells (PSCs) from panels of genetically diverse individuals is emerging as a powerful experimental system for characterizing the impact of natural genetic variation on developing cell types and tissues. Here, we establish new PSC lines and experimental approaches for modeling embryonic development in a genetically diverse, outbred mouse stock (Diversity Outbred mice). We show that a range of inbred and outbred PSC lines can be stably maintained in the primed pluripotent state (epiblast stem cells -- EpiSCs) and establish the contribution of genetic variation to phenotypic differences in gene regulation and directed differentiation. Using pooled in vitro fertilization, we generate and characterize a genetic reference panel of Diversity Outbred PSCs (n = 230). Finally, we demonstrate the feasibility of pooled culture of Diversity Outbred EpiSCs as "cell villages", which can facilitate the differentiation of large numbers of EpiSC lines for forward genetic screens. These data can complement and inform similar efforts within the stem cell biology and human genetics communities to model the impact of natural genetic variation on phenotypic variation and disease-risk.
RESUMEN
Directed differentiation of pluripotent stem cells (PSCs) is a powerful model system for deconstructing embryonic development. Although mice are the most advanced mammalian model system for genetic studies of embryonic development, state-of-the-art protocols for directed differentiation of mouse PSCs into defined lineages require additional steps and generates target cell types with lower purity than analogous protocols for human PSCs, limiting their application as models for mechanistic studies of development. Here, we examine the potential of mouse epiblast stem cells cultured in media containing Wnt pathway inhibitors as a starting point for directed differentiation. As a proof of concept, we focused our efforts on two specific cell/tissue types that have proven difficult to generate efficiently and reproducibly from mouse embryonic stem cells: definitive endoderm and neural organoids. We present new protocols for rapid generation of nearly pure definitive endoderm and forebrain-patterned neural organoids that model the development of prethalamic and hippocampal neurons. These differentiation models present new possibilities for combining mouse genetic tools with in vitro differentiation to characterize molecular and cellular mechanisms of embryonic development.
Asunto(s)
Endodermo , Células Madre Pluripotentes , Animales , Diferenciación Celular/fisiología , Endodermo/metabolismo , Femenino , Estratos Germinativos , Humanos , Mamíferos , Ratones , Organoides , Embarazo , ProsencéfaloRESUMEN
Inositol polyphosphates are vital metabolic and secondary messengers, involved in diverse cellular functions. Therefore, tight regulation of inositol polyphosphate metabolism is essential for proper cell physiology. Here, we describe an early-onset neurodegenerative syndrome caused by loss-of-function mutations in the multiple inositol-polyphosphate phosphatase 1 gene (MINPP1). Patients are found to have a distinct type of Pontocerebellar Hypoplasia with typical basal ganglia involvement on neuroimaging. We find that patient-derived and genome edited MINPP1-/- induced stem cells exhibit an inefficient neuronal differentiation combined with an increased cell death. MINPP1 deficiency results in an intracellular imbalance of the inositol polyphosphate metabolism. This metabolic defect is characterized by an accumulation of highly phosphorylated inositols, mostly inositol hexakisphosphate (IP6), detected in HEK293 cells, fibroblasts, iPSCs and differentiating neurons lacking MINPP1. In mutant cells, higher IP6 level is expected to be associated with an increased chelation of intracellular cations, such as iron or calcium, resulting in decreased levels of available ions. These data suggest the involvement of IP6-mediated chelation on Pontocerebellar Hypoplasia disease pathology and thereby highlight the critical role of MINPP1 in the regulation of human brain development and homeostasis.
Asunto(s)
Enfermedades Cerebelosas/metabolismo , Quelantes/metabolismo , Citoplasma/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismo , Ácido Fítico/metabolismo , Animales , Muerte Celular , Diferenciación Celular , Enfermedades Cerebelosas/diagnóstico por imagen , Enfermedades Cerebelosas/patología , Niño , Preescolar , Femenino , Técnicas de Inactivación de Genes , Células HEK293 , Homeostasis , Humanos , Lactante , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Mutación , Trastornos del Neurodesarrollo/metabolismo , Monoéster Fosfórico Hidrolasas/genética , Monoéster Fosfórico Hidrolasas/farmacología , Fosforilación , Células Madre/efectos de los fármacos , TranscriptomaRESUMEN
Proper brain development relies highly on protein N-glycosylation to sustain neuronal migration, axon guidance and synaptic physiology. Impairing the N-glycosylation pathway at early steps produces broad neurological symptoms identified in congenital disorders of glycosylation. However, little is known about the molecular mechanisms underlying these defects. We generated a cerebellum specific knockout mouse for Srd5a3, a gene involved in the initiation of N-glycosylation. In addition to motor coordination defects and abnormal granule cell development, Srd5a3 deletion causes mild N-glycosylation impairment without significantly altering ER homeostasis. Using proteomic approaches, we identified that Srd5a3 loss affects a subset of glycoproteins with high N-glycans multiplicity per protein and decreased protein abundance or N-glycosylation level. As IgSF-CAM adhesion proteins are critical for neuron adhesion and highly N-glycosylated, we observed impaired IgSF-CAM-mediated neurite outgrowth and axon guidance in Srd5a3 mutant cerebellum. Our results link high N-glycan multiplicity to fine-tuned neural cell adhesion during mammalian brain development.
Asunto(s)
Cerebelo/metabolismo , Neuronas/citología , Neuronas/metabolismo , Polisacáridos/metabolismo , 3-Oxo-5-alfa-Esteroide 4-Deshidrogenasa/deficiencia , 3-Oxo-5-alfa-Esteroide 4-Deshidrogenasa/metabolismo , Animales , Orientación del Axón , Adhesión Celular , Moléculas de Adhesión Celular/metabolismo , Diferenciación Celular , Membrana Celular/metabolismo , Cerebelo/embriología , Gránulos Citoplasmáticos/metabolismo , Eliminación de Gen , Glicosilación , Inmunoglobulinas/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Proteínas de la Membrana/deficiencia , Proteínas de la Membrana/metabolismo , Ratones Noqueados , Actividad Motora , Mutación/genética , Vías Nerviosas/metabolismo , Proteómica , Células de Purkinje/metabolismo , Reproducibilidad de los Resultados , Respuesta de Proteína DesplegadaRESUMEN
Cerebellar atrophy is a key neuroradiological finding usually associated with cerebellar ataxia and cognitive development defect in children. Unlike the adult forms, early onset cerebellar atrophies are classically described as mostly autosomal recessive conditions and the exact contribution of de novo mutations to this phenotype has not been assessed. In contrast, recent studies pinpoint the high prevalence of pathogenic de novo mutations in other developmental disorders such as intellectual disability, autism spectrum disorders and epilepsy. Here, we investigated a cohort of 47 patients with early onset cerebellar atrophy and/or hypoplasia using a custom gene panel as well as whole exome sequencing. De novo mutations were identified in 35% of patients while 27% had mutations inherited in an autosomal recessive manner. Understanding if these de novo events act through a loss or a gain of function effect is critical for treatment considerations. To gain a better insight into the disease mechanisms causing these cerebellar defects, we focused on CACNA1G, a gene not yet associated with the early-onset form. This gene encodes the Cav3.1 subunit of T-type calcium channels highly expressed in Purkinje neurons and deep cerebellar nuclei. We identified four patients with de novo CACNA1G mutations. They all display severe motor and cognitive impairment, cerebellar atrophy as well as variable features such as facial dysmorphisms, digital anomalies, microcephaly and epilepsy. Three subjects share a recurrent c.2881G>A/p.Ala961Thr variant while the fourth patient has the c.4591A>G/p.Met1531Val variant. Both mutations drastically impaired channel inactivation properties with significantly slower kinetics (â¼5 times) and negatively shifted potential for half-inactivation (>10 mV). In addition, these two mutations increase neuronal firing in a cerebellar nuclear neuron model and promote a larger window current fully inhibited by TTA-P2, a selective T-type channel blocker. This study highlights the prevalence of de novo mutations in early-onset cerebellar atrophy and demonstrates that A961T and M1531V are gain of function mutations. Moreover, it reveals that aberrant activity of Cav3.1 channels can markedly alter brain development and suggests that this condition could be amenable to treatment.
Asunto(s)
Canales de Calcio Tipo T/genética , Ataxia Cerebelosa/genética , Adolescente , Adulto , Atrofia/patología , Encéfalo/patología , Calcio/metabolismo , Canales de Calcio/genética , Canales de Calcio Tipo T/metabolismo , Ataxia Cerebelosa/fisiopatología , Enfermedades Cerebelosas/complicaciones , Cerebelo/patología , Niño , Preescolar , Estudios de Cohortes , Discapacidades del Desarrollo/genética , Femenino , Mutación con Ganancia de Función/genética , Humanos , Discapacidad Intelectual/genética , Masculino , Microcefalia/genética , Mutación , Linaje , Fenotipo , Células de Purkinje/patologíaRESUMEN
Microlissencephaly is a rare brain malformation characterized by congenital microcephaly and lissencephaly. Microlissencephaly is suspected to result from abnormalities in the proliferation or survival of neural progenitors. Despite the recent identification of six genes involved in microlissencephaly, the pathophysiological basis of this condition remains poorly understood. We performed trio-based whole exome sequencing in seven subjects from five non-consanguineous families who presented with either microcephaly or microlissencephaly. This led to the identification of compound heterozygous mutations in WDR81, a gene previously associated with cerebellar ataxia, intellectual disability and quadrupedal locomotion. Patient phenotypes ranged from severe microcephaly with extremely reduced gyration with pontocerebellar hypoplasia to moderate microcephaly with cerebellar atrophy. In patient fibroblast cells, WDR81 mutations were associated with increased mitotic index and delayed prometaphase/metaphase transition. Similarly, in vivo, we showed that knockdown of the WDR81 orthologue in Drosophila led to increased mitotic index of neural stem cells with delayed mitotic progression. In summary, we highlight the broad phenotypic spectrum of WDR81-related brain malformations, which include microcephaly with moderate to extremely reduced gyration and cerebellar anomalies. Our results suggest that WDR81 might have a role in mitosis that is conserved between Drosophila and humans.
Asunto(s)
Fibroblastos/citología , Microcefalia/genética , Microcefalia/patología , Mitosis/genética , Mutación/genética , Proteínas del Tejido Nervioso/genética , Células-Madre Neurales/citología , Animales , Animales Modificados Genéticamente , Encéfalo/diagnóstico por imagen , Encéfalo/patología , Células Cultivadas , Preescolar , Drosophila , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Femenino , Fibroblastos/patología , Regulación de la Expresión Génica/genética , Humanos , Antígeno Ki-67/metabolismo , Masculino , Microcefalia/diagnóstico por imagen , Células-Madre Neurales/patología , Interferencia de ARN/fisiología , Adulto JovenRESUMEN
BACKGROUND: Cerebellar atrophy and developmental delay are commonly associated features in large numbers of genetic diseases that frequently also include epilepsy. These defects are highly heterogeneous on both the genetic and clinical levels. Patients with these signs also typically present with non-specific neuroimaging results that can help prioritize further investigation but don't suggest a specific molecular diagnosis. METHODS: To genetically explore a cohort of 18 Egyptian families with undiagnosed cerebellar atrophy identified on MRI, we sequenced probands and some non-affected family members via high-coverage whole exome sequencing (WES; >97 % of the exome covered at least by 30x). Patients were mostly from consanguineous families, either sporadic or multiplex. We analyzed WES data and filtered variants according to dominant and recessive inheritance models. RESULTS: We successfully identified disease-causing mutations in half of the families screened (9/18). These mutations are located in seven different genes, PLA2G6 being the gene most frequently mutated (n = 3). We also identified a recurrent de novo mutation in the KIF1A gene and a molybdenum cofactor deficiency caused by the loss of the start codon in the MOCS2A open-reading frame in a mildly affected subject. CONCLUSIONS: This study illustrates the necessity of screening for dominant mutations in WES data from consanguineous families. Our identification of a patient with a mild and improving phenotype carrying a previously characterized severe loss of function mutation also broadens the clinical spectrum associated with molybdenum cofactor deficiency.