RESUMEN
BACKGROUND/AIM: Autosomal recessive primary microcephaly (MCPH) is a rare and genetically heterogeneous group of disorders characterized by intellectual disability and microcephaly at birth, classically without further organ involvement. MCPH3 is caused by biallelic variants in the cyclin-dependent kinase 5 regulatory subunit-associated protein 2 gene CDK5RAP2. In the corresponding Cdk5rap2 mutant or Hertwig's anemia mouse model, congenital microcephaly as well as defects in the hematopoietic system, germ cells and eyes have been reported. The reduction in brain volume, particularly affecting gray matter, has been attributed mainly to disturbances in the proliferation and survival of early neuronal progenitors. In addition, defects in dendritic development and synaptogenesis exist that affect the excitation-inhibition balance. Here, we studied proteomic changes in cerebral cortices of Cdk5rap2 mutant mice. MATERIAL AND METHODS: We used large-gel two-dimensional gel (2-DE) electrophoresis to separate cortical proteins. 2-DE gels were visualized by a trained observer on a light box. Spot changes were considered with respect to presence/absence, quantitative variation and altered mobility. RESULT: We identified a reduction in more than 30 proteins that play a role in processes such as cell cytoskeleton dynamics, cell cycle progression, ciliary functions and apoptosis. These proteome changes in the MCPH3 model can be associated with various functional and morphological alterations of the developing brain. CONCLUSION: Our results shed light on potential protein candidates for the disease-associated phenotype reported in MCPH3.
Asunto(s)
Microcefalia , Humanos , Ratones , Animales , Microcefalia/genética , Proteoma/genética , Proteómica , Proteínas de Ciclo Celular/genética , Mutación , Proteínas del Tejido Nervioso/genéticaRESUMEN
Hom ozygous variants in the peptidyl-tRNA hydrolase 2 gene (PTRH2) cause infantile-onset multisystem neurologic, endocrine, and pancreatic disease. The objective is to delineate the mechanisms underlying the core cerebellar phenotype in this disease. For this, we generated constitutive (Ptrh2LoxPxhCMVCre, Ptrh2-/- mice) and Purkinje cell (PC) specific (Ptrh2LoxPxPcp2Cre, Ptrh2ΔPCmice) Ptrh2 mutant mouse models and investigated the effect of the loss of Ptrh2 on cerebellar development. We show that Ptrh2-/- knockout mice had severe postnatal runting and lethality by postnatal day 14. Ptrh2ΔPC PC specific knockout mice survived until adult age; however, they showed progressive cerebellar atrophy and functional cerebellar deficits with abnormal gait and ataxia. PCs of Ptrh2ΔPC mice had reduced cell size and density, stunted dendrites, and lower levels of ribosomal protein S6, a readout of the mammalian target of rapamycin pathway. By adulthood, there was a marked loss of PCs. Thus, we identify a cell autonomous requirement for PTRH2 in PC maturation and survival. Loss of PTRH2 in PCs leads to downregulation of the mTOR pathway and PC atrophy. This suggests a molecular mechanism underlying the ataxia and cerebellar atrophy seen in patients with PTRH2 mutations leading to infantile-onset multisystem neurologic, endocrine, and pancreatic disease.
Asunto(s)
Ataxia Cerebelosa , Enfermedades Pancreáticas , Humanos , Ratones , Animales , Adulto , Ataxia/patología , Células de Purkinje/fisiología , Ratones Noqueados , Enfermedades Pancreáticas/genética , Enfermedades Pancreáticas/metabolismo , Enfermedades Pancreáticas/patología , Diferenciación Celular , Atrofia/patología , MamíferosRESUMEN
BACKGROUND: Minichromosomal maintenance (MCM) complex components 2, 4, 5 and 6 have been linked to human disease with phenotypes including microcephaly and intellectual disability. The MCM complex has DNA helicase activity and is thereby important for the initiation and elongation of the replication fork and highly expressed in proliferating neural stem cells. METHODS: Whole-exome sequencing was applied to identify the genetic cause underlying the neurodevelopmental disease of the index family. The expression pattern of Mcm7 was characterised by performing quantitative real-time PCR, in situ hybridisation and immunostaining. To prove the disease-causative nature of identified MCM7, a proof-of-principle experiment was performed. RESULTS: We reported that the homozygous missense variant c.793G>A/p.A265T (g.7:99695841C>T, NM_005916.4) in MCM7 was associated with autosomal recessive primary microcephaly (MCPH), severe intellectual disability and behavioural abnormalities in a consanguineous pedigree with three affected individuals. We found concordance between the spatiotemporal expression pattern of Mcm7 in mice and a proliferative state: Mcm7 expression was higher in early mouse developmental stages and in proliferative zones of the brain. Accordingly, Mcm7/MCM7 levels were detectable particularly in undifferentiated mouse embryonal stem cells and human induced pluripotent stem cells compared with differentiated neurons. We further demonstrate that the downregulation of Mcm7 in mouse neuroblastoma cells reduces cell viability and proliferation, and, as a proof-of-concept, that this is counterbalanced by the overexpression of wild-type but not mutant MCM7. CONCLUSION: We report mutations of MCM7 as a novel cause of autosomal recessive MCPH and intellectual disability and highlight the crucial function of MCM7 in nervous system development.
Asunto(s)
Células Madre Pluripotentes Inducidas , Discapacidad Intelectual , Microcefalia , Malformaciones del Sistema Nervioso , Animales , Humanos , Discapacidad Intelectual/genética , Ratones , Microcefalia/complicaciones , Microcefalia/genética , Componente 7 del Complejo de Mantenimiento de Minicromosoma/genética , Mutación/genética , LinajeRESUMEN
Malfunction of pre-mRNA processing factors are linked to several human diseases including cancer and neurodegeneration. Here we report the identification of a de novo heterozygous missense mutation in the SNRPE gene (c.65T>C (p.Phe22Ser)) in a patient with non-syndromal primary (congenital) microcephaly and intellectual disability. SNRPE encodes SmE, a basal component of pre-mRNA processing U snRNPs. We show that the microcephaly-linked SmE variant is unable to interact with the SMN complex and as a consequence fails to assemble into U snRNPs. This results in widespread mRNA splicing alterations in fibroblast cells derived from this patient. Similar alterations were observed in HEK293 cells upon SmE depletion that could be rescued by the expression of wild type but not mutant SmE. Importantly, the depletion of SmE in zebrafish causes aberrant mRNA splicing alterations and reduced brain size, reminiscent of the patient microcephaly phenotype. We identify the EMX2 mRNA, which encodes a protein required for proper brain development, as a major mis-spliced down stream target. Together, our study links defects in the SNRPE gene to microcephaly and suggests that alterations of cellular splicing of specific mRNAs such as EMX2 results in the neurological phenotype of the disease.
Asunto(s)
Empalme Alternativo , Proteínas de Homeodominio/genética , Discapacidad Intelectual/genética , Microcefalia/genética , Mutación Missense , Factores de Transcripción/genética , Proteínas Nucleares snRNP/genética , Animales , Línea Celular , Modelos Animales de Enfermedad , Femenino , Células HEK293 , Humanos , Linaje , Empalme del ARN , ARN Mensajero/genética , Ribonucleoproteínas Nucleares Pequeñas/metabolismo , Secuenciación del Exoma , Pez Cebra , Proteínas Nucleares snRNP/química , Proteínas Nucleares snRNP/metabolismoRESUMEN
Mid-hindbrain malformations can occur during embryogenesis through a disturbance of transient and localized gene expression patterns within these distinct brain structures. Rho guanine nucleotide exchange factor (ARHGEF) family members are key for controlling the spatiotemporal activation of Rho GTPase, to modulate cytoskeleton dynamics, cell division, and cell migration. We identified, by means of whole exome sequencing, a homozygous frameshift mutation in the ARHGEF2 as a cause of intellectual disability, a midbrain-hindbrain malformation, and mild microcephaly in a consanguineous pedigree of Kurdish-Turkish descent. We show that loss of ARHGEF2 perturbs progenitor cell differentiation and that this is associated with a shift of mitotic spindle plane orientation, putatively favoring more symmetric divisions. The ARHGEF2 mutation leads to reduction in the activation of the RhoA/ROCK/MLC pathway crucial for cell migration. We demonstrate that the human brain malformation is recapitulated in Arhgef2 mutant mice and identify an aberrant migration of distinct components of the precerebellar system as a pathomechanism underlying the midbrain-hindbrain phenotype. Our results highlight the crucial function of ARHGEF2 in human brain development and identify a mutation in ARHGEF2 as novel cause of a neurodevelopmental disorder.
Asunto(s)
Movimiento Celular/genética , Mutación del Sistema de Lectura/genética , Discapacidad Intelectual/genética , Factores de Intercambio de Guanina Nucleótido Rho/genética , Animales , Citoesqueleto/genética , Exoma/genética , Femenino , Secuenciación de Nucleótidos de Alto Rendimiento , Homocigoto , Humanos , Discapacidad Intelectual/diagnóstico por imagen , Discapacidad Intelectual/patología , Imagen por Resonancia Magnética , Masculino , Mesencéfalo/diagnóstico por imagen , Mesencéfalo/patología , Ratones , Linaje , Rombencéfalo/diagnóstico por imagen , Rombencéfalo/patología , Transducción de Señal , Proteína de Unión al GTP rhoA/genéticaRESUMEN
Congenital microcephaly is highly associated with intellectual disability. Features of autosomal recessive primary microcephaly subtype 3 (MCPH3) also include hyperactivity and seizures. The disease is caused by biallelic mutations in the Cyclin-dependent kinase 5 regulatory subunit-associated protein 2 gene CDK5RAP2. In the mouse, Cdk5rap2 mutations similar to the human condition result in reduced brain size and a strikingly thin neocortex already at early stages of neurogenesis that persists through adulthood. The microcephaly phenotype in MCPH arises from a neural stem cell proliferation defect. Here, we report a novel role for Cdk5rap2 in the regulation of dendritic development and synaptogenesis of neocortical layer 2/3 pyramidal neurons. Cdk5rap2-deficient murine neurons show poorly branched dendritic arbors and an increased density of immature thin spines and glutamatergic synapses in vivo. Moreover, the excitatory drive is enhanced in ex vivo brain slice preparations of Cdk5rap2 mutant mice. Concurrently, we show that pyramidal neurons receive fewer inhibitory inputs. Together, these findings point towards a shift in the excitation - inhibition balance towards excitation in Cdk5rap2 mutant mice. Thus, MCPH3 is associated not only with a neural progenitor proliferation defect but also with altered function of postmitotic neurons and hence with altered connectivity.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Microcefalia/fisiopatología , Neocórtex/fisiopatología , Vías Nerviosas/fisiopatología , Neurogénesis/fisiología , Animales , Proteínas de Ciclo Celular/genética , Diferenciación Celular/fisiología , Ratones , Ratones Mutantes , Microcefalia/genética , Microcefalia/metabolismo , Mutación , Neocórtex/metabolismo , Vías Nerviosas/metabolismo , Células Piramidales/metabolismo , Células Piramidales/patología , Transmisión Sináptica/fisiologíaRESUMEN
Homozygous mutations in the cyclin-dependent kinase-5 regulatory subunit-associated protein 2 gene CDK5RAP2 cause primary autosomal recessive microcephaly (MCPH). MCPH is characterized by a pronounced reduction of brain volume, particularly of the cerebral cortex, and mental retardation. Though it is a rare developmental disorder, MCPH has moved into the spotlight of neuroscience because of its proposed central role in stem-cell biology and brain development. Investigation of the neural basis of genetically defined MCPH has been limited to animal studies and neuroimaging of affected patients as no neuropathological studies have been published. In the present study, we depict the spatiotemporal expression of CDK5RAP2 in the developing brain of mouse and human. We found intriguing concordance between regions of high CDK5RAP2 expression in the mouse and sites of pathology suggested by neuroimaging studies in humans and mouse. Our findings in human tissue confirm those in mouse tissues, underlining the function of CDK5RAP2 in cell proliferation and arguing for a conserved role of this protein in the development of the mammalian cerebral cortex.
Asunto(s)
Encéfalo/embriología , Encéfalo/metabolismo , Proteínas de Ciclo Celular/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Neuronas/metabolismo , Animales , Feto/metabolismo , Humanos , Ratones , Microcefalia/embriología , Microcefalia/metabolismoRESUMEN
Cyclin dependent kinase 5 regulatory subunit-associated protein 2 (CDK5RAP2) has gained attention in the last years following the discovery, in 2005, that recessive mutations cause primary autosomal recessive microcephaly. This disease is seen as an isolated developmental defect of the brain, particularly of the cerebral cortex, and was thus historically also referred to as microcephalia vera. Unraveling the pathomechanisms leading to this human disease is fascinating scientists because it can convey insight into basic mechanisms of physiologic brain development (particularly of cortex formation). It also finds itself in the spotlight because of its implication in trends in mammalian evolution with a massive increase in the size of the cerebral cortex in primates. Here, we provide a timely overview of the current knowledge on the function of CDK5RAP2 and mechanisms that might lead to disease in humans when the function of this protein is disturbed.
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteínas de Ciclo Celular , Centriolos/metabolismo , Técnicas de Silenciamiento del Gen , Humanos , Péptidos y Proteínas de Señalización Intracelular/química , Péptidos y Proteínas de Señalización Intracelular/genética , Microcefalia/genética , Microcefalia/patología , Mutación , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/genética , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Tubulina (Proteína)/química , Tubulina (Proteína)/metabolismoAsunto(s)
Consanguinidad , Predisposición Genética a la Enfermedad/genética , Microcefalia/genética , Mutación , Salud de la Familia , Femenino , Geografía , Humanos , Masculino , Microcefalia/patología , Proteínas Asociadas a Microtúbulos/genética , Proteínas del Tejido Nervioso/genética , Pakistán , Linaje , Secuenciación del Exoma/métodosRESUMEN
Pontine tegmental cap dysplasia is a rare hindbrain malformation syndrome with a hypoplastic pons, a tissue protrusion into the fourth ventricle, and cranial nerve dysfunction. We here report clinical, imaging, and genetic findings of the first extremely low-birth-weight preterm infant with pontine tegmental cap dysplasia born at 25 weeks of gestation and provide an overview of 29 sporadic cases. A prenatally diagnosed hypoplastic and rostrally shifted cerebellum was indicative of a hindbrain defect and later identified as an early sign of pontine tegmental cap dysplasia in our patient. The neonate exhibited severe muscle hypotonia, persistent thermolability, and clinical signs of an involvement of facial, cochlear, and hypoglossal nerves. Furthermore, paroxysmal episodes of agonizing pain with facial tics, tonic and clonic muscle contractions, blepharospasm, and singultus are highlighted as new phenotypic features of pontine tegmental cap dysplasia. With our report, we present a severe case of pontine tegmental cap dysplasia and provide a brief overview of current knowledge on this rare disease.
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Cerebelo/anomalías , Hipotonía Muscular/diagnóstico por imagen , Malformaciones del Sistema Nervioso/diagnóstico por imagen , Tegmento Pontino/anomalías , Cerebelo/diagnóstico por imagen , Discapacidades del Desarrollo/diagnóstico por imagen , Femenino , Humanos , Recien Nacido Extremadamente Prematuro , Recién Nacido , Imagen por Resonancia Magnética , Tegmento Pontino/diagnóstico por imagenRESUMEN
Infantile-onset multisystem neurologic, endocrine, and pancreatic disease (IMNEPD) has been recently linked to biallelic mutation of the peptidyl-tRNA hydrolase 2 gene PTRH2. Two index patients with IMNEPD in the original report had multiple neurological symptoms such as postnatal microcephaly, intellectual disability, developmental delay, sensorineural deafness, cerebellar atrophy, ataxia, and peripheral neuropathy. In addition, distal muscle weakness and abnormalities of thyroid, pancreas, and liver were found. Here, we report five further IMNEPD patients with a different homozygous PTRH2 mutation, broaden the phenotypic spectrum of the disease and differentiate common symptoms and interindividual variability in IMNEPD associated with a unique mutation. We thereby hope to better define IMNEPD and promote recognition and diagnosis of this novel disease entity.
Asunto(s)
Hidrolasas de Éster Carboxílico/genética , Hidrolasas de Éster Carboxílico/metabolismo , Regulación Enzimológica de la Expresión Génica , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Enfermedades Pancreáticas/genética , Enfermedades Pancreáticas/patología , Humanos , Mutación , LinajeRESUMEN
Autosomal recessive primary microcephaly (MCPH) is a rare neurodevelopmental disorder characterized by a pronounced reduction of brain volume and intellectual disability. A current model for the microcephaly phenotype invokes a stem cell proliferation and differentiation defect, which has moved the disease into the spotlight of stem cell biology and neurodevelopmental science. Homozygous mutations of the Cyclin-dependent kinase-5 regulatory subunit-associated protein 2 gene CDK5RAP2 are one genetic cause of MCPH. To further characterize the pathomechanism underlying MCPH, we generated a conditional Cdk5rap2 LoxP/hCMV Cre mutant mouse. Further analysis, initiated on account of a lack of a microcephaly phenotype in these mutant mice, revealed the presence of previously unknown splice variants of the Cdk5rap2 gene that are at least in part accountable for the lack of microcephaly in the mice.
Asunto(s)
Empalme Alternativo/genética , Proteínas de Ciclo Celular/genética , Animales , Secuencia de Bases , Encéfalo/crecimiento & desarrollo , Proliferación Celular , Predisposición Genética a la Enfermedad , Humanos , Imagen por Resonancia Magnética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microcefalia/genética , Proteínas del Tejido Nervioso/genética , Células-Madre Neurales/fisiología , Isoformas de Proteínas/genética , Análisis de Secuencia de ADNRESUMEN
Biallelic mutations in the gene encoding centrosomal CDK5RAP2 lead to autosomal recessive primary microcephaly (MCPH), a disorder characterized by pronounced reduction in volume of otherwise architectonical normal brains and intellectual deficit. The current model for the microcephaly phenotype in MCPH invokes a premature shift from symmetric to asymmetric neural progenitor-cell divisions with a subsequent depletion of the progenitor pool. The isolated neural phenotype, despite the ubiquitous expression of CDK5RAP2, and reports of progressive microcephaly in individual MCPH cases prompted us to investigate neural and non-neural differentiation of Cdk5rap2-depleted and control murine embryonic stem cells (mESC). We demonstrate an accumulating proliferation defect of neurally differentiating Cdk5rap2-depleted mESC and cell death of proliferative and early postmitotic cells. A similar effect does not occur in non-neural differentiation into beating cardiomyocytes, which is in line with the lack of non-central nervous system features in MCPH patients. Our data suggest that MCPH is not only caused by premature differentiation of progenitors, but also by reduced propagation and survival of neural progenitors.
Asunto(s)
Proteínas de Ciclo Celular/deficiencia , Diferenciación Celular/fisiología , Células Madre Embrionarias/metabolismo , Miocitos Cardíacos/metabolismo , Células-Madre Neurales/metabolismo , Animales , Línea Celular , Supervivencia Celular/fisiología , RatonesRESUMEN
OBJECTIVE: To identify the cause of a so-far unreported phenotype of infantile-onset multisystem neurologic, endocrine, and pancreatic disease (IMNEPD). METHODS: We characterized a consanguineous family of Yazidian-Turkish descent with IMNEPD. The two affected children suffer from intellectual disability, postnatal microcephaly, growth retardation, progressive ataxia, distal muscle weakness, peripheral demyelinating sensorimotor neuropathy, sensorineural deafness, exocrine pancreas insufficiency, hypothyroidism, and show signs of liver fibrosis. We performed whole-exome sequencing followed by bioinformatic analysis and Sanger sequencing on affected and unaffected family members. The effect of mutations in the candidate gene was studied in wild-type and mutant mice and in patient and control fibroblasts. RESULTS: In a consanguineous family with two individuals with IMNEPD, we identified a homozygous frameshift mutation in the previously not disease-associated peptidyl-tRNA hydrolase 2 (PTRH2) gene. PTRH2 encodes a primarily mitochondrial protein involved in integrin-mediated cell survival and apoptosis signaling. We show that PTRH2 is highly expressed in the developing brain and is a key determinant in maintaining cell survival during human tissue development. Moreover, we link PTRH2 to the mTOR pathway and thus the control of cell size. The pathology suggested by the human phenotype and neuroimaging studies is supported by analysis of mutant mice and patient fibroblasts. INTERPRETATION: We report a novel disease phenotype, show that the genetic cause is a homozygous mutation in the PTRH2 gene, and demonstrate functional effects in mouse and human tissues. Mutations in PTRH2 should be considered in patients with undiagnosed multisystem neurologic, endocrine, and pancreatic disease.
RESUMEN
BACKGROUND: Autosomal recessive primary microcephaly (MCPH) is a rare neurodevelopmental disease with severe microcephaly at birth due to a pronounced reduction in brain volume and intellectual disability. Biallelic mutations in the WD repeat-containing protein 62 gene WDR62 are the genetic cause of MCPH2. However, the exact underlying pathomechanism of MCPH2 remains to be clarified. METHODS/RESULTS: We characterized the clinical, radiological, and cellular features that add to the human MCPH2 phenotype. Exome sequencing followed by Sanger sequencing in a German family with two affected daughters with primary microcephaly revealed in the index patient the compound heterozygous mutations c.1313G>A (p.R438H) / c.2864-2867delACAG (p.D955Afs*112) of WDR62, the second of which is novel. Radiological examination displayed small frontal lobes, corpus callosum hypoplasia, simplified hippocampal gyration, and cerebellar hypoplasia. We investigated the cellular phenotype in patient-derived lymphoblastoid cells and compared it with that of healthy female controls. WDR62 expression in the patient's immortalized lymphocytes was deranged, and mitotic spindle defects as well as abnormal centrosomal protein localization were apparent. CONCLUSION: We propose that a disruption of centrosome integrity and/or spindle organization may play an important role in the development of microcephaly in MCPH2.
Asunto(s)
Centrosoma/metabolismo , Proteínas del Tejido Nervioso/genética , Huso Acromático/metabolismo , Proteínas de Ciclo Celular , Heterocigoto , Humanos , Microcefalia , MutaciónRESUMEN
BACKGROUND: Primary autosomal recessive microcephaly (MCPH) is a rare neurodevelopmental disorder that results in severe microcephaly at birth with pronounced reduction in brain volume, particularly of the neocortex, simplified cortical gyration and intellectual disability. Homozygous mutations in the Cyclin-dependent kinase 5 regulatory subunit-associated protein 2 gene CDK5RAP2 are the cause of MCPH3. Despite considerable interest in MCPH as a model disorder for brain development, the underlying pathomechanism has not been definitively established and only four pedigrees with three CDK5RAP2 mutations have been reported. Specifically for MCPH3, no detailed radiological or histological descriptions exist. METHODS/RESULTS: We sought to characterize the clinical and radiological features and pathological cellular processes that contribute to the human MCPH3 phenotype. Haplotype analysis using microsatellite markers around the MCPH1-7 and PNKP loci in an Italian family with two sons with primary microcephaly, revealed possible linkage to the MCPH3 locus. Sequencing of the coding exons and exon/intron splice junctions of the CDK5RAP2 gene identified homozygosity for the novel nonsense mutation, c.4441C > T (p.Arg1481*), in both affected sons. cMRI showed microcephaly, simplified gyral pattern and hypogenesis of the corpus callosum. The cellular phenotype was assessed in EBV-transformed lymphocyte cell lines established from the two affected sons and compared with healthy male controls. CDK5RAP2 protein levels were below detection level in immortalized lymphocytes from the patients. Moreover, mitotic spindle defects and disrupted γ-tubulin localization to the centrosome were apparent. CONCLUSION: These results suggest that spindle defects and a disruption of centrosome integrity play an important role in the development of microcephaly in MCPH3.
Asunto(s)
Genes Recesivos , Péptidos y Proteínas de Señalización Intracelular/genética , Microcefalia/genética , Mutación , Proteínas del Tejido Nervioso/genética , Western Blotting , Proteínas de Ciclo Celular , Línea Celular Transformada , Exones , Femenino , Haplotipos , Homocigoto , Humanos , Intrones , Imagen por Resonancia Magnética , Masculino , Microcefalia/patología , Linaje , Fenotipo , Reacción en Cadena de la PolimerasaRESUMEN
OBJECTIVES: Gene expression analysis via quantitative real-time PCR (qPCR) is a key approach in biological and medical research. Here, variations between runs and samples are compensated for by in-parallel analysis of reference genes, which require a most stable expression throughout all samples and experimental procedures to function as internal standards. In reality, there is no universal reference gene; but rather, assumed reference genes vary widely among various cell types. This demands an evaluation of reference genes for each specific experimental purpose, especially in the case of developmental studies. The aim of the present study was to identify suitable reference genes for gene expression analysis in the developing murine brain neocortex in vivo and in mouse embryonic stem cells (mESC) throughout differentiation in vitro. METHODS: The five candidate genes Actb, 18s, Gapdh, Hprt, and RpII were analyzed throughout development in vivo and in vitro using the quartiles of C(q) values, fold change, coefficient of variation (CV) and the difference between maximum minus twofold standard deviation and mean as the criteria to evaluate their expression stability. RESULTS: We found that RpII was the most stable expressed gene in mESC throughout differentiation, while in the developing murine neocortex Gapdh showed the highest expression stability. CONCLUSIONS: Based on our results, we suggest for gene expression analysis in the context of neurodevelopment the usage of RpII as a reference gene for mESC and Gapdh or Hprt for the murine neocortex.
Asunto(s)
Encéfalo/citología , Diferenciación Celular/genética , Células Madre Embrionarias/fisiología , Regulación del Desarrollo de la Expresión Génica , Estudios de Asociación Genética , Actinas/biosíntesis , Actinas/genética , Animales , Animales Recién Nacidos , Encéfalo/embriología , Encéfalo/fisiología , Células Cultivadas , Femenino , Genes Esenciales/genética , Estudios de Asociación Genética/métodos , Gliceraldehído-3-Fosfato Deshidrogenasa (Fosforilante)/biosíntesis , Gliceraldehído-3-Fosfato Deshidrogenasa (Fosforilante)/genética , Hipoxantina Fosforribosiltransferasa/biosíntesis , Hipoxantina Fosforribosiltransferasa/genética , Ratones , Ratones Endogámicos C57BL , Embarazo , ARN Polimerasa II/biosíntesis , ARN Polimerasa II/genética , ARN Ribosómico 18S/biosíntesis , ARN Ribosómico 18S/genéticaRESUMEN
Autosomal recessive primary microcephaly (MCPH), historically referred to as Microcephalia vera, is a genetically and clinically heterogeneous disease. Patients with MCPH typically exhibit congenital microcephaly as well as mental retardation, but usually no further neurological findings or malformations. Their microcephaly with grossly preserved macroscopic organization of the brain is a consequence of a reduced brain volume, which is evident particularly within the cerebral cortex and thus results to a large part from a reduction of grey matter. Some patients with MCPH further provide evidence of neuronal heterotopias, polymicrogyria or cortical dysplasia suggesting an associated neuronal migration defect. Genetic causes of MCPH subtypes 1-7 include mutations in genes encoding microcephalin, cyclin-dependent kinase 5 regulatory associated protein 2 (CDK5RAP2), abnormal spindle-like, microcephaly associated protein (ASPM), centromeric protein J (CENPJ), and SCL/TAL1-interrupting locus (STIL) as well as linkage to the two loci 19q13.1-13.2 and 15q15-q21. Here, we provide a timely overview of current knowledge on mechanisms leading to microcephaly in humans with MCPH and abnormalities in cell division/cell survival in corresponding animal models. Understanding the pathomechanisms leading to MCPH is of high importance not only for our understanding of physiologic brain development (particularly of cortex formation), but also for that of trends in mammalian evolution with a massive increase in size of the cerebral cortex in primates, of microcephalies of other etiologies including environmentally induced microcephalies, and of cancer formation.
Asunto(s)
Encéfalo/patología , Microcefalia/genética , Mutación/genética , Proteínas del Tejido Nervioso/genética , Animales , Genes Recesivos , Heterogeneidad Genética , HumanosRESUMEN
Mice deficient in transcription factor gene Emx2 show developmental alterations in the hippocampal dentate gyrus. Emx2, however, is also expressed in the region around the developing hippocampal fissure. The developing fissure contains a radial glial scaffolding, and is surrounded by the outer marginal zone and the dentate marginal zone, which become specifically colonized by neurons and differentiate into stratum lacunosum-moleculare and molecular layer of the dentate, respectively. In this study we show that the Emx2 mutant lacks the glial scaffolding of the fissure and has an outer marginal zone (precursor of the stratum lacunosum-moleculare), as well as a dentate marginal zone severely reduced in size while most of the reelin (Reln)-expressing cells that should occupy it fail to be generated. We have also identified a subpopulation of hippocampal Reln-expressing cells of the marginal zone, probably born in the hem, expressing a specific combination of markers, and for which Emx2 is not essentially required. Additionally, we show differential mutant phenotypes of both Emx2 and Pax6 in neocortical vs. hippocampal Reln-expressing cells, indicating differential development of both subpopulations.