RESUMEN
Schwann cells are the nerve ensheathing cells of the peripheral nervous system. Absence, loss and malfunction of Schwann cells or their myelin sheaths lead to peripheral neuropathies such as Charcot-Marie-Tooth disease in humans. During Schwann cell development and myelination chromatin is dramatically modified. However, impact and functional relevance of these modifications are poorly understood. Here, we analyzed histone H2B monoubiquitination as one such chromatin modification by conditionally deleting the Rnf40 subunit of the responsible E3 ligase in mice. Rnf40-deficient Schwann cells were arrested immediately before myelination or generated abnormally thin, unstable myelin, resulting in a peripheral neuropathy characterized by hypomyelination and progressive axonal degeneration. By combining sequencing techniques with functional studies we show that H2B monoubiquitination does not influence global gene expression patterns, but instead ensures selective high expression of myelin and lipid biosynthesis genes and proper repression of immaturity genes. This requires the specific recruitment of the Rnf40-containing E3 ligase by Egr2, the central transcriptional regulator of peripheral myelination, to its target genes. Our study identifies histone ubiquitination as essential for Schwann cell myelination and unravels new disease-relevant links between chromatin modifications and transcription factors in the underlying regulatory network.
Asunto(s)
Proteína 2 de la Respuesta de Crecimiento Precoz/fisiología , Neuropatía Hereditaria Motora y Sensorial/metabolismo , Histonas/metabolismo , Sistema Nervioso Periférico/metabolismo , Células de Schwann/metabolismo , Animales , Línea Celular Tumoral , Células HEK293 , Humanos , Ratones , Ratones Transgénicos , Sistema Nervioso Periférico/patología , Ratas , Células de Schwann/patología , Ubiquitina-Proteína Ligasas/genética , UbiquitinaciónRESUMEN
Oligodendrocytes are the myelinating glia of the central nervous system and ensure rapid saltatory conduction. Shortage or loss of these cells leads to severe malfunctions as observed in human leukodystrophies and multiple sclerosis, and their replenishment by reprogramming or cell conversion strategies is an important research aim. Using a transgenic approach we increased levels of the transcription factor Sox10 throughout the mouse embryo and thereby prompted Fabp7-positive glial cells in dorsal root ganglia of the peripheral nervous system to convert into cells with oligodendrocyte characteristics including myelin gene expression. These rarely studied and poorly characterized satellite glia did not go through a classic oligodendrocyte precursor cell stage. Instead, Sox10 directly induced key elements of the regulatory network of differentiating oligodendrocytes, including Olig2, Olig1, Nkx2.2 and Myrf. An upstream enhancer mediated the direct induction of the Olig2 gene. Unlike Sox10, Olig2 was not capable of generating oligodendrocyte-like cells in dorsal root ganglia. Our findings provide proof-of-concept that Sox10 can convert conducive cells into oligodendrocyte-like cells in vivo and delineates options for future therapeutic strategies.
Asunto(s)
Diferenciación Celular/genética , Sistema Nervioso Central/metabolismo , Esclerosis Múltiple/genética , Factores de Transcripción SOXE/genética , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Sistema Nervioso Central/patología , Embrión de Mamíferos , Proteína de Unión a los Ácidos Grasos 7 , Proteínas de Unión a Ácidos Grasos/genética , Proteína Homeobox Nkx-2.2 , Proteínas de Homeodominio/genética , Humanos , Ratones , Esclerosis Múltiple/patología , Proteínas del Tejido Nervioso/genética , Neuroglía , Proteínas Nucleares , Factor de Transcripción 2 de los Oligodendrocitos , Oligodendroglía/metabolismo , Factores de Transcripción SOXE/metabolismo , Médula Espinal/metabolismo , Médula Espinal/patología , Factores de Transcripción/genética , Proteínas de Pez CebraRESUMEN
Oligodendrocytes and Schwann cells are the myelinating glia of the vertebrate nervous system and by generation of myelin sheaths allow rapid saltatory conduction. Previous in vitro work had pointed to a role of the zinc finger containing specificity proteins Sp1 and Sp3 as major regulators of glial differentiation and myelination. Here, we asked whether such a role is also evident in vivo using mice with specific deletions of Sp1 or Sp3 in myelinating glia. We also studied glia-specific conditional Sp2- and constitutive Sp4-deficient mice to include all related glutamine-rich Sp factors into our analysis. Surprisingly, we did not detect developmental Schwann cell abnormalities in any of the mutant mice. Oligodendrocyte development and differentiation was also not fundamentally affected as oligodendrocytes were present in all mouse mutants and retained their ability to differentiate and initiate myelin gene expression. The most severe defect we observed was a 50% reduction in Mbp- and proteolipid protein 1 (Plp1)-positive differentiating oligodendrocytes in Sp2 mutants at birth. Unexpectedly, glial development appeared undisturbed even in the joint absence of Sp1 and Sp3. We conclude that Sp2 has a minor effect on the differentiation of myelinating glia, and that glutamine-rich Sp proteins are not essential regulators of the process.
Asunto(s)
Diferenciación Celular/fisiología , Glutamina/metabolismo , Vaina de Mielina/metabolismo , Neuroglía/metabolismo , Oligodendroglía/metabolismo , Factor de Transcripción Sp2/metabolismo , Animales , Células Cultivadas , Regulación del Desarrollo de la Expresión Génica/fisiología , Proteína Básica de Mielina/metabolismo , Ratas , Células de Schwann/efectos de los fármacos , Células de Schwann/metabolismoRESUMEN
The basic helix-loop-helix transcription factor Olig2 is a key determinant for the specification of neural precursor cells into oligodendrocyte progenitor cells. However, the functional role of Olig2 in oligodendrocyte migration and differentiation remains elusive both during developmental myelination and under demyelinating conditions of the adult central nervous system. To decipher Olig2 functions, we generated transgenic mice (TetOlig2:Sox10(rtTA/+)) overexpressing Olig2 in Sox10(+) oligodendroglial cells in a doxycycline inducible manner. We show that Olig2 overexpression increases the generation of differentiated oligodendrocytes, leading to precocious myelination of the central nervous system. Unexpectedly, we found that gain of Olig2 function in oligodendrocyte progenitor cells enhances their migration rate. To determine whether Olig2 overexpression in adult oligodendrocyte progenitor cells promotes oligodendrocyte regeneration for myelin repair, we induced lysophosphatidylcholine demyelination in the corpus callosum of TetOlig2:Sox10(rtTA/+) and control mice. We found that Olig2 overexpression enhanced oligodendrocyte progenitor cell differentiation and remyelination. To assess the relevance of these findings in demyelinating diseases, we also examined OLIG2 expression in multiple sclerosis lesions. We demonstrate that OLIG2 displays a differential expression pattern in multiple sclerosis lesions that correlates with lesion activity. Strikingly, OLIG2 was predominantly detected in NOGO-A(+) (now known as RTN4-A) maturing oligodendrocytes, which prevailed in active lesion borders, rather than chronic silent and shadow plaques. Taken together, our data provide proof of principle indicating that OLIG2 overexpression in oligodendrocyte progenitor cells might be a possible therapeutic mechanism for enhancing myelin repair.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Enfermedades Desmielinizantes/metabolismo , Vaina de Mielina/fisiología , Proteínas del Tejido Nervioso/metabolismo , Oligodendroglía/fisiología , Regeneración/genética , Médula Espinal/citología , Células Madre/fisiología , Animales , Animales Recién Nacidos , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Diferenciación Celular/genética , Células Cultivadas , Enfermedades Desmielinizantes/inducido químicamente , Enfermedades Desmielinizantes/patología , Modelos Animales de Enfermedad , Doxiciclina/farmacología , Embrión de Mamíferos , Regulación de la Expresión Génica/genética , Lisofosfatidilcolinas/toxicidad , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Esclerosis Múltiple/patología , Proteínas del Tejido Nervioso/genética , Factor de Transcripción 2 de los Oligodendrocitos , Oligodendroglía/patología , Oligodendroglía/ultraestructura , Regeneración/efectos de los fármacos , Factores de Transcripción SOXE/genética , Factores de Transcripción SOXE/metabolismo , Médula Espinal/patologíaRESUMEN
A variety of markers are invaluable for identifying and purifying stem/progenitor cells. Here we report the generation of a murine reporter line driven by Pw1 that reveals cycling and quiescent progenitor/stem cells in all adult tissues thus far examined, including the intestine, blood, testis, central nervous system, bone, skeletal muscle, and skin. Neurospheres generated from the adult PW1-reporter mouse show near 100% reporter-gene expression following a single passage. Furthermore, epidermal stem cells can be purified solely on the basis of reporter-gene expression. These cells are clonogenic, repopulate the epidermal stem-cell niches, and give rise to new hair follicles. Finally, we demonstrate that only PW1 reporter-expressing epidermal cells give rise to follicles that are capable of self-renewal following injury. Our data demonstrate that PW1 serves as an invaluable marker for competent self-renewing stem cells in a wide array of adult tissues, and the PW1-reporter mouse serves as a tool for rapid stem cell isolation and characterization.
Asunto(s)
Células Madre Adultas/citología , Células Madre Adultas/metabolismo , Factores de Transcripción de Tipo Kruppel/genética , Animales , Linaje de la Célula/genética , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Células Epidérmicas , Epidermis/metabolismo , Femenino , Regulación del Desarrollo de la Expresión Génica , Genes Reporteros , Marcadores Genéticos , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Hibridación in Situ , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , EmbarazoRESUMEN
The KN Motif and AnKyrin Repeat Domain 1 (KANK1) is proposed as a tumour suppressor gene, as its expression is reduced or absent in several types of tumour tissue, and over-expressing the protein inhibited the proliferation of tumour cells in solid cancer models. We report a novel germline loss of heterozygosity mutation encompassing the KANK1 gene in a young patient diagnosed with myelodysplastic neoplasm (MDS) with no additional disease-related genomic aberrations. To study the potential role of KANK1 in haematopoiesis, we generated a new transgenic mouse model with a confirmed loss of KANK1 expression. KANK1 knockout mice did not develop any haematological abnormalities; however, the loss of its expression led to alteration in the colony forming and proliferative potential of bone marrow (BM) cells and a decrease in hematopoietic stem and progenitor cells (HSPCs) population frequency. A comprehensive marker expression analysis of lineage cell populations indicated a role for Kank1 in lymphoid cell development, and total protein analysis suggests the involvement of Kank1 in BM cells' cytoskeleton formation and mobility.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Neoplasias , Animales , Humanos , Ratones , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Repetición de Anquirina/genética , Proteínas del Citoesqueleto/metabolismo , Modelos Animales de EnfermedadRESUMEN
The basic helix-loop-helix transcription factors Olig1 and Olig2 are required for oligodendrocyte specification and differentiation during central nervous system (CNS) development but the effects of overexpression of these factors in murine development are not well understood. To test whether Olig1 and Olig2 may reprogram CNS stem/progenitors toward an oligodendroglial fate for myelination, we generated transgenic mice with doxycycline (Dox)-inducible expression of Olig1 or Olig2 in nestin-expressing stem/progenitor cells of the CNS. Overexpression of Olig1 or Olig2 from E8.5 to E12.5 was sufficient to promote the generation of platelet-derived growth factor receptor alpha + oligodendrocyte precursors (OPCs) in the spinal cord. We also demonstrated that overexpression of Olig2, but not Olig1, enhanced the stem/progenitor cell proliferation and generation of motoneuron precursors and inhibited the development of V3 interneurons. In the postnatal brain, Dox-inducible expression of Olig2 but not Olig1 in nestin+ stem/progenitors of the subventricular zone increased the generation of OPCs that migrated and differentiated into mature oligodendrocytes in the corpus callosum, cortex and olfactory bulb, leading to increased and precocious myelination. Altogether, our data indicate that Olig2 is a potential therapeutic target to enhance myelination and remyelination in the CNS.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Encéfalo/metabolismo , Diferenciación Celular , Linaje de la Célula , Células Madre Embrionarias/metabolismo , Vaina de Mielina/metabolismo , Oligodendroglía/metabolismo , Médula Espinal/metabolismo , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Encéfalo/embriología , Encéfalo/crecimiento & desarrollo , Movimiento Celular , Proliferación Celular , Edad Gestacional , Interneuronas/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Neuronas Motoras/metabolismo , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Médula Espinal/embriologíaRESUMEN
Despite progress, our understanding of psychiatric and neurological illnesses remains poor, at least in part due to the inability to access neurons directly from patients. Currently, there are in vitro models available but significant work remains, including the search for a less invasive, inexpensive and rapid method to obtain neuronal-like cells with the capacity to deliver reproducible results. Here, we present a new protocol to transdifferentiate human circulating monocytes into neuronal-like cells in 20 days and without the need for viral insertion or reprograming. We have thoroughly characterized these monocyte-derived-neuronal-like cells (MDNCs) through various approaches including immunofluorescence (IF), flow cytometry, qRT-PCR, single cell mRNA sequencing, electrophysiology and pharmacological techniques. These MDNCs resembled human neurons early in development, expressed a variety of neuroprogenitor and neuronal genes as well as several neuroprogenitor and neuronal proteins and also presented electrical activity. In addition, when these neuronal-like cells were exposed to either dopamine or colchicine, they responded similarly to neurons by retracting their neuronal arborizations. More importantly, MDNCs exhibited reproducible differentiation rates, arborizations and expression of dopamine 1 receptors (DR1) on separate sequential samples from the same individual. Differentiation efficiency measured by cell morphology was on average 11.9 ± 1.4% (mean, SEM, n = 38,819 cells from 15 donors). To provide context and help researchers decide which in vitro model of neuronal development is best suited to address their scientific question,we compared our results with those of other in vitro models currently available and exposed advantages and disadvantages of each paradigm.
RESUMEN
Clonal lineage information is fundamental in revealing cell fate choices. Using genetic single-cell labeling in utero, we investigated lineage segregations during anteroposterior axis formation in mouse. We show that while endoderm and surface ectoderm segregate during gastrulation, neural ectoderm and mesoderm share a common progenitor persisting through all stages of axis elongation. These data challenge the paradigm that the three germ layers, formed by gastrulation, constitute the primary branchpoints in differentiation of the pluripotent epiblast toward tissue-specific precursors. Bipotent neuromesodermal progenitors show self-renewing characteristics and may represent the cellular substrate coupling sustained axial elongation and coordinated differentiation of these tissues. These findings have important implications for the interpretation of the phenotypic defects of several mouse mutants and the directed differentiation of embryonic stem (ES) cells in vitro.