RESUMO
The nuclei are the main output structures of the cerebellum. Each and every cerebellar cortical computation reaches several areas of the brain by means of cerebellar nuclei processing and integration. Nevertheless, our knowledge of these structures is still limited compared to the cerebellar cortex. Here, we present a mouse genetic inducible fate-mapping study characterizing rhombic lip-derived glutamatergic neurons of the nuclei, the most conspicuous family of long-range cerebellar efferent neurons. Glutamatergic neurons mainly occupy dorsal and lateral territories of the lateral and interposed nuclei, as well as the entire medial nucleus. In mice, they are born starting from about embryonic day 9.5, with a peak between 10.5 and 12.5, and invade the nuclei with a lateral-to-medial progression. While some markers label a heterogeneous population of neurons sharing a common location (BRN2), others appear to be lineage specific (TBR1, LMX1a, and MEIS2). A comparative analysis of TBR1 and LMX1a distributions reveals an incomplete overlap in their expression domains, in keeping with the existence of separate efferent subpopulations. Finally, some tagged glutamatergic progenitors are not labeled by any of the markers used in this study, disclosing further complexity. Taken together, our results obtained in late embryonic nuclei shed light on the heterogeneity of the excitatory neuron pool, underlying the diversity in connectivity and functions of this largely unexplored cerebellar territory. Our findings contribute to laying the groundwork for a comprehensive functional analysis of nuclear neuron subpopulations.
RESUMO
Amyotrophic lateral sclerosis (ALS) is a progressive, lethal neurodegenerative disease mostly affecting people around 50-60 years of age. TDP-43, an RNA-binding protein involved in pre-mRNA splicing and controlling mRNA stability and translation, forms neuronal cytoplasmic inclusions in an overwhelming majority of ALS patients, a phenomenon referred to as TDP-43 proteinopathy. These cytoplasmic aggregates disrupt mRNA transport and localization. The axon, like dendrites, is a site of mRNA translation, permitting the local synthesis of selected proteins. This is especially relevant in upper and lower motor neurons, whose axon spans long distances, likely accentuating their susceptibility to ALS-related noxae. In this work we have generated and characterized two cellular models, consisting of virtually pure populations of primary mouse cortical neurons expressing a human TDP-43 fusion protein, wt or carrying an ALS mutation. Both forms facilitate cytoplasmic aggregate formation, unlike the corresponding native proteins, giving rise to bona fide primary culture models of TDP-43 proteinopathy. Neurons expressing TDP-43 fusion proteins exhibit a global impairment in axonal protein synthesis, an increase in oxidative stress, and defects in presynaptic function and electrical activity. These changes correlate with deregulation of axonal levels of polysome-engaged mRNAs playing relevant roles in the same processes. Our data support the emerging notion that deregulation of mRNA metabolism and of axonal mRNA transport may trigger the dying-back neuropathy that initiates motor neuron degeneration in ALS.
RESUMO
Mutations in the PARK2 gene encoding the protein parkin cause autosomal recessive juvenile parkinsonism (ARJP), a neurodegenerative disease characterized by early dysfunction and loss of dopamine (DA) neurons in the substantia nigra pars compacta (SNc). No therapy is currently available to prevent or slow down the neurodegeneration in ARJP patients. Preclinical models are key to clarifying the early events that lead to neurodegeneration and reveal the potential of novel neuroprotective strategies. ParkinQ311X is a transgenic mouse model expressing in DA neurons a mutant parkin variant found in ARJP patients. This model was previously reported to show the neuropathological hallmark of the disease, i.e., the progressive loss of DA neurons. However, the early dysfunctions that precede neurodegeneration have never been investigated. Here, we analyzed SNc DA neurons in parkinQ311X mice and found early features of mitochondrial dysfunction, extensive cytoplasmic vacuolization, and dysregulation of spontaneous in vivo firing activity. These data suggest that the parkinQ311X mouse recapitulates key features of ARJP and provides a useful tool for studying the neurodegenerative mechanisms underlying the human disease and for screening potential neuroprotective drugs.
RESUMO
Mutations in the PARK2 gene encoding the protein parkin cause autosomal recessive juvenile Parkinsonism (ARJP), a neurodegenerative disease characterized by dysfunction and death of dopamine (DA) neurons in the substantia nigra pars compacta (SNc). Since a neuroprotective therapy for ARJP does not exist, research efforts aimed at discovering targets for neuroprotection are critically needed. A previous study demonstrated that loss of parkin function or expression of parkin mutants associated with ARJP causes an accumulation of glutamate kainate receptors (KARs) in human brain tissues and an increase of KAR-mediated currents in neurons in vitro. Based on the hypothesis that such KAR hyperactivation may contribute to the death of nigral DA neurons, we investigated the effect of KAR antagonism on the DA neuron dysfunction and death that occur in the parkinQ311X mouse, a model of human parkin-induced toxicity. We found that early accumulation of KARs occurs in the DA neurons of the parkinQ311X mouse, and that chronic administration of the KAR antagonist UBP310 prevents DA neuron loss. This neuroprotective effect is associated with the rescue of the abnormal firing rate of nigral DA neurons and downregulation of GluK2, the key KAR subunit. This study provides novel evidence of a causal role of glutamate KARs in the DA neuron dysfunction and loss occurring in a mouse model of human parkin-induced toxicity. Our results support KAR as a potential target in the development of neuroprotective therapy for ARJP.
Assuntos
Alanina/análogos & derivados , Neurônios Dopaminérgicos/efeitos dos fármacos , Neurônios Dopaminérgicos/metabolismo , Doença de Parkinson/tratamento farmacológico , Doença de Parkinson/metabolismo , Receptores de Ácido Caínico/antagonistas & inibidores , Timina/análogos & derivados , Alanina/farmacologia , Animais , Modelos Animais de Doenças , Neurônios Dopaminérgicos/patologia , Regulação para Baixo , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Doença de Parkinson/genética , Doença de Parkinson/patologia , Receptores de Ácido Caínico/metabolismo , Timina/farmacologia , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Receptor de GluK2 CainatoRESUMO
The choroid plexus (ChP) is a secretory tissue that produces cerebrospinal fluid (CSF) secreted into the ventricular system. It is a monolayer of secretory, multiciliated epithelial cells derived from neuroepithelial progenitors and overlying a stroma of mesenchymal cells of mesodermal origin. Zfp423, which encodes a Kruppel-type zinc-finger transcription factor essential for cerebellar development and mutated in rare cases of cerebellar vermis hypoplasia/Joubert syndrome and other ciliopathies, is expressed in the hindbrain roof plate, from which the IV ventricle ChP arises, and, later, in mesenchymal cells, which give rise to the stroma and leptomeninges. Mouse Zfp423 mutants display a marked reduction of the hindbrain ChP (hChP), which: (1) fails to express established markers of its secretory function and genes implicated in its development and maintenance (Lmx1a and Otx2); (2) shows a perturbed expression of signaling pathways previously unexplored in hChP patterning (Wnt3); and (3) displays a lack of multiciliated epithelial cells and a profound dysregulation of master genes of multiciliogenesis (Gmnc). Our results propose that Zfp423 is a master gene and one of the earliest known determinants of hChP development.
Assuntos
Plexo Corióideo/embriologia , Proteínas de Ligação a DNA/metabolismo , Rombencéfalo/embriologia , Fatores de Transcrição/metabolismo , Animais , Plexo Corióideo/citologia , Proteínas de Ligação a DNA/genética , Proteínas com Homeodomínio LIM/genética , Proteínas com Homeodomínio LIM/metabolismo , Camundongos , Camundongos Mutantes , Fatores de Transcrição Otx/genética , Fatores de Transcrição Otx/metabolismo , Rombencéfalo/citologia , Fatores de Transcrição/genética , Proteína Wnt3/genética , Proteína Wnt3/metabolismoRESUMO
Somatic sensation is defined by the existence of a diversity of primary sensory neurons with unique biological features and response profiles to external and internal stimuli. However, there is no coherent picture about how this diversity of cell states is transcriptionally generated. Here, we use deep single cell analysis to resolve fate splits and molecular biasing processes during sensory neurogenesis in mice. Our results identify a complex series of successive and specific transcriptional changes in post-mitotic neurons that delineate hierarchical regulatory states leading to the generation of the main sensory neuron classes. In addition, our analysis identifies previously undetected early gene modules expressed long before fate determination although being clearly associated with defined sensory subtypes. Overall, the early diversity of sensory neurons is generated through successive bi-potential intermediates in which synchronization of relevant gene modules and concurrent repression of competing fate programs precede cell fate stabilization and final commitment.
Assuntos
Neurogênese/genética , Células Receptoras Sensoriais/citologia , Células Receptoras Sensoriais/fisiologia , Análise de Sequência de RNA/métodos , Análise de Célula Única/métodos , Animais , Diferenciação Celular , Subunidade alfa 3 de Fator de Ligação ao Core/genética , Modelos Animais de Doenças , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Redes Reguladoras de Genes , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios/fisiologia , Células-TroncoRESUMO
The collier/Olf1/EBF family genes encode helix-loop-helix transcription factors (TFs) highly conserved in evolution, initially characterized for their roles in the immune system and in various aspects of neural development. The Early B cell Factor 2 (Ebf2) gene plays an important role in the establishment of cerebellar cortical topography and in Purkinje cell (PC) subtype specification. In the adult cerebellum, Ebf2 is expressed in zebrin II (ZII)-negative PCs, where it suppresses the ZII+ molecular phenotype. However, it is not clear whether Ebf2 is restricted to a PC subset from the onset of its expression or is initially distributed in all PCs and silenced only later in the prospective ZII+ subtype. Moreover, the dynamic distribution and role of Ebf2 in the differentiation of other cerebellar cells remain unclarified. In this paper, by genetic fate mapping, we determine that Ebf2 mRNA is initially found in all PC progenitors, suggesting that unidentified upstream factors silence its expression before completion of embryogenesis. Moreover we show Ebf2 activation in an early born subset of granule cell (GC) precursors homing in the anterior lobe. Conversely, Ebf2 transcription is repressed in other cerebellar cortex interneurons. Last, we show that, although Ebf2 only labels the medial cerebellar nuclei (CN) in the adult cerebellum, the gene is expressed prenatally in projection neurons of all CN. Importantly, in Ebf2 nulls, fastigial nuclei are severely hypocellular, mirroring the defective development of anterior lobe PCs. Our findings further clarify the roles of this terminal selector gene in cerebellar development.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/biossíntese , Cerebelo/embriologia , Cerebelo/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Sobrevivência Celular/fisiologia , Cerebelo/crescimento & desenvolvimento , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Células de Purkinje/metabolismoRESUMO
Neural crest cells are embryonic progenitors that generate numerous cell types in vertebrates. With single-cell analysis, we show that mouse trunk neural crest cells become biased toward neuronal lineages when they delaminate from the neural tube, whereas cranial neural crest cells acquire ectomesenchyme potential dependent on activation of the transcription factor Twist1. The choices that neural crest cells make to become sensory, glial, autonomic, or mesenchymal cells can be formalized as a series of sequential binary decisions. Each branch of the decision tree involves initial coactivation of bipotential properties followed by gradual shifts toward commitment. Competing fate programs are coactivated before cells acquire fate-specific phenotypic traits. Determination of a specific fate is achieved by increased synchronization of relevant programs and concurrent repression of competing fate programs.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Células-Tronco Mesenquimais/citologia , Crista Neural/citologia , Crista Neural/embriologia , Células-Tronco Neurais/citologia , Neurogênese/genética , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Linhagem da Célula , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Mutantes , Proteínas do Tecido Nervoso/metabolismo , Crista Neural/metabolismo , Células-Tronco Neurais/metabolismo , Tubo Neural/citologia , Tubo Neural/embriologia , Neuroglia/citologia , Neurônios/citologia , Proteínas Nucleares/metabolismo , Análise de Célula Única , Proteína 1 Relacionada a Twist/metabolismoRESUMO
The sensation of pain is essential for the preservation of the functional integrity of the body. However, the key molecular regulators necessary for the initiation of the development of pain-sensing neurons have remained largely unknown. Here, we report that, in mice, inactivation of the transcriptional regulator PRDM12, which is essential for pain perception in humans, results in a complete absence of the nociceptive lineage, while proprioceptive and touch-sensitive neurons remain. Mechanistically, our data reveal that PRDM12 is required for initiation of neurogenesis and activation of a cascade of downstream pro-neuronal transcription factors, including NEUROD1, BRN3A, and ISL1, in the nociceptive lineage while it represses alternative fates other than nociceptors in progenitor cells. Our results thus demonstrate that PRDM12 is necessary for the generation of the entire lineage of pain-initiating neurons.
Assuntos
Proteínas de Transporte/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Neurogênese/fisiologia , Neurônios/fisiologia , Nociceptores/fisiologia , Animais , Proteínas de Transporte/genética , Linhagem da Célula , Galinhas , Feminino , Perfilação da Expressão Gênica , Imuno-Histoquímica , Masculino , Camundongos , Proteínas do Tecido Nervoso/genética , Neurogênese/genética , Nociceptividade/fisiologia , Fatores de Transcrição/metabolismoRESUMO
BACKGROUND: Mammalian motor circuits display remarkable cellular diversity with hundreds of motor neuron (MN) subtypes innervating hundreds of different muscles. Extensive research on limb muscle-innervating MNs has begun to elucidate the genetic programs that control animal locomotion. In striking contrast, the molecular mechanisms underlying the development of axial muscle-innervating MNs, which control breathing and spinal alignment, are poorly studied. METHODS: Our previous studies indicated that the function of the Collier/Olf/Ebf (COE) family of transcription factors (TFs) in axial MN development may be conserved from nematodes to simple chordates. Here, we examine the expression pattern of all four mouse COE family members (mEbf1-mEbf4) in spinal MNs and employ genetic approaches in both nematodes and mice to investigate their function in axial MN development. RESULTS: We report that mEbf1 and mEbf2 are expressed in distinct MN clusters (termed "columns") that innervate different axial muscles. Mouse Ebf1 is expressed in MNs of the hypaxial motor column (HMC), which is necessary for breathing, while mEbf2 is expressed in MNs of the medial motor column (MMC) that control spinal alignment. Our characterization of Ebf2 knock-out mice uncovered a requirement for Ebf2 in the differentiation program of a subset of MMC MNs and revealed for the first time molecular diversity within MMC neurons. Intriguingly, transgenic expression of mEbf1 or mEbf2 can rescue axial MN differentiation and locomotory defects in nematodes (Caenorhabditis elegans) lacking unc-3, the sole C. elegans ortholog of the COE family, suggesting functional conservation among mEbf1, mEbf2 and nematode UNC-3. CONCLUSIONS: These findings support the hypothesis that genetic programs controlling axial MN development are deeply conserved across species, and further advance our understanding of such programs by revealing an essential role for Ebf2 in mouse axial MNs. Because human mutations in COE orthologs lead to neurodevelopmental disorders characterized by motor developmental delay, our findings may advance our understanding of these human conditions.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/fisiologia , Proteínas de Caenorhabditis elegans/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Neurônios Motores/fisiologia , Músculo Esquelético/fisiologia , Medula Espinal/metabolismo , Transativadores/fisiologia , Fatores de Transcrição/fisiologia , Animais , Animais Geneticamente Modificados , Caenorhabditis elegans , Embrião de Mamíferos , Camundongos , Camundongos Knockout , Neurônios Motores/metabolismoRESUMO
The Zfp423/ZNF423 gene encodes a 30-zinc-finger transcription factor involved in key developmental pathways. Although null Zfp423 mutants develop cerebellar malformations, the underlying mechanism remains unknown. ZNF423 mutations are associated with Joubert Syndrome, a ciliopathy causing cerebellar vermis hypoplasia and ataxia. ZNF423 participates in the DNA-damage response (DDR), raising questions regarding its role as a regulator of neural progenitor cell cycle progression in cerebellar development. To characterize in vivo the function of ZFP423 in neurogenesis, we analyzed allelic murine mutants in which distinct functional domains are deleted. One deletion impairs mitotic spindle orientation, leading to premature cell cycle exit and Purkinje cell (PC) progenitor pool deletion. The other deletion impairs PC differentiation. In both mutants, cell cycle progression is remarkably delayed and DDR markers are upregulated in cerebellar ventricular zone progenitors. Our in vivo evidence sheds light on the domain-specific roles played by ZFP423 in different aspects of PC progenitor development, and at the same time strengthens the emerging notion that an impaired DDR may be a key factor in the pathogenesis of JS and other ciliopathies.
Assuntos
Ciclo Celular , Proteínas de Ligação a DNA/fisiologia , Células-Tronco Neurais/citologia , Neurônios/citologia , Células de Purkinje/citologia , Fatores de Transcrição/fisiologia , Anormalidades Múltiplas/genética , Alelos , Animais , Diferenciação Celular , Divisão Celular , Proliferação de Células , Cerebelo/anormalidades , Dano ao DNA , Anormalidades do Olho/genética , Deleção de Genes , Doenças Renais Císticas/genética , Camundongos , Mutação , Domínios Proteicos , Retina/anormalidades , Fuso Acromático/metabolismo , Dedos de ZincoRESUMO
A core principle of nervous system organization is the diversification of neuron classes into subclasses that share large sets of features but differ in select traits. We describe here a molecular mechanism necessary for motor neurons to acquire subclass-specific traits in the nematode Caenorhabditis elegans. Cholinergic motor neuron classes of the ventral nerve cord can be subdivided into subclasses along the anterior-posterior (A-P) axis based on synaptic connectivity patterns and molecular features. The conserved COE-type terminal selector UNC-3 not only controls the expression of traits shared by all members of a neuron class, but is also required for subclass-specific traits expressed along the A-P axis. UNC-3, which is not regionally restricted, requires region-specific cofactors in the form of Hox proteins to co-activate subclass-specific effector genes in post-mitotic motor neurons. This intersectional gene regulatory principle for neuronal subclass diversification may be conserved from nematodes to mice.
Assuntos
Caenorhabditis elegans/embriologia , Diferenciação Celular , Regulação da Expressão Gênica no Desenvolvimento , Neurônios Motores/fisiologia , Animais , Variação Biológica da População , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Homeodomínio/metabolismo , Camundongos , Fatores de Transcrição/metabolismoRESUMO
The Contactin-1 axonal glycoprotein (formerly F3/Contactin) plays a relevant role in cerebellar ontogenesis, as shown in Contactin-1 KO-mice and in transgenic mice misexpressing the corresponding cDNA from a heterologous promoter. Likewise, null mutant mice for the Collier/Olf1/Early B-cell family transcription factor EBF2, in which Purkinje neuron development is primarily affected, exhibit abnormalities in cerebellar corticogenesis. Here, to evaluate the contribution to the Ebf2 null phenotype of changes in the profile of Contactin-1, we study its expression in Ebf2 null mice. In addition, we explore the activation profile of the Cntn1 gene promoter upon transferring the Ebf2 mutation to transgenic mice expressing an enhanced green fluorescent protein reporter under control of Cntn1 gene regulatory sequences. In Ebf2 null mice, Contactin-1 protein expression and Cntn1 gene promoter activity are both downregulated during embryonic and early postnatal cerebellar development, both in the rostral and caudal folia, while in the latter an upregulation is observed at postnatal day 8. In vitro, vectors driving EBF1,2,3 transcription factors from a cytomegalovirus (CMV) promoter transactivate a Cntn1-Choline acetyltransferse (CAT) promoter-reporter construct in cotransfection assays and, accordingly, by chromatin immunoprecipitation, we show that the Cntn1 gene 5' flanking region is bound by the EBF2 transcription factor, consistent with the evidence that this region bears the cognate deoxyribonucleic acid (DNA) consensus sequences. These data indicate that Contactin-1 expression is dependent upon EBF factors, suggesting that the Cntn1 gene belongs to the expanding regulatory cascade driven by these transcriptional regulators so that changes in its activation may contribute to the phenotype of Ebf2 null mutant mice.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Cerebelo/crescimento & desenvolvimento , Cerebelo/metabolismo , Contactina 1/metabolismo , Transativadores/metabolismo , Animais , Sequência de Bases , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Western Blotting , Proliferação de Células/fisiologia , Cerebelo/citologia , Colina O-Acetiltransferase/genética , Colina O-Acetiltransferase/metabolismo , Contactina 1/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Imuno-Histoquímica , Imunoprecipitação , Camundongos Transgênicos , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Alinhamento de Sequência , TransfecçãoRESUMO
The intracellular mechanisms driving postmitotic development of cortical γ-aminobutyric acid (GABA)ergic interneurons are poorly understood. We have addressed the function of Rac GTPases in cortical and hippocampal interneuron development. Developing neurons express both Rac1 and Rac3. Previous work has shown that Rac1 ablation does not affect the development of migrating cortical interneurons. Analysis of mice with double deletion of Rac1 and Rac3 shows that these GTPases are required during postmitotic interneuron development. The number of parvalbumin-positive cells was affected in the hippocampus and cortex of double knockout mice. Rac depletion also influences the maturation of interneurons that reach their destination, with reduction of inhibitory synapses in both hippocampal CA1 and cortical pyramidal cells. The decreased number of cortical migrating interneurons and their altered morphology indicate a role of Rac1 and Rac3 in regulating the motility of cortical interneurons, thus interfering with their final localization. While electrophysiological passive and active properties of pyramidal neurons including membrane capacity, resting potential, and spike amplitude and duration were normal, these cells showed reduced spontaneous inhibitory currents and increased excitability. Our results show that Rac1 and Rac3 contribute synergistically to postmitotic development of specific populations of GABAergic cells, suggesting that these proteins regulate their migration and differentiation.
Assuntos
Córtex Cerebral/citologia , Neurônios GABAérgicos/fisiologia , Hipocampo/citologia , Proteínas rac de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , 4-Aminopiridina/farmacologia , Animais , Animais Recém-Nascidos , Bicuculina/farmacologia , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Antagonistas de Aminoácidos Excitatórios/farmacologia , Antagonistas de Receptores de GABA-A/farmacologia , Neurônios GABAérgicos/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento/genética , Potenciais Pós-Sinápticos Inibidores/genética , Interneurônios/efeitos dos fármacos , Interneurônios/fisiologia , Camundongos , Camundongos Knockout , Piperazinas/farmacologia , Bloqueadores dos Canais de Potássio/farmacologia , Proteínas Vesiculares de Transporte de Aminoácidos Inibidores/metabolismo , Proteínas rac de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/genéticaRESUMO
Mice deficient for the transcription factor Ebf2 lose a subset of Purkinje cells during development and have a hypotrophic cerebellar cortex. Related motor symptoms and the function of Purkinje cells surviving in the adult have not been studied so far. Ebf2 null mice presented locomotor impairment and a deficiency of motor coordination and motor learning. Ebf2 null Purkinje cells of the anterior lobe, relative to wild-type controls, were patch-clamp recorded in acutely prepared slices. While immature Purkinje cells (10-20 postnatal days) of Ebf2 null mice showed no significant difference relative to wild-types, in the adult they featured a higher input resistance, increased anomalous rectification, decreased first spike latency, higher initial firing frequency, lower voltage threshold and reduced afterhyperpolarizations and post-burst hyperpolarizations. These parameters indicate a difference in the response to both hyperpolarizing and depolarizing stimuli, corresponding to an altered cerebellar cortical output signaling. In contrast, adult climbing fibers attained a normal monoinnervation pattern and the parallel fiber-Purkinje cell synapse showed evoked postsynaptic currents and paired-pulse facilitation functionally indistinguishable from wild-type PCs. These results suggest that the motor deficits exhibited by Ebf2 null mice could be due, at least in part, to an impairment of the firing properties of surviving Purkinje cells. These findings indicate that Ebf2 is important for the development and maintenance of normal Purkinje cell discharge properties.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Cerebelo/fisiologia , Atividade Motora/fisiologia , Células de Purkinje/fisiologia , Potenciais de Ação/fisiologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/deficiência , Aprendizagem/fisiologia , Camundongos , Camundongos Knockout , Técnicas de Patch-ClampRESUMO
By serving as the sole output of the cerebellar cortex, integrating a myriad of afferent stimuli, Purkinje cells (PCs) constitute the principal neuron in cerebellar circuits. Several neurodegenerative cerebellar ataxias feature a selective cell-autonomous loss of PCs, warranting the development of regenerative strategies. To date, very little is known as to the regulatory cascades controlling PC development. During central nervous system development, the proneural gene neurogenin 2 (Neurog2) contributes to many distinct neuronal types by specifying their fate and/or dictating development of their morphological features. By analyzing a mouse knock-in line expressing Cre recombinase under the control of Neurog2 cis-acting sequences we show that, in the cerebellar primordium, Neurog2 is expressed by cycling progenitors cell-autonomously fated to become PCs, even when transplanted heterochronically. During cerebellar development, Neurog2 is expressed in G1 phase by progenitors poised to exit the cell cycle. We demonstrate that, in the absence of Neurog2, both cell-cycle progression and neuronal output are significantly affected, leading to an overall reduction of the mature cerebellar volume. Although PC fate identity is correctly specified, the maturation of their dendritic arbor is severely affected in the absence of Neurog2, as null PCs develop stunted and poorly branched dendrites, a defect evident from the early stages of dendritogenesis. Thus, Neurog2 represents a key regulator of PC development and maturation.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/fisiologia , Ciclo Celular , Cerebelo/crescimento & desenvolvimento , Dendritos/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Células de Purkinje/fisiologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Linhagem da Célula , Cerebelo/fisiologia , Feminino , Técnicas de Introdução de Genes , Camundongos , Camundongos Endogâmicos C57BL , Proteínas do Tecido Nervoso/genética , Neurogênese/fisiologia , Gravidez , Transplante de Células-Tronco , Células-Tronco/fisiologiaRESUMO
Medulloblastoma arises from mutations occurring in stem/progenitor cells located in restricted hindbrain territories. Here we report that the mouse postnatal ventricular zone lining the IV ventricle also harbors bona fide stem cells that, remarkably, share the same molecular profile with cerebellar white matter-derived neural stem cells (NSC). To identify novel molecular mediators involved in medulloblastomagenesis, we compared these distinct postnatal hindbrain-derived NSC populations, which are potentially tumor initiating, with murine compound Ptch/p53 mutant medulloblastoma cancer stem cells (CSC) that faithfully phenocopy the different variants of human medulloblastoma in vivo. Transcriptome analysis of both hindbrain NSCs and medulloblastoma CSCs resulted in the generation of well-defined gene signatures, each reminiscent of a specific human medulloblastoma molecular subclass. Most interestingly, medulloblastoma CSCs upregulated developmentally related genes, such as Ebfs, that were shown to be highly expressed in human medulloblastomas and play a pivotal role in experimental medullo-blastomagenesis. These data indicate that gene expression analysis of medulloblastoma CSCs holds great promise not only for understanding functional differences between distinct CSC populations but also for identifying meaningful signatures that might stratify medulloblastoma patients beyond histopathologic staging.
Assuntos
Neoplasias Cerebelares/genética , Perfilação da Expressão Gênica , Meduloblastoma/genética , Animais , Animais Recém-Nascidos , Neoplasias Cerebelares/classificação , Neoplasias Cerebelares/patologia , Humanos , Meduloblastoma/classificação , Meduloblastoma/patologia , Camundongos , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neurais/metabolismo , Rombencéfalo/citologiaRESUMO
Cajal-Retzius (CR) cells play a crucial role in the formation of the cerebral cortex, yet the molecules that control their development are largely unknown. Here, we show that Ebf transcription factors are expressed in forebrain signalling centres-the septum, cortical hem and the pallial-subpallial boundary-known to generate CR cells. We identified Ebf2, through fate mapping studies, as a novel marker for cortical hem- and septum-derived CR cells. Loss of Ebf2 in vivo causes a transient decrease in CR cell numbers on the cortical surface due to a migratory defect in the cortical hem, and is accompanied by upregulation of Ebf3 in this and other forebrain territories that produce CR cells, without affecting proper cortical lamination. Accordingly, using in vitro preparations, we demonstrated that both Ebf2 and Ebf3, singly or together, control the migration of CR cells arising in the cortical hem. These findings provide evidence that Ebfs directly regulate CR cell development.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/fisiologia , Linhagem da Célula , Córtex Cerebral/embriologia , Neurônios , Fatores de Transcrição/fisiologia , Animais , Diferenciação Celular , Movimento Celular/fisiologia , Córtex Cerebral/citologia , Camundongos , Neurônios/citologia , Neurônios/fisiologiaRESUMO
Zinc finger protein 423 encodes a 30 Zn-finger transcription factor involved in cerebellar and olfactory development. ZFP423 is a known interactor of SMAD1-SMAD4 and of Collier/Olf-1/EBF proteins, and acts as a modifier of retinoic acid-induced differentiation. In the present article, we show that ZFP423 interacts with the Notch1 intracellular domain in mammalian cell lines and in Xenopus neurula embryos, to activate the expression of the Notch1 target Hes5/ESR1. This effect is antagonized by EBF transcription factors, both in cultured cells and in Xenopus embryos, and amplified in vitro by BMP4, suggesting that ZFP423 acts to integrate BMP and Notch signaling, selectively promoting their convergence onto the Hes5 gene promoter.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/biossíntese , Proteína Morfogenética Óssea 4/metabolismo , Proteínas de Ligação a DNA/metabolismo , Embrião de Mamíferos/metabolismo , Embrião não Mamífero/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Receptor Notch1/metabolismo , Proteínas Repressoras/biossíntese , Transdução de Sinais/fisiologia , Fatores de Transcrição/metabolismo , Proteínas de Xenopus/biossíntese , Proteínas de Xenopus/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Proteína Morfogenética Óssea 4/genética , Células COS , Chlorocebus aethiops , Proteínas de Ligação a DNA/genética , Embrião de Mamíferos/citologia , Embrião não Mamífero/citologia , Humanos , Camundongos , Receptor Notch1/genética , Proteínas Repressoras/genética , Fatores de Transcrição/genética , Regulação para Cima/fisiologia , Proteínas de Xenopus/genética , Xenopus laevisRESUMO
Autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) is a focal form of epilepsy characterized by seizures occurring during non-REM sleep. We have developed and characterized the first mouse model for ADNFLE type III carrying the V287L mutation of the beta2 subunit of neuronal nicotinic receptor. Mice expressing mutant receptors show a spontaneous epileptic phenotype by electroencephalography with very frequent interictal spikes and seizures. Expression of the mutant beta2 subunit is driven by a neuronal-specific tetracycline-controlled promoter, which allows planned silencing of transgene expression in a reversible fashion and tracking the involvement of mutant receptor in crucial phases of epileptogenesis. We found that restricted silencing during development is sufficient to prevent the occurrence of epileptic seizures in adulthood. Our data indicate that mutant nicotinic receptors are responsible for abnormal formation of neuronal circuits and/or long-lasting alteration of network assembly in the developing brain, thus leading to epilepsy.