RESUMO
Astrocytes are the most abundant cells in the CNS, and have many essential functions, including maintenance of blood-brain barrier integrity, and CNS water, ion, and glutamate homeostasis. Mammalian astrogliogenesis has generally been considered to be completed soon after birth, and to be reactivated in later life only under pathological circumstances. Here, by using genetic fate-mapping, we demonstrate that new corpus callosum astrocytes are continuously generated from nestin(+) subventricular zone (SVZ) neural progenitor cells (NPCs) in normal adult mice. These nestin fate-mapped corpus callosum astrocytes are uniformly postmitotic, express glutamate receptors, and form aquaporin-4(+) perivascular endfeet. The entry of new astrocytes from the SVZ into the corpus callosum appears to be balanced by astroglial apoptosis, because overall numbers of corpus callosum astrocytes remain constant during normal adulthood. Nestin fate-mapped astrocytes also flow anteriorly from the SVZ in association with the rostral migratory stream, but do not penetrate into the deeper layers of the olfactory bulb. Production of new astrocytes from nestin(+) NPCs is absent in the normal adult cortex, striatum, and spinal cord. Our study is the first to demonstrate ongoing SVZ astrogliogenesis in the normal adult mammalian forebrain.
Assuntos
Astrócitos/fisiologia , Corpo Caloso/citologia , Corpo Caloso/fisiologia , Ventrículos Laterais/citologia , Ventrículos Laterais/fisiologia , Animais , Encéfalo/citologia , Encéfalo/crescimento & desenvolvimento , Movimento Celular , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Nestina/fisiologia , Células-Tronco Neurais/fisiologia , Neurogênese/fisiologia , Medula Espinal/citologia , Medula Espinal/crescimento & desenvolvimento , Proteínas Vesiculares de Transporte de Glutamato/metabolismoRESUMO
Alteration in mitochondrial dynamics has been implicated in many neurodegenerative diseases. Mitochondrial apoptosis inducing factor (AIF) plays a key role in multiple cellular and disease processes. Using immunoblotting and flow cytometry analysis with Harlequin mutant mice that have a proviral insertion in the AIF gene, we first revealed that mitofusion 1 (Mfn1), a key mitochondrial fusion protein, is significantly diminished in Purkinje cells of the Harlequin cerebellum. Next, we investigated the cerebellar pathology of Harlequin mice in an age-dependent fashion, and identified a striking process of progressive and patterned Purkinje cell degeneration. Using immunohistochemistry with zebrin II, the most studied compartmentalization marker in the cerebellum, we found that zebrin II-negative Purkinje cells first started to degenerate at 7 months of age. By 11 months of age, almost half of the Purkinje cells were degenerated. Subsequently, most of the Purkinje cells disappeared in the Harlequin cerebellum. The surviving Purkinje cells were concentrated in cerebellar lobules IX and X, where these cells were positive for heat shock protein 25 and resistant to degeneration. We further showed that the patterned Purkinje cell degeneration was dependent on caspase but not poly(ADP-ribose) polymerase-1 (PARP-1) activation, and confirmed the marked decrease of Mfn1 in the Harlequin cerebellum. Our results identified a previously unrecognized role of AIF in Purkinje cell degeneration, and revealed that AIF deficiency leads to altered mitochondrial fusion and caspase-dependent cerebellar Purkinje cell loss in Harlequin mice. This study is the first to link AIF and mitochondrial fusion, both of which might play important roles in neurodegeneration.
Assuntos
Fator de Indução de Apoptose/genética , Doenças Cerebelares/genética , Doenças Cerebelares/patologia , GTP Fosfo-Hidrolases/biossíntese , GTP Fosfo-Hidrolases/deficiência , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/patologia , Células de Purkinje/patologia , Animais , Fator de Indução de Apoptose/deficiência , Doenças Cerebelares/metabolismo , Modelos Animais de Doenças , Feminino , Masculino , Camundongos , Camundongos Mutantes Neurológicos , Camundongos Transgênicos , Doenças Neurodegenerativas/metabolismo , Células de Purkinje/metabolismoRESUMO
Despite the general uniformity in cellular composition of the adult cerebellar cortex, there is a complex underlying pattern of parasagittal stripes of Purkinje cells with characteristic molecular phenotypes and patterns of connectivity. It is not known whether interneuron processes are restricted at stripe boundaries. To begin to address the issue, three strategies were used to explore how cerebellar Golgi cell dendrites are organized with respect to parasagittal stripes: first, double immunofluorescence staining combining anti-neurogranin to identify Golgi cell dendrites with the Purkinje cell compartmentation antigens zebrin II/aldolase C, HNK-1, and phospholipase Cbeta4; second, zebrin II immunohistochemistry combined with a rapid Golgi-Cox impregnation procedure to reveal Golgi cell dendritic arbors; third, stripe antigen expression was used on sections of a GlyT2-EGFP transgenic mouse in which reporter expression is prominent in Golgi cell dendrites. In each case, the dendritic projections of Golgi cells were studied in the vicinity of Purkinje cell stripe boundaries. The data presented here show that the dendrites of a cerebellar interneuron, the Golgi cell, respect the fundamental cerebellar stripe cytoarchitecture.
Assuntos
Córtex Cerebelar/química , Córtex Cerebelar/citologia , Dendritos/química , Animais , Córtex Cerebelar/metabolismo , Dendritos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Células de Purkinje/química , Células de Purkinje/citologia , Células de Purkinje/metabolismoRESUMO
Unipolar brush cells (UBCs) are glutamatergic cerebellar interneurons of the granular layer. Previous studies have shown that there are two distinct subsets of UBCs present in the mice cerebellar cortex: calcium-binding protein calretinin (CR) positive and metabotropic glutamate receptor (mGluR)1alpha positive. In this study, we identify phospholipase C (PLC) beta4 as an antigenic marker of a novel subset of UBCs. Double immunolabeling reveals that none of the CR+ subset expresses PLCbeta4. In contrast, most members of the mGluR1alpha subset also express PLCbeta4. In addition, 65% of the PLCbeta4+ subset does not express mGluR1alpha. Thus, there are three distinct UBC subsets in the mouse cerebellum: CR+/PLCbeta4-/mGluR1alpha-, PLCbeta4+/mGluR1alpha-/CR-, and mGluR1alpha+/PLCbeta4+/CR-. Each has a different topographical distribution, both between lobules and mediolaterally within the vermis. The development of PLCbeta4 expression in UBCs is exclusively postnatal--first seen only at P12 and mature at about 3 weeks. A distinct subset of PLCbeta4+ UBCs is also present in primary cerebellar cultures.
Assuntos
Cerebelo/citologia , Interneurônios/metabolismo , Inibição Neural/fisiologia , Fosfolipase C beta/metabolismo , Animais , Animais Recém-Nascidos , Calbindina 2 , Embrião de Mamíferos , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Proteínas de Fluorescência Verde/genética , Interneurônios/classificação , Proteínas Luminescentes/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Técnicas de Cultura de Órgãos , Receptores de Glutamato Metabotrópico/metabolismo , Proteína G de Ligação ao Cálcio S100/metabolismoRESUMO
In this study, we have developed highly expandable neural stem cells (NSCs) from HESCs and iPSCs that artificially express the oligodendrocyte (OL) specific transcription factor gene Zfp488. This is enough to restrict them to an exclusive oligodendrocyte progenitor cell (OPC) fate during differentiation in vitro and in vivo. During CNS development, Zfp488 is induced during the early stages of OL generation, and then again during terminal differentiation of OLs. Interestingly, the human ortholog Znf488, crucial for OL development in human, has been recently identified to function as a dorsoventral pattering regulator in the ventral spinal cord for the generation of P1, P2/pMN, and P2 neural progenitor domains. Forced expression of Zfp488 gene in human NSCs led to the robust generation of OLs and suppression of neuronal and astrocyte fate in vitro and in vivo. Zfp488 expressing NSC derived oligodendrocytes are functional and can myelinate rat dorsal root ganglion neurons in vitro, and form myelin in Shiverer mice brain in vivo. After transplantation near a site of demyelination, Zfp488 expressing hNSCs migrated to the lesion and differentiated into premyelinating OLs. A certain fraction also homed in the subventricular zone (SVZ). Zfp488-ZsGreen1-hNSC derived OLs formed compact myelin in Shiverer mice brain seen under the electron microscope. Transplanted human neural stem cells (NSC) that have the potential to differentiate into functional oligodendrocytes in response to remyelinating signals can be a powerful therapeutic intervention for disorders where oligodendrocyte (OL) replacement is beneficial.
Assuntos
Diferenciação Celular , Células-Tronco Neurais/citologia , Neurogênese , Neuroglia/citologia , Oligodendroglia/citologia , Animais , Linhagem Celular , Células Cultivadas , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Camundongos , Bainha de Mielina/metabolismo , Células-Tronco Neurais/metabolismo , Células-Tronco Neurais/fisiologia , Neurônios/citologia , Neurônios/metabolismo , Células Precursoras de Oligodendrócitos/citologia , Células Precursoras de Oligodendrócitos/metabolismo , Oligodendroglia/metabolismo , Oligodendroglia/fisiologia , Ratos , Transplante de Células-Tronco/métodos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Despite the apparent uniformity in cellular composition of the adult mammalian cerebellar cortex, it is actually highly compartmentalized into transverse zones, and within each zone the cortex is further subdivided into a reproducible array of parasagittal stripes. The most extensively studied compartmentation antigen is zebrin II/aldolase c, which is expressed by a subset of Purkinje cells forming parasagittal stripes. Gamma-aminobutyric acid B receptors (GABABRs) are G-protein-coupled receptors that mediate a slow, prolonged form of inhibition in many brain areas. This study examines the localization of GABABR2 in the mouse cerebellum by using whole mount and section immunohistochemistry. The data reveal that GABABR2 immunoreactivity is expressed strongly in the dendrites of a subset of Purkinje cells that form a reproducible array of transverse zones and parasagittal stripes. By using double immunostaining, the striped pattern of GABABR2 expression was shown to be identical to that revealed by anti-zebrin II and complementary to that of phospholipase Cbeta4. This finding supports previous functional studies showing that inhibitory neurotransmission is highly patterned in the cerebellar cortex.
Assuntos
Córtex Cerebelar/fisiologia , Dendritos/fisiologia , Células de Purkinje/fisiologia , Receptores de GABA-B/genética , Transmissão Sináptica/fisiologia , Animais , Imuno-Histoquímica , Camundongos , Receptores de GABA-B/metabolismoRESUMO
Given the importance of sensory innervation in tooth vitality, the identification of signals that control nerve regeneration and the cellular events they induce is essential. Previous studies demonstrated that the complement system, a major component of innate immunity and inflammation, is activated at the injured site of human carious teeth and plays an important role in dental-pulp regeneration via interaction of the active Complement C5a fragment with pulp progenitor cells. In this study, we further determined the role of the active fragment complement C5a receptor (C5aR) in dental nerve regeneration in regards to local secretion of nerve growth factor (NGF) upon carious injury. Using ELISA and AXIS co-culture systems, we demonstrate that C5aR is critically implicated in the modulation of NGF secretion by LTA-stimulated pulp fibroblasts. The NGF secretion by LTA-stimulated pulp fibroblasts, which is negatively regulated by C5aR activation, has a role in the control of the neurite outgrowth length in our axon regeneration analysis. Our data provide a scientific step forward that can guide development of future therapeutic tools for innovative and incipient interventions targeting the dentin-pulp regeneration process by linking the neurite outgrowth to human pulp fibroblast through complement system activation.
Assuntos
Polpa Dentária , Fibroblastos/metabolismo , Fator de Crescimento Neural/metabolismo , Neuritos/metabolismo , Receptor da Anafilatoxina C5a/metabolismo , Cárie Radicular/metabolismo , Técnicas de Cocultura , Polpa Dentária/inervação , Polpa Dentária/metabolismo , Polpa Dentária/patologia , Fibroblastos/patologia , Humanos , Neuritos/patologia , Cárie Radicular/patologiaRESUMO
Astroglia are a major cellular constituent of the central nervous system (CNS) and play crucial roles in brain development, function, and integrity. Increasing evidence demonstrates that astroglia dysfunction occurs in a variety of neurological disorders ranging from CNS injuries to genetic diseases and chronic degenerative conditions. These new insights herald the concept that transplantation of astroglia could be of therapeutic value in treating the injured or diseased CNS. Recent technological advances in the generation of human astroglia from stem and progenitor cells have been prominent. We propose that a better understanding of the suitability of astroglial cells in transplantation as well as of their therapeutic effects in animal models may lead to the establishment of astroglia-based therapies to treat neurological diseases.
Assuntos
Astrócitos/transplante , Doenças do Sistema Nervoso/terapia , Células-Tronco/citologia , Animais , Astrócitos/citologia , Técnicas de Cultura de Células/métodos , Terapia Baseada em Transplante de Células e Tecidos/métodos , HumanosRESUMO
Cyclin-dependent kinase 5 (Cdk5) is primarily associated with brain development but it is also implicated in lens and muscle differentiation. We found that Cdk5 is also expressed in mouse ovary, and explored the possibility that it plays a role in that tissue. We show by Western blotting and immunohistochemistry that the known Cdk5 activator, p35, is also present in the mouse ovary. Cdk5 and p35 were detected in oocytes at all stages of the follicle. While Cdk5 was present in the cytoplasm and nucleus of the oocyte, p35 was observed only in the cytoplasm. Both proteins were detected in the cytoplasm of luteinized cells in the corpus luteum. Immunoprecipitation and histone H1 kinase assays revealed that they form an ovarian complex with considerable kinase activity. Phosphorylation assays showed that several ovarian proteins are substrates for Cdk5/p35 in vitro. Together our findings suggest that p35-associated Cdk5 activity plays an important role in the ovary, where it may regulate cell differentiation and apoptosis as it does in the brain.
Assuntos
Quinases Ciclina-Dependentes/biossíntese , Proteínas do Tecido Nervoso/biossíntese , Ovário/metabolismo , Animais , Western Blotting , Diferenciação Celular , Núcleo Celular/metabolismo , Quinase 5 Dependente de Ciclina , Citoplasma/metabolismo , Feminino , Histonas/metabolismo , Immunoblotting , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos C57BL , Oócitos/metabolismo , Proteínas Quinases/metabolismo , Fatores de TempoRESUMO
The present study describes the distribution of tyrosine hydroxylase (TH)-immunoreactive (IR) elements in the olfactory bulb of the common marmoset monkey (Callithrix jacchus), a primate species by immunohistochemistry. We identified six layers of the olfactory bulb of the common marmoset monkey in sections stained with cresyl violet. The majority of TH-IR cells were found in the glomerular layer. A few TH-IR cells were present in the external plexiform and granule cell layers. TH-IR fibers were identified in all layers of the olfactory bulb. The density of these nerve fibers was high in the internal plexiform and granule cell layers. The results in the olfactory bulb of the common marmoset monkey are generally similar to previous reports in some mammals. These data suggest that TH in the olfactory bulb of the common marmoset monkey may play a role in olfactory transmission via the glomeruli like in other mammals.
Assuntos
Callithrix/anatomia & histologia , Dopamina/análise , Neurônios/citologia , Bulbo Olfatório/anatomia & histologia , Animais , Imuno-Histoquímica , Bulbo Olfatório/citologia , Tirosina 3-Mono-Oxigenase/análiseRESUMO
Secretion of neurotransmitters is initiated by voltage-gated calcium influx through presynaptic, voltage- gated N-type calcium channels. However, little is known about their cellular distribution in the mouse cerebellum. In the cerebellum, alpha1B immunoreactivity is found mainly on the cell bodies of all Purkinje cells. In addition, the immunoreactivity was detected on a subset of Purkinje cell dendrites, clustered to form a parasagittal array of bands. In the anterior lobe vermis, immunoreactive Purkinje cell dendrites form narrow stripes separated by broad bands of unstained dendrites. Moving caudally through the vermis, these stripes become thicker as a larger fraction of the Purkinje cell dendrites become immunoreactive. This localization study of the alpha1B pore-forming subunits in mouse cerebellum may guide future investigations of the role of calcium channels in neurological pathways.
Assuntos
Canais de Cálcio Tipo N/metabolismo , Cerebelo/metabolismo , Animais , Cerebelo/citologia , Dendritos/metabolismo , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos BALB C , Células de Purkinje/metabolismoRESUMO
The Pogo mouse is an autosomal recessive ataxic mutant that arose spontaneously in the inbred KJR/MsKist strain derived originally from Korean wild mice. The ataxic phenotype is characterized by difficulty in maintaining posture and side to side stability, faulty coordination between limbs and trunk, and the consequent inability to walk straight. In the present study, the cerebellar concentrations of glutamate and GABA were analyzed, since glutamate is a most prevalent excitatory neurotransmitter whereas gamma-aminobutyric acid (GABA) is one of the most abundant inhibitory neurotransmitters, which may be the main neurotransmitters related with the ataxia and epilepsy. The concentration of glutamate of cerebellum decreased significantly in ataxic mutant Pogo mouse compared to those of control mouse. However, GABA concentration was not decrease. These results suggested that the decrease in glutamate concentration may contribute to ataxia in mutant Pogo mouse.
Assuntos
Cerebelo/metabolismo , Marcha Atáxica/metabolismo , Ácido Glutâmico/metabolismo , Ácido gama-Aminobutírico/metabolismo , Animais , Calbindinas , Cerebelo/patologia , Marcha Atáxica/patologia , Imuno-Histoquímica , Camundongos , Camundongos Mutantes , Proteína G de Ligação ao Cálcio S100/metabolismoRESUMO
Human pluripotent stem cells (hPSCs) have been differentiated to astroglia, but the utilization of hPSC-derived astroglia as cell therapy for neurological diseases has not been well studied. Astroglia are heterogeneous, and not all astroglia are equivalent in promoting neural repair. A prerequisite for cell therapy is to derive defined cell populations with superior therapeutic effects. Here we use an Olig2-GFP human embryonic stem cell (hESC) reporter to demonstrate that hESC-derived Olig2(+) progenitors generate a subtype of previously uncharacterized astroglia (Olig2PC-Astros). These Olig2PC-Astros differ substantially from astroglia differentiated from Olig2-negative hESC-derived neural progenitor cells (NPC-Astros), particularly in their neuroprotective properties. When grafted into brains subjected to global ischaemia, Olig2PC-Astros exhibit superior neuroprotective effects and improved behavioural outcome compared to NPC-Astros. Thus, this new paradigm of human astroglial differentiation is useful for studying the heterogeneity of human astroglia, and the unique Olig2PC-Astros may constitute a new cell therapy for treating cerebral ischaemia and other neurological diseases.
Assuntos
Astrócitos/citologia , Astrócitos/transplante , Isquemia Encefálica/terapia , Células-Tronco Embrionárias/citologia , Animais , Astrócitos/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Biomarcadores/metabolismo , Isquemia Encefálica/metabolismo , Isquemia Encefálica/patologia , Diferenciação Celular , Linhagem da Célula , Células Cultivadas , Células-Tronco Embrionárias/metabolismo , Expressão Gênica , Genes Reporter , Proteínas de Fluorescência Verde , Hipocampo/metabolismo , Hipocampo/patologia , Humanos , Injeções Intraventriculares , Masculino , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Fator de Transcrição 2 de Oligodendrócitos , Técnicas de Patch-Clamp , Ratos , Ratos Sprague-Dawley , Técnicas Estereotáxicas , Transplante HeterólogoRESUMO
BACKGROUND: The cerebellum is composed of a diverse array of neuronal subtypes. Here we have used a candidate approach to identify Zac1, a tumor suppressor gene encoding a zinc finger transcription factor, as a new player in the transcriptional network required for the development of a specific subset of cerebellar nuclei and a population of Golgi cells in the cerebellar cortex. RESULTS: We found that Zac1 has a complex expression profile in the developing cerebellum, including in two proliferating progenitor populations; the cerebellar ventricular zone and the external granular layer overlying posterior cerebellar lobules IX and X. Zac1 is also expressed in some postmitotic cerebellar neurons, including a subset of GABAergic interneurons in the medial cerebellar nuclei. Notably, GABAergic interneurons in the cerebellar nuclei are derived from the cerebellar ventricular zone, where Zac1 is also expressed, consistent with a lineage relationship between these two Zac1+ populations. Zac1 is also expressed in a small subset of cells in the posterior vermis, including some neurogranin-immunoreactive (NG+) Golgi cells, which, based on short-term birthdating, are derived from the EGL, where Zac1 is also expressed. However, Zac1+ cells and NG+ Golgi cells in the cerebellar cortex also display unique properties, as they are generated within different, albeit overlapping, time windows. Finally, consistent with the expression profile of Zac1, two conspicuous abnormalities were found in the cerebellum of Zac1 null mice: the medial cerebellar nuclei, and not the others, were significantly reduced in size; and the number of Golgi cells in cerebellar lobule IX was reduced by approximately 60% compared to wild-type littermates. CONCLUSIONS: The data presented here indicate that the tumor suppressor gene Zac1 is expressed in a complex fashion in the developing cerebellum, including in two dividing progenitor populations and in specific subsets of postmitotic neurons, including Golgi cells and GABAergic neurons in the medial nuclei, which require Zac1 for their differentiation. We thus conclude that Zac1 is a critical regulator of normal cerebellar development, adding a new transcriptional regulator to the growing list of factors involved in generating neuronal diversity in the developing cerebellum.
Assuntos
Proteínas de Ciclo Celular/fisiologia , Cerebelo , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Genes Supressores de Tumor/fisiologia , Neurônios/classificação , Neurônios/fisiologia , Fatores de Transcrição/fisiologia , Fatores Etários , Animais , Animais Recém-Nascidos , Contagem de Células/métodos , Células Cultivadas , Cerebelo/citologia , Cerebelo/embriologia , Cerebelo/crescimento & desenvolvimento , Proteínas de Ligação a DNA/metabolismo , Embrião de Mamíferos , Regulação da Expressão Gênica no Desenvolvimento/genética , Ácido Glutâmico/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurogranina/metabolismo , Proteínas com Domínio T , Fatores de Transcrição/deficiência , Ácido gama-Aminobutírico/metabolismoRESUMO
Unipolar brush cells (UBCs) are excitatory interneurons with their somata located in the granular layer. Recently, T-brain factor 2 (Tbr2) was shown to be expressed in a subset of UBCs in mouse cerebellum. Scrambler mice exhibit severe cerebellum abnormalities, including the failure of embryonic Purkinje cell dispersal and a complete absence of foliation due to a mutation in the disabled-1 adaptor protein. Since most UBC markers are expressed postnatally, it has proven difficult to identify the relationship between developing Purkinje cell clusters and migrating UBCs. Because scrambler mice closely mimic normal embryonic day 18 cerebellum, we examined whether Tbr2-positive UBCs are associated with Purkinje cell cluster markers such as zebrin II, which is the most studied compartmentation marker in the cerebellum. We investigated the distribution of Tbr2-positive UBCs in this mutant by using anti-Tbr2 immunocytochemistry. The data revealed that Tbr2 immunoreactivity was exclusively present in the nucleus of UBCs in scrambler cerebellum. Based on expression data, a Tbr2-positive UBC map was constructed. In addition, Tbr2-positive UBCs are found associated with ectopic zebrin II-immunoreactive Purkinje cell clusters in scrambler cerebellum. These data suggest that UBCs use Purkinje cell compartmentation to migrate into their final position through interactions with the embryonic array of specific Purkinje cell subtypes.
RESUMO
Pax6, a paired homeobox DNA binding protein, has been found to be expressed in the cerebellum in both granule cells and their precursors in the external granular layer (EGL). In this study we have traced Pax6 expression through embryonic development in mice by using a polyclonal antibody against Pax6 and used it to study the cellular dispersal pattern of the EGL. During dispersal the EGL was thicker and Pax6 expression was more intense on the rostral side of the lateral corners of the cerebellum. Pax6 immunoreactive cells were found to be migrating from the EGL during the early stage of EGL dispersal, which suggested the early inward migration of granule cells. Double staining with various markers confirmed that the early-migrating cells are not Purkinje cells, interneurons or glia. Although the Pax6 immunoreactive cells within the cerebellum were not apparently proliferating, NeuN, a marker for postmitotic granule cells, was not expressed in these cells until E16. Furthermore, granule cells were observed migrating inwards from the EGL both during and after EGL dispersal. These early migrating granule cells populated the whole cerebellum. These findings offer novel views on specific stages of granule cell dispersal and migration.
RESUMO
The chick is a well-understood developmental model of cerebellar pattern formation,but we know much less about the patterning of the adult chicken cerebellum. Therefore an expression study of two Purkinje cell stripe antigens-zebrin II/aldolase C and phospholipase Cbeta4 (PLCbeta4)-has been carried out in the adult chicken (Gallus domesticus). The mammalian cerebellar cortex is built around transverse expression domains ("transverse zones"), each of which is further subdivided into parasagittally oriented stripes. The results from the adult chicken reveal a similar pattern. Five distinct transverse domains were identified. In the anterior lobe a uniformly zebrin II-immunopositive/PLCbeta4-immunonegative lingular zone (LZ; lobule I) and a striped anterior zone (AZ; lobules II-VIa) were distinguished. A central zone (CZ; approximately lobules VIa-VIIIa,b) and a posterior zone (PZ; approximately lobules VIIIa,b-IXc,d) were distinguished in the posterior lobe. Finally, the nodular zone (NZ; lobule X) is uniformly zebrin II-immunoreactive and is innervated by vestibular mossy fibers. Lobule IXc,d is considered as a transitional region between the PZ and the NZ, because the vestibular mossy fiber projection extends into these lobules and because they receive optokinetic mossy and climbing fiber input. It is proposed that the zebrin II-immunonegative P3- stripe corresponds to the lateral vermal B zone of the mammalian cerebellum and that the border between the avian homologs of the mammalian vermis and hemispheres is located immediately lateral to P3-. Thus, there seem to be transverse zones in chicken that are plausible homologs of those identified in mammals, together with an LZ that is characteristic of birds.
Assuntos
Proteínas Aviárias/metabolismo , Córtex Cerebelar/metabolismo , Galinhas/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Fosfolipase C beta/metabolismo , Envelhecimento , Animais , Antígenos/metabolismo , Western Blotting , Calbindinas , Córtex Cerebelar/anatomia & histologia , Galinhas/anatomia & histologia , Imuno-Histoquímica , Proteína G de Ligação ao Cálcio S100/metabolismo , Especificidade da EspécieRESUMO
Transverse boundaries divide the mammalian cerebellar cortex into transverse zones, and within each zone the cortex is further subdivided into a symmetrical array of parasagittal stripes. This topography is highly conserved across the Mammalia. Bats have a remarkable cerebellum with presumed adaptations to flight and to echolocation, but nothing is known of its compartmentation. We have therefore used two Purkinje cell compartmentation antigens, zebrin II/aldolase C and phospholipase Cbeta4, to reveal the topography of the cerebellum in microchiropteran bats. Three species of bat were studied, Lasiurus cinereus, Lasionycteris noctivagans, and Eptesicus fuscus. A reproducible pattern of zones and stripes was revealed that is similar across the three species. The architecture of the bat cerebellum conforms to the ground plan of other mammals. However, two exceptions to the highly conserved mammalian architectural plan were revealed. First, many Purkinje cells in lobule I express zebrin II. A zebrin II-immunopositive lobule I has not been seen previously in mammals but is characteristic of the avian cerebellum. Second, lobules VI-VII comprise the large central zone. Within the central zone two subdomains are evident, a small anterior subdomain (lobule VI) in which Purkinje cells are predominantly zebrin II-immunopositive/PLCbeta4-immunonegative, as in other mammals, and a posterior subdomain (lobule VII), in which alternating zebrin II/phospholipase Cbeta4 stripes are prominent.
Assuntos
Cerebelo/citologia , Quirópteros/anatomia & histologia , Células de Purkinje/metabolismo , Animais , Calbindinas , Quirópteros/classificação , Proteínas do Tecido Nervoso/metabolismo , Fosfolipase C beta/metabolismo , Proteína G de Ligação ao Cálcio S100/metabolismo , Especificidade da EspécieRESUMO
The gross anatomical distribution of an antigen is typically mapped using a combination of serial sectioning, immunocytochemistry, and three-dimensional reconstruction. This is a tedious and time-consuming procedure, which introduces an array of potential alignment and differential shrinkage errors and requires considerable experience and specialized equipment. In particular, it is unsuited for routine screening applications. To circumvent these problems, this unit presents a routine whole-mount immunocytochemistry protocol that can be used to map many antigenic distributions in the developing and adult brain. The technique can also be easily adapted to detect anterograde and retrograde transport tracers.
Assuntos
Encéfalo/anatomia & histologia , Encéfalo/metabolismo , Imuno-Histoquímica/métodos , Animais , Biotina/análogos & derivados , Biotina/metabolismo , Dextranos/metabolismo , Técnicas de Preparação Histocitológica/instrumentação , Técnicas de Preparação Histocitológica/métodos , Imuno-Histoquímica/instrumentação , Camundongos , Neurônios/metabolismoRESUMO
Early B-cell factor 2 (EBF2) is one of four mammalian members of an atypical helix-loop-helix transcription factor family (COE). COE proteins have been implicated in various aspects of nervous and immune system development. We and others have generated and described mice carrying a null mutation of Ebf2, a gene previously characterized in the context of Xenopus laevis primary neurogenesis and neuronal differentiation. In addition to deficits in neuroendocrine and olfactory development, and peripheral nerve maturation, Ebf2 null mice feature an ataxic gait and obvious motor deficits associated with clear-cut abnormalities of cerebellar development. The number of Purkinje cells (PCs) in the Ebf2 null is markedly decreased, resulting in a small cerebellum with notable foliation defects, particularly in the anterior vermis. We show that this stems from the defective migration of a molecularly defined PC subset that subsequently dies by apoptosis. Part of the striped cerebellar topography is disrupted due to cell death and, in addition, many of the surviving PCs, that would normally adopt a zebrin II-negative phenotype, transdifferentiate to Zebrin II-positive, an unprecedented finding suggesting that Ebf2 is required for the establishment of a proper cerebellar cortical map.