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Traumatic brain injury (TBI) is a main cause of death and disabilities in young adults. Although learning and memory impairments are a major clinical manifestation of TBI, the consequences of TBI on the hippocampus are still not well understood. In particular, how lesions to the sensorimotor cortex damage the hippocampus, to which it is not directly connected, is still elusive. Here, we study the effects of sensorimotor cortex ablation (SCA) on the hippocampal dentate gyrus, by applying a highly sensitive gray-level co-occurrence matrix (GLCM) analysis. Using GLCM analysis of granule neurons, we discovered, in our TBI paradigm, subtle changes in granule cell (GC) morphology, including textual uniformity, contrast, and variance, which is not detected by conventional microscopy. We conclude that sensorimotor cortex trauma leads to specific changes in the hippocampus that advance our understanding of the cellular underpinnings of cognitive impairments in TBI. Moreover, we identified GLCM analysis as a highly sensitive method to detect subtle changes in the GC layers that is expected to significantly improve further studies investigating the impact of TBI on hippocampal neuropathology.
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Lesões Encefálicas , Giro Denteado/lesões , Giro Denteado/patologia , Hipocampo/patologia , Neurônios/patologia , Animais , Modelos Animais de Doenças , Masculino , Fotomicrografia , Ratos WistarRESUMO
The embryonic formation of midbrain dopaminergic (mDA) neurons in vivo provides critical guidelines for the in vitro differentiation of mDA neurons from stem cells, which are currently being developed for Parkinson's disease cell replacement therapy. Bone morphogenetic protein (BMP)/SMAD inhibition is routinely used during early steps of stem cell differentiation protocols, including for the generation of mDA neurons. However, the function of the BMP/SMAD pathway for in vivo specification of mammalian mDA neurons is virtually unknown. Here, we report that BMP5/7-deficient mice (Bmp5-/-; Bmp7-/-) lack mDA neurons due to reduced neurogenesis in the mDA progenitor domain. As molecular mechanisms accounting for these alterations in Bmp5-/-; Bmp7-/- mutants, we have identified expression changes of the BMP/SMAD target genes MSX1/2 (msh homeobox 1/2) and SHH (sonic hedgehog). Conditionally inactivating SMAD1 in neural stem cells of mice in vivo (Smad1Nes) hampered the differentiation of progenitor cells into mDA neurons by preventing cell cycle exit, especially of TH+SOX6+ (tyrosine hydroxylase, SRY-box 6) and TH+GIRK2+ (potassium voltage-gated channel subfamily-J member-6) substantia nigra neurons. BMP5/7 robustly increased the in vitro differentiation of human induced pluripotent stem cells and induced neural stem cells to mDA neurons by up to threefold. In conclusion, we have identified BMP/SMAD signaling as a novel critical pathway orchestrating essential steps of mammalian mDA neurogenesis in vivo that balances progenitor proliferation and differentiation. Moreover, we demonstrate the potential of BMPs to improve the generation of stem-cell-derived mDA neurons in vitro, highlighting the importance of sequential BMP/SMAD inhibition and activation in this process.SIGNIFICANCE STATEMENT We identify bone morphogenetic protein (BMP)/SMAD signaling as a novel essential pathway regulating the development of mammalian midbrain dopaminergic (mDA) neurons in vivo and provide insights into the molecular mechanisms of this process. BMP5/7 regulate MSX1/2 (msh homeobox 1/2) and SHH (sonic hedgehog) expression to direct mDA neurogenesis. Moreover, the BMP signaling component SMAD1 controls the differentiation of mDA progenitors, particularly to substantia nigra neurons, by directing their cell cycle exit. Importantly, BMP5/7 increase robustly the differentiation of human induced pluripotent and induced neural stem cells to mDA neurons. BMP/SMAD are routinely inhibited in initial stages of stem cell differentiation protocols currently being developed for Parkinson's disease cell replacement therapies. Therefore, our findings on opposing roles of the BMP/SMAD pathway during in vitro mDA neurogenesis might improve these procedures significantly.
Assuntos
Proteínas Morfogenéticas Ósseas/fisiologia , Neurônios Dopaminérgicos/fisiologia , Mesencéfalo/fisiologia , Células-Tronco Neurais , Neurogênese/fisiologia , Células-Tronco Pluripotentes , Transdução de Sinais/fisiologia , Proteínas Smad/fisiologia , Animais , Proteína Morfogenética Óssea 5/genética , Proteína Morfogenética Óssea 5/metabolismo , Proteína Morfogenética Óssea 7/genética , Proteína Morfogenética Óssea 7/metabolismo , Proliferação de Células , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Fator de Transcrição MSX1/genética , Fator de Transcrição MSX1/metabolismo , Mesencéfalo/citologia , Camundongos , Camundongos Knockout , Proteína Smad1/genética , Proteína Smad1/metabolismoRESUMO
Midbrain dopaminergic (mDA) neurons modulate various motor and cognitive functions, and their dysfunction or degeneration has been implicated in several psychiatric diseases. Both Sonic Hedgehog (Shh) and Wnt signaling pathways have been shown to be essential for normal development of mDA neurons. Primary cilia are critical for the development of a number of structures in the brain by serving as a hub for essential developmental signaling cascades, but their role in the generation of mDA neurons has not been examined. We analyzed mutant mouse lines deficient in the intraflagellar transport protein IFT88, which is critical for primary cilia function. Conditional inactivation of Ift88 in the midbrain after E9.0 results in progressive loss of primary cilia, a decreased size of the mDA progenitor domain, and a reduction in mDA neurons. We identified Shh signaling as the primary cause of these defects, since conditional inactivation of the Shh signaling pathway after E9.0, through genetic ablation of Gli2 and Gli3 in the midbrain, results in a phenotype basically identical to the one seen in Ift88 conditional mutants. Moreover, the expansion of the mDA progenitor domain observed when Shh signaling is constitutively activated does not occur in absence of Ift88. In contrast, clusters of Shh-responding progenitors are maintained in the ventral midbrain of the hypomorphic Ift88 mouse mutant, cobblestone. Despite the residual Shh signaling, the integrity of the mDA progenitor domain is severely disturbed, and consequently very few mDA neurons are generated in cobblestone mutants. Our results identify for the first time a crucial role of primary cilia in the induction of mDA progenitors, define a narrow time window in which Shh-mediated signaling is dependent upon normal primary cilia function for this purpose, and suggest that later Wnt signaling-dependent events act independently of primary cilia.
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Cílios/metabolismo , Neurônios Dopaminérgicos/metabolismo , Embrião de Mamíferos/citologia , Proteínas Hedgehog/metabolismo , Mesencéfalo/citologia , Mesencéfalo/embriologia , Neurogênese , Animais , Cílios/ultraestrutura , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Fatores de Transcrição Kruppel-Like/metabolismo , Camundongos , Mutação/genética , Proteínas do Tecido Nervoso/metabolismo , Neurogênese/genética , Neuroglia/metabolismo , Fenótipo , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais/genética , Receptor Smoothened , Células-Tronco/citologia , Células-Tronco/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Proteínas Wnt/metabolismo , Proteína Gli2 com Dedos de Zinco , Proteína Gli3 com Dedos de ZincoRESUMO
Complex patterns and networks of genes coordinate rhombomeric identities, hindbrain segmentation and neuronal differentiation and are responsible for later brainstem functions. Pax6 is a highly conserved transcription factor crucial for neuronal development, yet little is known regarding its early roles during hindbrain segmentation. We show that Pax6 expression is highly dynamic in rhombomeres, suggesting an early function in the hindbrain. Utilization of multiple gain- and loss-of-function approaches in chick and mice revealed that loss of Pax6 disrupts the sharp expression borders of Krox20, Kreisler, Hoxa2, Hoxb1 and EphA and leads to their expansion into adjacent territories, whereas excess Pax6 reduces these expression domains. A mutual negative cross-talk between Pax6 and Krox20 allows these genes to be co-expressed in the hindbrain through regulation of the Krox20-repressor gene Nab1 by Pax6. Rhombomere boundaries are also distorted upon Pax6 manipulations, suggesting a mechanism by which Pax6 acts to set hindbrain segmentation. Finally, FGF signaling acts upstream of the Pax6-Krox20 network to regulate Pax6 segmental expression. This study unravels a novel role for Pax6 in the segmental organization of the early hindbrain and provides new evidence for its significance in regional organization along the central nervous system.
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Encéfalo/embriologia , Proteínas do Olho/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/fisiologia , Fatores de Transcrição Box Pareados/fisiologia , Proteínas Repressoras/fisiologia , Animais , Encéfalo/metabolismo , Mapeamento Encefálico/métodos , Proliferação de Células , Embrião de Galinha , Cicloeximida/farmacologia , Proteína 2 de Resposta de Crescimento Precoce/metabolismo , Eletroporação/métodos , Proteínas do Olho/genética , Genótipo , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Hibridização In Situ , Fator de Transcrição MafB/metabolismo , Camundongos , Fator de Transcrição PAX6 , Fatores de Transcrição Box Pareados/genética , Proteínas Repressoras/genética , Rombencéfalo/embriologia , Rombencéfalo/metabolismo , Transdução de Sinais , Fatores de TempoRESUMO
Mandible shape in the mouse is a complex trait that is influenced by many genetic factors. However, little is known about the action of single genes on adult mandible shape so far, since most developmentally relevant genes are already required during embryogenesis, i.e., knockouts lead to embryonic death or severe deformations, before the mandible is fully formed. We employ here a geometric morphometric approach to identify subtle phenotypic differences caused by dosage effects of candidate genes. We use mouse strains with specific gene modifications (knockouts and knockins) to compare heterozygous animals with controls from the same stock, which is expected to be equivalent to a change of gene expression of the respective locus. Such differences in expression level are also likely to occur as part of the natural variation. We focus on Bmp pathway genes (Bmp4, its antagonist Noggin, and combinations of Bmp5-7 genotypes), but include also two other developmental control genes suspected to affect mandible development in some way (Egfr and Irf6). In addition, we study the effects of Hoxd13, as well as an extracellular matrix constituent (Col2a1). We find that subtle but significant shape differences are caused by differences in gene dosage of several of these genes. The changes seen for Bmp4 and Noggin are partially compatible with the action of these genes known from birds and fish. We find significant shape changes also for Hoxd13, although this gene has so far only been implicated in skeletal patterning processes of the limbs. Comparing the effect sizes of gene dosage changes to the variation found in natural populations of mice as well as quantitative trait loci (QTL) effects on mandible shape, we find that the effect sizes caused by gene dosage changes are at the lower end of the spectrum of natural variation, but larger than the average additive effects found in QTL studies. We conclude that studying gene dosage effects have the potential to provide new insights into aspects of craniofacial development, variation, and evolution.
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Dosagem de Genes , Mandíbula/anatomia & histologia , Camundongos/anatomia & histologia , Camundongos/genética , Locos de Características Quantitativas , Transdução de Sinais , Animais , Proteínas Morfogenéticas Ósseas/metabolismo , Mandíbula/fisiologia , FenótipoRESUMO
Parkinson's disease (PD) is characterized by degeneration of neurons, particularly dopaminergic neurons in the substantia nigra. PD brains show accumulation of α-synuclein in Lewy bodies and accumulation of dysfunctional mitochondria. However, the mechanisms leading to mitochondrial pathology in sporadic PD are poorly understood. PINK1 is a key for mitophagy activation and recycling of unfit mitochondria. The activation of mitophagy depends on the accumulation of uncleaved PINK1 at the outer mitochondrial membrane and activation of a cascade of protein ubiquitination at the surface of the organelle. We have now found that SIAH3, a member of the SIAH proteins but lacking ubiquitin-ligase activity, is increased in PD brains and cerebrospinal fluid and in neurons treated with α-synuclein preformed fibrils (α-SynPFF). We also observed that SIAH3 is aggregated together with PINK1 in the mitochondria of PD brains. SIAH3 directly interacts with PINK1, leading to their intra-mitochondrial aggregation in cells and neurons and triggering a cascade of toxicity with PINK1 inactivation along with mitochondrial depolarization and neuronal death. We also found that SIAH1 interacts with PINK1 and promotes ubiquitination and proteasomal degradation of PINK1. Similar to the dimerization of SIAH1/SIAH2, SIAH3 interacts with SIAH1, promoting its translocation to mitochondria and preventing its ubiquitin-ligase activity toward PINK1. Our results support the notion that the increase in SIAH3 and intra-mitochondrial aggregation of SIAH3-PINK1 may mediate α-synuclein pathology by promoting proteotoxicity and preventing the elimination of dysfunctional mitochondria. We consider it possible that PINK1 activity is decreased in sporadic PD, which impedes proper mitochondrial renewal in the disease.
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Doença de Parkinson , alfa-Sinucleína , Humanos , alfa-Sinucleína/metabolismo , Doença de Parkinson/metabolismo , Proteínas Quinases/metabolismo , Mitofagia , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , UbiquitinaRESUMO
The locus coeruleus (LC) which is the major noradrenergic nucleus in the brain develops under the influence of Bmps secreted by the roof plate and Fgf8 emitted from the mid-hindbrain organizer. We studied the development of the LC in different Bmp mouse mutants and report the absence of this nucleus in Bmp5(-/-);Bmp7(-/-) double knockouts. Notably, genes marking organizers and neuronal populations adjacent to the LC precursor field are unperturbed in Bmp5(-/-);Bmp7(-/-) animals. In addition, we found that in En1(+/Otx2) mutants in which the caudal Otx2 expression domain and thereby the mid-hindbrain organizer are shifted caudally, LC neurons are concomitantly reduced along with Bmp5/7. Complementing these results, Otx1(-/-);Otx2(+/-) mutants, in which the mid-hinbrain organizer is shifted rostrally, show a rostrally extended Bmp5 expression area and an increase in LC neurons. Taken together, our data indicate that LC development requires either Bmp5 or Bmp7, and one is able to compensate for the loss of the other. In addition, we conclude that the position of the mid-hindbrain organizer determines the size of the LC and propose that Bmp5/7 play an important role in mediating this organizer function.
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Proteína Morfogenética Óssea 5/metabolismo , Proteína Morfogenética Óssea 7/metabolismo , Locus Cerúleo/citologia , Locus Cerúleo/embriologia , Mesencéfalo/fisiologia , Norepinefrina/metabolismo , Rombencéfalo/fisiologia , Animais , Apoptose , Fator 8 de Crescimento de Fibroblasto/genética , Fator 8 de Crescimento de Fibroblasto/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Locus Cerúleo/metabolismo , Mesencéfalo/citologia , Camundongos , Camundongos Knockout , Neurônios/citologia , Neurônios/fisiologia , Fatores de Transcrição Otx/genética , Fatores de Transcrição Otx/metabolismo , Rombencéfalo/citologia , Células-Tronco/citologia , Células-Tronco/fisiologiaRESUMO
Oxidopamine (6-hydroxydopamine, 6-OHDA) is a toxin commonly used for the creation of experimental animal models of Parkinson's disease, attention-deficit hyperactivity disorder, and Lesch-Nyhan syndrome. Its exact mechanism of action is not completely understood, although there are many indications that it is related to the generation of reactive oxygen species (ROS), primarily in dopaminergic neurons. In certain experimental conditions, oxidopamine may also cause programmed cell death via various signaling pathways. Oxidopamine may also have a significant impact on chromatin structure and nuclear structural organization in some cells. Today, many researchers use oxidopamine-associated oxidative damage to evaluate different antioxidant-based pharmacologically active compounds as drug candidates for various neurological and non-neurological diseases. Additional research is needed to clarify the exact biochemical pathways associated with oxidopamine toxicity, related ROS generation and apoptosis. In this short review, we focus on the recent research in experimental physiology and pharmacology, related to the cellular and animal experimental models of oxidopamine - mediated toxicity.
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Estresse Oxidativo/efeitos dos fármacos , Oxidopamina/farmacologia , Animais , Humanos , Espécies Reativas de Oxigênio/metabolismoRESUMO
Release of Zn(2+) from presynaptic glutamatergic terminals has long been considered the principle challenge necessitating the existence of zinc homeostatic proteins (ZHP) in the mammalian nervous system. It is now known that neural cells also possess an intracellular zinc pool, termed here [Zn(2+)](i), which functions in a cell signaling context. A major challenge is characterizing the interaction of these two populations of zinc ions. To assess the relationship of this Zn(2+) pool to cellular ZHP production, we employed immunofluorescence and immunoblot analysis to compare the expression of ZHP's ZnT-1 and MT-I/II in olfactory bulb and hippocampus of wild-type and ZnT-3 KO mice, which lack synaptic Zn(2+). In both areas, the respective distribution and concentration of ZnT-1 and MT-I/II were identical in ZnT-3 KO and control animals. We subsequently examined ZHP content in ZnT-3 KO and WT mice treated with a membrane-permeable Zn(2+) chelator. In both olfactory bulb and hippocampus of the KO mice, the ZHP content was significantly reduced 15 h after chelation of [Zn(2+)](i) compared to WT controls. Our findings support the conclusion that ZHP expression is regulated by crosstalk between synaptic and intracellular pools of Zn(2+).
Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Sistema Nervoso Central/metabolismo , Proteínas de Membrana/metabolismo , Zinco/metabolismo , Animais , Proteínas de Transporte/genética , Proteínas de Transporte de Cátions/genética , Sistema Nervoso Central/anatomia & histologia , Quelantes/metabolismo , Imunofluorescência , Homeostase , Proteínas de Membrana/genética , Proteínas de Membrana Transportadoras , Metalotioneína/genética , Metalotioneína/metabolismo , Camundongos , Camundongos Knockout , Transdução de Sinais/fisiologia , Sinapses/metabolismoRESUMO
The levels and activity of the enzyme paraoxonase 1 affect the vulnerability to the teratogenic effects of organophosphate pesticides. Mutant mice lacking the gene for paraoxonase1 (PON1-/-) are more susceptible to the toxic effects of chlorpyrifos, and were hypothesized to be more vulnerable to social behavior deficits induced by exposure to chlorpyrifos during gestation. Three experiments were performed comparing PON1-/- mice to PON1+/+ mice born to dams treated with 0.5 mg/kg chlorpyrifos or cornoil vehicle on gestational days 12-15. Chlofpyrifos-exposed male PON1-/- mouse pups had delayed development of reflexes in in the first experiment. In the second experiment, adult male and female PON1-/- mice and the female PON1+/+ mice all displayed lower social preference than the male vehicle-treated PON1+/+ mice. The PON1-/- mice and the female PON1+/+ mice displayed lower social preference compared to the PON1+/+ male mice. Male adult mice that had been exposed in utero to chlorpyrifos showed less conditioned social preference regardless of genotype. In the third study, the delayed reflex development was replicated in male and female PON1-/- mice, but chlorpyrifos did not augment this effect. Nest Odor Preference, a test of early social attachment to dam and siblings, was lower in PON1-/- mouse pups compared to PON1+/+ pups. This study shows for the first time that PON1-/- mice have a behavioral phenotype that indicates impaired reflex development and social behavior. Chlorpyrifos exposure during gestation tended to augment some of these effects.
Assuntos
Arildialquilfosfatase/genética , Clorpirifos/toxicidade , Inibidores da Colinesterase/toxicidade , Deficiências do Desenvolvimento/genética , Efeitos Tardios da Exposição Pré-Natal/genética , Comportamento Social , Teratogênicos/toxicidade , Animais , Arildialquilfosfatase/deficiência , Feminino , Masculino , Camundongos , Gravidez , Efeitos Tardios da Exposição Pré-Natal/etiologia , ReflexoRESUMO
The mesodiencephalic dopaminergic (mdDA) neurons, including the nigrostriatal subset that preferentially degenerates in Parkinson's Disease (PD), strongly depend on an accurately balanced Wingless-type MMTV integration site family member 1 (WNT1)/beta-catenin signaling pathway during their development. Loss of this pathway abolishes the generation of these neurons, whereas excessive WNT1/b-catenin signaling prevents their correct differentiation. The identity of the cells responding to this pathway in the developing mammalian ventral midbrain (VM) as well as the precise progression of WNT/b-catenin action in these cells are still unknown. We show that strong WNT/b-catenin signaling inhibits the differentiation of WNT/b-catenin-responding mdDA progenitors into PITX3+ and TH+ mdDA neurons by repressing the Pitx3 gene in mice. This effect is mediated by RSPO2, a WNT/b-catenin agonist, and lymphoid enhancer binding factor 1 (LEF1), an essential nuclear effector of the WNT/b-catenin pathway, via conserved LEF1/T-cell factor binding sites in the Pitx3 promoter. LEF1 expression is restricted to a caudolateral mdDA progenitor subset that preferentially responds to WNT/b-catenin signaling and gives rise to a fraction of all mdDA neurons. Our data indicate that an attenuation of WNT/b-catenin signaling in mdDA progenitors is essential for their correct differentiation into specific mdDA neuron subsets. This is an important consideration for stem cell-based regenerative therapies and in vitro models of neuropsychiatric diseases.
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Dopamine-synthesizing neurons located in the mammalian ventral midbrain are at the center stage of biomedical research due to their involvement in severe human neuropsychiatric and neurodegenerative disorders, most prominently Parkinson's Disease (PD). The induction of midbrain dopaminergic (mDA) neurons depends on two important signaling centers of the mammalian embryo: the ventral midline or floor plate (FP) of the neural tube, and the isthmic organizer (IsO) at the mid-/hindbrain boundary (MHB). Cells located within and close to the FP secrete sonic hedgehog (SHH), and members of the wingless-type MMTV integration site family (WNT1/5A), as well as bone morphogenetic protein (BMP) family. The IsO cells secrete WNT1 and the fibroblast growth factor 8 (FGF8). Accordingly, the FGF8, SHH, WNT, and BMP signaling pathways play crucial roles during the development of the mDA neurons in the mammalian embryo. Moreover, these morphogens are essential for the generation of stem cell-derived mDA neurons, which are critical for the modeling, drug screening, and cell replacement therapy of PD. This review summarizes our current knowledge about the functions and crosstalk of these signaling pathways in mammalian mDA neuron development in vivo and their applications in stem cell-based paradigms for the efficient derivation of these neurons in vitro.
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[This corrects the article on p. 372 in vol. 10, PMID: 29170629.].
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Hydrocephalus can occur in children alone or in combination with other neurodevelopmental disorders that are often associated with brain overgrowth. Despite the severity of these disorders, the molecular and cellular mechanisms underlying these pathologies and their comorbidity are poorly understood. Here, we studied the consequences of genetically inactivating in mice dual-specificity phosphatase 16 (Dusp16), which is known to negatively regulate mitogen-activated protein kinases (MAPKs) and which has never previously been implicated in brain development and disorders. Mouse mutants lacking a functional Dusp16 gene (Dusp16-/-) developed fully-penetrant congenital obstructive hydrocephalus together with brain overgrowth. The midbrain aqueduct in Dusp16-/- mutants was obstructed during mid-gestation by an expansion of neural progenitors, and during later gestational stages by neurons resulting in a blockage of cerebrospinal fluid (CSF) outflow. In contrast, the roof plate and ependymal cells developed normally. We identified a delayed cell cycle exit of neural progenitors in Dusp16-/- mutants as a cause of progenitor overproliferation during mid-gestation. At later gestational stages, this expanded neural progenitor pool generated an increased number of neurons associated with enlarged brain volume. Taken together, we found that Dusp16 plays a critical role in neurogenesis by balancing neural progenitor cell proliferation and neural differentiation. Moreover our results suggest that a lack of functional Dusp16 could play a central role in the molecular mechanisms linking brain overgrowth and hydrocephalus.
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Midbrain dopaminergic and hindbrain serotonergic neurons play an important role in the modulation of behavior and are involved in a series of neuropsychiatric disorders. Despite the importance of these cells, little is known about the molecular mechanisms governing their development. During embryogenesis, midbrain dopaminergic neurons are specified rostral to the midbrain-hindbrain organizer (MHO), and hindbrain serotonergic neurons are specified caudal to it. We report that in transgenic mice in which Otx2 and accordingly the MHO are shifted caudally, the midbrain dopaminergic neuronal population expands to the ectopically positioned MHO and is enlarged. Complementary, the extension of the hindbrain serotonergic cell group is decreased. These changes are preserved in adulthood, and the additional, ectopic dopaminergic neurons project to the striatum, which is a proper dopaminergic target area. In addition, in mutants in which Otx2 and the MHO are shifted rostrally, dopaminergic and serotonergic neurons are relocated at the newly positioned MHO. However, in these mice, the size ratio between these two cell populations is changed in favor of the serotonergic cell population. To investigate whether the position of the MHO during embryogenesis is also of functional relevance for adult behavior, we tested mice with a caudally shifted MHO and report that these mutants show a higher locomotor activity. Together, we provide evidence that the position of the MHO determines the location and size of midbrain dopaminergic and hindbrain serotonergic cell populations in vivo. In addition, our data suggest that the position of the MHO during embryogenesis can modulate adult locomotor activity.
Assuntos
Dopamina , Mesencéfalo/anatomia & histologia , Mesencéfalo/fisiologia , Neurônios/citologia , Neurônios/fisiologia , Organizadores Embrionários/anatomia & histologia , Organizadores Embrionários/fisiologia , Rombencéfalo/anatomia & histologia , Rombencéfalo/fisiologia , Serotonina , Animais , Comportamento Animal/fisiologia , Mapeamento Encefálico/métodos , Dopamina/fisiologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento/genética , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/fisiologia , Masculino , Mesencéfalo/citologia , Camundongos , Camundongos Congênicos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Camundongos Endogâmicos , Camundongos Transgênicos , Proteínas do Tecido Nervoso/deficiência , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/fisiologia , Organizadores Embrionários/citologia , Fatores de Transcrição Otx , Rombencéfalo/citologia , Serotonina/fisiologia , Transativadores/deficiência , Transativadores/genética , Transativadores/fisiologiaRESUMO
Subtle mood fluctuations are normal emotional experiences, whereas drastic mood swings can be a manifestation of bipolar disorder (BPD). Despite their importance for normal and pathological behavior, the mechanisms underlying endogenous mood instability are largely unknown. During embryogenesis, the transcription factor Otx2 orchestrates the genetic networks directing the specification of dopaminergic (DA) and serotonergic (5-HT) neurons. Here we behaviorally phenotyped mouse mutants overexpressing Otx2 in the hindbrain, resulting in an increased number of DA neurons and a decreased number of 5-HT neurons in both developing and mature animals. Over the course of 1 month, control animals exhibited stable locomotor activity in their home cages, whereas mutants showed extended periods of elevated or decreased activity relative to their individual average. Additional behavioral paradigms, testing for manic- and depressive-like behavior, demonstrated that mutants showed an increase in intra-individual fluctuations in locomotor activity, habituation, risk-taking behavioral parameters, social interaction, and hedonic-like behavior. Olanzapine, lithium, and carbamazepine ameliorated the behavioral alterations of the mutants, as did the mixed serotonin receptor agonist quipazine and the specific 5-HT2C receptor agonist CP-809101. Testing the relevance of the genetic networks specifying monoaminergic neurons for BPD in humans, we applied an interval-based enrichment analysis tool for genome-wide association studies. We observed that the genes specifying DA and 5-HT neurons exhibit a significant level of aggregated association with BPD but not with schizophrenia or major depressive disorder. The results of our translational study suggest that aberrant development of monoaminergic neurons leads to mood fluctuations and may be associated with BPD.
Assuntos
Monoaminas Biogênicas/metabolismo , Transtorno Bipolar/etiologia , Transtorno Bipolar/patologia , Deficiências do Desenvolvimento/complicações , Transtornos do Humor/etiologia , Neurônios/patologia , Animais , Antidepressivos/uso terapêutico , Transtorno Bipolar/tratamento farmacológico , Adaptação à Escuridão/efeitos dos fármacos , Adaptação à Escuridão/genética , Deficiências do Desenvolvimento/genética , Deficiências do Desenvolvimento/patologia , Modelos Animais de Doenças , Comportamento Exploratório/efeitos dos fármacos , Proteínas de Homeodomínio/genética , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Camundongos , Camundongos Transgênicos , Transtornos do Humor/tratamento farmacológico , Atividade Motora/efeitos dos fármacos , Mutação/genética , Neurônios/metabolismo , Fatores de Transcrição Otx/metabolismo , Serotoninérgicos/uso terapêuticoRESUMO
Studying the development of mesodiencephalic dopaminergic (mdDA) neurons provides an important basis for better understanding dopamine-associated brain functions and disorders and is critical for establishing cell replacement therapy for Parkinson's disease. The transcription factors Otx2 and Lmx1b play a key role in the development of mdDA neurons. However, little is known about the genes downstream of Otx2 and Lmx1b in the pathways controlling the formation of mdDA neurons in vivo. Here we report on our investigation of Lmx1b as downstream target of Otx2 in the formation of mdDA neurons. Mouse mutants expressing Otx2 under the control of the En1 promoter (En1+/Otx2) showed increased Otx2 expression in the mid-hindbrain region, resulting in upregulation of Lmx1b and expansion of mdDA neurons there. In contrast, Lmx1b-/- mice showed decreased expression of Otx2 and impairments in several aspects of mdDA neuronal formation. To study the functional interaction between Otx2 and Lmx1b, we generated compound mutants in which Otx2 expression was restored in mice lacking Lmx1b (En1+/Otx2;Lmx1b-/-). In these animals Otx2 was not sufficient to rescue any of the aberrations in the formation of mdDA neurons caused by the loss of Lmx1b, but rescued the loss of ocular motor neurons. Gene expression studies in Lmx1b-/- embryos indicated that in these mutants Wnt1, En1 and Fgf8 expression are induced but subsequently lost in the mdDA precursor domain and the mid-hindbrain organizer in a specific, spatio-temporal manner. In summary, we demonstrate that Otx2 critically depends on Lmx1b for the formation of mdDA neurons, but not for the generation of ocular motor neurons. Moreover, our data suggest that Lmx1b precisely maintains the expression pattern of Wnt1, Fgf8 and En1, which are essential for mid-hindbrain organizer function and the formation of mdDA neurons.
Assuntos
Neurônios Dopaminérgicos/fisiologia , Proteínas com Homeodomínio LIM/genética , Mesencéfalo/fisiologia , Fatores de Transcrição Otx/genética , Fatores de Transcrição/genética , Animais , Padronização Corporal/genética , Diferenciação Celular/genética , Dopamina/genética , Embrião de Mamíferos/fisiologia , Feminino , Fator 8 de Crescimento de Fibroblasto/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Proteínas de Homeodomínio/genética , Camundongos , Proteínas do Tecido Nervoso/genética , Rombencéfalo/fisiologia , Proteína Wnt1/genéticaRESUMO
During late developmental phases individual sympathetic neurons undergo a switch from noradrenergic to cholinergic neurotransmission. This phenomenon of plasticity depends on target-derived signals in vivo and is triggered by neurotrophic factors in neuronal cultures. To analyze genome-wide expression differences between the two transmitter phenotypes we employed DNA microarrays. RNA expression profiles were obtained from chick paravertebral sympathetic ganglia, treated with neurotrophin 3, glial cell line-derived neurotrophic factor or ciliary neurotrophic factor, all of which stimulate cholinergic differentiation. Results were compared with the effect of nerve growth factor, which functions as a pro-noradrenergic stimulus. The gene set common to all three comparisons defined the noradrenergic and cholinergic synexpression groups. Several functional categories, such as signal transduction, G-protein-coupled signaling, cation transport, neurogenesis and synaptic transmission, were enriched in these groups. Experiments based on the prediction that some of the identified genes play a role in the neurotransmitter switch identified bone morphogenetic protein signaling as an inhibitor of cholinergic differentiation.
Assuntos
Gânglios Simpáticos/citologia , Gânglios Simpáticos/embriologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Neurônios/fisiologia , Neurotransmissores/metabolismo , Fenótipo , Animais , Embrião de Galinha , Perfilação da Expressão Gênica/métodos , Hibridização In Situ/métodos , Neurotransmissores/genética , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Técnicas de Cultura de Órgãos , RNA Mensageiro/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodosRESUMO
Midbrain neurons synthesizing the neurotransmitter dopamine play a central role in the modulation of different brain functions and are associated with major neurological and psychiatric disorders. Despite the importance of these cells, the molecular mechanisms controlling their development are still poorly understood. The secreted glycoprotein Wnt1 is expressed in close vicinity to developing midbrain dopaminergic neurons. Here, we show that Wnt1 regulates the genetic network, including Otx2 and Nkx2-2, that is required for the establishment of the midbrain dopaminergic progenitor domain during embryonic development. In addition, Wnt1 is required for the terminal differentiation of midbrain dopaminergic neurons at later stages of embryogenesis. These results identify Wnt1 as a key molecule in the development of midbrain dopaminergic neurons in vivo. They also suggest the Wnt1-controlled signaling pathway as a promising target for new therapeutic strategies in the treatment of Parkinson's disease.