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1.
Sci Adv ; 8(13): eabl9156, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-35363520

RESUMO

Recent studies using single-cell RNA-sequencing have revealed cellular heterogeneity in the developing mammalian cerebellum, yet the regulatory logic underlying this cellular diversity remains to be elucidated. Using integrated single-cell RNA and ATAC analyses, we resolved developmental trajectories of cerebellar progenitors and identified putative trans- and cis-elements that control cell state transition. We reverse engineered gene regulatory networks (GRNs) of each cerebellar cell type. Through in silico simulations and in vivo experiments, we validated the efficacy of GRN analyses and uncovered the molecular control of a posterior transitory zone (PTZ), a distinct progenitor zone residing immediately anterior to the morphologically defined rhombic lip (RL). We showed that perturbing cell fate specification in the PTZ and RL causes posterior cerebellar vermis hypoplasia, the most common cerebellar birth defect in humans. Our study provides a foundation for comprehensive studies of developmental programs of the mammalian cerebellum.


Assuntos
Malformações do Sistema Nervoso , Transcriptoma , Animais , Diferenciação Celular/genética , Cerebelo/metabolismo , Epigênese Genética , Mamíferos/genética , Camundongos , Malformações do Sistema Nervoso/genética , Malformações do Sistema Nervoso/metabolismo
2.
J Gastroenterol Hepatol ; 35(12): 2241-2247, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32386240

RESUMO

BACKGROUND AND AIM: Mitochondrial damage is commonly involved in liver injury. We have previously shown that normal mitochondria can be coated with a carrier protein to form complexes that are specifically taken up by liver cells in culture. The aim of the current study was to determine whether mitochondrial complexes could be specifically delivered to the livers of living rats by intravenous injection. METHODS: Mitochondria were harvested from fresh mouse liver, mixed with an asialoglycoprotein-based carrier, asialoorosomucoid-polylysine (AsOR-PL), and purified to form complexes. To facilitate the release of internalized mitochondria from endosomes, an endosomolytic peptide, listeriolysin O (LLO), was coupled to AsOR to form AsOR-LLO. Mitochondria alone, mitochondrial complexes with AsOR-PL, and mitochondrial complexes plus AsOR-LLO conjugate all containing the same number of mitochondria were injected intravenously. Animals were killed, and organs were removed and analyzed by quantitative polymerase chain reaction of mouse mitochondrial DNA, electron microscopy (EM), and in situ polymerase chain reaction and hybridization followed by immunohistochemical analyses. RESULTS: Calculations revealed that approximately 27% of the total injected mitochondria was detected in the liver, while less than 2% was found in spleen, and < 1% in lungs. Immunohistochemistry showed that mouse mitochondrial DNA staining was minimal with mitochondrial complexes alone, strong periportal with mitochondrial complexes co-injected with AsOR-LLO, and absent with mitochondria alone. CONCLUSIONS: Targetable mitochondrial complexes can be delivered to rat liver, and the efficiency of that process is greatly enhanced by co-injection of a targetable endosomal release agent, AsOR-LLO.


Assuntos
Assialoglicoproteínas/administração & dosagem , Toxinas Bacterianas/administração & dosagem , Transplante de Células/métodos , Proteínas de Choque Térmico/administração & dosagem , Proteínas Hemolisinas/administração & dosagem , Fígado , Mitocôndrias Hepáticas/transplante , Orosomucoide/análogos & derivados , Polilisina/administração & dosagem , Animais , Proteínas de Transporte , Endossomos , Feminino , Hepatócitos/citologia , Injeções Intravenosas , Camundongos Endogâmicos , Orosomucoide/administração & dosagem , Ratos Sprague-Dawley
3.
Neural Dev ; 13(1): 3, 2018 03 08.
Artigo em Inglês | MEDLINE | ID: mdl-29519242

RESUMO

BACKGROUND: Most oligodendrocytes of the spinal cord originate from ventral progenitor cells of the pMN domain, characterized by expression of the transcription factor Olig2. A minority of oligodendrocytes is also recognized to emerge from dorsal progenitors during fetal development. The prevailing view is that generation of ventral oligodendrocytes depends on Sonic hedgehog (Shh) while dorsal oligodendrocytes develop under the influence of Fibroblast Growth Factors (FGFs). RESULTS: Using the well-established model of the chicken embryo, we show that ventral spinal progenitor cells activate FGF signaling at the onset of oligodendrocyte precursor cell (OPC) generation. Inhibition of FGF receptors at that time appears sufficient to prevent generation of ventral OPCs, highlighting that, in addition to Shh, FGF signaling is required also for generation of ventral OPCs. We further reveal an unsuspected interplay between Shh and FGF signaling by showing that FGFs serve dual essential functions in ventral OPC specification. FGFs are responsible for timely induction of a secondary Shh signaling center, the lateral floor plate, a crucial step to create the burst of Shh required for OPC specification. At the same time, FGFs prevent down-regulation of Olig2 in pMN progenitor cells as these cells receive higher threshold of the Shh signal. Finally, we bring arguments favoring a key role of newly differentiated neurons acting as providers of the FGF signal required to trigger OPC generation in the ventral spinal cord. CONCLUSION: Altogether our data reveal that the FGF signaling pathway is activated and required for OPC commitment in the ventral spinal cord. More generally, our data may prove important in defining strategies to produce large populations of determined oligodendrocyte precursor cells from undetermined neural progenitors, including stem cells. In the long run, these new data could be useful in attempts to stimulate the oligodendrocyte fate in residing neural stem cells.


Assuntos
Fatores de Crescimento de Fibroblastos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Proteínas Hedgehog/metabolismo , Oligodendroglia/metabolismo , Transdução de Sinais/fisiologia , Medula Espinal/citologia , Animais , Embrião de Galinha , Eletroporação , Fatores de Crescimento de Fibroblastos/genética , Fatores de Crescimento de Fibroblastos/farmacologia , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Proteínas Hedgehog/genética , Técnicas In Vitro , Proteínas do Tecido Nervoso , Fator de Transcrição 2 de Oligodendrócitos/metabolismo , Técnicas de Cultura de Órgãos , Medula Espinal/embriologia , Células-Tronco/fisiologia
4.
Development ; 141(6): 1392-403, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24595292

RESUMO

In the ventral spinal cord, generation of neuronal and glial cell subtypes is controlled by Sonic hedgehog (Shh). This morphogen contributes to cell diversity by regulating spatial and temporal sequences of gene expression during development. Here, we report that establishing Shh source cells is not sufficient to induce the high-threshold response required to specify sequential generation of ventral interneurons and oligodendroglial cells at the right time and place in zebrafish. Instead, we show that Shh-producing cells must repeatedly upregulate the secreted enzyme Sulfatase1 (Sulf1) at two critical time points of development to reach their full inductive capacity. We provide evidence that Sulf1 triggers Shh signaling activity to establish and, later on, modify the spatial arrangement of gene expression in ventral neural progenitors. We further present arguments in favor of Sulf1 controlling Shh temporal activity by stimulating production of active forms of Shh from its source. Our work, by pointing out the key role of Sulf1 in regulating Shh-dependent neural cell diversity, highlights a novel level of regulation, which involves temporal evolution of Shh source properties.


Assuntos
Proteínas Hedgehog/metabolismo , Medula Espinal/embriologia , Medula Espinal/metabolismo , Sulfatases/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/metabolismo , Animais , Animais Geneticamente Modificados , Padronização Corporal/genética , Padronização Corporal/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Regulação Enzimológica da Expressão Gênica , Técnicas de Silenciamento de Genes , Proteínas Hedgehog/deficiência , Proteínas Hedgehog/genética , Camundongos , Células-Tronco Neurais/classificação , Células-Tronco Neurais/metabolismo , Neurogênese/genética , Neurogênese/fisiologia , Transdução de Sinais , Medula Espinal/citologia , Sulfatases/genética , Sulfotransferases/genética , Sulfotransferases/metabolismo , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/deficiência , Proteínas de Peixe-Zebra/genética
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