RESUMO
KEY POINTS: The embryonic PHOX2B-progenitor domain generates neuronal and glial cells which together are involved in chemosensory control of breathing and sleep homeostasis. Ablating PHOX2B-derived astrocytes significantly contributes to secondary hypoxic respiratory depression as well as abnormalities in sleep homeostasis. PHOX2B-derived astrocyte ablation results in axonal pathologies in the retrotrapezoid nucleus. ABSTRACT: We identify in mice a population of â¼800 retrotrapezoid nucleus (RTN) astrocytes derived from PHOX2B-positive, OLIG3-negative progenitor cells, that interact with PHOX2B-expressing RTN chemosensory neurons. PHOX2B-derived astrocyte ablation during early life results in adult-onset O2 chemoreflex deficiency. These animals also display changes in sleep homeostasis, including fragmented sleep and disturbances in delta power after sleep deprivation, all without observable changes in anxiety or social behaviours. Ultrastructural evaluation of the RTN demonstrates that PHOX2B-derived astrocyte ablation results in features characteristic of degenerative neuro-axonal dystrophy, including abnormally dilated axon terminals and increased amounts of synapses containing autophagic vacuoles/phagosomes. We conclude that PHOX2B-derived astrocytes are necessary for maintaining a functional O2 chemosensory reflex in the adult, modulate sleep homeostasis, and are key regulators of synaptic integrity in the RTN region, which is necessary for the chemosensory control of breathing. These data also highlight how defects in embryonic development may manifest as neurodegenerative pathology in an adult.
Assuntos
Astrócitos/fisiologia , Proteínas de Homeodomínio/fisiologia , Respiração , Sono/fisiologia , Fatores de Transcrição/fisiologia , Animais , Diferenciação Celular , Células-Tronco Embrionárias/citologia , Homeostase , Camundongos Transgênicos , Neurônios/fisiologiaRESUMO
Safety concerns and/or the stochastic nature of current transduction approaches have hampered nuclear reprogramming's clinical translation. We report a novel non-viral nanotechnology-based platform permitting deterministic large-scale transfection with single-cell resolution. The superior capabilities of our technology are demonstrated by modification of the well-established direct neuronal reprogramming paradigm using overexpression of the transcription factors Brn2, Ascl1, and Myt1l (BAM). Reprogramming efficiencies were comparable to viral methodologies (up to ~9-12%) without the constraints of capsid size and with the ability to control plasmid dosage, in addition to showing superior performance relative to existing non-viral methods. Furthermore, increased neuronal complexity could be tailored by varying BAM ratio and by including additional proneural genes to the BAM cocktail. Furthermore, high-throughput NEP allowed easy interrogation of the reprogramming process. We discovered that BAM-mediated reprogramming is regulated by AsclI dosage, the S-phase cyclin CCNA2, and that some induced neurons passed through a nestin-positive cell stage. FROM THE CLINICAL EDITOR: In the field of regenerative medicine, the ability to direct cell fate by nuclear reprogramming is an important facet in terms of clinical application. In this article, the authors described their novel technique of cell reprogramming through overexpression of the transcription factors Brn2, Ascl1, and Myt1l (BAM) by in situ electroporation through nanochannels. This new technique could provide a platform for further future designs.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Reprogramação Celular , Proteínas de Ligação a DNA/genética , DNA/administração & dosagem , Proteínas do Tecido Nervoso/genética , Neurônios/citologia , Fatores do Domínio POU/genética , Fatores de Transcrição/genética , Transfecção/métodos , Animais , Linhagem Celular , DNA/genética , Eletroporação/métodos , Fibroblastos/citologia , Fibroblastos/metabolismo , Humanos , Camundongos , Neurônios/metabolismo , Plasmídeos/administração & dosagem , Plasmídeos/genética , Regulação para CimaRESUMO
The mammalian genome encodes two A-type cyclins, which are considered potentially redundant yet essential regulators of the cell cycle. Here, we tested requirements for cyclin A1 and cyclin A2 function in cerebellar development. Compound conditional loss of cyclin A1/A2 in neural progenitors resulted in severe cerebellar hypoplasia, decreased proliferation of cerebellar granule neuron progenitors (CGNP), and Purkinje (PC) neuron dyslamination. Deletion of cyclin A2 alone showed an identical phenotype, demonstrating that cyclin A1 does not compensate for cyclin A2 loss in neural progenitors. Cyclin A2 loss lead to increased apoptosis at early embryonic time points but not at post-natal time points. In contrast, neural progenitors of the VZ/SVZ did not undergo increased apoptosis, indicating that VZ/SVZ-derived and rhombic lip-derived progenitor cells show differential requirements to cyclin A2. Conditional knockout of cyclin A2 or the SHH proliferative target Nmyc in CGNP also resulted in PC neuron dyslamination. Although cyclin E1 has been reported to compensate for cyclin A2 function in fibroblasts and is upregulated in cyclin A2 null cerebella, cyclin E1 expression was unable to compensate for loss-of cyclin A2 function.
Assuntos
Córtex Cerebral/embriologia , Ciclina A2/fisiologia , Animais , Proliferação de Células , Córtex Cerebral/citologia , Córtex Cerebral/metabolismo , Ciclina A2/genética , Ciclina A2/metabolismo , Hibridização In Situ , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Células-Tronco Neurais/metabolismoRESUMO
The postnatal period in mammals represents a developmental epoch of significant change in the autonomic nervous system (ANS). This study focuses on postnatal development of the area postrema, a crucial ANS structure that regulates temperature, breathing, and satiety, among other activities. We find that the human area postrema undergoes significant developmental changes during postnatal development. To characterize these changes further, we used transgenic mouse reagents to delineate neuronal circuitry. We discovered that, although a well-formed ANS scaffold exists early in embryonic development, the area postrema shows a delayed maturation. Specifically, postnatal days 0-7 in mice show no significant change in area postrema volume or synaptic input from PHOX2B-derived neurons. In contrast, postnatal days 7-20 show a significant increase in volume and synaptic input from PHOX2B-derived neurons. We conclude that key ANS structures show unexpected dynamic developmental changes during postnatal development. These data provide a basis for understanding ANS dysfunction and disease predisposition in premature and postnatal humans.
Assuntos
Área Postrema/crescimento & desenvolvimento , Rede Nervosa/crescimento & desenvolvimento , Animais , Animais Recém-Nascidos , Área Postrema/química , Feminino , Humanos , Recém-Nascido , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Rede Nervosa/química , Especificidade da EspécieRESUMO
We sought to determine the contribution of scaffold topography to the migration and morphology of neural stem cells by mimicking anatomical features of scaffolds found in vivo. We mimicked two types of central nervous system scaffolds encountered by neural stem cells during development in vitro by constructing different diameter electrospun polycaprolactone (PCL) fiber mats, a substrate that we have shown to be topographically similar to brain scaffolds. We compared the effects of large fibers (made to mimic blood vessel topography) with those of small-diameter fibers (made to mimic radial glial process topography) on the migration and differentiation of neural stem cells. Neural stem cells showed differential migratory and morphological reactions with laminin in different topographical contexts. We demonstrate, for the first time, that neural stem cell biological responses to laminin are dependent on topographical context. Large-fiber topography without laminin prevented cell migration, which was partially reversed by treatment with rock inhibitor. Cell morphology complexity assayed by fractal dimension was inhibited in nocodazole- and cytochalasin-D-treated neural precursor cells in large-fiber topography, but was not changed in small-fiber topography with these inhibitors. These data indicate that cell morphology has different requirements on cytoskeletal proteins dependent on the topographical environment encountered by the cell. We propose that the physical structure of distinct scaffolds induces unique signaling cascades that regulate migration and morphology in embryonic neural precursor cells. J. Comp. Neurol. 524:3485-3502, 2016. © 2016 Wiley Periodicals, Inc.
Assuntos
Movimento Celular/fisiologia , Células-Tronco Neurais/fisiologia , Neurogênese/fisiologia , Neurônios/fisiologia , Actinas/antagonistas & inibidores , Actinas/metabolismo , Animais , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/metabolismo , Feminino , Humanos , Laminina/metabolismo , Masculino , Camundongos , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Modelos Neurológicos , Células-Tronco Neurais/citologia , Células-Tronco Neurais/efeitos dos fármacos , Neurogênese/efeitos dos fármacos , Neurônios/citologia , Neurônios/efeitos dos fármacos , Poliésteres , Propriedades de Superfície , Alicerces Teciduais , Quinases Associadas a rho/antagonistas & inibidores , Quinases Associadas a rho/metabolismoRESUMO
Various stem cell niches of the brain have differential requirements for Cyclin A2. Cyclin A2 loss results in marked cerebellar dysmorphia, whereas forebrain growth is retarded during early embryonic development yet achieves normal size at birth. To understand the differential requirements of distinct brain regions for Cyclin A2, we utilized neuroanatomical, transgenic mouse, and mathematical modeling techniques to generate testable hypotheses that provide insight into how Cyclin A2 loss results in compensatory forebrain growth during late embryonic development. Using unbiased measurements of the forebrain stem cell niche, we parameterized a mathematical model whereby logistic growth instructs progenitor cells as to the cell-types of their progeny. Our data was consistent with prior findings that progenitors proliferate along an auto-inhibitory growth curve. The growth retardation inCCNA2-null brains corresponded to cell cycle lengthening, imposing a developmental delay. We hypothesized that Cyclin A2 regulates DNA repair and that CCNA2-null progenitors thus experienced lengthened cell cycle. We demonstrate that CCNA2-null progenitors suffer abnormal DNA repair, and implicate Cyclin A2 in double-strand break repair. Cyclin A2's DNA repair functions are conserved among cell lines, neural progenitors, and hippocampal neurons. We further demonstrate that neuronal CCNA2 ablation results in learning and memory deficits in aged mice.
Assuntos
Envelhecimento/fisiologia , Encéfalo/metabolismo , Ciclo Celular/genética , Ciclina A2/metabolismo , Neurônios/metabolismo , Animais , Comportamento Animal/fisiologia , Condicionamento Psicológico/fisiologia , Ciclina A2/genética , Reparo do DNA , Força da Mão/fisiologia , Camundongos , Camundongos Transgênicos , Modelos Biológicos , Destreza Motora/fisiologia , Comportamento Social , Nicho de Células-TroncoRESUMO
Late embryonic and postnatal cerebellar folial surface area expansion promotes cerebellar cortical cytoarchitectural lamination. We developed a streamlined sampling scheme to generate unbiased estimates of murine cerebellar surface area and volume using stereologic principles. We demonstrate that, during the proliferative phase of the external granular layer (EGL) and folial surface area expansion, EGL thickness does not change and thus is a topological proxy for progenitor self-renewal. The topological constraints indicate that, during proliferative phases, migration out of the EGL is balanced by self-renewal. Progenitor self-renewal must, therefore, include mitotic events yielding 2 cells in the same layer to increase surface area (ß events) and mitotic events yielding 2 cells, with 1 cell in a superficial layer and 1 cell in a deeper layer (α events). As the cerebellum grows, therefore, ß events lie upstream of α events. Using a mathematical model constrained by the measurements of volume and surface area, we could quantify intermitotic times for ß events on a per-cell basis in postnatal mouse cerebellum. Furthermore, we found that loss of CCNA2, which decreases EGL proliferation and secondarily induces cerebellar cortical dyslamination, shows preserved α-type events. Thus, CCNA2-null cerebellar granule progenitor cells are capable of self-renewal of the EGL stem cell niche; this is concordant with prior findings of extensive apoptosis in CCNA2-null mice. Similar methodologies may provide another layer of depth to the interpretation of results from stereologic studies.