RESUMO
The prevalence of obesity is increasing in older adults and contributes to age-related decline. Caloric restriction (CR) alleviates obesity phenotypes and delays the onset of age-related changes. However, how late in life organisms benefit from switching from a high-(H) to a low-calorie (L) diet is unclear. We transferred male flies from a H to a L (HL) diet or vice versa (LH) at different times during life. Both shifts immediately change fly rate of aging even when applied late in life. HL shift rapidly reduces fly mortality rate to briefly lower rate than in flies on a constant L diet, and extends lifespan. Transcriptomic analysis uncovers that flies aged on H diet have acquired increased stress response, which may have temporal advantage over flies aged on L diet and leads to rapid decrease in mortality rate after HL switch. Conversely, a LH shift increases mortality rate, which is temporarily higher than in flies aged on a H diet, and shortens lifespan. Unexpectedly, more abundant transcriptomic changes accompanied LH shift, including increase in ribosome biogenesis, stress response and growth. These changes reflect protection from sudden release of ROS, energy storage, and use of energy to growth, which all likely contribute to higher mortality rate. As the beneficial effects of CR on physiology and lifespan are conserved across many organisms, our study provides framework to study underlying mechanisms of CR interventions that counteract the detrimental effects of H diets and reduce rate of aging even when initiated later in life.
Assuntos
Ingestão de Energia , Longevidade , Animais , Masculino , Longevidade/fisiologia , Envelhecimento/fisiologia , Restrição Calórica , Drosophila melanogaster/fisiologia , ObesidadeRESUMO
The embryonic diencephalon forms integration centers and relay stations in the forebrain. Anecdotal expression studies suggest that the diencephalon contains multiple developmental compartments and subdivisions. Here, we utilized single cell RNA sequencing to profile transcriptomes of dissociated cells from the diencephalon of E12.5 mouse embryos. We identified the divergence of different progenitors, intermediate progenitors, and emerging neurons. By mapping the identified cell groups to their spatial origins, we characterized the molecular features of cell types and cell states arising from various diencephalic domains. Furthermore, we reconstructed the developmental trajectory of distinct cell lineages, and thereby identified the genetic cascades and gene regulatory networks underlying the progression of the cell cycle, neurogenesis and cellular diversification. The analysis provides new insights into the molecular mechanisms underlying the amplification of intermediate progenitor cells in the thalamus. The single cell-resolved trajectories not only confirm a close relationship between the rostral thalamus and prethalamus, but also uncover an unexpected close relationship between the caudal thalamus, epithalamus and rostral pretectum. Our data provide a useful resource for systematic studies of cell heterogeneity and differentiation kinetics within the diencephalon.
Assuntos
Epitálamo/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Neurônios/citologia , Área Pré-Tectal/embriologia , Análise de Célula Única/métodos , Tálamo/embriologia , Animais , Padronização Corporal , Diferenciação Celular , Linhagem da Célula , Perfilação da Expressão Gênica , Redes Reguladoras de Genes , Proteínas de Homeodomínio/genética , Camundongos , Neurogênese , Análise de Sequência de RNA/métodos , Células-Tronco , Análise Serial de TecidosRESUMO
Existing methodologies to produce human neural stem cells and neurons from embryonic stem cells frequently involve multistep processes and the use of complex and expensive media components, cytokines or small molecules. Here, we report a simple technique to generate human neuroepithelial progenitors and neurons by periodic mechanical dissection and adherent-cell depletion on regular cell-culture grade plastic surfaces. This neural induction technique does not employ growth factors, small molecules or peptide inhibitors, apart from those present in serum-free supplements. Suggestive of their central nervous system origin, we found that neural progenitors formed by this technique expressed radial glia markers, and, when differentiated, expressed TUBB3, RBFOX3 (NeuN) and serotonin, but not markers for peripheral neurons. With these data, we postulate that incorporation of periodic mechanical stimuli and plastic surface-mediated cell selection could improve and streamline existing human neuron production protocols.
Assuntos
Técnicas de Cultura de Células/métodos , Células-Tronco Neurais/citologia , Neurônios/citologia , Animais , Adesão Celular , Diferenciação Celular , Linhagem Celular , Células-Tronco Embrionárias Humanas/citologia , Humanos , CamundongosRESUMO
The embryonic tectum displays an anteroposterior gradient in development and produces the superior colliculus and inferior colliculus. Studies suggest that partition of the tectum is controlled by different strengths and durations of FGF signals originated from the so-called isthmic organizer at the mid/hindbrain junction; however, the underlying mechanism is unclear. We show that deleting Ptpn11, which links FGF with the ERK pathway, prevents inferior colliculus formation by depleting a previously uncharacterized stem cell zone. The stem-zone loss is attributed to shortening of S phase and acceleration of cell cycle exit and neurogenesis. Expression of a constitutively active Mek1 (Mek1DD), the known ERK activator, restores the tectal stem zone and the inferior colliculus without Ptpn11. By contrast, Mek1DD expression fails to rescue the tectal stem zone and the inferior colliculus in the absence of Fgf8 and the isthmic organizer, indicating that FGF and Mek1DD initiate qualitatively and/or quantitatively distinctive signaling. Together, our data show that the formation of the inferior colliculus relies on the provision of new cells from the tectal stem zone. Furthermore, distinctive ERK signaling mediates Fgf8 in the control of cell survival, tissue polarity and cytogenetic gradient during the development of the tectum.
Assuntos
MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Fatores de Crescimento de Fibroblastos/metabolismo , Colículos Inferiores/citologia , Colículos Inferiores/metabolismo , Células-Tronco/citologia , Células-Tronco/metabolismo , Animais , Apoptose/genética , Apoptose/fisiologia , Axônios/metabolismo , Padronização Corporal/genética , Padronização Corporal/fisiologia , Ciclo Celular/genética , Ciclo Celular/fisiologia , Morte Celular/genética , Morte Celular/fisiologia , MAP Quinases Reguladas por Sinal Extracelular/genética , Fator 8 de Crescimento de Fibroblasto/genética , Fator 8 de Crescimento de Fibroblasto/metabolismo , Fatores de Crescimento de Fibroblastos/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , MAP Quinase Quinase 1/genética , MAP Quinase Quinase 1/metabolismo , Mesencéfalo/citologia , Mesencéfalo/metabolismo , Camundongos Knockout , Neurogênese/genética , Neurogênese/fisiologia , Proteína Tirosina Fosfatase não Receptora Tipo 11/genética , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismoRESUMO
Evolution of complex behaviors in higher vertebrates and primates require the development of sophisticated neuronal circuitry and the expansion of brain surface area to accommodate the vast number of neuronal and glial populations. To achieve these goals, the neocortex in primates and the cerebellum in amniotes have developed specialized types of basal progenitors to aid the folding of their cortices. In the cerebellum, Bergmann glia constitute such a basal progenitor population, having a distinctive morphology and playing a critical role in cerebellar corticogenesis. Here, we review recent studies on the induction of Bergmann glia and their crucial role in mediating folding of the cerebellar cortex. These studies uncover a key function of FGF-ERK-ETV signaling cascade in the transformation of Bergmann glia from radial glia in the ventricular zone. Remarkably, in the neocortex, the same signaling axis operates to facilitate the transformation of ventricular radial glia into basal radial glia, a Bergmann glia-like basal progenitor population, which have been implicated in the establishment of neocortical gyri. These new findings draw a striking similarity in the function and ontogeny of the two basal progenitor populations born in distinct brain compartments.
Assuntos
Cerebelo/citologia , Cerebelo/fisiologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Neuroglia/fisiologia , Transdução de Sinais/fisiologia , Animais , Humanos , Proteína Tirosina Fosfatase não Receptora Tipo 11/genética , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismoRESUMO
The thalamus and habenula, two important nodes of the forebrain circuitry, are derived from a single developmental compartment, called prosomere 2, in the diencephalon. Habenular and thalamic neurons display distinct molecular identity, neurochemistry, and connectivity. Furthermore, their progenitors exhibit distinctive neurogenic patterns with a marked delay in the onset of neurogenesis in the thalamus. However, the progenitors in prosomere 2 express many common developmental regulators and the mechanism underlying the specification and differentiation of these two populations of neurons remains unknown. Gbx2, coding for a homeodomain transcription factor, is initially expressed in thalamic neuronal precursors that have just exited the cell cycle, and its expression is maintained in many mature thalamic neurons in adults. Deletion of Gbx2 severely disrupts histogenesis of the thalamus and abolishes thalamocortical projections in mice. Here, by using genome-wide transcriptional profiling, we show that Gbx2 promotes thalamic but inhibits habenular molecular characters. Remarkably, although Gbx2 is expressed in postmitotic neuronal precursors, deletion of Gbx2 changes gene expression and cell proliferation in dividing progenitors in the developing thalamus. These defects are partially rescued by the mosaic presence of wild-type cells, demonstrating a cell non-autonomous role of Gbx2 in regulating the development of thalamic progenitors. Our results suggest that Gbx2 is essential for the acquisition of the thalamic neuronal identity by repressing habenular identity through a feedback signaling from postmitotic neurons to progenitors.
Assuntos
Habenula/embriologia , Proteínas de Homeodomínio/fisiologia , Neurônios/citologia , Tálamo/embriologia , Animais , Proteínas de Homeodomínio/genética , Camundongos , Fatores de Transcrição/genética , TranscriptomaRESUMO
Wnt1 and Wnt3a secreted from the dorsal neural tube were previously shown to regulate a gene expression program in the dorsal otic vesicle that is necessary for vestibular morphogenesis (Riccomagno et al., 2005. Genes Dev. 19, 1612-1623). Unexpectedly, Wnt1(-/-); Wnt3a(-/-) embryos also displayed a pronounced defect in the outgrowth of the ventrally derived cochlear duct. To determine how Wnt signaling in the dorsal otocyst contributes to cochlear development we performed a series of genetic fate mapping experiments using two independent Wnt responsive driver strains (TopCreER and Gbx2(CreER)) that when crossed to inducible responder lines (Rosa(lacZ) or Rosa(zsGreen)) permanently labeled dorsomedial otic progenitors and their derivatives. Tamoxifen time course experiments revealed that most vestibular structures showed some degree of labeling when recombination was induced between E7.75 and E12.5, consistent with continuous Wnt signaling activity in this tissue. Remarkably, a population of Wnt responsive cells in the dorsal otocyst was also found to contribute to the sensory epithelium of the cochlear duct, including auditory hair and support cells. Similar results were observed with both TopCreER and Gbx2(CreER) strains. The ventral displacement of Wnt responsive cells followed a spatiotemporal sequence that initiated in the anterior otic cup at, or immediately prior to, the 17-somite stage (E9) and then spread progressively to the posterior pole of the otic vesicle by the 25-somite stage (E9.5). These lineage-tracing experiments identify the earliest known origin of auditory sensory progenitors within a population of Wnt responsive cells in the dorsomedial otic cup.
Assuntos
Cóclea/metabolismo , Orelha Interna/metabolismo , Epitélio/metabolismo , Via de Sinalização Wnt/genética , Animais , Linhagem da Célula/genética , Movimento Celular/genética , Proliferação de Células/genética , Cóclea/citologia , Cóclea/embriologia , Orelha Interna/citologia , Orelha Interna/embriologia , Embrião de Mamíferos/efeitos dos fármacos , Embrião de Mamíferos/embriologia , Embrião de Mamíferos/metabolismo , Epitélio/embriologia , Antagonistas de Estrogênios/farmacologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Imuno-Histoquímica , Hibridização In Situ , Masculino , Camundongos Transgênicos , Microscopia Confocal , Morfogênese/efeitos dos fármacos , Morfogênese/genética , Tamoxifeno/farmacologia , Fatores de TempoRESUMO
Folding of the cortex and the persistence of radial glia (RG)-like cells called Bergmann glia (BG) are hallmarks of the mammalian cerebellum. Similar to basal RG in the embryonic neocortex, BG maintain only basal processes and continuously express neural stem cell markers. Past studies had focused on the function of BG in granule cell migration and how granule cell progenitors (GCP) regulate cerebellar foliation. The molecular control of BG generation and its role in cerebellar foliation are less understood. Here, we have analyzed the function of the protein tyrosine phosphatase Shp2 in mice by deleting its gene Ptpn11 in the entire cerebellum or selectively in the GCP lineage. Deleting Ptpn11 in the entire cerebellum by En1-cre blocks transformation of RG into BG but preserves other major cerebellar cell types. In the absence of BG, inward invagination of GCP persists but is uncoupled from the folding of the Purkinje cell layer and the basement membrane, leading to disorganized lamination and an absence of cerebellar folia. In contrast, removing Ptpn11 in the GCP lineage by Atoh1-cre has no effect on cerebellar development, indicating that Shp2 is not cell autonomously required in GCP. Furthermore, we demonstrate that Ptpn11 interacts with Fgf8 and is essential for ERK activation in RG and nascent BG. Finally, expressing constitutively active MEK1 rescues BG formation and cerebellar foliation in Shp2-deficient cerebella. Our results demonstrate an essential role of Shp2 in BG specification via fibroblast growth factor/extracellular signal-regulated protein kinase signaling, and reveal a crucial function of BG in organizing cerebellar foliation.
Assuntos
Cerebelo/citologia , Cerebelo/fisiologia , Sistema de Sinalização das MAP Quinases/fisiologia , Neuroglia/fisiologia , Proteína Tirosina Fosfatase não Receptora Tipo 11/fisiologia , Animais , Células Ependimogliais/fisiologia , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Proteína Tirosina Fosfatase não Receptora Tipo 11/deficiênciaRESUMO
Combinatorial expression of transcription factors forms transcriptional codes to confer neuronal identities and connectivity. However, how these intrinsic factors orchestrate the spatiotemporal expression of guidance molecules to dictate the responsiveness of axons to guidance cues is less understood. Thalamocortical axons (TCAs) represent the major input to the neocortex and modulate cognitive functions, consciousness and alertness. TCAs travel a long distance and make multiple target choices en route to the cortex. The homeodomain transcription factor Gbx2 is essential for TCA development, as loss of Gbx2 abolishes TCAs in mice. Using a novel TCA-specific reporter, we have discovered that thalamic axons are mostly misrouted to the ventral midbrain and dorsal midline of the diencephalon in Gbx2-deficient mice. Furthermore, conditionally deleting Gbx2 at different embryonic stages has revealed a sustained role of Gbx2 in regulating TCA navigation and targeting. Using explant culture and mosaic analyses, we demonstrate that Gbx2 controls the intrinsic responsiveness of TCAs to guidance cues. The guidance defects of Gbx2-deficient TCAs are associated with abnormal expression of guidance receptors Robo1 and Robo2. Finally, we demonstrate that Gbx2 controls Robo expression by regulating LIM-domain transcription factors through three different mechanisms: Gbx2 and Lhx2 compete for binding to the Lmo3 promoter and exert opposing effects on its transcription; repressing Lmo3 by Gbx2 is essential for Lhx2 activity to induce Robo2; and Gbx2 represses Lhx9 transcription, which in turn induces Robo1. Our findings illustrate the transcriptional control of differential expression of Robo1 and Robo2, which may play an important role in establishing the topography of TCAs.
Assuntos
Axônios/fisiologia , Proteínas de Homeodomínio/fisiologia , Proteínas com Homeodomínio LIM/genética , Proteínas do Tecido Nervoso/genética , Tálamo/embriologia , Animais , Axônios/metabolismo , Células Cultivadas , Córtex Cerebral/embriologia , Córtex Cerebral/metabolismo , Córtex Cerebral/fisiologia , Embrião de Mamíferos , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Proteínas com Homeodomínio LIM/metabolismo , Camundongos , Camundongos Transgênicos , Proteínas do Tecido Nervoso/metabolismo , Neurogênese/genética , Neurogênese/fisiologia , Gravidez , Receptores Imunológicos/genética , Receptores Imunológicos/metabolismo , Tálamo/metabolismo , Tálamo/fisiologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas RoundaboutRESUMO
Sensory and endoneurocrine tissues as diverse as the lens, the olfactory epithelium, the inner ear, the cranial sensory ganglia, and the anterior pituitary arise from a common pool of progenitors in the preplacodal ectoderm (PPE). Around late gastrulation, the PPE forms at the border surrounding the anterior neural plate, and expresses a unique set of evolutionarily conserved transcription regulators including Six1, Eya 1 and Eya2. Here, we describe the first report to generate and characterize the SIX1(+) PPE cells from human embryonic stem (ES) cells by adherent differentiation. Before forming PPE cells, differentiating cultures first expressed the non-neural ectoderm specific transcriptional factors TFAP2A, GATA2, GATA3, DLX3, and DLX5, which are crucial in establishing the PPE competence. We demonstrated that bone morphogenetic protein (BMP) activity plays a transient but essential role in inducing expression of these PPE competence factors and eventually the PPE cells. Interestingly, we found that attenuating BMP signaling after establishing the competence state induces anterior placode precursors. By manipulating BMP and hedgehog signaling pathways, we further differentiate these precursors into restricted lineages including the lens placode and the oral ectoderm (pituitary precursor) cells. Finally, we also show that sensory neurons can be generated from human PPE cells, demonstrating the multipotency of the human ES-derived PPE cells.
Assuntos
Proteína Morfogenética Óssea 4/metabolismo , Diferenciação Celular/fisiologia , Ectoderma/embriologia , Células-Tronco Embrionárias/fisiologia , Transdução de Sinais/fisiologia , Amidas , Análise de Variância , Animais , Proteína Morfogenética Óssea 4/farmacologia , Proteínas de Transporte , Bovinos , Diferenciação Celular/efeitos dos fármacos , Córnea/citologia , Primers do DNA/genética , Ectoderma/citologia , Células-Tronco Embrionárias/citologia , Proteínas de Homeodomínio/metabolismo , Humanos , Imuno-Histoquímica , Pirazóis , Piridinas , Pirimidinas , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Receptoras Sensoriais/citologia , Transdução de Sinais/genética , TretinoínaRESUMO
In vertebrates, the common expression border of two homeobox genes, Otx2 and Gbx2, demarcates the prospective midbrain-hindbrain border (MHB) in the neural plate at the end of gastrulation. The presence of a compartment boundary at the MHB has been demonstrated, but the mechanism and timing of its formation remain unclear. We show by genetic inducible fate mapping using a Gbx2(CreER) knock-in mouse line that descendants of Gbx2(+) cells as early as embryonic day (E) 7.5 do not cross the MHB. Without Gbx2, hindbrain-born cells abnormally populate the entire midbrain, demonstrating that Gbx2 is essential for specifying hindbrain fate. Gbx2(+) and Otx2(+) cells segregate from each other, suggesting that mutually exclusive expression of Otx2 and Gbx2 in midbrain and hindbrain progenitors is responsible for cell sorting in establishing the MHB. The MHB organizer gene Fgf8, which is expressed as a sharp transverse band immediately posterior to the lineage boundary at the MHB, is crucial in maintaining the lineage-restricted boundary after E7.5. Partial deletion of Fgf8 disrupts MHB lineage separation. Activation of FGF pathways has a cell-autonomous effect on cell sorting in midbrain progenitors. Therefore, Fgf8 from the MHB may signal the nearby mesencephalic cells to impart distinct cell surface characteristics or induce local cell-cell signaling, which consequently prevents cell movements across the MHB. Our findings reveal the distinct function of Gbx2 and Fgf8 in a stepwise process in the development of the compartment boundary at the MHB and that Fgf8, in addition to its organizer function, plays a crucial role in maintaining the lineage boundary at the MHB by restricting cell movement.
Assuntos
Fator 8 de Crescimento de Fibroblasto/metabolismo , Proteínas de Homeodomínio/metabolismo , Mesencéfalo/embriologia , Mesencéfalo/metabolismo , Rombencéfalo/embriologia , Rombencéfalo/metabolismo , Animais , Linhagem da Célula , Fator 8 de Crescimento de Fibroblasto/genética , Deleção de Genes , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Mesencéfalo/citologia , Camundongos , Camundongos Transgênicos , Rombencéfalo/citologia , Transdução de SinaisRESUMO
The assembly of neural circuits is dependent upon the generation of specific neuronal subtypes, each subtype displaying unique properties that direct the formation of selective connections with appropriate target cells. Actions of transcription factors in neural progenitors and postmitotic cells are key regulators in this process. LIM-homeodomain transcription factors control crucial aspects of neuronal differentiation, including subtype identity and axon guidance. Nonetheless, their regulation during development is poorly understood and the identity of the downstream molecular effectors of their activity remains largely unknown. Here, we demonstrate that the Lhx2 transcription factor is dynamically regulated in distinct pools of thalamic neurons during the development of thalamocortical connectivity in mice. Indeed, overexpression of Lhx2 provokes defective thalamocortical axon guidance in vivo, while specific conditional deletion of Lhx2 in the thalamus produces topographic defects that alter projections from the medial geniculate nucleus and from the caudal ventrobasal nucleus in particular. Moreover, we demonstrate that Lhx2 influences axon guidance and the topographical sorting of axons by regulating the expression of Robo1 and Robo2 guidance receptors, which are essential for these axons to establish correct connections in the cerebral cortex. Finally, augmenting Robo1 function restores normal axon guidance in Lhx2-overexpressing neurons. By regulating axon guidance receptors, such as Robo1 and Robo2, Lhx2 differentially regulates the axon guidance program of distinct populations of thalamic neurons, thus enabling the establishment of specific neural connections.
Assuntos
Axônios/fisiologia , Córtex Cerebral/fisiologia , Proteínas com Homeodomínio LIM/fisiologia , Proteínas do Tecido Nervoso/biossíntese , Neurogênese/fisiologia , Receptores Imunológicos/biossíntese , Tálamo/fisiologia , Fatores de Transcrição/fisiologia , Animais , Córtex Cerebral/crescimento & desenvolvimento , Córtex Cerebral/metabolismo , Embrião de Mamíferos , Deleção de Genes , Regulação da Expressão Gênica no Desenvolvimento , Proteínas com Homeodomínio LIM/metabolismo , Camundongos , Camundongos Endogâmicos ICR , Camundongos Knockout , Camundongos Transgênicos , Vias Neurais/crescimento & desenvolvimento , Vias Neurais/metabolismo , Vias Neurais/fisiologia , Transdução de Sinais/fisiologia , Tálamo/crescimento & desenvolvimento , Tálamo/metabolismo , Fatores de Transcrição/metabolismo , Proteínas RoundaboutRESUMO
Evaluations of benefits of implementing low impact development (LID) stormwater management techniques can extend up to a watershed scale. This presents a challenge for representing them in watershed models, since they are typically orders of magnitude smaller in size. This paper presents an approach that is focused on trying to evaluate the benefits of implementing LIDs on a lot level. The methodology uses the concept of urban hydrological response Unit and results in developing and applying performance curves that are a function of lot properties to estimate the potential benefit of large-scale LID implementation. Lot properties are determined using a municipal geographic information system database and processed to determine groups of lots with similar properties. A representative lot from each group is modeled over a typical rainfall year using USEPA Stormwater Management Model to develop performance functions that relate the lot properties and the change in annual runoff volume and corresponding phosphorus loading with different LIDs implemented. The results of applying performance functions on all urban areas provide the potential locations, benefit and cost of implementation of all LID techniques, guiding future decisions for LID implementation by watershed area municipalities.
Assuntos
Drenagem Sanitária , Modelos Teóricos , CidadesRESUMO
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/metabolismoRESUMO
Mammalian forebrain cholinergic neurons are composed of local circuit neurons in the striatum and projection neurons in the basal forebrain. These neurons are known to arise from a common pool of progenitors that primarily resides in the medial ganglionic eminence (MGE). However, little is known about the genetic programs that differentiate these two types of cholinergic neurons. Using inducible genetic fate mapping, here we examined the developmental fate of cells that express the homeodomain transcription factor Gbx2 in the MGE. We show that the Gbx2 lineage-derived cells that undergo tangential migration exclusively give rise to almost all cholinergic interneurons in the striatum, whereas those undergoing radial migration mainly produce noncholinergic neurons in the basal forebrain. Deletion of Gbx2 throughout the mouse embryo or specifically in the MGE results in abnormal distribution and significant reduction of cholinergic neurons in the striatum. We show that early-born (before embryonic day 12.5) cholinergic interneurons preferentially populate the lateral aspect of the striatum and mature earlier than late-born (after embryonic day 12.5) neurons, which normally reside in the medial part of the striatum. In the absence of Gbx2, early-born striatal cholinergic precursors display abnormal neurite outgrowth and increased complexity, and abnormally contribute to the medial part of the caudate-putamen, whereas late-born striatal cholinergic interneurons are mostly missing. Together, our data demonstrate that Gbx2 is required for the development of striatal cholinergic interneurons, perhaps by regulating tangential migration of the striatal cholinergic precursors.
Assuntos
Acetilcolina/fisiologia , Diferenciação Celular/genética , Proteínas de Homeodomínio/genética , Interneurônios/fisiologia , Neostriado/embriologia , Células-Tronco Neurais/fisiologia , Animais , Linhagem da Célula/genética , Movimento Celular/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento/genética , Técnicas de Introdução de Genes/métodos , Proteínas de Homeodomínio/fisiologia , Interneurônios/citologia , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Neostriado/citologia , Neostriado/metabolismo , Células-Tronco Neurais/citologia , Neurogênese/genéticaRESUMO
The single Fgf8 gene in mice produces eight protein isoforms (Fgf8a-h) with different N-termini by alternative splicing. Gain-of-function studies have demonstrated that Fgf8a and Fgf8b have distinct activities in the developing midbrain and hindbrain (MHB) due to their different binding affinities with FGF receptors. Here we have performed loss-of-function analyses to determine the in vivo requirement for these two Fgf8 spliceforms during MHB development. We showed that deletion of Fgf8b-containing spliceforms (b, d, f and h) leads to loss of multiple key regulatory genes, including Fgf8 itself, in the MHB region. Therefore, specific inactivation of Fgf8b-containing spliceforms, similar to the loss of Fgf8, in MHB progenitors results in deletion of the midbrain, isthmus, and cerebellum. We also created a splice-site mutation abolishing Fgf8a-containing spliceforms (a, c, e, and g). Mice lacking Fgf8a-containing spliceforms exhibit growth retardation and postnatal lethality, and the phenotype is variable in different genetic backgrounds, suggesting that the Fgf8a-containing spliceforms may play a role in modulating the activity of Fgf8. Surprisingly, no discernable defect was detected in the midbrain and cerebellum of Fgf8a-deficient mice. To determine if Fgf17, which is expressed in the MHB region and possesses similar activities to Fgf8a based on gain-of-function studies, may compensate for the loss of Fgf8a, we generated Fgf17 and Fgf8a double mutant mice. Mice lacking both Fgf8a-containing spliceforms and Fgf17 display the same defect in the posterior midbrain and anterior cerebellum as Fgf17 mutant mice. Therefore, Fgf8b-containing spliceforms, but not Fgf8a, are essential for the function of Fgf8 during the development of the midbrain and cerebellum.
Assuntos
Cerebelo/crescimento & desenvolvimento , Fator 8 de Crescimento de Fibroblasto/fisiologia , Mesencéfalo/crescimento & desenvolvimento , Processamento Alternativo/fisiologia , Animais , Fator 8 de Crescimento de Fibroblasto/genética , Fatores de Crescimento de Fibroblastos , Camundongos , Camundongos Knockout , MutaçãoRESUMO
Soluble growth factors play an important role in the coordination and integration of cell proliferation, differentiation, fate determination, and morphogenesis during development of multicellular organisms. Fibroblast growth factors (FGFs) are a large family of polypeptide growth factors that are present in organisms ranging from nematodes to humans. RNA alternative splicing of FGFs and their receptors further enhances the complexity of this ligand-receptor system. The mouse Fgf8 gene produces eight splice variants, which encode isoform proteins with different N-termini and distinct receptor-binding affinity and biological activity. In this article, we review the roles of Fgf8 in vertebrate development and summarize the recent findings on the in vivo function of different Fgf8 splice variants. We propose that multiple Fgf8 isoform proteins act in concert to regulate the overall function of Fgf8 and account for the diverse and essential role of Fgf8 during vertebrate development.
Assuntos
Encéfalo/metabolismo , Fator 8 de Crescimento de Fibroblasto/metabolismo , Transdução de Sinais , Animais , Encéfalo/embriologia , Encéfalo/crescimento & desenvolvimento , Diferenciação Celular , Proliferação de Células , Fator 8 de Crescimento de Fibroblasto/genética , Fatores de Crescimento de Fibroblastos/metabolismo , Humanos , Camundongos , Morfogênese , Ligação Proteica , Isoformas de Proteínas , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Proteínas de Peixe-Zebra/metabolismoRESUMO
Single-cell transcriptional and epigenomics profiles have been applied in a variety of tissues and diseases for discovering new cell types, differentiation trajectories, and gene regulatory networks. Many methods such as Monocle 2/3, URD, and STREAM have been developed for tree-based trajectory building. Here, we propose a fast and flexible trajectory learning method, LISA2, for single-cell data analysis. This new method has two distinctive features: (1) LISA2 utilizes specified leaves and root to reduce the complexity for building the developmental trajectory, especially for some special cases such as rare cell populations and adjacent terminal cell states; and (2) LISA2 is applicable for both transcriptomics and epigenomics data. LISA2 visualizes complex trajectories using 3D Landmark ISOmetric feature MAPping (L-ISOMAP). We apply LISA2 to simulation and real datasets in cerebellum, diencephalon, and hematopoietic stem cells including both single-cell transcriptomics data and single-cell assay for transposase-accessible chromatin data. LISA2 is efficient in estimating single-cell trajectory and expression trends for different kinds of molecular state of cells.
RESUMO
The mouse homeobox gene, Gbx2, is expressed in discreet domains in the neural tube and plays a key role in forebrain and hindbrain development. Previous studies have demonstrated that mutual inhibition between Gbx2 and Otx2, which are respectively expressed in the anterior and posterior parts of the neural plate, positions the prospective midbrain-hindbrain junction. We describe here a conditional Gbx2 gain-of-function transgenic mouse line, Gbx2-GOF, which expresses Gbx2 and red fluorescence protein, mCherry, upon Cre-mediated recombination. In the absence of Cre, beta-galactosidase is broadly expressed in mouse embryos and adult brains carrying the transgene. By combining Gbx2-GOF and En1(Cre) knock-in allele, we activated expression of Gbx2 and mCherry throughout the mesencephalon (mes) and rhombomere 1 (r1). The ectopic expression of Gbx2 causes an anterior shift of the mes/r1 junction at embryonic day 10.5. Interestingly, we found that persistent expression of Gbx2 throughout the mes/r1 region largely abolishes expression of the isthmic organizer gene Fgf8, leading to deletion of the midbrain and cerebellum at later stages. Our data suggest that the juxtaposition of the expression domains of Gbx2 and Otx2 within the mes/r1 area is essential for the maintenance of Fgf8 expression. Furthermore, the Gbx2-GOF transgenic line is suitable for functional study of Gbx2 during development.
Assuntos
Cerebelo/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Mesencéfalo/metabolismo , Transgenes , Animais , Cerebelo/embriologia , Proteínas de Homeodomínio/metabolismo , Mesencéfalo/embriologia , Camundongos , Camundongos TransgênicosRESUMO
CONTEXT: Cancer cell lines are used extensively in various research. Knowledge of genetic alterations in these lines is important for understanding mechanisms underlying their biology. However, since paired normal tissues are usually unavailable for comparison, precisely determining genetic alterations in cancer cell lines is difficult. To address this issue, a highly efficient and reliable method is developed. AIMS: Establishing a highly efficient and reliable experimental system for genetic profiling of cell lines. MATERIALS AND METHODS: A widely used breast cancer cell line, MCF-7, was genetically profiled with 4,396 single nucleotide polymorphisms (SNPs) spanning 11 whole chromosomes and two other small regions using a newly developed high-throughput multiplex genotyping approach. RESULTS: The fractions of homozygous SNPs in MCF-7 (13.3%) were significantly lower than those in the control cell line and in 24 normal human individuals (25.1% and 27.4%, respectively). Homozygous SNPs in MCF-7 were found in clusters. The sizes of these clusters were significantly larger than the expected based on random allelic combination. Fourteen such regions were found on chromosomes 1p, 1q, 2q, 6q, 13, 15q, 16q, 17q and 18p in MCF-7 and two in the small regions. CONCLUSIONS: These results are generally concordant with those obtained using different approaches but are better in defining their chromosomal positions. The used approach provides a reliable way to detecting possible genetic alterations in cancer cell lines without paired normal tissues.