RESUMO
Eviction of histones from nucleosomes and their exchange with newly synthesized or alternative variants is a central epigenetic determinant. Here, we define the genome-wide occupancy and exchange pattern of canonical and non-canonical histone variants in mouse embryonic stem cells by genetically encoded exchange sensors. While exchange of all measured variants scales with transcription, we describe variant-specific associations with transcription elongation and Polycomb binding. We found considerable exchange of H3.1 and H2B variants in heterochromatin and repeat elements, contrasting the occupancy and little exchange of H3.3 in these regions. This unexpected association between H3.3 occupancy and exchange of canonical variants is also evident in active promoters and enhancers, and further validated by reduced H3.1 dynamics following depletion of H3.3-specific chaperone, HIRA. Finally, analyzing transgenic mice harboring H3.1 or H3.3 sensors demonstrates the vast potential of this system for studying histone exchange and its impact on gene expression regulation in vivo.
Assuntos
Histonas , Células-Tronco Embrionárias Murinas , Animais , Camundongos , Histonas/genética , Histonas/metabolismo , Células-Tronco Embrionárias Murinas/metabolismo , Nucleossomos/genética , Sequências Reguladoras de Ácido Nucleico , Regulação da Expressão GênicaRESUMO
Mice deficient for all ten-eleven translocation (TET) genes exhibit early gastrulation lethality. However, separating cause and effect in such embryonic failure is challenging. To isolate cell-autonomous effects of TET loss, we used temporal single-cell atlases from embryos with partial or complete mutant contributions. Strikingly, when developing within a wild-type embryo, Tet-mutant cells retain near-complete differentiation potential, whereas embryos solely comprising mutant cells are defective in epiblast to ectoderm transition with degenerated mesoderm potential. We map de-repressions of early epiblast factors (e.g., Dppa4 and Gdf3) and failure to activate multiple signaling from nascent mesoderm (Lefty, FGF, and Notch) as likely cell-intrinsic drivers of TET loss phenotypes. We further suggest loss of enhancer demethylation as the underlying mechanism. Collectively, our work demonstrates an unbiased approach for defining intrinsic and extrinsic embryonic gene function based on temporal differentiation atlases and disentangles the intracellular effects of the demethylation machinery from its broader tissue-level ramifications.
Assuntos
Gastrulação , Mesoderma , Animais , Diferenciação Celular/genética , Embrião de Mamíferos/metabolismo , Gastrulação/genética , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Proteínas Nucleares/metabolismo , Transdução de SinaisRESUMO
Mouse embryonic development is a canonical model system for studying mammalian cell fate acquisition. Recently, single-cell atlases comprehensively charted embryonic transcriptional landscapes, yet inference of the coordinated dynamics of cells over such atlases remains challenging. Here, we introduce a temporal model for mouse gastrulation, consisting of data from 153 individually sampled embryos spanning 36 h of molecular diversification. Using algorithms and precise timing, we infer differentiation flows and lineage specification dynamics over the embryonic transcriptional manifold. Rapid transcriptional bifurcations characterize the commitment of early specialized node and blood cells. However, for most lineages, we observe combinatorial multi-furcation dynamics rather than hierarchical transcriptional transitions. In the mesoderm, dozens of transcription factors combinatorially regulate multifurcations, as we exemplify using time-matched chimeric embryos of Foxc1/Foxc2 mutants. Our study rejects the notion of differentiation being governed by a series of binary choices, providing an alternative quantitative model for cell fate acquisition.
Assuntos
Desenvolvimento Embrionário/fisiologia , Gastrulação/fisiologia , Animais , Diferenciação Celular , Linhagem da Célula , Embrião de Mamíferos/citologia , Desenvolvimento Embrionário/genética , Feminino , Expressão Gênica , Camundongos/embriologia , Camundongos Endogâmicos C57BL , Células-Tronco Embrionárias Murinas , Gravidez , Análise de Sequência de RNA/métodos , Análise de Célula Única/métodosRESUMO
The causes and contributing factors of autism spectrum disorders (ASD) are poorly understood. One gene associated with increased risk for ASD is methylenetetrahydrofolate-reductase (MTHFR), which encodes a key enzyme in one carbon (C1) metabolism. The MTHFR 677C > T polymorphism reduces the efficiency of methyl group production with possible adverse downstream effects on gene expression. In this study, the effects of prenatal and/or postnatal diets enriched in C1 nutrients on ASD-like behavior were evaluated in Mthfr-deficient mice. Differences in intermediate pathways between the mice with and without ASD-like behaviors were tested. The findings indicate that maternal and offspring Mthfr deficiency increased the risk for an ASD-like phenotype in the offspring. The risk of ASD-like behavior was reduced in Mthfr-deficient mice supplemented with C1 nutrients prenatally. Specifically, among offspring of Mthfr+/- dams, prenatal diet supplementation was protective against ASD-like symptomatic behavior compared to the control diet with an odds ratio of 0.18 (CI:0.035, 0.970). Changes in major C1 metabolites, such as the ratios between betaine/choline and SAM/SAH in the cerebral-cortex, were associated with ASD-like behavior. Symptomatic mice presenting ASD-like behavior showed decreased levels of GABA pathway proteins such as GAD65/67 and VGAT and altered ratios of the glutamate receptor subunits GluR1/GluR2 in males and NR2A/NR2B in females. The altered ratios, in turn, favor receptor subunits with higher sensitivity to neuronal activity. Our study suggests that MTHFR deficiency can increase the risk of ASD-like behavior in mice and that prenatal dietary intervention focused on MTHFR genotypes can reduce the risk of ASD-like behavior.
RESUMO
Implementing ovarian tissue engineering for the maturation of primordial follicles, the most abundant follicle population in the ovary, holds great potential for women fertility preservation. Here, we evaluated whether macroporous alginate scaffolds with affinity-bound bone morphogenetic protein-4 (BMP-4) could mimic the ovary microenvironment and support the culture and growth of primordial follicles seeded with supporting ovarian cells. Porcine primordial follicles developed in the alginate scaffolds up to the pre-antral stage within 21 days. Affinity-bound BMP-4 significantly contributed to follicular maturation, as evident by the 5-fold increase in the number of developing follicles and enhanced estradiol secretion in these cultures compared to when BMP-4 was added to cultures with no affinity binding. After 21 days in culture, an increase in GDF-9/AMH gene expression, which is correlated with follicular development, was statistically significant when BMP-4 was affinity bound, compared to all other scaffold groups. When developed in-vivo, after xeno-transplantation of the follicle devices supplemented with additional angiogenic factors, the follicles reached antral size and secreted hormones at levels leading to restoration of ovarian function in ovariectomized severe combined immunodeficiency (SCID) mice. Altogether, our results provide first affirmation for the applicability of macroporous alginate scaffolds as a suitable platform for promoting follicle maturation in-vitro and in-vivo, and lay the foundations for the advantageous use of affinity binding presentation of growth factors to cultured follicles.
Assuntos
Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Ovário/efeitos dos fármacos , Alicerces Teciduais/química , Alginatos/farmacologia , Animais , Proteína Morfogenética Óssea 4/farmacologia , Células Cultivadas , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Hormônios/sangue , Humanos , Camundongos , Folículo Ovariano/efeitos dos fármacos , Folículo Ovariano/crescimento & desenvolvimento , Porosidade , Sulfatos/farmacologia , Suínos , Sobrevivência de Tecidos/efeitos dos fármacosRESUMO
There is currently a demand for new highly efficient and specific drugs to treat osteoporosis, a chronic bone disease affecting millions of people worldwide. We have developed a combinatorial strategy for engineering bispecific inhibitors that simultaneously target the unique combination of c-FMS and αvß3 integrin, which act in concert to facilitate bone resorption by osteoclasts. Using functional fluorescence-activated cell sorting (FACS)-based screening assays of random mutagenesis macrophage colony-stimulating factor (M-CSF) libraries against c-FMS and αvß3 integrin, we engineered dual-specific M-CSF mutants with high affinity to both receptors. These bispecific mutants act as functional antagonists of c-FMS and αvß3 integrin activation and hence of osteoclast differentiation in vitro and osteoclast activity in vivo. This study thus introduces a versatile platform for the creation of new-generation therapeutics with high efficacy and specificity for osteoporosis and other bone diseases. It also provides new tools for studying molecular mechanisms and the cell signaling pathways that mediate osteoclast differentiation and function.
Assuntos
Conservadores da Densidade Óssea/farmacologia , Reabsorção Óssea/prevenção & controle , Integrina alfaVbeta3/antagonistas & inibidores , Fator Estimulador de Colônias de Macrófagos/farmacologia , Osteoclastos/efeitos dos fármacos , Osteoporose/tratamento farmacológico , Receptor de Fator Estimulador de Colônias de Macrófagos/antagonistas & inibidores , Animais , Sítios de Ligação , Conservadores da Densidade Óssea/química , Conservadores da Densidade Óssea/metabolismo , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Células da Medula Óssea/patologia , Reabsorção Óssea/genética , Reabsorção Óssea/metabolismo , Reabsorção Óssea/patologia , Diferenciação Celular , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Regulação da Expressão Gênica , Humanos , Integrina alfaVbeta3/química , Integrina alfaVbeta3/genética , Integrina alfaVbeta3/metabolismo , Fator Estimulador de Colônias de Macrófagos/química , Fator Estimulador de Colônias de Macrófagos/genética , Fator Estimulador de Colônias de Macrófagos/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Macrófagos/patologia , Camundongos , Simulação de Acoplamento Molecular , Mutação , Osteoclastos/metabolismo , Osteoclastos/patologia , Osteoporose/genética , Osteoporose/metabolismo , Osteoporose/patologia , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Engenharia de Proteínas , Domínios e Motivos de Interação entre Proteínas , Receptor de Fator Estimulador de Colônias de Macrófagos/química , Receptor de Fator Estimulador de Colônias de Macrófagos/genética , Receptor de Fator Estimulador de Colônias de Macrófagos/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transdução de SinaisRESUMO
Several conditions related to the intrauterine environment are associated with neuropsychiatric conditions in later life. In humans, approximately 2% of infants are exposed to perinatal hypoxia-ischemia or prolonged anoxic insult, a condition to which very low birth weight preterm infants exhibit the highest susceptibility. Analyses of postmortem tissue link some presentations of these conditions to changes in GABA pathway functionality in the brains of affected subjects. Using animal models of early-life hypoxia-ischemia, losses of particular interneuron populations were reported. We hypothesize that the origin of GABAergic cell loss is in the mispositioning of neurons during the formation of the cerebral cortex. Here we report that in C57 black mice exposed to hypoxic conditions (9% O2; 3% CO2), 22-26% of cell loss was detected in the cortical plate as early as four days after the hypoxic event. Moreover, the surviving cells failed to populate the proper layers in the developing cortex. Differential sensitivities were observed in neurons that originated from different germinal zones. A significant effect of GABAergic cell location along the anterior-posterior and medio-lateral axes on neuron sensitivity to hypoxia was detected. Finally, changes in guidance molecules in the developing cortex, including increases in hypoxia-inducible factor 1-alpha levels and intracellular distribution, decreases in reelin levels in the cortical plate and altered organization of radial glia, were observed. These changes in the molecular landscape of the immediate environment of the immature neurons may contribute to the observed outcomes in neuronal migration to, and establishment in, the correct cortical layers. We suggest that the interneuron loss may be related to these early events.
Assuntos
Córtex Cerebral/patologia , Neurônios GABAérgicos/patologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Hipóxia/patologia , Animais , Movimento Celular/fisiologia , Córtex Cerebral/metabolismo , Neurônios GABAérgicos/metabolismo , Hipóxia/metabolismo , Camundongos , Neurogênese/fisiologia , Proteína ReelinaRESUMO
Changes in bone matrix composition are frequently found with bone diseases and may be associated with increased fracture risk. Bone is rich in the trace element zinc. Zinc was established to play a significant role in the growth, development, and maintenance of healthy bones; however, the mechanisms underlying zinc effects on the integrity of the skeleton are poorly understood. Here, we show that the zinc receptor (ZnR)/Gpr39 is required for normal bone matrix deposition by osteoblasts. Initial analysis showed that Gpr39-deficient ( Gpr39-/-) mice had weaker bones as a result of altered bone composition. Fourier transform infrared spectroscopy analysis showed high mineral-to-matrix ratios in the bones of Gpr39-/- mice. Histologic analysis showed abnormally high numbers of active osteoblasts but normal osteoclast numbers on the surfaces of bones from Gpr39-/- mice. Furthermore, Gpr39-/- osteoblasts had disorganized matrix deposition in vitro with cultures exhibiting abnormally low collagen and high mineral contents, findings that demonstrate a cell-intrinsic role for ZnR/Gpr39 in these cells. We show that both collagen synthesis and deposition by Gpr39-/- osteoblasts are perturbed. Finally, the expression of the zinc transporter Zip13 and a disintegrin and metalloproteinase with thrombospondin motifs family of zinc-dependent metalloproteases that regulate collagen processing was downregulated in Gpr39-/- osteoblasts. Altogether, our results suggest that zinc sensing by ZnR/Gpr39 affects the expression levels of zinc-dependent enzymes in osteoblasts and regulates collagen processing and deposition.-Jovanovic, M., Schmidt, F. N., Guterman-Ram, G., Khayyeri, H., Hiram-Bab, S., Orenbuch, A., Katchkovsky, S., Aflalo, A., Isaksson, H., Busse, B., Jähn, K., Levaot, N. Perturbed bone composition and integrity with disorganized osteoblast function in zinc receptor/Gpr39-deficient mice.
Assuntos
Densidade Óssea , Matriz Óssea/metabolismo , Osteoblastos/metabolismo , Receptores Acoplados a Proteínas G/deficiência , Animais , Matriz Óssea/patologia , Proteínas de Transporte de Cátions/biossíntese , Proteínas de Transporte de Cátions/genética , Colágeno/biossíntese , Colágeno/genética , Regulação da Expressão Gênica , Camundongos , Camundongos Knockout , Osteoblastos/patologia , Osteoclastos/metabolismo , Osteoclastos/patologia , Receptores Acoplados a Proteínas G/metabolismoRESUMO
Monocyte fusion into osteoclasts, bone resorbing cells, plays a key role in bone remodeling and homeostasis; therefore, aberrant cell fusion may be involved in a variety of debilitating bone diseases. Research in the last decade has led to the discovery of genes that regulate osteoclast fusion, but the basic molecular and cellular regulatory mechanisms underlying the fusion process are not completely understood. Here, we reveal a role for Dyrk2 in osteoclast fusion. We demonstrate that Dyrk2 down regulation promotes osteoclast fusion, whereas its overexpression inhibits fusion. Moreover, Dyrk2 also promotes the fusion of foreign-body giant cells, indicating that Dyrk2 plays a more general role in cell fusion. In an earlier study, we showed that fusion is a cell heterotypic process initiated by fusion-founder cells that fuse to fusion-follower cells, the latter of which are unable to initiate fusion. Here, we show that Dyrk2 limits the expansion of multinucleated founder cells through the suppression of the fusion competency of follower cells.
Assuntos
Fusão Celular , Osteoclastos/enzimologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Animais , Reabsorção Óssea , Diferenciação Celular , Proliferação de Células , Regulação Enzimológica da Expressão Gênica , Células Gigantes de Corpo Estranho/enzimologia , Camundongos , Camundongos Endogâmicos C57BL , Fatores de Transcrição NFATC/genética , Fatores de Transcrição NFATC/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Tirosina Quinases/genética , Células RAW 264.7 , Interferência de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais , Fatores de Tempo , Transfecção , Quinases DyrkRESUMO
Presynaptic terminals are specialized sites for information transmission where vesicles fuse with the plasma membrane and are locally recycled. Recent work has extended this classical view, with the observation that a subset of functional vesicles is dynamically shared between adjacent terminals by lateral axonal transport. Conceptually, such transport would be expected to disrupt vesicle retention around the active zone, yet terminals are characterized by a high-density vesicle cluster, suggesting that counteracting stabilizing mechanisms must operate against this tendency. The synapsins are a family of proteins that associate with synaptic vesicles and determine vesicle numbers at the terminal, but their specific function remains controversial. Here, using multiple quantitative fluorescence-based approaches and electron microscopy, we show that synapsin is instrumental for resisting vesicle dispersion and serves as a regulatory element for controlling lateral vesicle sharing between synapses. Deleting synapsin disrupts the organization of presynaptic vesicle clusters, making their boundaries hard to define. Concurrently, the fraction of vesicles amenable to transport is increased, and more vesicles are translocated to the axon. Importantly, in neurons from synapsin knock-out mice the resting and recycling pools are equally mobile. Synapsin, when present, specifically restricts the mobility of resting pool vesicles without affecting the division of vesicles between these pools. Specific expression of synapsin IIa, the sole isoform affecting synaptic depression, rescues the knock-out phenotype. Together, our results show that synapsin is pivotal for maintaining synaptic vesicle cluster integrity and that it contributes to the regulated sharing of vesicles between terminals.
Assuntos
Hipocampo/citologia , Neurônios/fisiologia , Terminações Pré-Sinápticas/fisiologia , Sinapsinas/metabolismo , Vesículas Sinápticas/fisiologia , 6-Ciano-7-nitroquinoxalina-2,3-diona/farmacologia , Animais , Animais Recém-Nascidos , Células Cultivadas , Antagonistas de Aminoácidos Excitatórios/farmacologia , Recuperação de Fluorescência Após Fotodegradação , Regulação da Expressão Gênica/genética , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Eletrônica de Transmissão , Proteínas do Tecido Nervoso/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/ultraestrutura , Terminações Pré-Sinápticas/efeitos dos fármacos , Terminações Pré-Sinápticas/ultraestrutura , Inibidores de Proteínas Quinases/farmacologia , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/genética , Purinas/farmacologia , Compostos de Piridínio/metabolismo , Compostos de Amônio Quaternário/metabolismo , Roscovitina , Estatísticas não Paramétricas , Sinapsinas/deficiência , Vesículas Sinápticas/efeitos dos fármacos , Vesículas Sinápticas/ultraestrutura , Fatores de Tempo , Transfecção/métodos , Valina/análogos & derivados , Valina/farmacologia , Proteína 2 Associada à Membrana da Vesícula/metabolismoRESUMO
The synaptic vesicle cycle encompasses the pre-synaptic events that drive neurotransmission. Influx of calcium leads to the fusion of synaptic vesicles with the plasma membrane and the release of neurotransmitter, closely followed by endocytosis. Vacated release sites are repopulated with vesicles which are then primed for release. When activity is intense, reserve vesicles may be mobilized to counteract an eventual decline in transmission. Recently, interplay between endocytosis and repopulation of the readily releasable pool of vesicles has been identified. In this study, we show that exo-endocytosis is necessary to enable detachment of synapsin from reserve pool vesicles during synaptic activity. We report that blockage of exocytosis in cultured mouse hippocampal neurons, either by tetanus toxin or by the deletion of munc13, inhibits the activity-dependent redistribution of synapsin from the pre-synaptic terminal into the axon. Likewise, perturbation of endocytosis with dynasore or by a dynamin dominant-negative mutant fully prevents synapsin redistribution. Such inhibition of synapsin redistribution occurred despite the efficient phosphorylation of synapsin at its protein kinase A/CaMKI site, indicating that disengagement of synapsin from the vesicles requires exocytosis and endocytosis in addition to phosphorylation. Our results therefore reveal hitherto unidentified feedback within the synaptic vesicle cycle involving the synapsin-managed reserve pool.