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1.
Nat Commun ; 11(1): 4654, 2020 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-32943640

RESUMO

The shift from maternal to embryonic control is a critical developmental milestone in preimplantation development. Widespread transcriptomic and epigenetic remodeling facilitate this transition from terminally differentiated gametes to totipotent blastomeres, but the identity of transcription factors (TF) and genomic elements regulating embryonic genome activation (EGA) are poorly defined. The timing of EGA is species-specific, e.g., the timing of murine and human EGA differ significantly. To deepen our understanding of mammalian EGA, here we profile changes in open chromatin during bovine preimplantation development. Before EGA, open chromatin is enriched for maternal TF binding, similar to that observed in humans and mice. During EGA, homeobox factor binding becomes more prevalent and requires embryonic transcription. A cross-species comparison of open chromatin during preimplantation development reveals strong similarity in the regulatory circuitry underlying bovine and human EGA compared to mouse. Moreover, TFs associated with murine EGA are not enriched in cattle or humans, indicating that cattle may be a more informative model for human preimplantation development than mice.


Assuntos
Montagem e Desmontagem da Cromatina/fisiologia , Embrião de Mamíferos/metabolismo , Desenvolvimento Embrionário/genética , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Genoma , Animais , Blastômeros , Bovinos/embriologia , Cromatina/metabolismo , Fertilização , Humanos , Camundongos , Oócitos , Especificidade da Espécie , Fatores de Transcrição/metabolismo
2.
PLoS One ; 15(8): e0237933, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32822407

RESUMO

Defining the relationship between maternal care, sensory development and brain gene expression in neonates is important to understand the impact of environmental challenges during sensitive periods in early life. In this study, we used a selection approach to test the hypothesis that variation in maternal licking and grooming (LG) during the first week of life influences sensory development in Wistar rat pups. We tracked the onset of the auditory brainstem response (ABR), the timing of eye opening (EO), middle ear development with micro-CT X-ray tomography, and used qRT-PCR to monitor changes in gene expression of the hypoxia-sensitive pathway and neurotrophin signaling in pups reared by low-LG or high-LG dams. The results show the first evidence that the transcription of genes involved in the hypoxia-sensitive pathway and neurotrophin signaling is regulated during separate sensitive periods that occur before and after hearing onset, respectively. Although the timing of ABR onset, EO, and the relative mRNA levels of genes involved in the hypoxia-sensitive pathway did not differ between pups from different LG groups, we found statistically significant increases in the relative mRNA levels of four genes involved in neurotrophin signaling in auditory brain regions from pups of different LG backgrounds. These results suggest that sensitivity to hypoxic challenge might be widespread in the auditory system of neonate rats before hearing onset, and that maternal LG may affect the transcription of genes involved in experience-dependent neuroplasticity.


Assuntos
Comportamento Animal/fisiologia , Encéfalo/crescimento & desenvolvimento , Encéfalo/metabolismo , Potenciais Evocados Auditivos do Tronco Encefálico/fisiologia , Asseio Animal/fisiologia , Comportamento Materno/fisiologia , Fatores de Crescimento Neural/metabolismo , Animais , Animais Recém-Nascidos , Olho/crescimento & desenvolvimento , Regulação da Expressão Gênica no Desenvolvimento/genética , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Audição , Hipóxia/genética , Hipóxia/metabolismo , Fatores de Crescimento Neural/genética , Plasticidade Neuronal/fisiologia , Ratos , Ratos Wistar , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Microtomografia por Raio-X
3.
Nat Cell Biol ; 22(9): 1116-1129, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32807903

RESUMO

How allelic asymmetry is generated remains a major unsolved problem in epigenetics. Here we model the problem using X-chromosome inactivation by developing "BioRBP", an enzymatic RNA-proteomic method that enables probing of low-abundance interactions and an allelic RNA-depletion and -tagging system. We identify messenger RNA-decapping enzyme 1A (DCP1A) as a key regulator of Tsix, a noncoding RNA implicated in allelic choice through X-chromosome pairing. DCP1A controls Tsix half-life and transcription elongation. Depleting DCP1A causes accumulation of X-X pairs and perturbs the transition to monoallelic Tsix expression required for Xist upregulation. While ablating DCP1A causes hyperpairing, forcing Tsix degradation resolves pairing and enables Xist upregulation. We link pairing to allelic partitioning of CCCTC-binding factor (CTCF) and show that tethering DCP1A to one Tsix allele is sufficient to drive monoallelic Xist expression. Thus, DCP1A flips a bistable switch for the mutually exclusive determination of active and inactive Xs.


Assuntos
Endorribonucleases/metabolismo , RNA/metabolismo , Transativadores/metabolismo , Cromossomo X/metabolismo , Alelos , Animais , Fator de Ligação a CCCTC/metabolismo , Linhagem Celular , Feminino , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , RNA Mensageiro/metabolismo , Transcrição Genética/fisiologia , Regulação para Cima/fisiologia , Inativação do Cromossomo X/fisiologia
4.
Proc Natl Acad Sci U S A ; 117(26): 15305-15315, 2020 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-32541052

RESUMO

Small RNAs are abundant in plant reproductive tissues, especially 24-nucleotide (nt) small interfering RNAs (siRNAs). Most 24-nt siRNAs are dependent on RNA Pol IV and RNA-DEPENDENT RNA POLYMERASE 2 (RDR2) and establish DNA methylation at thousands of genomic loci in a process called RNA-directed DNA methylation (RdDM). In Brassica rapa, RdDM is required in the maternal sporophyte for successful seed development. Here, we demonstrate that a small number of siRNA loci account for over 90% of siRNA expression during B. rapa seed development. These loci exhibit unique characteristics with regard to their copy number and association with genomic features, but they resemble canonical 24-nt siRNA loci in their dependence on RNA Pol IV/RDR2 and role in RdDM. These loci are expressed in ovules before fertilization and in the seed coat, embryo, and endosperm following fertilization. We observed a similar pattern of 24-nt siRNA expression in diverse angiosperms despite rapid sequence evolution at siren loci. In the endosperm, siren siRNAs show a marked maternal bias, and siren expression in maternal sporophytic tissues is required for siren siRNA accumulation. Together, these results demonstrate that seed development occurs under the influence of abundant maternal siRNAs that might be transported to, and function in, filial tissues.


Assuntos
Brassica rapa/embriologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Regulação da Expressão Gênica de Plantas/fisiologia , RNA de Plantas , Sementes/crescimento & desenvolvimento , Alelos , Arabidopsis/metabolismo , Brassica rapa/genética , Brassica rapa/crescimento & desenvolvimento , Brassica rapa/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , RNA Interferente Pequeno , Sementes/genética , Sementes/metabolismo
5.
J Anim Sci ; 98(6)2020 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-32452520

RESUMO

A novel, non-terminal surgical procedure to remove a single placentome from the pregnant ewe for gene expression and histological analyses was recently developed in our laboratory. This technique allows for evaluation of nutritional insults on placental development at more than one stage of gestation using a single animal. Early attempts to develop a similar technique in cattle were met with complications due to inaccessibility of the gravid uterine horn because of its location and mass. One alternative is to collect a placentome from the contralateral uterine horn; however, the question remains as to whether gene expression varies among placentomes based on location relative to the fetus. Pregnant heifers were maintained on forage during early gestation and later moved into pens with a Calan gate system (American Calan, Northwood, NH). On gestational day (GD) 158, five heifers were assigned to receive a hay-based diet formulated to meet 100% of NRC requirements, and five heifers were fed 70% of NRC requirements until necropsy on GD244. At necropsy, a single representative placentome was selected for analysis from the antimesometrial side: (1) of the gravid uterine horn central to the amnion, (2) over the allantois immediately adjacent to the amnion, (3) in the tip of the gravid uterine horn, and (4) in the tip of the contralateral uterine horn. Mean placentome weight was greater (P < 0.05) for locations central to the amnion and allantois compared to locations within the tips of the ipsilateral and contralateral horns, respectively. Gene expression for angiogenic factors (FGF2, ODC1, VEGFA, and FLT1), nutrient transporters (SLC7A1 and SLC2A1), and factors associated with hormone action (ESR1, IGF1, IGFBP3, CSH1, and PAG1) were unaffected (P > 0.05) by dietary treatment or location of the placentome. Results indicate that location of the placentome in relation to the fetus does not impact gene expression, enhancing the efficacy of nonterminal methodologies for sampling gene expression in placentomes.


Assuntos
Bovinos/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Placenta/metabolismo , Prenhez , Útero/fisiologia , Animais , Dieta/veterinária , Feminino , Nutrientes , Placentação , Gravidez
6.
Nat Cell Biol ; 22(5): 534-545, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32367046

RESUMO

Following implantation, the naive pluripotent epiblast of the mouse blastocyst generates a rosette, undergoes lumenogenesis and forms the primed pluripotent egg cylinder, which is able to generate the embryonic tissues. How pluripotency progression and morphogenesis are linked and whether intermediate pluripotent states exist remain controversial. We identify here a rosette pluripotent state defined by the co-expression of naive factors with the transcription factor OTX2. Downregulation of blastocyst WNT signals drives the transition into rosette pluripotency by inducing OTX2. The rosette then activates MEK signals that induce lumenogenesis and drive progression to primed pluripotency. Consequently, combined WNT and MEK inhibition supports rosette-like stem cells, a self-renewing naive-primed intermediate. Rosette-like stem cells erase constitutive heterochromatin marks and display a primed chromatin landscape, with bivalently marked primed pluripotency genes. Nonetheless, WNT induces reversion to naive pluripotency. The rosette is therefore a reversible pluripotent intermediate whereby control over both pluripotency progression and morphogenesis pivots from WNT to MEK signals.


Assuntos
Células-Tronco Embrionárias/fisiologia , Células-Tronco Pluripotentes/fisiologia , Animais , Blastocisto/metabolismo , Blastocisto/fisiologia , Diferenciação Celular/fisiologia , Cromatina/metabolismo , Células-Tronco Embrionárias/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Camadas Germinativas/metabolismo , Camadas Germinativas/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Morfogênese/fisiologia , Fatores de Transcrição Otx/metabolismo , Células-Tronco Pluripotentes/metabolismo
7.
Proc Natl Acad Sci U S A ; 117(21): 11503-11512, 2020 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-32398375

RESUMO

To explain the evolutionary origin of vertebrate teeth from odontodes, it has been proposed that competent epithelium spread into the oropharyngeal cavity via the mouth and other possible channels such as the gill slits [Huysseune et al., 2009, J. Anat. 214, 465-476]. Whether tooth formation deep inside the pharynx in extant vertebrates continues to require external epithelia has not been addressed so far. Using zebrafish we have previously demonstrated that cells derived from the periderm penetrate the oropharyngeal cavity via the mouth and via the endodermal pouches and connect to periderm-like cells that subsequently cover the entire endoderm-derived pharyngeal epithelium [Rosa et al., 2019, Sci. Rep. 9, 10082]. We now provide conclusive evidence that the epithelial component of pharyngeal teeth in zebrafish (the enamel organ) is derived from medial endoderm, as hitherto assumed based on position deep in the pharynx. Yet, dental morphogenesis starts only after the corresponding endodermal pouch (pouch 6) has made contact with the skin ectoderm, and only after periderm-like cells have covered the prospective tooth-forming endodermal epithelium. Manipulation of signaling pathways shown to adversely affect tooth development indicates they act downstream of these events. We demonstrate that pouch-ectoderm contact and the presence of a periderm-like layer are both required, but not sufficient, for tooth initiation in the pharynx. We conclude that the earliest interactions to generate pharyngeal teeth encompass those between different epithelial populations (skin ectoderm, endoderm, and periderm-like cells in zebrafish), in addition to the epithelial-mesenchymal interactions that govern the formation of all vertebrate teeth.


Assuntos
Epitélio/fisiologia , Camadas Germinativas , Odontogênese/fisiologia , Faringe/fisiologia , Dente/crescimento & desenvolvimento , Animais , Evolução Biológica , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Camadas Germinativas/citologia , Camadas Germinativas/fisiologia , Transdução de Sinais/fisiologia , Peixe-Zebra
8.
Proc Natl Acad Sci U S A ; 117(17): 9413-9422, 2020 04 28.
Artigo em Inglês | MEDLINE | ID: mdl-32291340

RESUMO

Astrogenesis is repressed in the early embryonic period and occurs in the late embryonic period. A variety of external and internal signals contribute to the sequential differentiation of neural stem cells. Here, we discovered that immune-related CD93 plays a critical negative role in the regulation of astrogenesis in the mouse cerebral cortex. We show that CD93 expression is detected in neural stem cells and neurons but not in astrocytes and declines as differentiation proceeds. Cd93 knockout increases astrogenesis at the expense of neuron production during the late embryonic period. CD93 responds to the extracellular matrix protein Multimerin 2 (MMRN2) to trigger the repression of astrogenesis. Mechanistically, CD93 delivers signals to ß-Catenin through a series of phosphorylation cascades, and then ß-Catenin transduces these signals to the nucleus to activate Zfp503 transcription. The transcriptional repressor ZFP503 inhibits the transcription of glial fibrillary acidic protein (Gfap) by binding to the Gfap promoter with the assistance of Grg5. Furthermore, Cd93 knockout mice exhibit autism-like behaviors. Taken together, our results reveal that CD93 is a negative regulator of the onset of astrogenesis and provide insight into therapy for psychiatric disorders.


Assuntos
Astrócitos/fisiologia , Proteínas da Matriz Extracelular/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Glicoproteínas de Membrana/metabolismo , Subfamília D de Receptores Semelhantes a Lectina de Células NK/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Animais , Transtorno Autístico , Córtex Cerebral/citologia , Córtex Cerebral/embriologia , Eletroporação , Proteínas da Matriz Extracelular/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Inflamação , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos ICR , Subfamília D de Receptores Semelhantes a Lectina de Células NK/genética , Proteínas do Tecido Nervoso/genética , Neurogênese , Neuroglia , Gravidez
9.
Am J Physiol Endocrinol Metab ; 318(6): E901-E919, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32286880

RESUMO

Lack of GABAB receptors in GABAB1 knockout mice decreases neonatal ARC kisspeptin 1 (Kiss1) expression in the arcuate nucleus of the hypothalamus (ARC) in females, which show impaired reproduction as adults. Our aim was to selectively impair GABAB signaling during a short postnatal period to evaluate its impact on the reproductive system. Neonatal male and female mice were injected with the GABAB antagonist CGP 55845 (CGP, 1 mg/kg body wt sc) or saline from postnatal day 2 (PND2) to PND6, three times per day (8 AM, 1 PM, and 6 PM). One group was killed on PND6 for collection of blood samples (hormones by radioimmunoassay), brains for gene expression in the anteroventral periventricular nucleus-periventricular nucleus continuum (AVPV/PeN), and ARC micropunches [quantitative PCR (qPCR)] and gonads for qPCR, hormone contents, and histology. A second group of mice was injected with CGP (1 mg/kg body wt sc) or saline from PND2 to PND6, three times per day (8 AM, 1 PM, and 6 PM), and left to grow to adulthood. We measured body weight during development and parameters of sexual differentiation, puberty onset, and estrous cycles. Adult mice were killed, and trunk blood (hormones), brains for qPCR, and gonads for qPCR and hormone contents were obtained. Our most important findings on PND6 include the CGP-induced decrease in ARC Kiss1 and increase in neurokinin B (Tac2) in both sexes; the decrease in AVPV/PeN tyrosine hydroxylase (Th) only in females; the increase in gonad estradiol content in both sexes; and the increase in primordial follicles and decrease in primary and secondary follicles. Neonatally CGP-treated adults showed decreased ARC Kiss1 and ARC gonadotropin-releasing hormone (Gnrh1) and increased ARC glutamic acid decarboxylase 67 (Gad1) only in males; increased ARC GABAB receptor subunit 1 (Gabbr1) in both sexes; and decreased AVPV/PeN Th only in females. We demonstrate that ARC Kiss1 expression is chronically downregulated in males and that the normal sex difference in AVPV/PeN Th expression is abolished. In conclusion, neonatal GABAergic input through GABAB receptors shapes gene expression of factors critical to reproduction.


Assuntos
Núcleo Arqueado do Hipotálamo/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Hipotálamo Anterior/metabolismo , Receptores de GABA-B/metabolismo , Animais , Animais Recém-Nascidos , Núcleo Arqueado do Hipotálamo/efeitos dos fármacos , Estradiol/metabolismo , Feminino , Hormônio Foliculoestimulante/metabolismo , Antagonistas de Receptores de GABA-B/farmacologia , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Glutamato Descarboxilase/genética , Glutamato Descarboxilase/metabolismo , Hormônio Liberador de Gonadotropina/genética , Hormônio Liberador de Gonadotropina/metabolismo , Hipotálamo Anterior/efeitos dos fármacos , Kisspeptinas/genética , Kisspeptinas/metabolismo , Hormônio Luteinizante/metabolismo , Masculino , Camundongos , Ovário/efeitos dos fármacos , Ovário/metabolismo , Ácidos Fosfínicos/farmacologia , Propanolaminas/farmacologia , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , Puberdade/efeitos dos fármacos , Puberdade/genética , Receptores Estrogênicos/genética , Receptores Estrogênicos/metabolismo , Receptores de GABA-B/genética , Receptores de Progesterona/genética , Receptores de Progesterona/metabolismo , Reprodução/efeitos dos fármacos , Reprodução/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Diferenciação Sexual/efeitos dos fármacos , Diferenciação Sexual/genética , Taquicininas/genética , Taquicininas/metabolismo , Testículo/efeitos dos fármacos , Testículo/metabolismo , Testosterona/metabolismo , Tirosina 3-Mono-Oxigenase/genética , Tirosina 3-Mono-Oxigenase/metabolismo
10.
Development ; 147(7)2020 04 06.
Artigo em Inglês | MEDLINE | ID: mdl-32156756

RESUMO

Wnt/ß-catenin signalling has been implicated in the terminal asymmetric divisions of neuronal progenitors in vertebrates and invertebrates. However, the role of Wnt ligands in this process remains poorly characterized. Here, we used the terminal divisions of the embryonic neuronal progenitors in C. elegans to characterize the role of Wnt ligands during this process, focusing on a lineage that produces the cholinergic interneuron AIY. We observed that, during interphase, the neuronal progenitor is elongated along the anteroposterior axis, then divides along its major axis, generating an anterior and a posterior daughter with different fates. Using time-controlled perturbations, we show that three Wnt ligands, which are transcribed at higher levels at the posterior of the embryo, regulate the orientation of the neuronal progenitor and its asymmetric division. We also identify a role for a Wnt receptor (MOM-5) and a cortical transducer APC (APR-1), which are, respectively, enriched at the posterior and anterior poles of the neuronal progenitor. Our study establishes a role for Wnt ligands in the regulation of the shape and terminal asymmetric divisions of neuronal progenitors, and identifies downstream components.


Assuntos
Divisão Celular Assimétrica/genética , Caenorhabditis elegans/embriologia , Células-Tronco Neurais/citologia , Proteínas Wnt/fisiologia , Animais , Animais Geneticamente Modificados , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Divisão Celular/genética , Polaridade Celular , Embrião não Mamífero , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Ligantes , Células-Tronco Neurais/fisiologia , Neurônios/citologia , Neurônios/fisiologia , Proteínas Wnt/genética , Proteínas Wnt/metabolismo , Via de Sinalização Wnt/fisiologia , beta Catenina/metabolismo
11.
Dev Cell ; 53(1): 9-26.e4, 2020 04 06.
Artigo em Inglês | MEDLINE | ID: mdl-32197068

RESUMO

The mouse embryo undergoes compaction at the 8-cell stage, and its transition to 16 cells generates polarity such that the outer apical cells are trophectoderm (TE) precursors and the inner cell mass (ICM) gives rise to the embryo. Here, we report that this first cell fate specification event is controlled by glucose. Glucose does not fuel mitochondrial ATP generation, and glycolysis is dispensable for blastocyst formation. Furthermore, glucose does not help synthesize amino acids, fatty acids, and nucleobases. Instead, glucose metabolized by the hexosamine biosynthetic pathway (HBP) allows nuclear localization of YAP1. In addition, glucose-dependent nucleotide synthesis by the pentose phosphate pathway (PPP), along with sphingolipid (S1P) signaling, activates mTOR and allows translation of Tfap2c. YAP1, TEAD4, and TFAP2C interact to form a complex that controls TE-specific gene transcription. Glucose signaling has no role in ICM specification, and this process of developmental metabolism specifically controls TE cell fate.


Assuntos
Diferenciação Celular/fisiologia , Embrião de Mamíferos/metabolismo , Glucose/metabolismo , Glicólise/fisiologia , Proteínas de Homeodomínio/metabolismo , Animais , Blastocisto/metabolismo , Desenvolvimento Embrionário/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Camundongos , Fatores de Transcrição/metabolismo
12.
Cell Mol Life Sci ; 77(16): 3177-3194, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32095869

RESUMO

The degradation of maternally provided molecules is a very important process during early embryogenesis. However, the vast majority of studies deals with mRNA degradation and protein degradation is only a very little explored process yet. The aim of this article was to summarize current knowledge about the protein degradation during embryogenesis of mammals. In addition to resuming of known data concerning mammalian embryogenesis, we tried to fill the gaps in knowledge by comparison with facts known about protein degradation in early embryos of non-mammalian species. Maternal protein degradation seems to be driven by very strict rules in terms of specificity and timing. The degradation of some maternal proteins is certainly necessary for the normal course of embryonic genome activation (EGA) and several concrete proteins that need to be degraded before major EGA have been already found. Nevertheless, the most important period seems to take place even before preimplantation development-during oocyte maturation. The defects arisen during this period seems to be later irreparable.


Assuntos
Embrião de Mamíferos/metabolismo , Embrião de Mamíferos/fisiologia , Embrião não Mamífero/metabolismo , Embrião não Mamífero/fisiologia , Desenvolvimento Embrionário/fisiologia , Proteínas/metabolismo , Animais , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Genoma/fisiologia , Humanos , Oócitos/metabolismo , Oócitos/fisiologia
13.
Artigo em Inglês | MEDLINE | ID: mdl-32048872

RESUMO

Morphological and behavioral evidence suggests that vasoinhibin is present in the central nervous system (CNS), triggering neuroendocrine and behavioral responses to stress. Moreover, vasoinhibin reduces neuronal survival and differentiation of primary sensory neurons of the peripheral nervous system. To address the functional role played by vasoinhibin at the CNS, and to better understand the underlying mechanisms involved in its actions, we treated primary cultured hippocampal neurons obtained from embryonic day 16 (E16) mice with a human recombinant vasoinhibin. We examined the resulting cellular changes, focusing on neuronal cell death, and explored the local generation of vasoinhibin within the hippocampus. Our results show that vasoinhibin significantly reduced neuronal cell density and increased immunoreactive activated caspase-3 and TUNEL-positive staining at 72, 16, and 24 h, respectively. Furthermore, vasoinhibin increased the expression of proapoptotic genes BAX, BAD, BIM, and PUMA and decreased that of the antiapoptotic gene BCL-2 at 24 h, as assessed by quantitative real-time reverse transcription-polymerase chain reaction. Vasoinhibin effects were blocked by coincubation with a vasoinhibin antibody or with prolactin. Immunoreactive bands consistent with vasoinhibin were observed in hippocampal extracts by Western blot analysis, and a prolactin standard was cleaved to vasoinhibin by a hippocampal lysate in a heat- and cathepsin D inhibitor pepstatin A-dependent fashion. Taken together, these data support the notion that vasoinhibin is locally produced by cathepsin D within the embryonic mouse hippocampus, a brain region that plays a critical role in emotional regulation, resulting in decreased neuronal cell viability via the activation of the intrinsic apoptosis pathway.


Assuntos
Apoptose/fisiologia , Hipocampo/metabolismo , Neurônios/fisiologia , Animais , Proteínas de Ciclo Celular/metabolismo , Regulação para Baixo , Embrião de Mamíferos , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Hipocampo/embriologia , Camundongos , Prolactina/metabolismo , Regulação para Cima
14.
J Mol Histol ; 51(1): 99-107, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-32095972

RESUMO

Tooth formation is accomplished under strict genetic control procedures. Therefore, exploring the gene network system of tooth development has a very positive practical significance for the study of tooth tissue regeneration and the prevention and treatment of tooth abnormalities. Early bell stage is the initial phase of odontoblast formation and dentin matrix deposition in the process of tooth development. Through RNA sequencing and differential gene analysis of the rat tooth germ samples at cap stage and early bell stage, we found that the bile secretion pathway was the most significant difference signal pathway during the development between cap stage and bell stage, which mainly included ABCC3, AQP4, SLC10A1, SLC2A1, SLC4A4, ADCY5, AQP9, CFTR, ATP1A2, ATP1B1 and ATP1A1, totally 11genes. Immunostaining revealed that SLC2A1, SLC4A4, ADCY5 and ATP1B1were mainly expressed in epithelium in bud stage and inner and outer enamel epithelium during the embryonic phase. In the postnatal 1 and postnatal 7, SLC2A1, SLC4A4 and ABCC3 were highly expressed in ameloblasts and odontoblasts while ADCY5, ATP1B1 and SLC10A1was expressed moderately only in odontoblasts. This finding illustrated that the bile secretion pathway related genes may participate in the development of tooth germ.


Assuntos
Bile , Proteínas de Transporte/biossíntese , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Odontogênese , Via Secretória/fisiologia , Germe de Dente/embriologia , Animais , Ratos , Ratos Sprague-Dawley , Germe de Dente/citologia
15.
Nat Protoc ; 15(3): 840-876, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31969721

RESUMO

Simultaneous observation of 3D chromatin organization and transcription at the single-cell level and with high spatial resolution may hold the key to unveiling the mechanisms regulating embryonic development, cell differentiation and even disease. We recently developed Hi-M, a technology that enables the sequential labeling, 3D imaging and localization of multiple genomic DNA loci, together with RNA expression, in single cells within whole, intact Drosophila embryos. Importantly, Hi-M enables simultaneous detection of RNA expression and chromosome organization without requiring sample unmounting and primary probe rehybridization. Here, we provide a step-by-step protocol describing the design of probes, the preparation of samples, the stable immobilization of embryos in microfluidic chambers, and the complete procedure for image acquisition. The combined RNA/DNA fluorescence in situ hybridization procedure takes 4-5 d, including embryo collection. In addition, we describe image analysis software to segment nuclei, detect genomic spots, correct for drift and produce Hi-M matrices. A typical Hi-M experiment takes 1-2 d to complete all rounds of labeling and imaging and 4 additional days for image analysis. This technology can be easily expanded to investigate cell differentiation in cultured cells or organization of chromatin within complex tissues.


Assuntos
Cromossomos , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Processamento de Imagem Assistida por Computador , Transcrição Genética/fisiologia , Animais , Cromatina , DNA/química , DNA/genética , DNA/metabolismo , Drosophila/embriologia , Corantes Fluorescentes , Hibridização in Situ Fluorescente/métodos , RNA/química , RNA/genética , RNA/metabolismo
16.
Arch Insect Biochem Physiol ; 103(3): e21609, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31385626

RESUMO

Insect metamorphosis is regulated by two main hormones: ecdysone (20E), which promotes molting, and juvenile hormone (JH), which inhibits adult morphogenesis. The transduction mechanisms for the respective hormonal signals include the transcription factors Krüppel homolog 1 (Kr-h1) and E93, which are JH- and 20E-dependent, respectively. Kr-h1 is the main effector of the antimetamorphic action of JH, while E93 is a key promoter of metamorphosis. The ancestral regulatory axis of metamorphosis, which operates in insects with hemimetabolan (gradual) metamorphosis and is known as the MEKRE93 pathway, is based on Kr-h1 repression of E93. In the last juvenile stage, when the production of JH dramatically decreases, Kr-h1 expression is almost completely interrupted, E93 becomes upregulated and metamorphosis proceeds. The holometabolan (complete) metamorphosis mode of development includes the peculiar pupal stage, a sort of intermediate between the final larval instar and the adult stage. In holometabolan species, Broad-Complex (BR-C) transcription factors determine the pupal stage and E93 stimulates the expression of BR-C in the prepupa. The MEKRE93 pathway is conserved in holometabolan insects, which have added the E93/BR-C interaction loop to the ancestral (hemimetabolan) pathway during the evolution from hemimetaboly to holometaboly.


Assuntos
Proteínas de Drosophila/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Metamorfose Biológica/fisiologia , Fatores de Transcrição/metabolismo , Animais , Proteínas de Drosophila/genética , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição/genética
17.
Artigo em Inglês | MEDLINE | ID: mdl-31669373

RESUMO

Geckos possess strong adhesion ability, even can climb on smooth surface. Previous studies have shown that the setae of geckos play a crucial role in their ability to climb on vertical walls. But the biological molecular mechanism of their adhesion ability remains unclear. In the present study, the expression patterns of corneous beta proteins (CBPs) genes related to claws, scales, and feathers development (named as ge-gprp-9, ge-gprp-10, ge-gprp-11, ge-gprp-12, ge-gprp-13, ge-gprp-14, ge-gprp-15, and ge-gprp-16 respectively) in the developing pad lamellae of different embryonic stages (stage 34, stage 36, stage 39, and stage 42) of the Japanese gecko Gekko japonicus were detected using fluorescence quantitative PCR approach. The results showed that there were significant up-regulated expression of CBPs mRNA at embryonic stage 39 with the embryonic continuous maturation and the highest expression level was detected at embryonic stage 39 or stage 42. Moreover, the expression levels of four CBPs genes ge-gprp-9, ge-gprp-10, ge-gprp-11, and ge-gprp-12 in the embryonic and adult development of gecko were detected by fluorescence in situ hybridization technique. The results from in situ hybridization detection revealed that the positive signals of these CBPs genes expression were the same in the developing pad lamellae of G. japonicus. The positive signals of eight CBPs genes were mainly found in the setae tissue, oberhautchen, and ß layer, which suggests these CBPs genes are involved in the growth of setae.


Assuntos
Embrião não Mamífero/embriologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Lagartos/embriologia , Proteínas de Répteis/biossíntese , Sensilas/embriologia , Animais , Lagartos/genética , Proteínas de Répteis/genética
18.
Arch Insect Biochem Physiol ; 103(3): e21614, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31498475

RESUMO

Histone acetylation is an evolutionarily conserved epigenetic mechanism of eukaryotic gene regulation which is tightly controlled by the opposing activities of histone acetyltransferases (HATs) and histone deacetylases (HDACs). In insects, life-history traits such as longevity and fecundity are severely affected by the suppression of HAT/HDAC activity, which can be achieved by RNA-mediated gene silencing or the application of chemical inhibitors. We used both experimental approaches to investigate the effect of HAT/HDAC inhibition in the pea aphid (Acyrthosiphon pisum) a model insect often used to study complex life-history traits. The silencing of HAT genes (kat6b, kat7, and kat14) promoted survival or increased the number of offspring, whereas targeting rpd3 (HDAC) reduced the number of viviparous offspring but increased the number of premature nymphs, suggesting a role in embryogenesis and eclosion. Specific chemical inhibitors of HATs/HDACs showed a remarkably severe impact on life-history traits, reducing survival, delaying development, and limiting the number of offspring. The selective inhibition of HATs and HDACs also had opposing effects on aphid body weight. The suppression of HAT/HDAC activity in aphids by RNA interference or chemical inhibition revealed similarities and differences compared to the reported role of these enzymes in other insects. Our data suggest that gene expression in A. pisum is regulated by multiple HATs/HDACs, as indicated by the fitness costs triggered by inhibitors that suppress several of these enzymes simultaneously. Targeting multiple HATs or HDACs with combined effects on gene regulation could, therefore, be a promising approach to discover novel targets for the management of aphid pests.


Assuntos
Afídeos/enzimologia , Fertilidade/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Histona Acetiltransferases/metabolismo , Histona Desacetilases/metabolismo , Histonas/metabolismo , Acetilação , Animais , Afídeos/crescimento & desenvolvimento , Afídeos/metabolismo , Afídeos/fisiologia , Histona Acetiltransferases/genética , Histona Desacetilases/genética , Longevidade , Processamento de Proteína Pós-Traducional
19.
J Cell Physiol ; 235(1): 151-165, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31187491

RESUMO

Slc25a17 is known as a peroxisomal solute carrier, but the in vivo role of the protein has not been demonstrated. We found that the zebrafish genome contains two slc25a17 genes that function redundantly, but additively. Notably, peroxisome function in slc25a17 knockdown embryos is severely compromised, resulting in an altered lipid composition. Along the defects found in peroxisome-associated phenotypic presentations, we highlighted that development of the swim bladder is also highly dependent on Slc25a17 function. As Slc25a17 showed substrate specificity towards coenzyme A (CoA), injecting CoA, but not NAD+ , rescued the defective swim bladder induced by slc25a17 knockdown. These results indicated that Slc25a17 acts as a CoA transporter, involved in the maintenance of functional peroxisomes that are essential for the development of multiple organs during zebrafish embryogenesis. Given high homology in protein sequences, the role of zebrafish Slc25a17 may also be applicable to the mammalian system.


Assuntos
Coenzima A/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Proteínas de Membrana/metabolismo , Sacos Aéreos/crescimento & desenvolvimento , Sacos Aéreos/metabolismo , Sequência de Aminoácidos , Animais , Coenzima A/genética , Sequência Conservada , Evolução Molecular , Proteínas de Membrana/genética , Peixe-Zebra
20.
J Cell Physiol ; 235(1): 128-140, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31187500

RESUMO

Intervertebral disc degeneration and associated back pain are relatively common but sparsely understood conditions, affecting over 70% of the population during some point of life. Disc degeneration is often associated with a loss of nucleus pulposus (NP) cells. Genetic mouse models offer convenient avenues to understand the cellular and molecular regulation of the disc during its formation, growth, maintenance, and aging. However, due to the lack of inducible driver lines to precisely target NP cells in the postnatal mouse disc, progress in this area of research has been moderate. NP cells are known to express cytokeratin 19 (Krt19), and tamoxifen (Tam)-inducible Krt19CreERT allele is available. The current study describes the characterization of Krt19CreERT allele to specifically and efficiently target NP cells in neonatal, skeletally mature, middle-aged, and aged mice using two independent fluorescent reporter lines. The efficiency of recombination at all ages was validated by immunostaining for KRT19. Results show that following Tam induction, Krt19CreERT specifically drives recombination of NP cells in the spine of neonatal and aged mice, while no recombination was detected in the surrounding tissues. Knee joints from skeletally mature Tam-treated Krt19CreERT/+ ; R26tdTOM mouse show the absence of recombination in all tissues and cells of the knee joint. Thus, this study provides evidence for the use of Krt19CreERT allele for genetic characterization of NP cells at different stages of the mouse life.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Disco Intervertebral/metabolismo , Queratina-19/metabolismo , Envelhecimento , Alelos , Animais , Animais Recém-Nascidos , Genótipo , Queratina-19/genética , Camundongos , Camundongos Transgênicos , Mutação
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