RESUMO
Oocytes develop in the germline cyst, a cellular organization in which germ cells are tightly interconnected and surrounded by somatic cells. The cyst produces oocytes for follicle formation and is a hub for essential processes in meiosis and oocyte differentiation. However, the formation and organization of the cyst, and their contribution to oocyte production in vertebrates remain unclear. Here, we provide tools for three-dimensional and functional in vivo analyses of the germline cyst in the zebrafish ovary. We describe the use of serial block-face scanning electron microscopy (SBF-SEM) to resolve the three-dimensional architecture of cells and organelles in the cyst at ultrastructural resolution. We present a deep learning-based pipeline for high-throughput quantitative analysis of three-dimensional confocal datasets of cysts in vivo. We provide a method for laser ablation of cellular components for manipulating cyst cells in ovaries. These methods will facilitate the investigation of the cyst cellular organization, expand the toolkit for the study of the zebrafish ovary, and advance our understanding of female developmental reproduction. They could also be further applied to the investigation of other developmental systems.
Assuntos
Oogênese , Peixe-Zebra , Animais , Feminino , Oócitos , Ovário , Células Germinativas/ultraestruturaRESUMO
The Balbiani body (Bb) is the first marker of polarity in vertebrate oocytes. The Bb is a conserved structure found in diverse animals including insects, fish, amphibians, and mammals. During early zebrafish oogenesis, the Bb assembles as a transient aggregate of mRNA, proteins, and membrane-bound organelles at the presumptive vegetal side of the oocyte. As the early oocyte develops, the Bb appears to grow slowly, until at the end of stage I of oogenesis it disassembles and deposits its cargo of localized mRNAs and proteins. In fish and frogs, this cargo includes the germ plasm as well as gene products required to specify dorsal tissues of the future embryo. We demonstrate that the Bb is a stable, solid structure that forms a size exclusion barrier similar to other biological hydrogels. Despite its central role in oocyte polarity, little is known about the mechanism behind the Bb's action. Analysis of the few known protein components of the Bb is insufficient to explain how the Bb assembles, translocates, and disassembles. We isolated Bbs from zebrafish oocytes and performed mass spectrometry to define the Bb proteome. We successfully identified 77 proteins associated with the Bb sample, including known Bb proteins and novel RNA-binding proteins. In particular, we identified Cirbpa and Cirbpb, which have both an RNA-binding domain and a predicted self-aggregation domain. In stage I oocytes, Cirbpa and Cirbpb localize to the Bb rather than the nucleus (as in somatic cells), indicating that they may have a specialized function in the germ line. Both the RNA-binding domain and the self-aggregation domain are sufficient to localize to the Bb, suggesting that Cirbpa and Cirbpb interact with more than just their mRNA targets within the Bb. We propose that Cirbp proteins crosslink mRNA cargo and proteinaceous components of the Bb as it grows. Beyond Cirbpa and Cirbpb, our proteomics dataset presents many candidates for further study, making it a valuable resource for building a comprehensive mechanism for Bb function at a protein level.
Assuntos
Proteínas de Peixe-Zebra , Peixe-Zebra , Animais , Polaridade Celular/genética , Mamíferos/metabolismo , Oócitos/metabolismo , Oogênese/genética , Organelas/metabolismo , Proteômica , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
The COVID-19 pandemic presented serious risks to the health and financial wellbeing of millions of people across the world. While many individuals adapted to these challenges through a variety of prosocial and protective behaviors (e.g., social distancing, working from home), many others also engaged in dishonest behaviors (e.g., lying to obtain vaccines or furlough payments). Hence, the COVID-19 pandemic provided a unique context in which to obtain a better understanding of the relationship between risk and dishonesty. Across three preregistered studies, we assessed whether objective risk and perceived risk influenced the decision to behave dishonestly in order to gain access to vaccines and furlough payments during a pandemic. We also assessed the extent to which such dishonesty was deterred by the probability of the dishonesty being detected. We found that heightened health risk perceptions were positively related with lying to obtain a vaccine (Studies 1 and 2), but found no evidence of the same relationship between financial risk perceptions and lying to access furlough payments (Study 2). We also found that the probability of dishonesty being detected had a negative relationship with dishonest behavior (Study 3). In addition, across the three studies, we found that (i) dishonesty was consistently evident in approximately one-third of all of our samples, and (ii) greater dishonesty was associated with older age. We discuss how our findings could be utilized by policy makers to better deter and detect dishonest behaviors during future similar crises.
RESUMO
During amphibian development, neural patterning occurs via a two-step process. Spemann's organizer secretes BMP antagonists that induce anterior neural tissue. A subsequent caudalizing step re-specifies anterior fated cells to posterior fates such as hindbrain and spinal cord. The neural patterning paradigm suggests that a canonical Wnt-signaling gradient acts along the anteroposterior axis to pattern the nervous system. Wnt activity is highest in the posterior, inducing spinal cord, at intermediate levels in the trunk, inducing hindbrain, and is lowest in anterior fated forebrain, while BMP-antagonist levels are constant along the axis. Our results in Xenopus laevis challenge this paradigm. We find that inhibition of canonical Wnt signaling or its downstream transcription factors eliminates hindbrain, but not spinal cord fates, an observation not compatible with a simple high-to-low Wnt gradient specifying all fates along the neural anteroposterior axis. Additionally, we find that BMP activity promotes posterior spinal cord cell fate formation in an FGF-dependent manner, while inhibiting hindbrain fates. These results suggest a need to re-evaluate the paradigms of neural anteroposterior pattern formation during vertebrate development.
Assuntos
Padronização Corporal , Proteínas Morfogenéticas Ósseas/metabolismo , Sistema Nervoso/embriologia , Sistema Nervoso/metabolismo , Neurogênese , Transdução de Sinais , Proteínas Wnt/metabolismo , Animais , Proteínas Morfogenéticas Ósseas/genética , Ectoderma/embriologia , Ectoderma/metabolismo , Fatores de Crescimento de Fibroblastos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Mesoderma/embriologia , Mesoderma/metabolismo , Rombencéfalo/embriologia , Rombencéfalo/metabolismo , Medula Espinal/embriologia , Medula Espinal/metabolismo , Xenopus laevisRESUMO
BACKGROUND: Occasionally, low-grade appendiceal mucinous neoplasms (LAMN) present with mucinous peritoneal deposits (MPD) localized to periappendiceal tissue or diffused throughout the peritoneum. OBJECTIVE: This study was aimed at evaluating the relevance of mucin cellularity for predicting outcomes of LAMN with remote MPD. METHODS: The records of patients with LAMN and remote MPD who underwent initial assessment at a comprehensive cancer center from 1990 to 2015 were reviewed, and diagnostic procedures, treatments, and outcomes were analyzed. RESULTS: Of 48 patients included in the analysis, 19 had cellular MPD (CMPD) and 29 had acellular MPD. Of 33 patients who underwent cytoreductive surgery, 30 had a complete cytoreduction; the 3 patients with an incomplete cytoreduction had CMPD. In the follow-up period (median, 4 years), 6 patients died of the disease, all of whom had CMPD. Of 11 patients who had progression of disease, 10 had CMPD. CONCLUSION: Cellularity of remote MPD is an important determinant of disease outcome in LAMN. Approaches such as active surveillance may have a role in selected patients with LAMN and AMPD.
Assuntos
Adenocarcinoma Mucinoso/mortalidade , Neoplasias do Apêndice/mortalidade , Procedimentos Cirúrgicos de Citorredução/mortalidade , Neoplasias Peritoneais/mortalidade , Adenocarcinoma Mucinoso/patologia , Adenocarcinoma Mucinoso/cirurgia , Idoso , Neoplasias do Apêndice/patologia , Neoplasias do Apêndice/cirurgia , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Gradação de Tumores , Neoplasias Peritoneais/patologia , Neoplasias Peritoneais/cirurgia , Taxa de SobrevidaRESUMO
The source of symmetry breaking in vertebrate oocytes is unknown. Animal-vegetal oocyte polarity is established by the Balbiani body (Bb), a conserved structure found in all animals examined that contains an aggregate of specific mRNAs, proteins, and organelles. The Bb specifies the oocyte vegetal pole, which is key to forming the embryonic body axes as well as the germline in most vertebrates. How Bb formation is regulated and how its asymmetric position is established are unknown. Using quantitative image analysis, we trace oocyte symmetry breaking in zebrafish to a nuclear asymmetry at the onset of meiosis called the chromosomal bouquet. The bouquet is a universal feature of meiosis where all telomeres cluster to one pole on the nuclear envelope, facilitating chromosomal pairing and meiotic recombination. We show that Bb precursor components first localize with the centrosome to the cytoplasm adjacent to the telomere cluster of the bouquet. They then aggregate around the centrosome in a specialized nuclear cleft that we identified, assembling the early Bb. We show that the bouquet nuclear events and the cytoplasmic Bb precursor localization are mechanistically coordinated by microtubules. Thus the animal-vegetal axis of the oocyte is aligned to the nuclear axis of the bouquet. We further show that the symmetry breaking events lay upstream to the only known regulator of Bb formation, the Bucky ball protein. Our findings link two universal features of oogenesis, the Bb and the chromosomal bouquet, to oocyte polarization. We propose that a meiotic-vegetal center couples meiosis and oocyte patterning. Our findings reveal a novel mode of cellular polarization in meiotic cells whereby cellular and nuclear polarity are aligned. We further reveal that in zygotene nests, intercellular cytoplasmic bridges remain between oocytes and that the position of the cytoplasmic bridge coincides with the location of the centrosome meiotic-vegetal organizing center. These results suggest that centrosome positioning is set by the last mitotic oogonial division plane. Thus, oocytes are polarized in two steps: first, mitotic divisions preset the centrosome with no obvious polarization yet, then the meiotic-vegetal center forms at zygotene bouquet stages, when symmetry is, in effect, broken.
Assuntos
Polaridade Celular , Cromossomos/fisiologia , Meiose , Oócitos/fisiologia , Animais , Centrossomo/fisiologia , Microtúbulos/fisiologia , Peixe-ZebraRESUMO
A mechanistic dissection of early oocyte differentiation in vertebrates is key to advancing our knowledge of germline development, reproductive biology, the regulation of meiosis, and all of their associated disorders. Recent advances in the field include breakthroughs in the identification of germline stem cells in Medaka, in the cellular architecture of the germline cyst in mice, in a mechanistic dissection of chromosomal pairing and bouquet formation in meiosis in mice, in tracing oocyte symmetry breaking to the chromosomal bouquet of meiosis in zebrafish, and in the biology of the Balbiani body, a universal oocyte granule. Many of the major events in early oogenesis are universally conserved, and some are co-opted for species-specific needs. The chromosomal events of meiosis are of tremendous consequence to gamete formation and have been extensively studied. New light is now being shed on other aspects of early oocyte differentiation, which were traditionally considered outside the scope of meiosis, and their coordination with meiotic events. The emerging theme is of meiosis as a common groundwork for coordinating multifaceted processes of oocyte differentiation. In an accompanying manuscript we describe methods that allowed for investigations in the zebrafish ovary to contribute to these breakthroughs. Here, we review these advances mostly from the zebrafish and mouse. We discuss oogenesis concepts across established model organisms, and construct an inclusive paradigm for early oocyte differentiation in vertebrates.
Assuntos
Diferenciação Celular/fisiologia , Polaridade Celular/fisiologia , Meiose/fisiologia , Mitose/fisiologia , Oogênese/fisiologia , Vertebrados/fisiologia , Células-Tronco Germinativas Adultas/citologia , Animais , Cromossomos/ultraestrutura , Cílios/fisiologia , Feminino , Camundongos , Modelos Biológicos , Organelas/fisiologia , Organelas/ultraestrutura , Oryzias/fisiologia , Ovário/citologia , Ovário/crescimento & desenvolvimento , Óvulo/citologia , Telômero/fisiologia , Xenopus laevis/fisiologia , Peixe-Zebra/fisiologiaRESUMO
Oocyte differentiation is a highly dynamic and intricate developmental process whose mechanistic understanding advances female reproduction, fertility, and ovarian cancer biology. Despite the many attributes of the zebrafish model, it has yet to be fully exploited for the investigation of early oocyte differentiation and ovarian development. This is partly because the properties of the adult zebrafish ovary make it technically challenging to access early stage oocytes. As a result, characterization of these stages has been lacking and tools for their analysis have been insufficient. To overcome these technical hurdles, we took advantage of the juvenile zebrafish ovary, where early stage oocytes can readily be found in high numbers and progress in a predictable manner. We characterized the earliest stages of oocyte differentiation and ovarian development and defined accurate staging criteria. We further developed protocols for quantitative microscopy, live time-lapse imaging, ovarian culture, and isolation of stage-specific oocytes for biochemical analysis. These methods have recently provided us with an unprecedented view of early oogenesis, allowing us to study formation of the Balbiani body, a universal oocyte granule that is associated with oocyte survival in mice and required for oocyte and egg polarity in fish and frogs. Despite its tremendous developmental significance, the Bb has been little investigated and how it forms was unknown in any species for over two centuries. We were able to trace Balbiani body formation and oocyte symmetry breaking to the onset of meiosis. Through this investigation we revealed novel cytoskeletal structures in oocytes and the contribution of specialized cellular organization to differentiation. Overall, the juvenile zebrafish ovary arises as an exciting model for studies of cell and developmental biology. We review these and other recent advances in vertebrate oogenesis in an accompanying manuscript in this issue of Developmental Biology. Here, we describe the protocols for ovarian investigation that we developed in the zebrafish, including all experimental steps that will easily allow others to reproduce such analysis. This juvenile ovary toolbox also contributes to establishing the zebrafish as a model for post-larval developmental stages.
Assuntos
Oogênese , Óvulo/fisiologia , Manejo de Espécimes/métodos , Imagem com Lapso de Tempo/métodos , Animais , Células Cultivadas , Citoesqueleto/ultraestrutura , DNA/análise , Feminino , Genes Reporter , Hibridização in Situ Fluorescente/métodos , Proteínas Luminescentes/análise , Proteínas Luminescentes/genética , Meiose , Microscopia Confocal/instrumentação , Microscopia Confocal/métodos , Oócitos/fisiologia , Oócitos/ultraestrutura , Organelas/fisiologia , Organelas/ultraestrutura , Ovário/citologia , Ovário/crescimento & desenvolvimento , Óvulo/química , Óvulo/ultraestrutura , RNA Mensageiro/análise , Processos de Determinação Sexual , Coloração e Rotulagem/métodos , Peixe-ZebraRESUMO
Cell polarity generates intracellular asymmetries and functional regionalization in tissues and morphogenetic processes. Cell polarity in development often relies on mechanisms of RNA localization to specific subcellular domains to define the identity of future developing tissues. The totipotent egg of most animals illustrates in a grand way the importance of cell polarity and RNA localization in regulating multiple crucial developmental events. The polarization of the egg arises during its development in oogenesis. RNAs localize asymmetrically in the early oocyte defining its animal-vegetal (AV) axis, which upon further elaboration in mid- and late-oogenesis stages produces a mature egg with specific localized factors along its AV axis. These localized factors will define the future anterior-posterior (AP) and dorsal-ventral (DV) axes of the embryo. Furthermore, AV polarity confines germ cell determinants to the vegetal pole, from where they redistribute to the cleavage furrows of the 2- and 4-cell stage embryo, ultimately specifying the primordial germ cells (PGCs). The sperm entry region during fertilization is also defined by the AV axis. In frogs and fish, sperm enters through the animal pole, similar to the mouse where it enters predominantly in the animal half. Thus, AV polarity establishment and RNA localization are involved in all the major events of early embryonic development. In this chapter, we will review the RNA localization mechanisms in vertebrate oocytes that are key to embryonic patterning, referring to some of the groundbreaking studies in frog oocytes and incorporating the current genetic evidence from the zebrafish.
Assuntos
Polaridade Celular/genética , Desenvolvimento Embrionário/genética , Oogênese/genética , RNA/genética , Animais , Fertilização/genética , Células Germinativas/crescimento & desenvolvimento , Células Germinativas/metabolismo , Camundongos , Morfogênese/genética , Oócitos/crescimento & desenvolvimento , Xenopus/embriologia , Peixe-Zebra/embriologiaRESUMO
During development, early inducing programs must later be counterbalanced for coordinated tissue maturation. In Xenopus laevis embryos, activation of the Meis3 transcription factor by a mesodermal Wnt3a signal lies at the core of the hindbrain developmental program. We now identify a hindbrain restricting circuit, surprisingly comprising the hindbrain inducers Wnt3a and Meis3, and Tsh1 protein. Functional and biochemical analyses show that upon Tsh1 induction by strong Wnt3a/Meis3 feedback loop activity, the Meis3-Tsh1 transcription complex represses the Meis3 promoter, allowing cell cycle exit and neuron differentiation. Meis3 protein exhibits a conserved dual-role in hindbrain development, both inducing neural progenitors and maintaining their proliferative state. In this regulatory circuit, the Tsh1 co-repressor controls transcription factor gene expression that modulates cell cycle exit, morphogenesis and differentiation, thus coordinating neural tissue maturation. This newly identified Wnt/Meis/Tsh circuit could play an important role in diverse developmental and disease processes.
Assuntos
Proteínas Correpressoras/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/metabolismo , Fatores de Transcrição/metabolismo , Proteína Wnt3A/metabolismo , Proteínas de Xenopus/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Animais , Diferenciação Celular , Perfilação da Expressão Gênica , Neurônios/metabolismo , Fases de Leitura Aberta , Plasmídeos/metabolismo , Rombencéfalo/metabolismo , Transcrição Gênica , Xenopus , Peixe-Zebra , beta Catenina/metabolismoRESUMO
In vertebrates, canonical Wnt signaling controls posterior neural cell lineage specification. Although Wnt signaling to the neural plate is sufficient for posterior identity, the source and timing of this activity remain uncertain. Furthermore, crucial molecular targets of this activity have not been defined. Here, we identify the endogenous Wnt activity and its role in controlling an essential downstream transcription factor, Meis3. Wnt3a is expressed in a specialized mesodermal domain, the paraxial dorsolateral mesoderm, which signals to overlying neuroectoderm. Loss of zygotic Wnt3a in this region does not alter mesoderm cell fates, but blocks Meis3 expression in the neuroectoderm, triggering the loss of posterior neural fates. Ectopic Meis3 protein expression is sufficient to rescue this phenotype. Moreover, Wnt3a induction of the posterior nervous system requires functional Meis3 in the neural plate. Using ChIP and promoter analysis, we show that Meis3 is a direct target of Wnt/beta-catenin signaling. This suggests a new model for neural anteroposterior patterning, in which Wnt3a from the paraxial mesoderm induces posterior cell fates via direct activation of a crucial transcription factor in the overlying neural plate.
Assuntos
Proteínas de Homeodomínio/metabolismo , Mesoderma/embriologia , Placa Neural/embriologia , Transdução de Sinais/fisiologia , Proteínas Wnt/metabolismo , Proteínas de Xenopus/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Animais , Imunoprecipitação da Cromatina , Proteínas de Homeodomínio/genética , Hibridização In Situ , Técnicas In Vitro , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/genética , Proteínas Wnt/genética , Proteína Wnt3 , Proteína Wnt3A , Proteínas de Xenopus/genética , Xenopus laevis , Proteínas de Peixe-Zebra/genética , beta Catenina/genética , beta Catenina/metabolismoRESUMO
Meiotic chromosomal pairing is facilitated by a conserved cytoskeletal organization. Telomeres associate with perinuclear microtubules via Sun/KASH complexes on the nuclear envelope (NE) and dynein. Telomere sliding on perinuclear microtubules contributes to chromosome homology searches and is essential for meiosis. Telomeres ultimately cluster on the NE, facing the centrosome, in a configuration called the chromosomal bouquet. Here, we discuss novel components and functions of the bouquet microtubule organizing center (MTOC) in meiosis, but also broadly in gamete development. The cellular mechanics of chromosome movements and the bouquet MTOC dynamics are striking. The newly identified zygotene cilium mechanically anchors the bouquet centrosome and completes the bouquet MTOC machinery in zebrafish and mice. We hypothesize that various centrosome anchoring strategies evolved in different species. Evidence suggests that the bouquet MTOC machinery is a cellular organizer, linking meiotic mechanisms with gamete development and morphogenesis. We highlight this cytoskeletal organization as a new platform for creating a holistic understanding of early gametogenesis, with direct implications to fertility and reproduction.
Assuntos
Meiose , Peixe-Zebra , Animais , Camundongos , Telômero , Centro Organizador dos Microtúbulos , Centrossomo , Reprodução , Células GerminativasRESUMO
The longevity of sperm in teleost such as zebrafish and medaka is short when isolated even in saline-balanced solution at a physiological temperature. In contrast, some internal fertilizers exhibit the long-term storage of sperm, >10 months, in the female reproductive tract. This evidence implies that sperm in teleost possesses the ability to survive for a long time under suitable conditions; however, these conditions are not well understood. In this study, we show that the sperm of zebrafish can survive and maintain fertility in L-15-based storage medium supplemented with bovine serum albumin, fetal bovine serum, glucose, and lactic acid for 28 days at room temperature. The fertilized embryos developed to normal fertile adults. This storage medium was effective in medaka sperm stored for 7 days at room temperature. These results suggest that sperm from external fertilizer zebrafish and medaka has the ability to survive for at least 4 and 1 week, respectively, in the body fluid-like medium at a physiological temperature. This sperm storage method allows researchers to ship sperm by low-cost methods and to investigate key factors for motility and fertile ability in those sperm.
Assuntos
Oryzias , Preservação do Sêmen , Masculino , Feminino , Animais , Peixe-Zebra , Oryzias/fisiologia , Temperatura , Sêmen , Espermatozoides/fisiologia , Preservação do Sêmen/veterinária , Preservação do Sêmen/métodos , Motilidade dos Espermatozoides/fisiologiaRESUMO
Oogenesis produces functional eggs and is essential for fertility, embryonic development, and reproduction. The zebrafish ovary is an excellent model to study oogenesis in vertebrates, and recent studies have identified multiple regulators in oocyte development through forward genetic screens, as well as reverse genetics by CRISPR mutagenesis. However, many developmental steps in oogenesis, in zebrafish and other species, remain poorly understood, and their underlying mechanisms are unknown. Here, we take a genomic approach to systematically uncover biological activities throughout oogenesis. We performed transcriptomic analysis on five stages of oogenesis, from the onset of oocyte differentiation through Stage III, which precedes oocyte maturation. These transcriptomes revealed thousands of differentially expressed genes across stages of oogenesis. We analyzed trends of gene expression dynamics along oogenesis, as well as their expression in pair-wise comparisons between stages. We determined their functionally enriched terms, identifying uniquely characteristic biological activities in each stage. These data identified two prominent developmental phases in oocyte differentiation and traced the accumulation of maternally deposited embryonic regulator transcripts in the developing oocyte. Our analysis provides the first molecular description for oogenesis in zebrafish, which we deposit online as a resource for the community. Further, the presence of multiple gene paralogs in zebrafish, and the exclusive curation by many bioinformatic tools of the single paralogs present in humans, challenge zebrafish genomic analyses. We offer an approach for converting zebrafish gene name nomenclature to the human nomenclature for supporting genomic analyses generally in zebrafish. Altogether, our work provides a valuable resource as a first step to uncover oogenesis mechanisms and candidate regulators and track accumulating transcripts of maternal regulators of embryonic development.
RESUMO
A hallmark of meiosis is chromosomal pairing, which requires telomere tethering and rotation on the nuclear envelope through microtubules, driving chromosome homology searches. Telomere pulling toward the centrosome forms the "zygotene chromosomal bouquet." Here, we identified the "zygotene cilium" in oocytes. This cilium provides a cable system for the bouquet machinery and extends throughout the germline cyst. Using zebrafish mutants and live manipulations, we demonstrate that the cilium anchors the centrosome to counterbalance telomere pulling. The cilium is essential for bouquet and synaptonemal complex formation, oogenesis, ovarian development, and fertility. Thus, a cilium represents a conserved player in zebrafish and mouse meiosis, which sheds light on reproductive aspects in ciliopathies and suggests that cilia can control chromosomal dynamics.
Assuntos
Pareamento Cromossômico , Cílios , Oócitos , Oogênese , Ovário , Animais , Centrômero/genética , Centrômero/fisiologia , Pareamento Cromossômico/genética , Pareamento Cromossômico/fisiologia , Cílios/fisiologia , Feminino , Fertilidade/fisiologia , Camundongos , Morfogênese , Oócitos/crescimento & desenvolvimento , Oogênese/genética , Oogênese/fisiologia , Ovário/crescimento & desenvolvimento , Telômero/genética , Telômero/fisiologia , Peixe-Zebra/genética , Peixe-Zebra/fisiologiaRESUMO
In Xenopus embryos, XMeis3 protein activity is required for normal hindbrain formation. Our results show that XMeis3 protein knock down also causes a loss of primary neuron and neural crest cell lineages, without altering expression of Zic, Sox or Pax3 genes. Knock down or inhibition of the Pax3, Zic1 or Zic5 protein activities extinguishes embryonic expression of the XMeis3 gene, as well as triggering the loss of hindbrain, neural crest and primary neuron cell fates. Ectopic XMeis3 expression can rescue the Zic knock down phenotype. HoxD1 is an XMeis3 direct-target gene, and ectopic HoxD1 expression rescues cell fate losses in either XMeis3 or Zic protein knock down embryos. FGF3 and FGF8 are direct target genes of XMeis3 protein and their expression is lost in XMeis3 morphant embryos. In the genetic cascade controlling embryonic neural cell specification, XMeis3 lies below general-neuralizing, but upstream of FGF and regional-specific genes. Thus, XMeis3 protein is positioned at a key regulatory point, simultaneously regulating multiple neural cell fates during early vertebrate nervous system development.
Assuntos
Linhagem da Célula , Proteínas de Homeodomínio/metabolismo , Sistema Nervoso/citologia , Sistema Nervoso/embriologia , Fatores de Transcrição Box Pareados/metabolismo , Fatores de Transcrição/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/embriologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Biomarcadores/metabolismo , Embrião não Mamífero/citologia , Embrião não Mamífero/metabolismo , Fator 3 de Crescimento de Fibroblastos/metabolismo , Fator 8 de Crescimento de Fibroblasto/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Genes Dominantes/genética , Proteínas de Homeodomínio/genética , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Sistema Nervoso/metabolismo , Crista Neural/citologia , Crista Neural/embriologia , Crista Neural/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Fator de Transcrição PAX3 , Fatores de Transcrição Box Pareados/genética , Fenótipo , Fatores de Transcrição/genética , Proteínas de Xenopus/genética , Xenopus laevis/genéticaRESUMO
Ample evidence has accumulated in recent years to establish that most, if not all, nuclear proto-oncogenes are in fact sequence-specific DNA-binding proteins that modulate gene expression. Their synthesis or activity is modulated by extracellular signals or by cross talk between different classes of transcription factors.
Assuntos
Núcleo Celular/fisiologia , Oncogenes , Proteínas Proto-Oncogênicas/genética , Proto-Oncogenes , Animais , Sequência de Bases , Oligonucleotídeos/metabolismo , Proteínas Tirosina Quinases/genética , Fatores de Transcrição/metabolismoRESUMO
Oocyte production is crucial for sexual reproduction. Recent findings in zebrafish and other established model organisms emphasize that the early steps of oogenesis involve the coordination of simultaneous and tightly sequential processes across cellular compartments and between sister cells. To fully understand the mechanistic framework of these coordinated processes, cellular and morphological analysis in high temporal resolution is required. Here, we provide a protocol for four-dimensional live time-lapse analysis of cultured juvenile zebrafish ovaries. We describe how multiple-stage oocytes can be simultaneously analyzed in single ovaries, and several ovaries can be processed in single experiments. In addition, we detail adequate conditions for quantitative image acquisition. Finally, we demonstrate that using this protocol, we successfully capture rapid meiotic chromosomal movements in early prophase for the first time in zebrafish oocytes, in four dimensions and in vivo. Our protocol expands the use of the zebrafish as a model system to understand germ cell and ovarian development in postembryonic stages.
Assuntos
Cromossomos/fisiologia , Meiose/fisiologia , Oogênese/fisiologia , Ovário/fisiologia , Imagem com Lapso de Tempo/métodos , Peixe-Zebra/fisiologia , Animais , Feminino , Oócitos , Diferenciação Sexual/fisiologiaRESUMO
The terminal components of the complement system contribute to host defense by forming the multiprotein membrane attack complex (MAC) which is responsible for cell lysis and several noncytotoxic effects. Most of the complement proteins are synthesized in the liver, but the mechanisms controlling their tissue-specific expression have not been elucidated. In this study we show that mice lacking the hepatic transcription factor hepatocyte nuclear factor 1alpha (HNF1alpha) fail to transcribe C5 and C8A complement genes. In addition, mRNAs encoding for several other terminal complement components or subunits are expressed at lower levels, including C8beta, C8gamma, and C9. We next used a reconstitution assay involving human sera with selective complement deficiencies to assess mouse complement activity. Sera from HNF1alpha-deficient mice showed negligible hemolytic activity of both C5 and C8alpha-gamma subunits. The activity of C8beta was severely affected despite only a 50% reduction in C8beta mRNA levels in the liver. This is reminiscent of C8alpha-gamma-deficient patients who accumulate extremely low levels of the C8beta subunit. Our results demonstrate that HNF1alpha plays a key role in the expression of C5 and C8A genes, two terminal complement component genes that are essential for the assembly of MAC as a result of complement activation.
Assuntos
Complemento C5/genética , Complemento C8/genética , Proteínas de Ligação a DNA , Regulação da Expressão Gênica , Proteínas Nucleares/fisiologia , Fatores de Transcrição/fisiologia , Animais , Sequência de Bases , Cromatina , Complemento C5/imunologia , Complemento C8/imunologia , DNA Complementar , Testes Genéticos , Fator 1 Nuclear de Hepatócito , Fator 1-alfa Nuclear de Hepatócito , Fator 1-beta Nuclear de Hepatócito , Humanos , Fígado/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Dados de Sequência Molecular , Proteínas Nucleares/genética , Fatores de Transcrição/genética , Transcrição GênicaRESUMO
Biochemical and genetic evidence suggest that the SWI/SNF complex is involved in the remodeling of chromatin during gene activation. We have used antibodies specific against three human subunits of this complex to study its subnuclear localization, as well as its potential association with active chromatin and the nuclear skeleton. Immunofluorescence studies revealed a punctate nuclear labeling pattern that was excluded from the nucleoli and from regions of condensed chromatin. Dual labeling failed to reveal significant colocalization of BRG1 or hBRM proteins with RNA polymerase II or with nuclear speckles involved in splicing. Chromatin fractionation experiments showed that both soluble and insoluble active chromatin are enriched in the hSWI/SNF proteins as compared with bulk chromatin. hSWI/SNF proteins were also found to be associated with the nuclear matrix or nuclear scaffold, suggesting that a fraction of the hSWI/SNF complex could be involved in the chromatin organization properties associated with matrix attachment regions.