RESUMO
To accommodate the large cells following zygote formation, early blastomeres employ modified cell divisions. Karyomeres are one such modification, mitotic intermediates wherein individual chromatin masses are surrounded by nuclear envelope; the karyomeres then fuse to form a single mononucleus. We identified brambleberry, a maternal-effect zebrafish mutant that disrupts karyomere fusion, resulting in formation of multiple micronuclei. As karyomeres form, Brambleberry protein localizes to the nuclear envelope, with prominent puncta evident near karyomere-karyomere interfaces corresponding to membrane fusion sites. brambleberry corresponds to an unannotated gene with similarity to Kar5p, a protein that participates in nuclear fusion in yeast. We also demonstrate that Brambleberry is required for pronuclear fusion following fertilization in zebrafish. Our studies provide insight into the machinery required for karyomere fusion and suggest that specialized proteins are necessary for proper nuclear division in large dividing blastomeres.
Assuntos
Embrião não Mamífero/metabolismo , Membrana Nuclear/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Sequência de Aminoácidos , Animais , Blastômeros/metabolismo , Ciclo Celular , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Embrião não Mamífero/citologia , Feminino , Humanos , Insetos/citologia , Insetos/embriologia , Insetos/metabolismo , Masculino , Mamíferos/embriologia , Mamíferos/metabolismo , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Alinhamento de Sequência , Fuso Acromático/metabolismo , Peixe-Zebra/metabolismo , Zigoto/citologia , Zigoto/metabolismoRESUMO
Maternally-loaded factors in the egg accumulate during oogenesis and are essential for the acquisition of oocyte and egg developmental competence to ensure the production of viable embryos. However, their molecular nature and functional importance remain poorly understood. Here, we present a collection of 9 recessive maternal-effect mutants identified in a zebrafish forward genetic screen that reveal unique molecular insights into the mechanisms controlling the vertebrate oocyte-to-embryo transition. Four genes, over easy, p33bjta, poached and black caviar, were found to control initial steps in yolk globule sizing and protein cleavage during oocyte maturation that act independently of nuclear maturation. The krang, kazukuram, p28tabj, and spotty genes play distinct roles in egg activation, including cortical granule biology, cytoplasmic segregation, the regulation of microtubule organizing center assembly and microtubule nucleation, and establishing the basic body plan. Furthermore, we cloned two of the mutant genes, identifying the over easy gene as a subunit of the Adaptor Protein complex 5, Ap5m1, which implicates it in regulating intracellular trafficking and yolk vesicle formation. The novel maternal protein Krang/Kiaa0513, highly conserved in metazoans, was discovered and linked to the function of cortical granules during egg activation. These mutant genes represent novel genetic entry points to decipher the molecular mechanisms functioning in the oocyte-to-embryo transition, fertility, and human disease. Additionally, our genetic adult screen not only contributes to the existing knowledge in the field but also sets the basis for future investigations. Thus, the identified maternal genes represent key players in the coordination and execution of events prior to fertilization.
Assuntos
Oócitos , Oogênese , Proteínas de Peixe-Zebra , Peixe-Zebra , Animais , Peixe-Zebra/genética , Oócitos/metabolismo , Oócitos/crescimento & desenvolvimento , Oogênese/genética , Feminino , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Herança Materna/genética , Mutação , Embrião não Mamífero , Desenvolvimento Embrionário/genéticaRESUMO
Pattern formation by bone morphogenetic proteins (BMPs) demonstrates remarkable plasticity and utility in several contexts, such as early embryonic development, tissue patterning and the maintenance of stem cell niches. BMPs pattern tissues over many temporal and spatial scales: BMP gradients as short as 1-2 cell diameters maintain the stem cell niche of the Drosophila germarium over a 24-h cycle, and BMP gradients of several hundred microns establish dorsal-ventral tissue specification in Drosophila, zebrafish and Xenopus embryos in timescales between 30â min and several hours. The mechanisms that shape BMP signaling gradients are also incredibly diverse. Although ligand diffusion plays a dominant role in forming the gradient, a cast of diffusible and non-diffusible regulators modulate gradient formation and confer robustness, including scale invariance and adaptability to perturbations in gene expression and growth. In this Review, we document the diverse ways that BMP gradients are formed and refined, and we identify the core principles that they share to achieve reliable performance.
Assuntos
Padronização Corporal/fisiologia , Desenvolvimento Ósseo , Proteínas Morfogenéticas Ósseas/metabolismo , Desenvolvimento Embrionário , Animais , Padronização Corporal/genética , Proteínas Morfogenéticas Ósseas/genética , Osso e Ossos , Drosophila/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Embrião não Mamífero/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Transdução de Sinais , Xenopus laevis/embriologia , Xenopus laevis/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/metabolismo , Proteínas de Peixe-ZebraRESUMO
In the 1990s, labs on both sides of the Atlantic performed the largest genetic mutagenesis screen at that time using an emerging model organism: the zebrafish. Led by Christiane Nüsslein-Volhard in Tübingen, Germany, and Wolfgang Driever in Boston, USA, these colossal screens culminated in 1996 with the publication of 37 articles in a special issue of Development, which remains the journal's largest issue to this day. To celebrate the anniversary of the zebrafish issue and reflect on the 25â years since its publication, five zebrafish researchers share what the issue means to them, how it has contributed to their career and its impact on the zebrafish community.
Assuntos
Modelos Animais , Mutagênese/genética , Peixe-Zebra/genética , Animais , HumanosRESUMO
Bone Morphogenetic Protein (BMP) patterns the dorsal-ventral (DV) embryonic axis in all vertebrates, but it is unknown how cells along the DV axis interpret and translate the gradient of BMP signaling into differential gene activation that will give rise to distinct cell fates. To determine the mechanism of BMP morphogen interpretation in the zebrafish gastrula, we identified 57 genes that are directly activated by BMP signaling. By using Seurat analysis of single-cell RNA sequencing (scRNA-seq) data, we found that these genes are expressed in at least 3 distinct DV domains of the embryo. We distinguished between 3 models of BMP signal interpretation in which cells activate distinct gene expression through interpretation of thresholds of (1) the BMP signaling gradient slope; (2) the BMP signal duration; or (3) the level of BMP signal activation. We tested these 3 models using quantitative measurements of phosphorylated Smad5 (pSmad5) and by examining the spatial relationship between BMP signaling and activation of different target genes at single-cell resolution across the embryo. We found that BMP signaling gradient slope or BMP exposure duration did not account for the differential target gene expression domains. Instead, we show that cells respond to 3 distinct levels of BMP signaling activity to activate and position target gene expression. Together, we demonstrate that distinct pSmad5 threshold levels activate spatially distinct target genes to pattern the DV axis.
Assuntos
Padronização Corporal/genética , Proteínas Morfogenéticas Ósseas/genética , Proteínas Morfogenéticas Ósseas/metabolismo , Animais , Animais Geneticamente Modificados , Embrião não Mamífero , Gástrula/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Fosforilação , Transdução de Sinais/genética , Proteína Smad5/metabolismo , Distribuição Tecidual/genética , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
A qualitative systematic review was conducted to evaluate pre-exposure prophylaxis (PrEP) interventions, describe characteristics of best practices for increasing PrEP use and persistence, and explore research gaps based on current PrEP interventions. We searched CDC's Prevention Research Synthesis (PRS) Project's cumulative HIV database (includes CINAHL, EMBASE, Global Health, MEDLINE, PsycInfo, and Sociological Abstracts) to identify PrEP intervention studies conducted in the U.S., published between 2000 and 2022 (last searched January 2023). Eligibility criteria include studies that evaluated PrEP interventions for persons testing negative for HIV infection, or for healthcare providers who prescribed PrEP; included comparisons between groups or pre/post; and reported at least one relevant PrEP outcome. Each eligible intervention was evaluated on the quality of study design, implementation, analysis, and strength of evidence (PROSPERO registration number: CRD42021256460). Of the 26 eligible interventions, the majority were focused on men who have sex with men (n = 18) and reported PrEP adherence outcomes (n = 12). Nine interventions met the criteria for Best Practices (i.e., evidence-based interventions, evidence-informed interventions). Five were digital health interventions while two implemented individual counseling, one offered motivational interviewing, and one provided integrated medical care with a PrEP peer navigator. Longer intervention periods may provide more time for intervention exposure to facilitate behavioral change, and engaging the community when developing, designing and implementing interventions may be key for effectiveness. For digital health interventions, two-way messaging may help participants feel supported. Research gaps included a lack of Best Practices for several populations (e.g., Black persons, Hispanic/Latino persons, persons who inject drugs, and women of color) and evidence for various intervention strategies (e.g., interventions for promoting provider's PrEP prescription behavior, peer support). These findings call for more collaborative work with communities to develop interventions that work and implement and disseminate Best Practices for increasing PrEP use and persistence in communities.
Assuntos
Fármacos Anti-HIV , Infecções por HIV , Profilaxia Pré-Exposição , Humanos , Infecções por HIV/prevenção & controle , Estados Unidos , Fármacos Anti-HIV/uso terapêutico , Fármacos Anti-HIV/administração & dosagem , Adesão à Medicação , Masculino , Feminino , Guias de Prática Clínica como AssuntoRESUMO
Heterodimeric TGF-ß ligands outperform homodimers in a variety of developmental, cell culture, and therapeutic contexts; however, the mechanisms underlying this increased potency remain uncharacterized. Here, we use dorsal-ventral axial patterning of the zebrafish embryo to interrogate the BMP2/7 heterodimer signaling mechanism. We demonstrate that differential interactions with BMP antagonists do not account for the reduced signaling ability of homodimers. Instead, we find that while overexpressed BMP2 homodimers can signal, they require two nonredundant type I receptors, one from the Acvr1 subfamily and one from the Bmpr1 subfamily. This implies that all BMP signaling within the zebrafish gastrula, even BMP2 homodimer signaling, requires Acvr1. This is particularly surprising as BMP2 homodimers do not bind Acvr1 in vitro. Furthermore, we find that the roles of the two type I receptors are subfunctionalized within the heterodimer signaling complex, with the kinase activity of Acvr1 being essential, while that of Bmpr1 is not. These results suggest that the potency of the Bmp2/7 heterodimer arises from the ability to recruit both Acvr1 and Bmpr1 into the same signaling complex.
Assuntos
Proteína Morfogenética Óssea 2/metabolismo , Proteína Morfogenética Óssea 7/metabolismo , Receptores de Proteínas Morfogenéticas Ósseas/metabolismo , Transdução de Sinais , Proteínas de Peixe-Zebra/metabolismo , Receptores de Ativinas Tipo I/metabolismo , Animais , Proteína Morfogenética Óssea 2/genética , Proteína Morfogenética Óssea 7/genética , Receptores de Proteínas Morfogenéticas Ósseas/genética , Gástrula/metabolismo , Mutação , Ligação Proteica , Multimerização Proteica , Peixe-Zebra , Proteínas de Peixe-Zebra/genéticaRESUMO
BACKGROUND: Proteins of the TGFß family, which are largely studied as homodimers, are also known to form heterodimers with biological activity distinct from their component homodimers. For instance, heterodimers of bone morphogenetic proteins, including BMP2/BMP7, BMP2/BMP6, and BMP9/BMP10, among others, have illustrated the importance of these heterodimeric proteins within the context of TGFß signaling. RESULTS: In this study, we have determined that mature GDF5 can be combined with mature BMP2 or BMP4 to form BMP2/GDF5 and BMP4/GDF5 heterodimer. Intriguingly, this combination of a BMP2 or BMP4 monomer, which exhibit high affinity to heparan sulfate characteristic to the BMP class, with a GDF5 monomer with low heparan sulfate affinity produces a heterodimer with an intermediate affinity. Using heparin affinity chromatography to purify the heterodimeric proteins, we then determined that both the BMP2/GDF5 and BMP4/GDF5 heterodimers consistently signaled potently across an array of cellular and in vivo systems, while the activities of their homodimeric counterparts were more context dependent. These differences were likely driven by an increase in the combined affinities for the type 1 receptors, Alk3 and Alk6. Furthermore, the X-ray crystal structure of BMP2/GDF5 heterodimer was determined, highlighting the formation of two asymmetric type 1 receptor binding sites that are both unique relative to the homodimers. CONCLUSIONS: Ultimately, this method of heterodimer production yielded a signaling molecule with unique properties relative to the homodimeric ligands, including high affinity to multiple type 1 and moderate heparan binding affinity.
Assuntos
Proteína Morfogenética Óssea 2 , Proteínas Morfogenéticas Ósseas , Proteínas Morfogenéticas Ósseas/metabolismo , Proteína Morfogenética Óssea 2/genética , Proteína Morfogenética Óssea 2/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Ligação Proteica , Proteínas de Transporte/metabolismo , Heparitina SulfatoRESUMO
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
Forward genetic screens remain at the forefront of biology as an unbiased approach for discovering and elucidating gene function at the organismal and molecular level. Past mutagenesis screens targeting maternal-effect genes identified a broad spectrum of phenotypes ranging from defects in oocyte development to embryonic patterning. However, earlier vertebrate screens did not reach saturation, anticipated classes of phenotypes were not uncovered, and technological limitations made it difficult to pinpoint the causal gene. In this study, we performed a chemically-induced maternal-effect mutagenesis screen in zebrafish and identified eight distinct mutants specifically affecting the cleavage stage of development and one cleavage stage mutant that is also male sterile. The cleavage-stage phenotypes fell into three separate classes: developmental arrest proximal to the mid blastula transition (MBT), irregular cleavage, and cytokinesis mutants. We mapped each mutation to narrow genetic intervals and determined the molecular basis for two of the developmental arrest mutants, and a mutation causing male sterility and a maternal-effect mutant phenotype. One developmental arrest mutant gene encodes a maternal specific Stem Loop Binding Protein, which is required to maintain maternal histone levels. The other developmental arrest mutant encodes a maternal-specific subunit of the Minichromosome Maintenance Protein Complex, which is essential for maintaining normal chromosome integrity in the early blastomeres. Finally, we identify a hypomorphic allele of Polo-like kinase-1 (Plk-1), which results in a male sterile and maternal-effect phenotype. Collectively, these mutants expand our molecular-genetic understanding of the maternal regulation of early embryonic development in vertebrates.
Assuntos
Divisão Celular/genética , Desenvolvimento Embrionário/genética , Herança Materna/genética , Mutação , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Alelos , Animais , Blástula/citologia , Blástula/embriologia , Blástula/metabolismo , Padronização Corporal/genética , Núcleo Celular , Citocinese/genética , Feminino , Infertilidade Masculina/genética , Masculino , Mutagênese , Fenótipo , Proteínas de Peixe-Zebra/genéticaRESUMO
Numerous stages of organismal development rely on the cellular interpretation of gradients of secreted morphogens including members of the Bone Morphogenetic Protein (BMP) family through transmembrane receptors. Early gradients of BMPs drive dorsal/ventral patterning throughout the animal kingdom in both vertebrates and invertebrates. Growing evidence in Drosophila, zebrafish, murine and other systems suggests that BMP ligand heterodimers are the primary BMP signaling ligand, even in systems in which mixtures of BMP homodimers and heterodimers are present. Signaling by heterodimers occurs through a hetero-tetrameric receptor complex comprising of two distinct type one BMP receptors and two type II receptors. To understand the system dynamics and determine whether kinetic assembly of heterodimer-heterotetramer BMP complexes is favored, as compared to other plausible BMP ligand-receptor configurations, we developed a kinetic model for BMP tetramer formation based on current measurements for binding rates and affinities. We find that contrary to a common hypothesis, heterodimer-heterotetramer formation is not kinetically favored over the formation of homodimer-tetramer complexes under physiological conditions of receptor and ligand concentrations and therefore other mechanisms, potentially including differential kinase activities of the formed heterotetramer complexes, must be the cause of heterodimer-heterotetramer signaling primacy. Further, although BMP complex assembly favors homodimer and homomeric complex formation over a wide range of parameters, ignoring these signals and instead relying on the heterodimer improves the range of morphogen interpretation in a broad set of conditions, suggesting a performance advantage for heterodimer signaling in patterning multiple cell types in a gradient.
Assuntos
Proteínas Morfogenéticas Ósseas/química , Proteínas Morfogenéticas Ósseas/metabolismo , Modelos Biológicos , Animais , Fenômenos Biofísicos , Receptores de Proteínas Morfogenéticas Ósseas/metabolismo , Biologia Computacional , Simulação por Computador , Ligantes , Modelos Moleculares , Morfogênese , Multimerização Proteica , Estrutura Quaternária de Proteína , Transdução de SinaisRESUMO
Hippo signaling is a critical pathway that integrates extrinsic and intrinsic mechanical cues to regulate organ size. Despite its essential role in organogenesis, little is known about its role in cell fate specification and differentiation. Here, we unravel a novel and unexpected role of the Hippo pathway effector Taz (wwtr1) in controlling the size, shape and fate of a unique cell in the zebrafish ovary. We show that wwtr1 mutant females are infertile. In teleosts, fertilization occurs through the micropyle, a funnel-like opening in the chorion, formed by a unique enlarged follicle cell, the micropylar cell (MC). We describe here, for the first time, the mechanism that underlies the differentiation of the MC. Our genetic analyses show that Taz is essential for MC fate acquisition and subsequent micropyle formation in zebrafish. We identify Taz as the first bona fide MC marker and show that Taz is specifically and strongly enriched in the MC precursor. Altogether, we performed the first genetic and molecular characterization of the MC and propose that Taz is a key regulator of MC fate.This article has an associated 'The people behind the papers' interview.
Assuntos
Fertilização , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Morfogênese , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/crescimento & desenvolvimento , Peixe-Zebra/metabolismo , Junções Aderentes/efeitos dos fármacos , Junções Aderentes/metabolismo , Animais , Biomarcadores/metabolismo , Polaridade Celular/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Citocalasina D/farmacologia , Feminino , Fertilização/efeitos dos fármacos , Infertilidade Feminina/genética , Infertilidade Feminina/patologia , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Modelos Biológicos , Morfogênese/efeitos dos fármacos , Mutação/genética , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Oócitos/patologia , Óvulo/efeitos dos fármacos , Óvulo/metabolismo , Serina-Treonina Quinase 3 , Junções Íntimas/efeitos dos fármacos , Junções Íntimas/metabolismo , Proteínas com Motivo de Ligação a PDZ com Coativador TranscricionalRESUMO
ABSTRACT: Cannabis dabbing refers to the recreational inhalation of extremely concentrated tetrahydrocannabinol, the main psychotropic cannabinoid derived from the marijuana plant. The practice carries significant health and legal risks. This article discusses what nurses need to know about dabbing and how they can educate patients who may be engaging in risky behavior.
Assuntos
Uso da Maconha/epidemiologia , Relações Enfermeiro-Paciente , Educação de Pacientes como Assunto , Administração por Inalação , Humanos , Uso da Maconha/efeitos adversos , Uso da Maconha/legislação & jurisprudência , Uso da Maconha/psicologia , Assunção de Riscos , Estados Unidos/epidemiologiaRESUMO
BACKGROUND: This article summarizes the results from systematic reviews of human immunodeficiency virus (HIV) prevention interventions for people who use drugs (PWUD). We performed an overview of reviews, meta-analysis, meta-epidemiology, and PROSPERO Registration CRD42017070117. METHODS: We conducted a comprehensive systematic literature search using the Centers for Disease Control and Prevention HIV/AIDS Prevention Research Synthesis Project database to identify quantitative systematic reviews of HIV public heath interventions with PWUD published during 2002-2017. We recombined results of US studies across reviews to quantify effects on HIV infections, continuum of HIV care, sexual risk, and 5 drug-related outcomes (sharing injection equipment, injection frequency, opioid use, general drug use, and participation in drug treatment). We conducted summary meta-analyses separately for reviews of randomized controlled trials (RCTs) and quasi-experiments. We stratified effects by 5 intervention types: behavioral-psychosocial (BPS), syringe service programs (SSP), opioid agonist therapy (OAT), financial and scheduling incentives (FSI), and case management (CM). RESULTS: We identified 16 eligible reviews includingâ >140 US studies withâ >55â 000 participants. Summary effects among US studies were significant and favorable for 4 of 5 outcomes measured under RCT (eg, reduced opioid use; odds ratio [OR]â =â 0.70, confidence interval [CI]â =â 0.56-0.89) and all 6 outcomes under quasi-experiments (eg, reduced HIV infection [ORâ =â 0.42, CIâ =â 0.27-0.63]; favorable continuum of HIV care [ORâ =â 0.68, CIâ =â 0.53-0.88]). Each intervention type showed effectiveness on 1-6 outcomes. Heterogeneity was moderate to none for RCT but moderate to high for quasi-experiments. CONCLUSIONS: Behavioral-psychosocial, SSP, OAT, FSI, and CM interventions are effective in reducing risk of HIV and sequelae of injection and other drug use, and they have a continuing role in addressing the opioid crisis and Ending the HIV Epidemic.
Assuntos
Usuários de Drogas/estatística & dados numéricos , Infecções por HIV/prevenção & controle , Serviços Preventivos de Saúde/organização & administração , Transtornos Relacionados ao Uso de Substâncias/reabilitação , Terapia Comportamental/métodos , Administração de Caso/organização & administração , Usuários de Drogas/psicologia , Infecções por HIV/epidemiologia , Infecções por HIV/transmissão , Humanos , Metanálise como Assunto , Uso Comum de Agulhas e Seringas , Tratamento de Substituição de Opiáceos/métodos , Epidemia de Opioides/prevenção & controle , Epidemia de Opioides/estatística & dados numéricos , Sistemas de Apoio Psicossocial , Ensaios Clínicos Controlados Aleatórios como Assunto , Transtornos Relacionados ao Uso de Substâncias/complicações , Transtornos Relacionados ao Uso de Substâncias/epidemiologia , Transtornos Relacionados ao Uso de Substâncias/psicologia , Revisões Sistemáticas como Assunto , Estados Unidos/epidemiologiaRESUMO
Bone Morphogenetic Proteins (BMPs) play an important role in dorsal-ventral (DV) patterning of the early zebrafish embryo. BMP signaling is regulated by a network of extracellular and intracellular factors that impact the range and signaling of BMP ligands. Recent advances in understanding the mechanism of pattern formation support a source-sink mechanism, however it is not clear how the source-sink mechanism shapes patterns in 3D, nor how sensitive the pattern is to biophysical rates and boundary conditions along both the anteroposterior (AP) and DV axes of the embryo. We propose a new three-dimensional growing Partial Differential Equation (PDE)-based model to simulate the BMP patterning process during the blastula stage. This model provides a starting point to elucidate how different mechanisms and components work together in 3D to create and maintain the BMP gradient in the embryo. We also show how the 3D model fits the BMP signaling gradient data at multiple time points along both axes. Furthermore, sensitivity analysis of the model suggests that the spatiotemporal patterns of Chordin and BMP ligand gene expression are dominant drivers of shape in 3D and more work is needed to quantify the spatiotemporal profiles of gene and protein expression to further refine the models.
Assuntos
Blástula/embriologia , Padronização Corporal/fisiologia , Proteínas Morfogenéticas Ósseas/metabolismo , Modelos Biológicos , Proteínas de Peixe-Zebra/metabolismo , Animais , Glicoproteínas/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Transdução de Sinais/fisiologia , Análise Espaço-Temporal , Peixe-Zebra/embriologia , Peixe-Zebra/genéticaRESUMO
Animal-vegetal (AV) polarity of most vertebrate eggs is established during early oogenesis through the formation and disassembly of the Balbiani Body (Bb). The Bb is a structure conserved from insects to humans that appears as a large granule, similar to a mRNP granule composed of mRNA and proteins, that in addition contains mitochondria, ER and Golgi. The components of the Bb, which have amyloid-like properties, include germ cell and axis determinants of the embryo that are anchored to the vegetal cortex upon Bb disassembly. Our lab discovered in zebrafish the only gene known to function in Bb disassembly, microtubule-actin crosslinking factor 1a (macf1a). Macf1 is a conserved, giant multi-domain cytoskeletal linker protein that can interact with microtubules (MTs), actin filaments (AF), and intermediate filaments (IF). In macf1a mutant oocytes the Bb fails to dissociate, the nucleus is acentric, and AV polarity of the oocyte and egg fails to form. The cytoskeleton-dependent mechanism by which Macf1a regulates Bb mRNP granule dissociation was unknown. We found that disruption of AFs phenocopies the macf1a mutant phenotype, while MT disruption does not. We determined that cytokeratins (CK), a type of IF, are enriched in the Bb. We found that Macf1a localizes to the Bb, indicating a direct function in regulating its dissociation. We thus tested if Macf1a functions via its actin binding domain (ABD) and plectin repeat domain (PRD) to integrate cortical actin and Bb CK, respectively, to mediate Bb dissociation at the oocyte cortex. We developed a CRISPR/Cas9 approach to delete the exons encoding these domains from the macf1a endogenous locus, while maintaining the open reading frame. Our analysis shows that Macf1a functions via its ABD to mediate Bb granule dissociation and nuclear positioning, while the PRD is dispensable. We propose that Macf1a does not function via its canonical mechanism of linking two cytoskeletal systems together in dissociating the Bb. Instead our results suggest that Macf1a functions by linking one cytoskeletal system, cortical actin, to another structure, the Bb, where Macf1a is localized. Through this novel linking process, it dissociates the Bb at the oocyte cortex, thus specifying the AV axis of the oocyte and future egg. To our knowledge, this is also the first study to use genome editing to unravel the module-dependent function of a cytoskeletal linker.
Assuntos
Polaridade Celular/genética , Oogênese/genética , Plaquinas/genética , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/genética , Citoesqueleto de Actina/genética , Animais , Células Germinativas/crescimento & desenvolvimento , Complexo de Golgi/genética , Humanos , Filamentos Intermediários/genética , Microtúbulos/genética , Microtúbulos/metabolismo , Oócitos/crescimento & desenvolvimento , Oócitos/metabolismo , Peixe-Zebra/crescimento & desenvolvimentoRESUMO
The vertebrate embryonic dorsoventral axis is established and patterned by Wnt and bone morphogenetic protein (BMP) signaling pathways, respectively. Whereas Wnt signaling establishes the dorsal side of the embryo and induces the dorsal organizer, a BMP signaling gradient patterns tissues along the dorsoventral axis. Early Wnt signaling is provided maternally, whereas BMP ligand expression in the zebrafish is zygotic, but regulated by maternal factors. Concomitant with BMP activity patterning dorsoventral axial tissues, the embryo also undergoes dramatic morphogenetic processes, including the cell movements of gastrulation, epiboly and dorsal convergence. Although the zygotic regulation of these cell migration processes is increasingly understood, far less is known of the maternal regulators of these processes. Similarly, the maternal regulation of dorsoventral patterning, and in particular the maternal control of ventral tissue specification, is poorly understood. We identified split top, a recessive maternal-effect zebrafish mutant that disrupts embryonic patterning upstream of endogenous BMP signaling. Embryos from split top mutant females exhibit a dorsalized embryonic axis, which can be rescued by BMP misexpression or by derepressing endogenous BMP signaling. In addition to dorsoventral patterning defects, split top mutants display morphogenesis defects that are both BMP dependent and independent. These morphogenesis defects include incomplete dorsal convergence, delayed epiboly progression and an early lysis phenotype during gastrula stages. The latter two morphogenesis defects are associated with disruption of the actin and microtubule cytoskeleton within the yolk cell and defects in the outer enveloping cell layer, which are both known mediators of epiboly movements. Through chromosomal mapping and RNA sequencing analysis, we identified the lysosomal endopeptidase cathepsin Ba (ctsba) as the gene deficient in split top embryos. Our results identify a novel role for Ctsba in morphogenesis and expand our understanding of the maternal regulation of dorsoventral patterning.
Assuntos
Padronização Corporal , Catepsina B/metabolismo , Morfogênese , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/metabolismo , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Animais , Biomarcadores/metabolismo , Proteínas Morfogenéticas Ósseas/metabolismo , Embrião não Mamífero/metabolismo , Feminino , Microtúbulos/metabolismo , Mutação/genética , Fenótipo , Análise de Sequência de RNA , Transdução de SinaisRESUMO
Vertebrate development begins with precise molecular, cellular, and morphogenetic controls to establish the basic body plan of the embryo. In zebrafish, these tightly regulated processes begin during oogenesis and proceed through gastrulation to establish and pattern the axes of the embryo. During oogenesis a maternal factor is localized to the vegetal pole of the oocyte that is a determinant of dorsal tissues. Following fertilization this vegetally localized dorsal determinant is asymmetrically translocated in the egg and initiates formation of the dorsoventral axis. Dorsoventral axis formation and patterning is then mediated by maternal and zygotic factors acting through Wnt, BMP (bone morphogenetic protein), Nodal, and FGF (fibroblast growth factor) signaling pathways, each of which is required to establish and/or pattern the dorsoventral axis. This review addresses recent advances in our understanding of the molecular factors and mechanisms that establish and pattern the dorsoventral axis of the zebrafish embryo, including establishment of the animal-vegetal axis as it relates to formation of the dorsoventral axis.
Assuntos
Padronização Corporal , Embrião não Mamífero/fisiologia , Peixe-Zebra/embriologia , Zigoto/fisiologia , Animais , Proteína Morfogenética Óssea 2/genética , Proteína Morfogenética Óssea 2/metabolismo , Embrião não Mamífero/citologia , Embrião não Mamífero/metabolismo , Fatores de Crescimento de Fibroblastos/genética , Fatores de Crescimento de Fibroblastos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Oócitos/metabolismo , Oócitos/fisiologia , Oogênese , Transporte Proteico , Transdução de Sinais , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Peixe-Zebra/fisiologia , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo , Zigoto/citologia , Zigoto/metabolismoRESUMO
PURPOSE: Osteogenesis imperfecta (OI) predisposes people to recurrent fractures, bone deformities, and short stature. There is a lack of large-scale systematic studies that have investigated growth parameters in OI. METHODS: Using data from the Linked Clinical Research Centers, we compared height, growth velocity, weight, and body mass index (BMI) in 552 individuals with OI. Height, weight, and BMI were plotted on Centers for Disease Control and Prevention normative curves. RESULTS: In children, the median z-scores for height in OI types I, III, and IV were -0.66, -6.91, and -2.79, respectively. Growth velocity was diminished in OI types III and IV. The median z-score for weight in children with OI type III was -4.55. The median z-scores for BMI in children with OI types I, III, and IV were 0.10, 0.91, and 0.67, respectively. Generalized linear model analyses demonstrated that the height z-score was positively correlated with the severity of the OI subtype (P < 0.001), age, bisphosphonate use, and rodding (P < 0.05). CONCLUSION: From the largest cohort of individuals with OI, we provide median values for height, weight, and BMI z-scores that can aid the evaluation of overall growth in the clinic setting. This study is an important first step in the generation of OI-specific growth curves.