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1.
PLoS Comput Biol ; 16(8): e1008049, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32822341

RESUMO

Tissue morphogenesis relies on repeated use of dynamic behaviors at the levels of intracellular structures, individual cells, and cell groups. Rapidly accumulating live imaging datasets make it increasingly important to formalize and automate the task of mapping recurrent dynamic behaviors (motifs), as it is done in speech recognition and other data mining applications. Here, we present a "template-based search" approach for accurate mapping of sub- to multi-cellular morphogenetic motifs using a time series data mining framework. We formulated the task of motif mapping as a subsequence matching problem and solved it using dynamic time warping, while relying on high throughput graph-theoretic algorithms for efficient exploration of the search space. This formulation allows our algorithm to accurately identify the complete duration of each instance and automatically label different stages throughout its progress, such as cell cycle phases during cell division. To illustrate our approach, we mapped cell intercalations during germband extension in the early Drosophila embryo. Our framework enabled statistical analysis of intercalary cell behaviors in wild-type and mutant embryos, comparison of temporal dynamics in contracting and growing junctions in different genotypes, and the identification of a novel mode of iterative cell intercalation. Our formulation of tissue morphogenesis using time series opens new avenues for systematic decomposition of tissue morphogenesis.


Assuntos
Biologia Computacional/métodos , Processamento de Imagem Assistida por Computador/métodos , Morfogênese/fisiologia , Algoritmos , Animais , Divisão Celular/fisiologia , Mineração de Dados/métodos , Drosophila/citologia , Drosophila/embriologia , Embrião não Mamífero/citologia , Embrião não Mamífero/embriologia , Feminino , Masculino , Microscopia Confocal , Fatores de Tempo
2.
Nat Commun ; 11(1): 3317, 2020 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-32620775

RESUMO

Oriented cell division is a fundamental mechanism to control asymmetric stem cell division, neural tube elongation and body axis extension, among other processes. During zebrafish gastrulation, when the body axis extends, dorsal epiblast cells display divisions that are robustly oriented along the animal-vegetal embryonic axis. Here, we use a combination of lipidomics, metabolic tracer analysis and quantitative image analysis to show that sphingolipids mediate spindle positioning during oriented division of epiblast cells. We identify the Wnt signaling as a regulator of sphingolipid synthesis that mediates the activity of serine palmitoyltransferase (SPT), the first and rate-limiting enzyme in sphingolipid production. Sphingolipids determine the palmitoylation state of the Anthrax receptor, which then positions the mitotic spindle of dividing epiblast cells. Our data show how Wnt signaling mediates sphingolipid-dependent oriented division and how sphingolipids determine Anthrax receptor palmitoylation, which ultimately controls the activation of Diaphanous to mediate spindle rotation and oriented mitosis.


Assuntos
Embrião não Mamífero/metabolismo , Mitose , Receptores de Peptídeos/metabolismo , Esfingolipídeos/metabolismo , Via de Sinalização Wnt , Sequência de Aminoácidos , Animais , Divisão Celular Assimétrica/genética , Embrião não Mamífero/citologia , Embrião não Mamífero/embriologia , Gastrulação , Regulação da Expressão Gênica no Desenvolvimento , Camadas Germinativas/citologia , Camadas Germinativas/embriologia , Camadas Germinativas/metabolismo , Lipoilação , Tubo Neural/citologia , Tubo Neural/embriologia , Tubo Neural/metabolismo , Receptores de Peptídeos/genética , Homologia de Sequência de Aminoácidos , Serina C-Palmitoiltransferase/genética , Serina C-Palmitoiltransferase/metabolismo , Fuso Acromático/metabolismo , Peixe-Zebra , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
3.
Nat Cell Biol ; 22(7): 803-814, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32572169

RESUMO

Cell shape is controlled by the submembranous cortex, an actomyosin network mainly generated by two actin nucleators: the Arp2/3 complex and the formin mDia1. Changes in relative nucleator activity may alter cortical organization, mechanics and cell shape. Here we investigate how nucleation-promoting factors mediate interactions between nucleators. In vitro, the nucleation-promoting factor SPIN90 promotes formation of unbranched filaments by Arp2/3, a process thought to provide the initial filament for generation of dendritic networks. Paradoxically, in cells, SPIN90 appears to favour a formin-dominated cortex. Our in vitro experiments reveal that this feature stems mainly from two mechanisms: efficient recruitment of mDia1 to SPIN90-Arp2/3 nucleated filaments and formation of a ternary SPIN90-Arp2/3-mDia1 complex that greatly enhances filament nucleation. Both mechanisms yield rapidly elongating filaments with mDia1 at their barbed ends and SPIN90-Arp2/3 at their pointed ends. Thus, in networks, SPIN90 lowers branching densities and increases the proportion of long filaments elongated by mDia1.


Assuntos
Citoesqueleto de Actina/fisiologia , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Actinas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Forminas/metabolismo , Melanoma/patologia , Proteínas Musculares/metabolismo , Complexo 2-3 de Proteínas Relacionadas à Actina/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Blástula/citologia , Blástula/metabolismo , Forma Celular , Embrião não Mamífero/citologia , Embrião não Mamífero/metabolismo , Forminas/genética , Humanos , Melanoma/genética , Melanoma/metabolismo , Proteínas Musculares/genética , Xenopus laevis/crescimento & desenvolvimento , Xenopus laevis/metabolismo
4.
Nat Commun ; 11(1): 3055, 2020 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-32546686

RESUMO

Recent studies combine two novel technologies, single-cell RNA-sequencing and CRISPR-Cas9 barcode editing for elucidating developmental lineages at the whole organism level. While these studies provided several insights, they face several computational challenges. First, lineages are reconstructed based on noisy and often saturated random mutation data. Additionally, due to the randomness of the mutations, lineages from multiple experiments cannot be combined to reconstruct a species-invariant lineage tree. To address these issues we developed a statistical method, LinTIMaT, which reconstructs cell lineages using a maximum-likelihood framework by integrating mutation and expression data. Our analysis shows that expression data helps resolve the ambiguities arising in when lineages are inferred based on mutations alone, while also enabling the integration of different individual lineages for the reconstruction of an invariant lineage tree. LinTIMaT lineages have better cell type coherence, improve the functional significance of gene sets and provide new insights on progenitors and differentiation pathways.


Assuntos
Algoritmos , Sistemas CRISPR-Cas , Linhagem da Célula/genética , Perfilação da Expressão Gênica/estatística & dados numéricos , Análise de Célula Única/métodos , Animais , Encéfalo/citologia , Caenorhabditis elegans/embriologia , Caenorhabditis elegans/genética , Diferenciação Celular/genética , Interpretação Estatística de Dados , Embrião não Mamífero/citologia , Perfilação da Expressão Gênica/métodos , Funções Verossimilhança , Mutação , Análise de Célula Única/estatística & dados numéricos , Peixe-Zebra/genética
5.
J Vis Exp ; (159)2020 05 24.
Artigo em Inglês | MEDLINE | ID: mdl-32510506

RESUMO

Culex quinquefasciatus is a vector of a diverse range of vector-borne diseases such as avian malaria, West Nile virus (WNV), Japanese encephalitis, Eastern equine encephalitis, lymphatic filariasis, and Saint Louis encephalitis. Notably, avian malaria has played a major role in the extinction of numerous endemic island bird species, while WNV has become an important vector-borne disease in the United States. To gain further insight into C. quinquefasciatus biology and expand their genetic control toolbox, we need to develop more efficient and affordable methods for genome engineering in this species. However, some biological traits unique to Culex mosquitoes, particularly their egg rafts, have made it difficult to perform microinjection procedures required for genome engineering. To address these challenges, we have developed an optimized embryo microinjection protocol that focuses on mitigating the technical obstacles associated with the unique characteristics of Culex mosquitoes. These procedures demonstrate optimized methods for egg collection, egg raft separation and other handling procedures essential for successful microinjection in C. quinquefasciatus. When coupled with the CRISPR/Cas9 genome editing technology, these procedures allow us to achieve site-specific, efficient and heritable germline mutations, which are required to perform advanced genome engineering and develop genetic control technologies in this important, but currently understudied, disease vector.


Assuntos
Culex/embriologia , Culex/genética , Edição de Genes , Microinjeções/métodos , Mosquitos Vetores/genética , Febre do Nilo Ocidental/veterinária , Vírus do Nilo Ocidental/patogenicidade , Animais , Culex/virologia , Embrião não Mamífero/citologia , Embrião não Mamífero/metabolismo , Embrião não Mamífero/virologia , Feminino , Mutagênese Sítio-Dirigida , Febre do Nilo Ocidental/imunologia , Febre do Nilo Ocidental/virologia
6.
Proc Natl Acad Sci U S A ; 117(21): 11444-11449, 2020 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-32381735

RESUMO

Morphogenetic flows in developmental biology are characterized by the coordinated motion of thousands of cells that organize into tissues, naturally raising the question of how this collective organization arises. Using only the kinematics of tissue deformation, which naturally integrates local and global mechanisms along cell paths, we identify the dynamic morphoskeletons behind morphogenesis, i.e., the evolving centerpieces of multicellular trajectory patterns. These features are model- and parameter-free, frame-invariant, and robust to measurement errors and can be computed from unfiltered cell-velocity data. We reveal the spatial attractors and repellers of the embryo by quantifying its Lagrangian deformation, information that is inaccessible to simple trajectory inspection or Eulerian methods that are local and typically frame-dependent. Computing these dynamic morphoskeletons in wild-type and mutant chick and fly embryos, we find that they capture the early footprint of known morphogenetic features, reveal new ones, and quantitatively distinguish between different phenotypes.


Assuntos
Embrião de Galinha/citologia , Embrião de Galinha/crescimento & desenvolvimento , Drosophila melanogaster/embriologia , Modelos Biológicos , Animais , Animais Geneticamente Modificados , Fenômenos Biomecânicos , Embrião de Galinha/efeitos dos fármacos , Simulação por Computador , Proteínas de Drosophila/genética , Embrião não Mamífero/citologia , Fatores de Crescimento de Fibroblastos/antagonistas & inibidores , Fatores de Crescimento de Fibroblastos/metabolismo , Gástrula/crescimento & desenvolvimento , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Indazóis/farmacologia , Microscopia/métodos , Morfogênese , Mutação , Proteína 1 Relacionada a Twist/genética
7.
PLoS Biol ; 18(3): e3000435, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32168317

RESUMO

The circadian clock is a cell-autonomous time-keeping mechanism established gradually during embryonic development. Here, we generated a transgenic zebrafish line carrying a destabilized fluorescent protein driven by the promoter of a core clock gene, nr1d1, to report in vivo circadian rhythm at the single-cell level. By time-lapse imaging of this fish line and 3D reconstruction, we observed the sequential initiation of the reporter expression starting at photoreceptors in the pineal gland, then spreading to the cells in other brain regions at the single-cell level. Even within the pineal gland, we found heterogeneous onset of nr1d1 expression, in which each cell undergoes circadian oscillation superimposed over a cell type-specific developmental trajectory. Furthermore, we found that single-cell expression of nr1d1 showed synchronous circadian oscillation under a light-dark (LD) cycle. Remarkably, single-cell oscillations were dramatically dampened rather than desynchronized in animals raised under constant darkness, while the developmental trend still persists. It suggests that light exposure in early zebrafish embryos has significant effect on cellular circadian oscillations.


Assuntos
Relógios Circadianos/genética , Membro 1 do Grupo D da Subfamília 1 de Receptores Nucleares/genética , Glândula Pineal/citologia , Peixe-Zebra/genética , Animais , Animais Geneticamente Modificados , Proteínas de Bactérias/genética , Encéfalo/citologia , Ritmo Circadiano/genética , Ritmo Circadiano/fisiologia , Embrião não Mamífero/citologia , Proteínas Luminescentes/genética , Fotoperíodo , Glândula Pineal/fisiologia , Regiões Promotoras Genéticas , Análise de Célula Única , Imagem com Lapso de Tempo , Peixe-Zebra/embriologia
8.
Dev Cell ; 52(4): 446-460.e5, 2020 02 24.
Artigo em Inglês | MEDLINE | ID: mdl-32032546

RESUMO

Hematopoietic stem and progenitor cells (HSPCs), first specified from hemogenic endothelium (HE) in the ventral dorsal aorta (VDA), support lifelong hematopoiesis. Their de novo production promises significant therapeutic value; however, current in vitro approaches cannot efficiently generate multipotent long-lived HSPCs. Presuming this reflects a lack of extrinsic cues normally impacting the VDA, we devised a human dorsal aorta-on-a-chip platform that identified Yes-activated protein (YAP) as a cyclic stretch-induced regulator of HSPC formation. In the zebrafish VDA, inducible Yap overexpression significantly increased runx1 expression in vivo and the number of CD41+ HSPCs downstream of HE specification. Endogenous Yap activation by lats1/2 knockdown or Rho-GTPase stimulation mimicked Yap overexpression and induced HSPCs in embryos lacking blood flow. Notably, in static human induced pluripotent stem cell (iPSC)-derived HE culture, compound-mediated YAP activation enhanced RUNX1 levels and hematopoietic colony-forming potential. Together, our findings reveal a potent impact of hemodynamic Rho-YAP mechanotransduction on HE fate, relevant to de novo human HSPC production.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Endotélio Vascular/citologia , Hematopoese , Células-Tronco Hematopoéticas/citologia , Células-Tronco Pluripotentes Induzidas/citologia , Mecanotransdução Celular , Fatores de Transcrição/metabolismo , Animais , Aorta/citologia , Aorta/embriologia , Proteínas de Ciclo Celular/genética , Diferenciação Celular , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Embrião não Mamífero/citologia , Embrião não Mamífero/metabolismo , Endotélio Vascular/metabolismo , Células-Tronco Hematopoéticas/fisiologia , Hemodinâmica , Humanos , Células-Tronco Pluripotentes Induzidas/fisiologia , Fatores de Transcrição/genética , Peixe-Zebra , Proteínas rho de Ligação ao GTP/metabolismo
9.
Dev Cell ; 52(4): 492-508.e10, 2020 02 24.
Artigo em Inglês | MEDLINE | ID: mdl-32059773

RESUMO

How tissues migrate robustly through changing guidance landscapes is poorly understood. Here, quantitative imaging is combined with inducible perturbation experiments to investigate the mechanisms that ensure robust tissue migration in vivo. We show that tissues exposed to acute "chemokine floods" halt transiently before they perfectly adapt, i.e., return to the baseline migration behavior in the continued presence of elevated chemokine levels. A chemokine-triggered phosphorylation of the atypical chemokine receptor Cxcr7b reroutes it from constitutive ubiquitination-regulated degradation to plasma membrane recycling, thus coupling scavenging capacity to extracellular chemokine levels. Finally, tissues expressing phosphorylation-deficient Cxcr7b migrate normally in the presence of physiological chemokine levels but show delayed recovery when challenged with elevated chemokine concentrations. This work establishes that adaptation to chemokine fluctuations can be "outsourced" from canonical GPCR signaling to an autonomously acting scavenger receptor that both senses and dynamically buffers chemokine levels to increase the robustness of tissue migration.


Assuntos
Movimento Celular , Quimiocinas/metabolismo , Embrião não Mamífero/metabolismo , Receptores CXCR4/metabolismo , Receptores CXCR/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/metabolismo , Animais , Comunicação Celular , Quimiocinas/genética , Embrião não Mamífero/citologia , Fosforilação , Receptores CXCR/genética , Receptores CXCR4/genética , Transdução de Sinais , Peixe-Zebra/genética , Peixe-Zebra/crescimento & desenvolvimento , Proteínas de Peixe-Zebra/genética
10.
Elife ; 92020 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-31909715

RESUMO

Partitioning of mRNAs into ribonucleoprotein (RNP) granules supports diverse regulatory programs within the crowded cytoplasm. At least two types of RNP granules populate the germ plasm, a cytoplasmic domain at the posterior of the Drosophila oocyte and embryo. Germ granules deliver mRNAs required for germline development to pole cells, the germ cell progenitors. A second type of RNP granule, here named founder granules, contains oskar mRNA, which encodes the germ plasm organizer. Whereas oskar mRNA is essential for germ plasm assembly during oogenesis, we show that it is toxic to pole cells. Founder granules mediate compartmentalized degradation of oskar during embryogenesis to minimize its inheritance by pole cells. Degradation of oskar in founder granules is temporally and mechanistically distinct from degradation of oskar and other mRNAs during the maternal-to-zygotic transition. Our results show how compartmentalization in RNP granules differentially controls fates of mRNAs localized within the same cytoplasmic domain.


Assuntos
Compartimento Celular , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/embriologia , Células Germinativas/citologia , Células Germinativas/metabolismo , Proteólise , Animais , Movimento Celular , Grânulos Citoplasmáticos/metabolismo , Drosophila melanogaster/metabolismo , Embrião não Mamífero/citologia , Embrião não Mamífero/metabolismo , Feminino , Fatores de Iniciação de Peptídeos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
11.
J Vis Exp ; (155)2020 01 12.
Artigo em Inglês | MEDLINE | ID: mdl-31984960

RESUMO

We describe a technique for retrograde labeling of motor neurons in Drosophila. We use an oil-dissolved lipophilic dye and deliver a small droplet to an embryonic fillet preparation by a microinjector. Each motor neuron whose membrane is contacted by the droplet can then be rapidly labeled. Individual motor neurons are continuously labeled, enabling fine structural details to be clearly visualized. Given that lipophilic dyes come in various colors, the technique also provides a means to get adjacent neurons labeled in multicolor. This tracing technique is therefore useful for studying neuronal morphogenesis and synaptic connectivity in the motor neuron system of Drosophila.


Assuntos
Drosophila melanogaster/embriologia , Embrião não Mamífero/citologia , Corantes Fluorescentes/metabolismo , Lipídeos/química , Neurônios Motores/citologia , Animais , Dendritos/metabolismo , Dissecação , Feminino , Injeções , Masculino , Neurogênese
12.
Biol Open ; 9(1)2020 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-31941702

RESUMO

The sodium osmotic gradient is necessary for the initiation of brain ventricle inflation, but a previous study predicted that organic and inorganic osmolytes play equivalently important roles in osmotic homeostasis in astrocytes. To test whether organic osmoregulation also plays a role in brain ventricle inflation, the core component for volume-regulated anion and organic osmolyte channel, lrrc8a, was investigated in the zebrafish model. RT-PCR and whole-mount in situ hybridization indicated that both genes were ubiquitously expressed through to 12 hpf, and around the ventricular layer of neural tubes and the cardiogenic region at 24 hpf. Knocking down either one lrrc8a paralog with morpholino oligos resulted in abnormalities in circulation at 32 hpf. Morpholino oligos or CRISPR interference against either paralog led to smaller brain ventricles at 24 hpf. Either lrrc8aa or lrrc8ab mRNA rescued the phenotypic penetrance in both lrrc8aa and lrrc8ab morphants. Supplementation of taurine in the E3 medium and overexpression csad mRNA also rescued lrrc8aa and lrrc8ab morphants. Our results indicate that the two zebrafish lrrc8a paralogs are maternal message genes and are ubiquitously expressed in early embryos. The two genes play redundant roles in the expansion of brain ventricles and the circulatory system and taurine contributes to brain ventricle expansion via the volume-regulated anion and organic osmolyte channels.


Assuntos
Encéfalo , Embrião não Mamífero , Regulação da Expressão Gênica no Desenvolvimento , Canais Iônicos , Osmorregulação/fisiologia , Proteínas de Peixe-Zebra , Peixe-Zebra , Animais , Astrócitos/citologia , Astrócitos/metabolismo , Encéfalo/citologia , Encéfalo/embriologia , Embrião não Mamífero/citologia , Embrião não Mamífero/embriologia , Canais Iônicos/biossíntese , Canais Iônicos/genética , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/biossíntese , Proteínas de Peixe-Zebra/genética
13.
Proc Natl Acad Sci U S A ; 117(4): 1853-1859, 2020 01 28.
Artigo em Inglês | MEDLINE | ID: mdl-31932426

RESUMO

Living systems are more robust, diverse, complex, and supportive of human life than any technology yet created. However, our ability to create novel lifeforms is currently limited to varying existing organisms or bioengineering organoids in vitro. Here we show a scalable pipeline for creating functional novel lifeforms: AI methods automatically design diverse candidate lifeforms in silico to perform some desired function, and transferable designs are then created using a cell-based construction toolkit to realize living systems with the predicted behaviors. Although some steps in this pipeline still require manual intervention, complete automation in future would pave the way to designing and deploying unique, bespoke living systems for a wide range of functions.


Assuntos
Algoritmos , Automação , Bioengenharia/métodos , Simulação por Computador , Embrião não Mamífero/fisiologia , Modelos Biológicos , Xenopus laevis/fisiologia , Animais , Células Artificiais , Embrião não Mamífero/citologia , Humanos
14.
PLoS One ; 15(1): e0226600, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31914136

RESUMO

Since the size of newly hatched larval fish is directly related to egg size, small differences in initial egg size can be critical to survival and further development of offspring. Underlying processes causing size variation in fish offspring are still not entirely understood. In this study we investigated whether the spatial position of an individual egg within a clutch affects size variation in two benthic spawning coral reef fishes, the clownfishes Amphiprion ocellaris and A. frenatus. To evaluate the effects of within-clutch position on embryonic development, egg growth metrics and protein content were analysed on day 2, 5 and 8 after deposition (adp). Additionally the activities of the key metabolic enzymes citrate synthase (CS) and lactate dehydrogenase (LDH) were investigated to evaluate the physiological status of the embryos. Central eggs of A. frenatus were significantly longer and heavier than peripheral eggs only on day 2 and 5 adp (2.07 mg, 2.59 mm vs. 1.84 mg, 2.49 mm). No significant differences were observed in A. ocellaris between eggs originating from a central or peripheral (5 mm from edge) position (1.33 mg, 2.26 mm vs. 1.15 mg, 2,18 mm). Diameter of the eyes did not differ between the two fish species nor between different positions, for any age group. The protein content of eggs (7.5% of wet weight) was independent of age, position and species. Enzymatic activity increased from 2 adp until peak activity was observed for both enzymes on day 8 adp, independent from position. The range of CS- and LDH-activity was 0.3-13.0 and 0.2-71.7 U g-1 wet weight, respectively. Significant differences in enzymatic activity were observed between age groups in both species, which in connection with significantly larger eggs of A. frenatus at day 2 and 5 adp could hint at a better O2 supply of central eggs. Potential implications for captive breeding are given.


Assuntos
Proteínas do Ovo/metabolismo , Embrião não Mamífero/citologia , Embrião não Mamífero/enzimologia , Desenvolvimento Embrionário , Óvulo/citologia , Perciformes/embriologia , Perciformes/metabolismo , Animais , Óvulo/enzimologia , Processamento Espacial
15.
Nat Commun ; 11(1): 539, 2020 Jan 27.
Artigo em Inglês | MEDLINE | ID: mdl-31988277

RESUMO

In the Caenorhabditis elegans zygote, PAR protein patterns, driven by mutual anatagonism, determine the anterior-posterior axis and facilitate the redistribution of proteins for the first cell division. Yet, the factors that determine the selection of the polarity axis remain unclear. We present a reaction-diffusion model in realistic cell geometry, based on biomolecular reactions and accounting for the coupling between membrane and cytosolic dynamics. We find that the kinetics of the phosphorylation-dephosphorylation cycle of PARs and the diffusive protein fluxes from the cytosol towards the membrane are crucial for the robust selection of the anterior-posterior axis for polarisation. The local ratio of membrane surface to cytosolic volume is the main geometric cue that initiates pattern formation, while the choice of the long-axis for polarisation is largely determined by the length of the aPAR-pPAR interface, and mediated by processes that minimise the diffusive fluxes of PAR proteins between cytosol and membrane.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Polaridade Celular , Animais , Divisão Celular Assimétrica , Caenorhabditis elegans/citologia , Caenorhabditis elegans/embriologia , Biologia Computacional , Citosol/metabolismo , Embrião não Mamífero/citologia , Embrião não Mamífero/metabolismo , Desenvolvimento Embrionário , Cinética , Modelos Biológicos , Fosforilação , Transdução de Sinais , Termodinâmica
16.
FASEB J ; 34(1): 1345-1361, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31914618

RESUMO

Cell morphology and tissue integrity are essential for embryogenesis. Caveolins are membrane proteins that induce the formation of surface pits called caveolae that serve as membrane reservoirs for cell and tissue protection during development. In vertebrates, caveolin 1 (Cav1) and caveolin 3 (Cav3) are required for caveola formation. However, the formation of caveola and the function of caveolins in invertebrates are largely unknown. In this study, three caveolins, Cav-a, Cav-b, and CavY, are identified in the genome of the invertebrate chordate Ciona spp. Based on phylogenetic analysis, Cav-a is found to be closely related to the vertebrate Cav1 and Cav3. In situ hybridization shows that Cav-a is expressed in Ciona embryonic notochord and muscle. Cell-free experiments, model cell culture systems, and in vivo experiments demonstrate that Ciona Cav-a has the ability to induce membrane curvature at the plasma membrane. Knockdown of Cav-a in Ciona embryos causes loss of invaginations in the plasma membrane and results in the failure of notochord elongation and lumenogenesis. Expression of a dominant-negative Cav-a point mutation causes cells to change shape and become displaced from the muscle and notochord to disrupt tissue integrity. Furthermore, we demonstrate that Cav-a vesicles show polarized trafficking and localize at the luminal membrane during notochord lumenogenesis. Taken together, these results show that the invertebrate chordate caveolin from Ciona plays crucial roles in tissue integrity and morphology by inducing membrane curvature and intracellular vesicle trafficking during embryogenesis.


Assuntos
Caveolinas/metabolismo , Membrana Celular/metabolismo , Ciona/embriologia , Embrião não Mamífero/metabolismo , Desenvolvimento Embrionário , Modelos Biológicos , Animais , Transporte Biológico Ativo , Caveolinas/genética , Membrana Celular/genética , Ciona/citologia , Embrião não Mamífero/citologia
17.
Gen Comp Endocrinol ; 285: 113230, 2020 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-31348955

RESUMO

During the ovarian ontogeny in birds, five fundamental events can be recognized: migration and colonization of the primordial germ cells, differentiation and proliferation of oogonies, an organization of germinal nests, beginning of the meiotic process and folliculogenesis. The knowledge of these events is fundamental for the interpretation of the processes involved in the differentiation of female gametes. However, there are only references for some model species such as Gallus gallus domesticus and Coturnix coturnix. In a previous study, the histological structure of embryonic ovaries of Columba livia was revealed. Therefore, the objective of this work is to characterize the processes of meiosis and folliculogenesis C. livia from the analysis of the expression of the GnRH receptor, the 3ßHSD enzyme and the cell proliferation protein PCNA in embryonic and postnatal ovaries. Therefore, the expression of GnRHR, 3ßHSD, and PCNA was revealed in histological testicular and ovarian preparations in embryos (stages 41-43) and neonates (2, 5, 7, 10 and 75 days post-hatching). The present study demonstrates that the fate of germline cells is dictated by their location during gonadal development. Thus, the germline cells located in the cortex of the left gonad enter meiosis, while those in the right gonad and those in the medulla of the left ovary fail to go into meiosis. This indicates that somatic signals, instead of an autonomous cellular mechanism, regulate the entry of the germline cells into meiosis in the C. livia embryo. Future studies will be focused on the analysis of proteins associated with meiotic events and folliculogenesis in embryonic and neonatal ovaries of C. livia, to evaluate the regulation of meiosis in vitro.


Assuntos
3-Hidroxiesteroide Desidrogenases/metabolismo , Columbidae/metabolismo , Meiose , Folículo Ovariano/crescimento & desenvolvimento , Receptores LHRH/metabolismo , Animais , Proliferação de Células , Columbidae/embriologia , Embrião não Mamífero/citologia , Feminino , Células Germinativas/metabolismo , Imuno-Histoquímica , Oócitos/citologia , Oócitos/metabolismo , Folículo Ovariano/metabolismo , Antígeno Nuclear de Célula em Proliferação/metabolismo
18.
Development ; 147(2)2020 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-31862845

RESUMO

The development of tissues and organs requires close interaction of cells. To achieve this, cells express adhesion proteins such as the neural cell adhesion molecule (NCAM) or its Drosophila ortholog Fasciclin 2 (Fas2). Both are members of the Ig-domain superfamily of proteins that mediate homophilic adhesion. These proteins are expressed as isoforms differing in their membrane anchorage and their cytoplasmic domains. To study the function of single isoforms, we have conducted a comprehensive genetic analysis of F as2 We reveal the expression pattern of all major Fas2 isoforms, two of which are GPI anchored. The remaining five isoforms carry transmembrane domains with variable cytoplasmic tails. We generated F as2 mutants expressing only single isoforms. In contrast to the null mutation, which causes embryonic lethality, these mutants are viable, indicating redundancy among the different isoforms. Cell type-specific rescue experiments showed that glial-secreted Fas2 can rescue the F as2 mutant phenotype to viability. This demonstrates that cytoplasmic Fas2 domains have no apparent essential functions and indicate that Fas2 has function(s) other than homophilic adhesion. In conclusion, our data suggest novel mechanistic aspects of a long-studied adhesion protein.


Assuntos
Moléculas de Adesão Celular Neuronais/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/metabolismo , Transdução de Sinais , Animais , Adesão Celular , Moléculas de Adesão Celular Neuronais/química , Moléculas de Adesão Celular Neuronais/genética , Movimento Celular , Drosophila melanogaster/embriologia , Drosophila melanogaster/genética , Embrião não Mamífero/citologia , Embrião não Mamífero/metabolismo , Edição de Genes , Regulação da Expressão Gênica no Desenvolvimento , Glicosilfosfatidilinositóis/metabolismo , Mutação/genética , Neuroglia/metabolismo , Domínios Proteicos , Isoformas de Proteínas/metabolismo , Traqueia/embriologia , Traqueia/metabolismo
19.
Methods Mol Biol ; 2041: 87-106, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31646482

RESUMO

Xenopus embryos are one of the most used animal models in developmental biology and are well suited for apprehending functions of signaling pathways during embryogenesis. To do so, it is necessary to be able to detect expression pattern of the key genes of these signaling pathways. Here we describe the whole-mount in situ hybridization technique to investigate the expression pattern of ectonucleotidases and purinergic receptors during embryonic development.


Assuntos
5'-Nucleotidase/metabolismo , Embrião não Mamífero/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Hibridização In Situ/métodos , Receptores Purinérgicos/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/metabolismo , 5'-Nucleotidase/genética , Animais , Embrião não Mamífero/citologia , Desenvolvimento Embrionário , Feminino , Sondas RNA , Receptores Purinérgicos/genética , Transdução de Sinais , Proteínas de Xenopus/genética , Xenopus laevis/embriologia
20.
Chem Biol Interact ; 316: 108928, 2020 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-31857089

RESUMO

OBJECTIVE: Zebrafish inflammation models were used to evaluate the anti-inflammatory activity of isoniazid (INH) and preliminarily investigate the underlying mechanism. METHODS: Local, acute, and systemic zebrafish inflammation models were established by tail cutting, copper sulfate (CuSO4), and lipopolysaccharide (LPS) endotoxin treatments, respectively, to evaluate the anti-inflammatory activity of INH. Zebrafish in the inflammatory state were exposed to different concentrations of INH (1, 2, and 4 mM) for 72 h to observe changes in the migration and accumulation of inflammatory cells and measure the reactive oxygen species (ROS) content in zebrafish after INH treatment. The transcription levels of inflammation-related genes in zebrafish from all groups were measured using real-time polymerase chain reaction (RT-PCR). RESULTS: Compared to those observed in the control inflammation model group, the numbers of migrated and accumulated inflammatory cells in zebrafish in the INH-treated group significantly decreased. INH significantly decreased the ROS content induced by LPS. Compared to that observed in the LPS model group, INH at 1 and 2 mM significantly increased the expression of PPARγ and inhibited the expression of NF-κB, iκbαa, and AP-1 as well as the inflammatory factors TNF-ɑ, TGF-ß, IL-1b, and COX-2. CONCLUSION: In this study, different zebrafish inflammation models were used to confirm that INH has anti-inflammatory activity. The associated mechanism may occur through the inhibition of ROS release, activation of PPARγ expression, inhibition of the transcriptional regulatory activity of NF-κB and AP-1, and reduction of INH inflammatory factor expression to relieve inflammation. The results of this study provide references for the clinical application of INH.


Assuntos
Citocinas/metabolismo , Isoniazida/farmacologia , Transdução de Sinais/efeitos dos fármacos , Animais , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/metabolismo , Citocinas/genética , Embrião não Mamífero/citologia , Embrião não Mamífero/efeitos dos fármacos , Embrião não Mamífero/metabolismo , Feminino , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Lipopolissacarídeos/toxicidade , Masculino , NF-kappa B/metabolismo , PPAR gama/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Fator de Transcrição AP-1/metabolismo , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismo , Peixe-Zebra/metabolismo
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