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The promyelocytic leukemia (PML) body is a phase-separated nuclear structure physically associated with chromatin, implying its crucial roles in genome functions. However, its role in transcriptional regulation is largely unknown. We developed APEX-mediated chromatin labeling and purification (ALaP) to identify the genomic regions proximal to PML bodies. We found that PML bodies associate with active regulatory regions across the genome and with â¼300 kb of the short arm of the Y chromosome (YS300) in mouse embryonic stem cells. The PML body association with YS300 is essential for the transcriptional activity of the neighboring Y-linked clustered genes. Mechanistically, PML bodies provide specific nuclear spaces that the de novo DNA methyltransferase DNMT3A cannot access, resulting in the steady maintenance of a hypo-methylated state at Y-linked gene promoters. Our study underscores a new mechanism for gene regulation in the 3D nuclear space and provides insights into the functional properties of nuclear structures for genome function.
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ADN (Citosina-5-)-Metiltransferasas/metabolismo , Regulación de la Expresión Génica , Cuerpos de Inclusión Intranucleares/genética , Cromosoma Y/genética , Animales , Línea Celular , Cromatina/genética , Cromatina/metabolismo , ARN Helicasas DEAD-box/genética , ADN (Citosina-5-)-Metiltransferasas/genética , Metilación de ADN , ADN Metiltransferasa 3A , ADN-(Sitio Apurínico o Apirimidínico) Liasa/genética , Células Madre Embrionarias/fisiología , Endonucleasas/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Cuerpos de Inclusión Intranucleares/metabolismo , Ratones Noqueados , Antígenos de Histocompatibilidad Menor/genética , Enzimas Multifuncionales/genética , Familia de Multigenes , Estrés Oxidativo , Proteína de la Leucemia Promielocítica/genética , Proteína de la Leucemia Promielocítica/metabolismo , Proteínas/genética , Factores de Transcripción/genética , Cromosoma Y/metabolismoRESUMEN
Plant cells are surrounded by a cell wall and do not migrate, which makes the regulation of cell division orientation crucial for development. Regulatory mechanisms controlling cell division orientation may have contributed to the evolution of body organization in land plants. The GRAS family of transcription factors was transferred horizontally from soil bacteria to an algal common ancestor of land plants. SHORTROOT (SHR) and SCARECROW (SCR) genes in this family regulate formative periclinal cell divisions in the roots of flowering plants, but their roles in nonflowering plants and their evolution have not been studied in relation to body organization. Here, we show that SHR cell autonomously inhibits formative periclinal cell divisions indispensable for leaf vein formation in the moss Physcomitrium patens, and SHR expression is positively and negatively regulated by SCR and the GRAS member LATERAL SUPPRESSOR, respectively. While precursor cells of a leaf vein lacking SHR usually follow the geometry rule of dividing along the division plane with the minimum surface area, SHR overrides this rule and forces cells to divide nonpericlinally. Together, these results imply that these bacterially derived GRAS transcription factors were involved in the establishment of the genetic regulatory networks modulating cell division orientation in the common ancestor of land plants and were later adapted to function in flowering plant and moss lineages for their specific body organizations.
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Proteínas de Arabidopsis , Arabidopsis , Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , División Celular/genética , Raíces de Plantas/metabolismo , Regulación de la Expresión Génica de las PlantasRESUMEN
Despite previous intensive investigations on epiblast cell migration in avian embryos during primitive streak development before stage (st.) 4, this migration at later stages of brain development has remained uninvestigated. By live imaging of epiblast cells sparsely labeled with green fluorescence protein, we investigated anterior epiblast cell migration to form individual brain portions. Anterior epiblast cells from a broad area migrated collectively towards the head axis during st. 5-7 at a rate of 70-110â µm/h, changing directions from diagonal to parallel and forming the brain portions and abutting head ectoderm. This analysis revised the previously published head portion precursor map in anterior epiblasts at st. 4/5. Grafting outside the brain precursor region of mCherry-expressing nodes producing anterior mesendoderm (AME) or isolated AME tissues elicited new cell migration towards ectopic AME tissues. These locally convergent cells developed into secondary brains with portions that depended on the ectopic AME position in the anterior epiblast. Thus, anterior epiblast cells are bipotent for brain/head ectoderm development with given brain portion specificities. A brain portion potential map is proposed, also accounting for previous observations.
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Gástrula , Estratos Germinativos , Animales , Aves , Encéfalo , Movimiento Celular , Ectodermo/metabolismoRESUMEN
Mechanical forces are critical for the emergence of diverse three-dimensional morphologies of multicellular systems. However, it remains unclear what kind of mechanical parameters at cellular level substantially contribute to tissue morphologies. This is largely due to technical limitations of live measurements of cellular forces. Here we developed a framework for inferring and modeling mechanical forces of cell-cell interactions. First, by analogy to coarse-grained models in molecular and colloidal sciences, we approximated cells as particles, where mean forces (i.e. effective forces) of pairwise cell-cell interactions are considered. Then, the forces were statistically inferred by fitting the mathematical model to cell tracking data. This method was validated by using synthetic cell tracking data resembling various in vivo situations. Application of our method to the cells in the early embryos of mice and the nematode Caenorhabditis elegans revealed that cell-cell interaction forces can be written as a pairwise potential energy in a manner dependent on cell-cell distances. Importantly, the profiles of the pairwise potentials were quantitatively different among species and embryonic stages, and the quantitative differences correctly described the differences of their morphological features such as spherical vs. distorted cell aggregates, and tightly vs. non-tightly assembled aggregates. We conclude that the effective pairwise potential of cell-cell interactions is a live measurable parameter whose quantitative differences can be a parameter describing three-dimensional tissue morphologies.
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Caenorhabditis elegans , Modelos Teóricos , Animales , Rastreo Celular , Desarrollo EmbrionarioRESUMEN
Embryo contour extraction is the initial step in the quantitative analysis of embryo morphology, and it is essential for understanding the developmental process. Recent developments in light-sheet microscopy have enabled the in toto time-lapse imaging of embryos, including zebrafish. However, embryo contour extraction from images generated via light-sheet microscopy is challenging owing to the large amount of data and the variable sizes, shapes, and textures of objects. In this report, we provide a workflow for extracting the contours of zebrafish blastula and gastrula without contour labeling of an embryo. This workflow is based on the edge detection method using a change point detection approach. We assessed the performance of the edge detection method and compared it with widely used edge detection and segmentation methods. The results showed that the edge detection accuracy of the proposed method was superior to those of the Sobel, Laplacian of Gaussian, adaptive threshold, Multi Otsu, and k-means clustering-based methods, and the noise robustness of the proposed method was superior to those of the Multi Otsu and k-means clustering-based methods. The proposed workflow was shown to be useful for automating small-scale contour extractions of zebrafish embryos that cannot be specifically labeled owing to constraints, such as the availability of microscopic channels. This workflow may offer an option for contour extraction when deep learning-based approaches or existing non-deep learning-based methods cannot be applied.
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Microscopía , Pez Cebra , Animales , Microscopía/métodos , Procesamiento de Imagen Asistido por Computador/métodos , AlgoritmosRESUMEN
The adherens junction (AJ) is an actin filament-anchoring junction. It plays a central role in epithelial morphogenesis through cadherin-based recognition and adhesion among cells. The stability and plasticity of AJs are required for the morphogenesis. An actin-binding α-catenin is an essential component of the cadherin-catenin complex and functions as a tension transducer that changes its conformation and induces AJ development in response to tension. Despite much progress in understanding molecular mechanisms of tension sensitivity of α-catenin, its significance on epithelial morphogenesis is still unknown. Here we show that the tension sensitivity of α-catenin is essential for epithelial cells to form round spheroids through proper multicellular rearrangement. Using a novel in vitro suspension culture model, we found that epithelial cells form round spheroids even from rectangular-shaped cell masses with high aspect ratios without using high tension and that increased tension sensitivity of α-catenin affected this morphogenesis. Analyses of AJ formation and cellular tracking during rounding morphogenesis showed cellular rearrangement, probably through AJ remodeling. The rearrangement occurs at the cell mass level, but not single-cell level. Hypersensitive α-catenin mutant-expressing cells did not show cellular rearrangement at the cell mass level, suggesting that the appropriate tension sensitivity of α-catenin is crucial for the coordinated round morphogenesis.Key words: α-catenin, vinculin, adherens junction, morphogenesis, mechanotransduction.
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Uniones Adherentes , Mecanotransducción Celular , Uniones Adherentes/metabolismo , Cadherinas , Morfogénesis , alfa Catenina/química , alfa Catenina/metabolismoRESUMEN
The most basic form of locomotion in limbed vertebrates consists of alternating activities of the flexor and extensor muscles within each limb coupled with left/right limb alternation. Although larval zebrafish are not limbed, their pectoral fin movements exhibit the following fundamental aspects of this basic movement: abductor/adductor alternation (corresponding to flexor/extensor alternation) and left/right fin alternation. Because of the simplicity of their movements and the compact neural organization of their spinal cords, zebrafish can serve as a good model to identify the neuronal networks of the central pattern generator (CPG) that controls rhythmic appendage movements. Here, we set out to investigate neuronal circuits underlying rhythmic pectoral fin movements in larval zebrafish, using transgenic fish that specifically express GFP in abductor or adductor motor neurons (MNs) and candidate CPG neurons. First, we showed that spiking activities of abductor and adductor MNs were essentially alternating. Second, both abductor and adductor MNs received rhythmic excitatory and inhibitory synaptic inputs in their active and inactive phases, respectively, indicating that the MN spiking activities are controlled in a push-pull manner. Further, we obtained the following evidence that dmrt3a-expressing commissural inhibitory neurons are involved in regulating the activities of abductor MNs: (1) strong inhibitory synaptic connections were found from dmrt3a neurons to abductor MNs; and (2) ablation of dmrt3a neurons shifted the spike timing of abductor MNs. Thus, in this simple system of abductor/adductor alternation, the last-order inhibitory inputs originating from the contralaterally located neurons play an important role in controlling the firing timings of MNs.SIGNIFICANCE STATEMENT Pectoral fin movements in larval zebrafish exhibit fundamental aspects of basic rhythmic appendage movement: alternation of the abductor and adductor (corresponding to flexor-extensor alternation) coupled with left-right alternation. We set out to investigate the neuronal circuits underlying rhythmic pectoral fin movements in larval zebrafish. We showed that both abductor and adductor MNs received rhythmic excitatory and inhibitory synaptic inputs in their active and inactive phases, respectively. This indicates that MN activities are controlled in a push-pull manner. We further obtained evidence that dmrt3a-expressing commissural inhibitory neurons exert an inhibitory effect on abductor MNs. The current study marks the first step toward the identification of central pattern generator organization for rhythmic fin movements.
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Aletas de Animales/fisiología , Generadores de Patrones Centrales/fisiología , Neuronas Motoras/fisiología , Movimiento , Aletas de Animales/inervación , Animales , Generadores de Patrones Centrales/metabolismo , Proteínas de Unión al ADN/metabolismo , Neuronas Motoras/metabolismo , Periodicidad , Factores de Transcripción/metabolismo , Pez Cebra , Proteínas de Pez Cebra/metabolismoRESUMEN
Neural progenitor cells (NPCs), which are apicobasally elongated and densely packed in the developing brain, systematically move their nuclei/somata in a cell cycle-dependent manner, called interkinetic nuclear migration (IKNM): apical during G2 and basal during G1. Although intracellular molecular mechanisms of individual IKNM have been explored, how heterogeneous IKNMs are collectively coordinated is unknown. Our quantitative cell-biological and in silico analyses revealed that tissue elasticity mechanically assists an initial step of basalward IKNM. When the soma of an M-phase progenitor cell rounds up using actomyosin within the subapical space, a microzone within 10 µm from the surface, which is compressed and elastic because of the apical surface's contractility, laterally pushes the densely neighboring processes of non-M-phase cells. The pressed processes then recoil centripetally and basally to propel the nuclei/somata of the progenitor's daughter cells. Thus, indirect neighbor-assisted transfer of mechanical energy from mother to daughter helps efficient brain development.
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División del Núcleo Celular/fisiología , Núcleo Celular/fisiología , Células-Madre Neurales/fisiología , Células Neuroepiteliales/fisiología , Actomiosina/química , Actomiosina/metabolismo , Animales , Fenómenos Biomecánicos , Ciclo Celular/efectos de los fármacos , Ciclo Celular/fisiología , Núcleo Celular/efectos de los fármacos , Núcleo Celular/ultraestructura , División del Núcleo Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Corteza Cerebral/citología , Corteza Cerebral/fisiología , Elasticidad , Embrión de Mamíferos , Transferencia de Energía , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Ratones , Ratones Endogámicos ICR , Movimiento/fisiología , Células-Madre Neurales/citología , Células-Madre Neurales/efectos de los fármacos , Células Neuroepiteliales/citología , Células Neuroepiteliales/efectos de los fármacos , Imagen de Lapso de TiempoRESUMEN
Epithelial tubules, consisting of the epithelial cell sheet with a central lumen, are the basic structure of many organs. Mechanical forces play an important role in epithelial tubulogenesis; however, little is known about the mechanisms controlling the mechanical forces during epithelial tubule morphogenesis. Solo (also known as ARHGEF40) is a RhoA-targeting guanine-nucleotide exchange factor that is involved in mechanical force-induced RhoA activation and stress fiber formation. Solo binds to keratin-8/keratin-18 (K8/K18) filaments, and this interaction plays a crucial role in mechanotransduction. In this study, we examined the roles of Solo and K8/K18 filaments in epithelial tubulogenesis using MDCK cells cultured in 3D collagen gels. Knockdown of either Solo or K18 resulted in rounder tubules with increased lumen size, indicating that Solo and K8/K18 filaments play critical roles in forming the elongated morphology of epithelial tubules. Moreover, knockdown of Solo or K18 decreased the level of diphosphorylated myosin light chain (a marker of contractile force) at the luminal and outer surfaces of tubules, suggesting that Solo and K8/K18 filaments are involved in the generation of the myosin II-mediated contractile force during epithelial tubule morphogenesis. In addition, K18 filaments were normally oriented along the long axis of the tubule, but knockdown of Solo perturbed their orientation. These results suggest that Solo plays crucial roles in forming the elongated morphology of epithelial tubules and in regulating myosin II activity and K18 filament organization during epithelial tubule formation.Key words: epithelial tubulogenesis, Solo, keratin, Rho-GEF, myosin.
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Factores de Intercambio de Guanina Nucleótido/metabolismo , Queratina-18/metabolismo , Queratina-8/metabolismo , Animales , Técnicas de Cultivo de Célula , Colágeno/química , Citoesqueleto/metabolismo , Perros , Factores de Intercambio de Guanina Nucleótido/antagonistas & inhibidores , Factores de Intercambio de Guanina Nucleótido/genética , Filamentos Intermedios/metabolismo , Queratina-18/antagonistas & inhibidores , Queratina-18/genética , Queratina-8/genética , Células de Riñón Canino Madin Darby , Microscopía Fluorescente , Unión Proteica , Interferencia de ARN , ARN Interferente Pequeño/metabolismoRESUMEN
Transcriptome analyses in eukaryotes, including mice and humans, have identified polyA-containing transcripts that lack long open reading frames (ORFs; >100 amino acids). These transcripts are believed most likely to function as non-coding RNAs, but their translational capacities and biological activities have not been characterized in detail. Here, we report that polished rice (pri), which was previously identified as a gene for a non-coding RNA in Drosophila, is in fact transcribed into a polycistronic mRNA that contains evolutionarily conserved short ORFs that encode 11 or 32 amino acid-long peptides. pri was expressed in all epithelial tissues during embryogenesis. The loss of pri function completely eliminated apical cuticular structures, including the epidermal denticles and tracheal taenidia, and also caused defective tracheal-tube expansion. We found that pri is essential for the formation of specific F-actin bundles that prefigures the formation of the denticles and taenidium. We provide evidences that pri acts non-cell autonomously and that four of the conserved pri ORFs are functionally redundant. These results demonstrate that pri has essential roles in epithelial morphogenesis by regulating F-actin organization.
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Actinas/metabolismo , Drosophila/embriología , Desarrollo Embrionario/genética , Epitelio/embriología , Péptidos/metabolismo , ARN Mensajero/genética , Animales , Secuencia de Bases , Diferenciación Celular/genética , Secuencia Conservada/genética , Drosophila/citología , Drosophila/metabolismo , Epitelio/metabolismo , Evolución Molecular , Regulación del Desarrollo de la Expresión Génica/genética , Genes/genética , Datos de Secuencia Molecular , Sistemas de Lectura Abierta/genética , Péptidos/genética , Homología de Secuencia de Ácido NucleicoRESUMEN
Measuring mechanical forces of cell-cell interactions is important for studying morphogenesis in multicellular organisms. We previously reported an image-based statistical method for inferring effective mechanical potentials of pairwise cell-cell interactions by fitting cell tracking data with a theoretical model. However, whether this method is applicable to tissues with non-cellular components such as cavities remains elusive. Here we evaluated the applicability of the method to cavity-harboring tissues. Using synthetic data generated by simulations, we found that the effect of expanding cavities was added to the pregiven potentials used in the simulations, resulting in the inferred effective potentials having an additional repulsive component derived from the expanding cavities. Interestingly, simulations by using the effective potentials reproduced the cavity-harboring structures. Then, we applied our method to the mouse blastocysts, and found that the inferred effective potentials can reproduce the cavity-harboring structures. Pairwise potentials with additional repulsive components were also detected in two-dimensional cell sheets, by which curved sheets including tubes and cups were simulated. We conclude that our inference method is applicable to tissues harboring cavities and cell sheets, and the resultant effective potentials are useful to simulate the morphologies.
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The specification of the embryonic central nervous system (CNS) into future brain (forebrain, midbrain, or hindbrain) and spinal cord (SC) regions is a critical step of CNS development. A previous chicken embryo study indicated that anterior epiblast cells marked by Sox2 N2 enhancer activity are specified to the respective brain regions during the transition phase of the epiblast to the neural plate-forming neural primordium. The present study showed that the SC precursors positioned posterior to the hindbrain precursors in the anterior epiblast migrated posteriorly in contrast to the anterior migration of brain precursors. The anteroposterior specification of the CNS precursors occurs at an analogous time (â¼E7.5) in mouse embryos, in which an anterior-to-posterior incremental gradient of Wnt signal strength was observed. To examine the possible Wnt signal contribution to the anteroposterior CNS primordium specification, we utilized mouse epiblast stem cell (EpiSC)-derived neurogenesis in culture. EpiSCs maintained in an activin- and FGF2-containing medium start neural development after the removal of activin, following a day in a transitory state. We placed activin-free EpiSCs in EGF- and FGF2-containing medium to arrest neural development and expand the cells into neural stem cells (NSCs). Simultaneously, a Wnt antagonist or agonist was added to the culture, with the anticipation that different levels of Wnt signals would act on the transitory cells to specify CNS regionality; then, the Wnt-treated cells were expanded as NSCs. Gene expression profiles of six NSC lines were analyzed using microarrays and single-cell RNA-seq. The NSC lines demonstrated anteroposterior regional specification in response to increasing Wnt signal input levels: forebrain-midbrain-, hindbrain-, cervical SC-, and thoracic SC-like lines. The regional coverage of these NSC lines had a range; for instance, the XN1 line expressed Otx2 and En2, indicating midbrain characteristics, but additionally expressed the SC-characteristic Hoxa5. The ranges in the anteroposterior specification of neural primordia may be narrowed as neural development proceeds. The thoracic SC is presumably the posterior limit of the contribution by anterior epiblast-derived neural progenitors, as the characteristics of more posterior SC regions were not displayed.
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Live imaging of migrating and interacting cells in developing embryos has opened a new means for deciphering fundamental principles in morphogenesis and patterning, which was not possible with classic approaches of experimental embryology. In our recent study, we devised a new genetic tool to sparsely label cells with a green-fluorescent protein in the broad field of chicken embryos, enabling the analysis of cell migration during the early stages of brain development. Trajectory analysis indicated that anterior epiblast cells from a broad area gather to the head axis to form the brain primordia or brain-abutting head ectoderm. Grafting the mCherry-labeled stage (st.) 4 node in an anterior embryonic region resulted in the anterior extension of the anterior mesendoderm (AME), the precursor for the prechordal plate and anterior notochord, from the node graft at st. 5. Grafting the st. 4 node or st. 5 AME at various epiblast positions that otherwise develop into the head ectoderm caused local cell gathering to the graft-derived AME. The node was not directly associated with this local epiblast-gathering activity. The gathered anterior epiblast cells developed into secondary brain tissue consisting of consecutive brain portions, e.g., forebrain and midbrain or midbrain and hindbrain, reflecting the brain portion specificities inherent to the epiblast cells. The observations indicated the bipotentiality of all anterior epiblast cells to develop into the brain or head ectoderm. Thus, a new epiblast brain field map is proposed, allowing the reinterpretation of classical node graft data, and the role of the AME is highlighted. The new model leads to the conclusion that the node does not directly participate in brain development.
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Developmental maturation occurs in slow swimming behavior in larval zebrafish; older larvae acquire the ability to perform slow swimming while keeping their head stable in the yaw dimension. A class of long-distance descending commissural excitatory V0v neurons, called MCoD neurons, are known to develop in a later phase of neurogenesis, and participate in slow swimming in older larvae. We hypothesized that these MCoD neurons play a role in coordinating the activities of trunk muscles in the diagonal dimension (e.g., the rostral left and the caudal right) to produce the S-shaped swimming form that contributes to the stability of the head. Here, we show that MCoD neurons do indeed play this role. In larvae in which MCoD neurons were laser-ablated, the swimming body form often adopted a one-sided (C-shaped) bend with reduced appearance of the normal S-shaped bend. With this change in swimming form, the MCoD-ablated larvae exhibited a greater degree of head yaw displacement during slow swimming. In mice, the long-distance descending commissural V0v neurons have been implicated in diagonal interlimb coordination during walking. Together with this, our study suggests that the long-distance descending commissural V0v neurons form an evolutionarily conserved pathway in the spinal locomotor circuits that coordinates the movements of the diagonal body/limb muscles.
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Natación , Pez Cebra , Animales , Larva/fisiología , Ratones , Neuronas/fisiología , Médula Espinal/fisiología , Natación/fisiología , Pez Cebra/fisiologíaRESUMEN
Behavioral responses to environmental factors at the planktonic larval stage can have a crucial influence on habitat selection and therefore adult distributions in many benthic organisms. Reef-building corals show strong patterns of zonation across depth or underwater topography, with different suites of species aggregating in different light environments. One potential mechanism driving this pattern is the response of free-swimming larvae to light. However, there is little experimental support for this hypothesis; in particular, there are few direct and quantitative observations of larval behavior in response to light. Here, we analyzed the swimming behavior of larvae of the common reef coral Acropora tenuis under various light conditions. Larvae exhibited a step-down photophobic response, i.e. a marked decrease in swimming speed, in response to a rapid attenuation (step-down) of light intensity. Observations of larvae under different wavelengths indicated that only the loss of blue light (wavelengths between 400 and 500 nm) produced a significant response. Mathematical simulations of this step-down photophobic response indicate that larvae will aggregate in the lighter areas of two-dimensional large rectangular fields. These results suggest that the step-down photophobic response of coral larvae may play an important role in determining where larval settle on the reef.
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Antozoos/crecimiento & desarrollo , Larva/fisiología , Luz , Animales , Ecosistema , Larva/efectos de la radiaciónRESUMEN
The formation of vascular tubes is driven by extensive changes in endothelial cell (EC) shape. Here, we have identified a role of the actin-binding protein, Marcksl1, in modulating the mechanical properties of EC cortex to regulate cell shape and vessel structure during angiogenesis. Increasing and depleting Marcksl1 expression level in vivo results in an increase and decrease, respectively, in EC size and the diameter of microvessels. Furthermore, endothelial overexpression of Marcksl1 induces ectopic blebbing on both apical and basal membranes, during and after lumen formation, that is suppressed by reduced blood flow. High resolution imaging reveals that Marcksl1 promotes the formation of linear actin bundles and decreases actin density at the EC cortex. Our findings demonstrate that a balanced network of linear and branched actin at the EC cortex is essential in conferring cortical integrity to resist the deforming forces of blood flow to regulate vessel structure.
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Vasos Sanguíneos/anatomía & histología , Vasos Sanguíneos/fisiología , Proteínas de Unión a Calmodulina/metabolismo , Células Endoteliales/metabolismo , Hemodinámica/fisiología , Proteínas de Microfilamentos/metabolismo , Actinas/metabolismo , Actomiosina/metabolismo , Animales , Animales Modificados Genéticamente , Vasos Sanguíneos/citología , Proteínas de Unión a Calmodulina/genética , Células Endoteliales/citología , Regulación del Desarrollo de la Expresión Génica , Proteínas de Microfilamentos/genética , Modelos Animales , Transcriptoma , Pez Cebra/embriologíaRESUMEN
In many plant species, roots maintain specific growth angles relative to the direction of gravity, known as gravitropic set point angles (GSAs). These contribute to the efficient acquisition of water and nutrients. AtLAZY1/LAZY1-LIKE (LZY) genes are involved in GSA control by regulating auxin flow toward the direction of gravity in Arabidopsis. Here, we demonstrate that RCC1-like domain (RLD) proteins, identified as LZY interactors, are essential regulators of polar auxin transport. We show that interaction of the CCL domain of LZY with the BRX domain of RLD is important for the recruitment of RLD from the cytoplasm to the plasma membrane by LZY. A structural analysis reveals the mode of the interaction as an intermolecular ß-sheet in addition to the structure of the BRX domain. Our results offer a molecular framework in which gravity signal first emerges as polarized LZY3 localization in gravity-sensing cells, followed by polar RLD1 localization and PIN3 relocalization to modulate auxin flow.
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Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiología , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Proteínas de Arabidopsis/genética , Transporte Biológico , Gravitropismo , Sensación de Gravedad , Ácidos Indolacéticos/metabolismo , Raíces de Plantas/genética , Raíces de Plantas/crecimiento & desarrollo , Raíces de Plantas/fisiología , Brotes de la Planta , Unión ProteicaRESUMEN
Neural stem cells, called radial glia, maintain epithelial structure during the early neocortical development. The prevailing view claims that when radial glia first proliferate, their symmetric divisions require strict spindle orientation; its perturbation causes precocious neurogenesis and apoptosis. Here, we show that despite this conventional view, radial glia at the proliferative stage undergo normal symmetric divisions by regenerating an apical endfoot even if it is lost by oblique divisions. We found that the Notch-R-Ras-integrin ß1 pathway promotes the regeneration of endfeet, whose leading edge bears ectopic adherens junctions and the Par-polarity complex. However, this regeneration ability gradually declines during the subsequent neurogenic stage and hence oblique divisions induce basal translocation of radial glia to form the outer subventricular zone, a hallmark of the development of the convoluted brain. Our study reveals that endfoot regeneration is a temporally changing cryptic property, which controls the radial glial state and its shift is essential for mammalian brain size expansion.
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Encéfalo/crecimiento & desarrollo , Diferenciación Celular/fisiología , Neurogénesis/fisiología , Neuroglía/citología , Uniones Adherentes/metabolismo , Animales , División Celular/fisiología , Ventrículos Laterales/crecimiento & desarrollo , Mamíferos/metabolismo , Ratones , Células-Madre Neurales/citología , Neuronas/citología , Regeneración/fisiologíaRESUMEN
Time-lapse imaging of fluorescent proteins in living cells has become an indispensable tool in biological sciences. However, its application at the organismal level still faces a number of obstacles, such as large specimen sizes preventing illumination of internal tissues, high background fluorescence and uncontrollable movement of target tissues or embryos. Here we describe our solutions for these issues to obtain 4-D fluorescent images from living Drosophila embryos using confocal microscopes. A computational procedure that detects and corrects the shift of moving objects to virtually stabilize them in time-lapse movies (iSEMS) is presented. We discuss the importance of postimaging treatment of raw image stacks for the discovery of novel phenotypes that have previously escaped attention from the analyses of fixed specimens.
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Biología Evolutiva/métodos , Drosophila/embriología , Drosophila/fisiología , Microscopía Confocal/métodos , Animales , Procesamiento de Imagen Asistido por Computador , Proteínas Luminiscentes/química , Fenotipo , Programas Informáticos , Factores de TiempoRESUMEN
cDNAs encoding a Daphnia magna homolog of aryl hydrocarbon receptor nuclear translocator (ARNT) were isolated and the structural and functional features as well as the expression pattern of their product, DmagARNT, were analyzed. Among the known bHLH-PAS proteins, the deduced amino acid sequences of DmagARNT showed the highest degree of identity to that of Drosophila ARNT (TGO). Expression of DmagARNT in ARNT-lacking mouse Hepa-c4 cells resulted in the compensation for the loss of hypoxia response, suggesting the formation of a dimer with mouse HIF-1alpha and that the resulting heterodimer binds to the hypoxia-responsive elements (HRE), leading to transcription of the downstream luciferase gene. Expression of D. magna ARNT was evident at the middle to late stages of embryonic development (about 25 h to 48 h after ovulation) in several tissues, including a pair of the 1st antenna, 2nd antenna, 2nd maxilla, five pairs of the thoracic limbs, the central nerve system, anus, dorsal organ, maxillary gland, and carapace. As observed in other species, the D. magna ARNT is likely to function broadly as an expressed dimerization partner in developmental processes. In contrast, expression of ARNT in adult D. magna was limited to the epipodites of thoracic limbs, suggesting that ARNT plays a role solely in hypoxia response in adult Daphnia.