RESUMEN
Mitosis in early embryos often proceeds at a rapid pace, but how this pace is achieved is not understood. Here, we show that cyclin B3 is the dominant driver of rapid embryonic mitoses in the C. elegans embryo. Cyclins B1 and B2 support slow mitosis (NEBD to anaphase â¼600 s), but the presence of cyclin B3 dominantly drives the approximately threefold faster mitosis observed in wildtype. Multiple mitotic events are slowed down in cyclin B1 and B2-driven mitosis, and cyclin B3-associated Cdk1 H1 kinase activity is â¼25-fold more active than cyclin B1-associated Cdk1. Addition of cyclin B1 to fast cyclin B3-only mitosis introduces an â¼60-s delay between completion of chromosome alignment and anaphase onset; this delay, which is important for segregation fidelity, is dependent on inhibitory phosphorylation of the anaphase activator Cdc20. Thus, cyclin B3 dominance, coupled to a cyclin B1-dependent delay that acts via Cdc20 phosphorylation, sets the rapid pace and ensures mitotic fidelity in the early C. elegans embryo.
Asunto(s)
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Ciclina B1 , Embrión no Mamífero , Mitosis , Animales , Caenorhabditis elegans/embriología , Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Ciclina B1/metabolismo , Ciclina B1/genética , Embrión no Mamífero/metabolismo , Fosforilación , Proteína Quinasa CDC2/metabolismo , Proteína Quinasa CDC2/genética , Ciclina B/metabolismo , Ciclina B/genética , Proteínas Cdc20/metabolismo , Proteínas Cdc20/genética , Ciclina B2/metabolismo , Ciclina B2/genéticaRESUMEN
As development varies greatly across the tree of life, it may seem difficult to suggest a model that proposes a single mechanism for understanding collective cell behaviors and the coordination of tissue formation. Here we propose a mechanism called differentiation waves, which unify many disparate results involving developmental systems from across the tree of life. We demonstrate how a relatively simple model of differentiation proceeds not from function-related molecular mechanisms, but from so-called differentiation waves. A phenotypic model of differentiation waves is introduced, and its relation to molecular mechanisms is proposed. These waves contribute to a differentiation tree, which is an alternate way of viewing cell lineage and local action of the molecular factors. We construct a model of differentiation wave-related molecular mechanisms (genome, epigenome, and proteome) based on bioinformatic data from the nematode Caenorhabditis elegans. To validate this approach across different modes of development, we evaluate protein expression across different types of development by comparing Caenorhabditis elegans with several model organisms: fruit flies (Drosophila melanogaster), yeast (Saccharomyces cerevisiae), and mouse (Mus musculus). Inspired by gene regulatory networks, two Models of Interactive Contributions (fully-connected MICs and ordered MICs) are used to suggest potential genomic contributions to differentiation wave-related proteins. This, in turn, provides a framework for understanding differentiation and development.
Asunto(s)
Caenorhabditis elegans , Diferenciación Celular , Drosophila melanogaster , Desarrollo Embrionario , Animales , Diferenciación Celular/genética , Caenorhabditis elegans/genética , Caenorhabditis elegans/embriología , Desarrollo Embrionario/genética , Desarrollo Embrionario/fisiología , Drosophila melanogaster/genética , Drosophila melanogaster/embriología , Ratones , Redes Reguladoras de Genes , Saccharomyces cerevisiae/genética , Modelos Biológicos , Regulación del Desarrollo de la Expresión Génica , Biología Computacional/métodosRESUMEN
Calcium is an important signaling molecule during the oocyte-to-embryo transition (OET) and early embryogenesis. The hermaphroditic nematode Caenorhabditis elegans provides several unique advantages for the study of the OET as it is transparent and has an ordered gonad that produces one mature oocyte every ~23 min at 20 °C. We have modified the genetically encoded calcium indicator jGCaMP7s to fluorescently indicate the moment of fertilization within a living organism. We have termed this reporter "CaFE" for Calcium during Fertilization in C. elegans. The CaFE reporter was engineered into a safe harbor locus in single copy, has no significant impact on physiology or fecundity, and produces a robust signal upon fertilization. Here, a series of protocols is presented for utilizing the CaFE reporter as an in vivo tool for dissecting the OET and embryogenesis. We include methods to synchronize worms, examine the effects of RNAi knockdown, mount worms for imaging, and to visualize calcium in oocytes and embryos. Additionally, we present the generation of additional worm strains to aid in this type of analysis. Demonstrating the utility of the CaFE reporter to visualize the timing of fertilization, we report that double ovulation occurs when ipp-5 is targeted by RNAi and that only the first oocyte undergoes immediate fertilization. Furthermore, the discovery of single-cell calcium transients during early embryogenesis is reported here, demonstrating that the CaFE reporter persists into early development. Importantly, the CaFE reporter in worms is simple enough to use for incorporation into course-based undergraduate research (CURE) laboratory classes. The CaFE reporter, coupled with the ordered gonad and ease of RNAi in worms, facilitates inquiry into the cell-cell dynamics required to regulate internal fertilization and early embryogenesis.
Asunto(s)
Caenorhabditis elegans , Calcio , Fertilización , Animales , Caenorhabditis elegans/embriología , Caenorhabditis elegans/genética , Fertilización/fisiología , Calcio/metabolismo , Femenino , Desarrollo Embrionario/fisiología , Oocitos/metabolismo , Señalización del Calcio/fisiologíaRESUMEN
The paramount importance of mechanical forces in morphogenesis and embryogenesis is widely recognized, but understanding the mechanism at the cellular and molecular level remains challenging. Because of its simple internal organization, Caenorhabditis elegans is a rewarding system of study. As demonstrated experimentally, after an initial period of steady elongation driven by the actomyosin network, muscle contractions operate a quasi-periodic sequence of bending, rotation, and torsion, that leads to the final fourfold size of the embryos before hatching. How actomyosin and muscles contribute to embryonic elongation is investigated here theoretically. A filamentary elastic model that converts stimuli generated by biochemical signals in the tissue into driving forces, explains embryonic deformation under actin bundles and muscle activity, and dictates mechanisms of late elongation based on the effects of energy conversion and dissipation. We quantify this dynamic transformation by stretches applied to a cylindrical structure that mimics the body shape in finite elasticity, obtaining good agreement and understanding of both wild-type and mutant embryos at all stages.
Asunto(s)
Actomiosina , Caenorhabditis elegans , Embrión no Mamífero , Contracción Muscular , Caenorhabditis elegans/embriología , Animales , Actomiosina/metabolismo , Contracción Muscular/fisiología , Embrión no Mamífero/fisiología , Embrión no Mamífero/metabolismo , Embrión no Mamífero/embriología , Desarrollo Embrionario , Morfogénesis , Modelos Biológicos , Fenómenos BiomecánicosRESUMEN
Hertwig's rule states that cells divide along their longest axis, usually driven by forces acting on the mitotic spindle. Here, we show that in contrast to this rule, microtubule-based pulling forces in early Caenorhabditis elegans embryos align the spindle with the short axis of the cell. We combine theory with experiments to reveal that in order to correct this misalignment, inward forces generated by the constricting cytokinetic ring rotate the entire cell until the spindle is aligned with the cell's long axis. Experiments with slightly compressed mouse zygotes indicate that this cytokinetic ring-driven mechanism of ensuring Hertwig's rule is general for cells capable of rotating inside a confining shell, a scenario that applies to early cell divisions of many systems.
Asunto(s)
Caenorhabditis elegans , Huso Acromático , Animales , Caenorhabditis elegans/embriología , Ratones , Huso Acromático/metabolismo , Microtúbulos/metabolismo , Citocinesis/fisiología , Rotación , Cigoto/metabolismo , Cigoto/citología , Cigoto/crecimiento & desarrollo , Embrión no Mamífero/citología , Desarrollo Embrionario/fisiología , Modelos BiológicosRESUMEN
In embryonic development and organogenesis, cells sharing identical genetic codes acquire diverse gene expression states in a highly reproducible spatial distribution, crucial for multicellular formation and quantifiable through positional information. To understand the spontaneous growth of complexity, we constructed a one-dimensional division-decision model, simulating the growth of cells with identical genetic networks from a single cell. Our findings highlight the pivotal role of cell division in providing positional cues, escorting the system toward states rich in information. Moreover, we pinpointed lateral inhibition as a critical mechanism translating spatial contacts into gene expression. Our model demonstrates that the spatial arrangement resulting from cell division, combined with cell lineages, imparts positional information, specifying multiple cell states with increased complexity-illustrated through examples in C.elegans. This study constitutes a foundational step in comprehending developmental intricacies, paving the way for future quantitative formulations to construct synthetic multicellular patterns.
Asunto(s)
Redes Reguladoras de Genes , Modelos Biológicos , Animales , Redes Reguladoras de Genes/genética , Caenorhabditis elegans/genética , Caenorhabditis elegans/embriología , Caenorhabditis elegans/crecimiento & desarrollo , División Celular/fisiología , División Celular/genética , Biología Computacional , Desarrollo Embrionario/fisiología , Desarrollo Embrionario/genética , Linaje de la Célula , Simulación por Computador , Regulación del Desarrollo de la Expresión Génica/genéticaRESUMEN
Computational super-resolution methods, including conventional analytical algorithms and deep learning models, have substantially improved optical microscopy. Among them, supervised deep neural networks have demonstrated outstanding performance, however, demanding abundant high-quality training data, which are laborious and even impractical to acquire due to the high dynamics of living cells. Here, we develop zero-shot deconvolution networks (ZS-DeconvNet) that instantly enhance the resolution of microscope images by more than 1.5-fold over the diffraction limit with 10-fold lower fluorescence than ordinary super-resolution imaging conditions, in an unsupervised manner without the need for either ground truths or additional data acquisition. We demonstrate the versatile applicability of ZS-DeconvNet on multiple imaging modalities, including total internal reflection fluorescence microscopy, three-dimensional wide-field microscopy, confocal microscopy, two-photon microscopy, lattice light-sheet microscopy, and multimodal structured illumination microscopy, which enables multi-color, long-term, super-resolution 2D/3D imaging of subcellular bioprocesses from mitotic single cells to multicellular embryos of mouse and C. elegans.
Asunto(s)
Caenorhabditis elegans , Microscopía Fluorescente , Animales , Caenorhabditis elegans/embriología , Microscopía Fluorescente/métodos , Ratones , Imagenología Tridimensional/métodos , Algoritmos , Procesamiento de Imagen Asistido por Computador/métodos , Aprendizaje ProfundoRESUMEN
Systematic functional profiling of the gene set that directs embryonic development is an important challenge. To tackle this challenge, we used 4D imaging of C. elegans embryogenesis to capture the effects of 500 gene knockdowns and developed an automated approach to compare developmental phenotypes. The automated approach quantifies features-including germ layer cell numbers, tissue position, and tissue shape-to generate temporal curves whose parameterization yields numerical phenotypic signatures. In conjunction with a new similarity metric that operates across phenotypic space, these signatures enabled the generation of ranked lists of genes predicted to have similar functions, accessible in the PhenoBank web portal, for â¼25% of essential development genes. The approach identified new gene and pathway relationships in cell fate specification and morphogenesis and highlighted the utilization of specialized energy generation pathways during embryogenesis. Collectively, the effort establishes the foundation for comprehensive analysis of the gene set that builds a multicellular organism.
Asunto(s)
Caenorhabditis elegans , Desarrollo Embrionario , Regulación del Desarrollo de la Expresión Génica , Animales , Caenorhabditis elegans/embriología , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Embrión no Mamífero/metabolismo , Perfilación de la Expresión Génica/métodos , Técnicas de Silenciamiento del Gen , FenotipoRESUMEN
Animal cell cytokinesis, or the physical division of one cell into two, is thought to be driven by constriction of an actomyosin contractile ring at the division plane. The mechanisms underlying cell type-specific differences in cytokinesis remain unknown. Germ cells are totipotent cells that pass genetic information to the next generation. Previously, using formincyk-1(ts) mutant Caenorhabditis elegans 4-cell embryos, we found that the P2 germ precursor cell is protected from cytokinesis failure and can divide with greatly reduced F-actin levels at the cell division plane. Here, we identified two canonical germ fate determinants required for P2-specific cytokinetic protection: PIE-1 and POS-1. Neither has been implicated previously in cytokinesis. These germ fate determinants protect P2 cytokinesis by reducing the accumulation of septinUNC-59 and anillinANI-1 at the division plane, which here act as negative regulators of cytokinesis. These findings may provide insight into the regulation of cytokinesis in other cell types, especially in stem cells with high potency.
Asunto(s)
Actinas , Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , División Celular , Citocinesis , Células Germinativas , Septinas , Animales , Citocinesis/fisiología , Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/embriología , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Septinas/metabolismo , Septinas/genética , Células Germinativas/metabolismo , Células Germinativas/citología , Actinas/metabolismo , Proteínas Contráctiles/metabolismo , Actomiosina/metabolismoRESUMEN
Different signaling mechanisms concur to ensure robust tissue patterning and cell fate instruction during animal development. Most of these mechanisms rely on signaling proteins that are produced, transported, and detected. The spatiotemporal dynamics of signaling molecules are largely unknown, yet they determine signal activity's spatial range and time frame. Here, we use the Caenorhabditis elegans embryo to study how Wnt ligands, an evolutionarily conserved family of signaling proteins, dynamically organize to establish cell polarity in a developing tissue. We identify how Wnt ligands, produced in the posterior half of the embryos, spread extracellularly to transmit information to distant target cells in the anterior half. With quantitative live imaging and fluorescence correlation spectroscopy, we show that Wnt ligands diffuse through the embryo over a timescale shorter than the cell cycle, in the intercellular space, and outside the tissue below the eggshell. We extracted diffusion coefficients of Wnt ligands and their receptor Frizzled and characterized their co-localization. Integrating our different measurements and observations in a simple computational framework, we show how fast diffusion in the embryo can polarize individual cells through a time integration of the arrival of the ligands at the target cells. The polarity established at the tissue level by a posterior Wnt source can be transferred to the cellular level. Our results support a diffusion-based long-range Wnt signaling, which is consistent with the dynamics of developing processes.
Asunto(s)
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Polaridad Celular , Embrión no Mamífero , Proteínas Wnt , Animales , Caenorhabditis elegans/embriología , Caenorhabditis elegans/metabolismo , Proteínas Wnt/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Embrión no Mamífero/metabolismo , Embrión no Mamífero/embriología , Ligandos , Vía de Señalización Wnt , DifusiónRESUMEN
During epithelial morphogenesis, the apical junctions connecting cells must remodel as cells change shape and make new connections with their neighbors. In the C. elegans embryo, new apical junctions form when epidermal cells migrate and seal with one another to encase the embryo in skin ('ventral enclosure'), and junctions remodel when epidermal cells change shape to squeeze the embryo into a worm shape ('elongation'). The junctional cadherin-catenin complex (CCC), which links epithelial cells to each other and to cortical actomyosin, is essential for C. elegans epidermal morphogenesis. RNAi genetic enhancement screens have identified several genes encoding proteins that interact with the CCC to promote epidermal morphogenesis, including the scaffolding protein Afadin (AFD-1), whose depletion alone results in only minor morphogenesis defects. Here, by creating a null mutation in afd-1, we show that afd-1 provides a significant contribution to ventral enclosure and elongation on its own. Unexpectedly, we find that afd-1 mutant phenotypes are strongly modified by diet, revealing a previously unappreciated parental nutritional input to morphogenesis. We identify functional interactions between AFD-1 and the CCC by demonstrating that E-cadherin is required for the polarized distribution of AFD-1 to cell contact sites in early embryos. Finally, we show that afd-1 promotes the enrichment of polarity regulator, and CCC-interacting protein, PAC-1/ARHGAP21 to cell contact sites, and we identify genetic interactions suggesting that afd-1 and pac-1 regulate epidermal morphogenesis at least in part through parallel mechanisms. Our findings reveal that C. elegans AFD-1 makes a significant contribution to epidermal morphogenesis and functionally interfaces with core and associated CCC proteins.
Asunto(s)
Cadherinas , Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Epidermis , Morfogénesis , Animales , Cadherinas/metabolismo , Cadherinas/genética , Caenorhabditis elegans/embriología , Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Cateninas/metabolismo , Cateninas/genética , Células Epidérmicas/metabolismo , Epidermis/metabolismo , Epidermis/embriología , Proteínas Activadoras de GTPasa/metabolismo , Proteínas Activadoras de GTPasa/genética , Proteínas de Microfilamentos/metabolismo , Proteínas de Microfilamentos/genéticaRESUMEN
Bub1 and Polo kinases are well-known multitasking regulators of mitosis. New work shows that they team up at kinetochores to determine the mitotic duration of embryonic divisions in nematodes. As is often the case, the key effector is Cdc20 activity.
Asunto(s)
Ciclo Celular , Embrión no Mamífero , Proteínas Serina-Treonina Quinasas , Animales , Proteínas Cdc20/metabolismo , Embrión no Mamífero/citología , Embrión no Mamífero/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Mitosis , Factores de Tiempo , Caenorhabditis elegans/citología , Caenorhabditis elegans/embriología , Caenorhabditis elegans/metabolismoRESUMEN
How paternal exposure to ionizing radiation affects genetic inheritance and disease risk in the offspring has been a long-standing question in radiation biology. In humans, nearly 80% of transmitted mutations arise in the paternal germline1, but the transgenerational effects of ionizing radiation exposure has remained controversial and the mechanisms are unknown. Here we show that in sex-separated Caenorhabditis elegans strains, paternal, but not maternal, exposure to ionizing radiation leads to transgenerational embryonic lethality. The offspring of irradiated males displayed various genome instability phenotypes, including DNA fragmentation, chromosomal rearrangement and aneuploidy. Paternal DNA double strand breaks were repaired by maternally provided error-prone polymerase theta-mediated end joining. Mechanistically, we show that depletion of an orthologue of human histone H1.0, HIS-24, or the heterochromatin protein HPL-1, could significantly reverse the transgenerational embryonic lethality. Removal of HIS-24 or HPL-1 reduced histone 3 lysine 9 dimethylation and enabled error-free homologous recombination repair in the germline of the F1 generation from ionizing radiation-treated P0 males, consequently improving the viability of the F2 generation. This work establishes the mechanistic underpinnings of the heritable consequences of paternal radiation exposure on the health of offspring, which may lead to congenital disorders and cancer in humans.
Asunto(s)
Caenorhabditis elegans , Daño del ADN , Reparación del ADN , Histonas , Animales , Humanos , Masculino , Caenorhabditis elegans/embriología , Caenorhabditis elegans/genética , Caenorhabditis elegans/efectos de la radiación , Daño del ADN/efectos de la radiación , Inestabilidad Genómica/efectos de la radiación , Histonas/metabolismo , Mutación , Radiación Ionizante , Pérdida del Embrión/genética , Femenino , Roturas del ADN de Doble Cadena/efectos de la radiación , Reparación del ADN por Unión de Extremidades , ADN Polimerasa thetaRESUMEN
A key event at the onset of development is the activation of a contractile actomyosin cortex during the oocyte-to-embryo transition1-3. Here we report on the discovery that, in Caenorhabditis elegans oocytes, actomyosin cortex activation is supported by the emergence of thousands of short-lived protein condensates rich in F-actin, N-WASP and the ARP2/3 complex4-8 that form an active micro-emulsion. A phase portrait analysis of the dynamics of individual cortical condensates reveals that condensates initially grow and then transition to disassembly before dissolving completely. We find that, in contrast to condensate growth through diffusion9, the growth dynamics of cortical condensates are chemically driven. Notably, the associated chemical reactions obey mass action kinetics that govern both composition and size. We suggest that the resultant condensate dynamic instability10 suppresses coarsening of the active micro-emulsion11, ensures reaction kinetics that are independent of condensate size and prevents runaway F-actin nucleation during the formation of the first cortical actin meshwork.
Asunto(s)
Actomiosina , Condensados Biomoleculares , Caenorhabditis elegans , Oocitos , Citoesqueleto de Actina/metabolismo , Proteína 2 Relacionada con la Actina/metabolismo , Proteína 3 Relacionada con la Actina/metabolismo , Actinas/metabolismo , Actomiosina/química , Actomiosina/metabolismo , Animales , Condensados Biomoleculares/química , Condensados Biomoleculares/metabolismo , Caenorhabditis elegans/embriología , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Emulsiones/química , Emulsiones/metabolismo , Oocitos/metabolismo , Proteína Neuronal del Síndrome de Wiskott-Aldrich/metabolismoRESUMEN
SignificanceIntracellular gradients have essential roles in cell and developmental biology, but their formation is not fully understood. We have developed a computational approach facilitating interpretation of protein dynamics and gradient formation. We have combined this computational approach with experiments to understand how Polo-Like Kinase 1 (PLK-1) forms a cytoplasmic gradient in Caenorhabditis elegans embryos. Although the PLK-1 gradient depends on the Muscle EXcess-5/6 (MEX-5/6) proteins, we reveal differences in PLK-1 and MEX-5 gradient formation that can be explained by a model with two components, PLK-1 bound to MEX-5 and unbound PLK-1. Our combined approach suggests that a weak coupling between PLK-1 and MEX-5 reaction-diffusion mechanisms dictates the dynamic exchange of PLK-1 with the cytoplasm, explaining PLK-1 high diffusivity and smooth gradient.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/embriología , Caenorhabditis elegans/metabolismo , Proteoma , Proteómica , Animales , Embrión no Mamífero , Modelos Biológicos , Método de Montecarlo , Morfogénesis , Proteínas Serina-Treonina Quinasas , Transporte de Proteínas , Proteómica/métodosRESUMEN
Single-guide RNAs can target exogenous CRISPR-Cas proteins to unique DNA locations, enabling genetic tools that are efficient, specific and scalable. Here we show that short synthetic guide Piwi-interacting RNAs (piRNAs) (21-nucleotide sg-piRNAs) expressed from extrachromosomal transgenes can, analogously, reprogram the endogenous piRNA pathway for gene-specific silencing in the hermaphrodite germline, sperm and embryos of Caenorhabditis elegans. piRNA-mediated interference ('piRNAi') is more efficient than RNAi and can be multiplexed, and auxin-mediated degradation of the piRNA-specific Argonaute PRG-1 allows conditional gene silencing. Target-specific silencing results in decreased messenger RNA levels, amplification of secondary small interfering RNAs and repressive chromatin modifications. Short (300 base pairs) piRNAi transgenes amplified from arrayed oligonucleotide pools also induce silencing, potentially making piRNAi highly scalable. We show that piRNAi can induce transgenerational epigenetic silencing of two endogenous genes (him-5 and him-8). Silencing is inherited for four to six generations after target-specific sg-piRNAs are lost, whereas depleting PRG-1 leads to essentially permanent epigenetic silencing.
Asunto(s)
Animales Modificados Genéticamente/genética , Caenorhabditis elegans/genética , Silenciador del Gen , Interferencia de ARN , ARN Interferente Pequeño/genética , Animales , Proteínas Argonautas/genética , Caenorhabditis elegans/embriología , Proteínas de Caenorhabditis elegans/genética , Proteínas de Ciclo Celular/genética , Embrión no Mamífero , Epigénesis Genética , Femenino , MasculinoRESUMEN
Contractile ring constriction during cytokinesis is thought to compact central spindle microtubules to form the midbody, an antiparallel microtubule bundle at the intercellular bridge. In Caenorhabditis elegans, central spindle microtubule assembly requires targeting of the CLASP family protein CLS-2 to the kinetochores in metaphase and spindle midzone in anaphase. CLS-2 targeting is mediated by the CENP-F-like HCP-1/2, but their roles in cytokinesis and midbody assembly are not known. We found that although HCP-1 and HCP-2 mostly function cooperatively, HCP-1 plays a more primary role in promoting CLS-2-dependent central spindle microtubule assembly. HCP-1/2 codisrupted embryos did not form central spindles but completed cytokinesis and formed functional midbodies capable of supporting abscission. These central spindle-independent midbodies appeared to form via contractile ring constriction-driven bundling of astral microtubules at the furrow tip. This work suggests that, in the absence of a central spindle, astral microtubules can support midbody assembly and that midbody assembly is more predictive of successful cytokinesis than central spindle assembly.
Asunto(s)
Huso Acromático/metabolismo , Animales , Caenorhabditis elegans/embriología , Proteínas de Caenorhabditis elegans/metabolismo , Embrión no Mamífero/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Microtúbulos/metabolismoRESUMEN
Morphogenesis is a precise and robust dynamic process during metazoan embryogenesis, consisting of both cell proliferation and cell migration. Despite the fact that much is known about specific regulations at molecular level, how cell proliferation and migration together drive the morphogenesis at cellular and organismic levels is not well understood. Using Caenorhabditis elegans as the model animal, we present a phase field model to compute early embryonic morphogenesis within a confined eggshell. With physical information about cell division obtained from three-dimensional time-lapse cellular imaging experiments, the model can precisely reproduce the early morphogenesis process as seen in vivo, including time evolution of location and morphology of each cell. Furthermore, the model can be used to reveal key cell-cell attractions critical to the development of C. elegans embryo. Our work demonstrates how genetic programming and physical forces collaborate to drive morphogenesis and provides a predictive model to decipher the underlying mechanism.
Asunto(s)
Caenorhabditis elegans/embriología , Embrión no Mamífero/fisiología , Desarrollo Embrionario/fisiología , Modelos Biológicos , Animales , Biología ComputacionalRESUMEN
Asymmetric cell division is an essential feature of normal development and certain pathologies. The process and its regulation have been studied extensively in the Caenorhabditis elegans embryo, particularly how symmetry of the actomyosin cortical cytoskeleton is broken by a sperm-derived signal at fertilization, upstream of polarity establishment. Diploscapter pachys is the closest parthenogenetic relative to C. elegans, and D. pachys one-cell embryos also divide asymmetrically. However how polarity is triggered in the absence of sperm remains unknown. In post-meiotic embryos, we find that the nucleus inhabits principally one embryo hemisphere, the future posterior pole. When forced to one pole by centrifugation, the nucleus returns to its preferred pole, although poles appear identical as concerns cortical ruffling and actin cytoskeleton. The location of the meiotic spindle also correlates with the future posterior pole and slight actin enrichment is observed at that pole in some early embryos along with microtubule structures emanating from the meiotic spindle. Polarized location of the nucleus is not observed in pre-meiotic D. pachys oocytes. All together our results are consistent with the idea that polarity of the D. pachys embryo is attained during meiosis, seemingly based on the location of the meiotic spindle, by a mechanism that may be present but suppressed in C. elegans.
Asunto(s)
División Celular Asimétrica/fisiología , Meiosis/fisiología , Oocitos/citología , Oocitos/fisiología , Partenogénesis/fisiología , Rhabditoidea/citología , Rhabditoidea/embriología , Animales , Caenorhabditis elegans/citología , Caenorhabditis elegans/embriología , Núcleo Celular/fisiología , Femenino , Microtúbulos/fisiología , Oviparidad/fisiología , Huso Acromático/fisiologíaRESUMEN
Confocal microscopy1 remains a major workhorse in biomedical optical microscopy owing to its reliability and flexibility in imaging various samples, but suffers from substantial point spread function anisotropy, diffraction-limited resolution, depth-dependent degradation in scattering samples and volumetric bleaching2. Here we address these problems, enhancing confocal microscopy performance from the sub-micrometre to millimetre spatial scale and the millisecond to hour temporal scale, improving both lateral and axial resolution more than twofold while simultaneously reducing phototoxicity. We achieve these gains using an integrated, four-pronged approach: (1) developing compact line scanners that enable sensitive, rapid, diffraction-limited imaging over large areas; (2) combining line-scanning with multiview imaging, developing reconstruction algorithms that improve resolution isotropy and recover signal otherwise lost to scattering; (3) adapting techniques from structured illumination microscopy, achieving super-resolution imaging in densely labelled, thick samples; (4) synergizing deep learning with these advances, further improving imaging speed, resolution and duration. We demonstrate these capabilities on more than 20 distinct fixed and live samples, including protein distributions in single cells; nuclei and developing neurons in Caenorhabditis elegans embryos, larvae and adults; myoblasts in imaginal disks of Drosophila wings; and mouse renal, oesophageal, cardiac and brain tissues.