Early Caenorhabditis Elegans Embryo Research Articles

A cytokinetic ring-driven cell rotation achieves Hertwig’s rule in early development

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.

Loss of the mitochondrial protein SPD-3 elevates PLK-1 levels and dysregulates mitotic events.

In metazoans, Polo-like kinase (PLK1) controls several mitotic events including nuclear envelope breakdown, centrosome maturation, spindle assembly and progression through mitosis. Here we show that a mutation in the mitochondria-localized protein SPD-3 affects mitotic events by inducing elevated levels of PLK-1 in early Caenorhabditis elegans embryos. SPD-3 mutant embryos contain abnormally positioned mitotic chromosomes, show a delay in anaphase onset and asymmetrically disassemble the nuclear lamina. We found that more PLK-1 accumulated on centrosomes, nuclear envelope, nucleoplasm, and chromatin before NEBD, suggesting that PLK-1 overexpression is responsible for some of the observed mitotic phenotypes. In agreement with this, the chromosome positioning defects of the spd-3(oj35) mutant could be rescued by reducing PLK-1 levels. Our data suggests that the mitochondrial SPD-3 protein affects chromosome positioning and nuclear envelope integrity by up-regulating the endogenous levels of PLK-1 during early embryogenesis in C. elegans This finding suggests a novel link between mitochondria and nuclear envelope dynamics and chromosome positioning by increasing the amount of a key mitotic regulator, PLK-1, providing a novel link between mitochondria and mitosis.

Cell shape characterization, alignment and comparison using FlowShape.

The shape of a cell is tightly controlled, and reflects important processes including actomyosin activity, adhesion properties, cell differentiation and polarization. Hence, it is informative to link cell shape to genetic and other perturbations. However, most currently used cell shape descriptors capture only simple geometric features such as volume and sphericity. We propose FlowShape, a new framework to study cell shapes in a complete and generic way. In our framework a cell shape is represented by measuring the curvature of the shape and mapping it onto a sphere in a conformal manner. This single function on the sphere is next approximated by a series expansion: the spherical harmonics decomposition. The decomposition facilitates many analyses, including shape alignment and statistical cell shape comparison. The new tool is applied to perform a complete, generic analysis of cell shapes, using the early Caenorhabditis elegans embryo as a model case. We distinguish and characterize the cells at the seven-cell stage. Next, a filter is designed to identify protrusions on the cell shape to highlight lamellipodia in cells. Further, the framework is used to identify any shape changes following a gene knockdown of the Wnt pathway. Cells are first optimally aligned using the fast Fourier transform, followed by calculating an average shape. Shape differences between conditions are next quantified and compared to an empirical distribution. Finally, we put forward a highly performant implementation of the core algorithm, as well as routines to characterize, align and compare cell shapes, through the open-source software package FlowShape. The data and code needed to recreate the results are freely available at https://doi.org/10.5281/zenodo.7778752. The most recent version of the software is maintained at https://bitbucket.org/pgmsembryogenesis/flowshape/. Supplementary data are available at Bioinformatics online.

Acquisition of the spindle assembly checkpoint and its modulation by cell fate and cell size in a chordate embryo.

The spindle assembly checkpoint (SAC) is a surveillance system that preserves genome integrity by delaying anaphase onset until all chromosomes are correctly attached to spindle microtubules. Recruitment of SAC proteins to unattached kinetochores generates an inhibitory signal that prolongs mitotic duration. Chordate embryos are atypical in that spindle defects do not delay mitotic progression during early development, implying that either the SAC is inactive or the cell-cycle target machinery is unresponsive. Here, we show that in embryos of the chordate Phallusia mammillata, the SAC delays mitotic progression from the 8th cleavage divisions. Unattached kinetochores are not recognized by the SAC machinery until the 7th cell cycle, when the SAC is acquired. After acquisition, SAC strength, which manifests as the degree of mitotic lengthening induced by spindle perturbations, is specific to different cell types and is modulated by cell size, showing similarity to SAC control in early Caenorhabditis elegans embryos. We conclude that SAC acquisition is a process that is likely specific to chordate embryos, while modulation of SAC efficiency in SAC proficient stages depends on cell fate and cell size, which is similar to non-chordate embryos.

Aurora A and cortical flows promote polarization and cytokinesis by inducing asymmetric ECT-2 accumulation.

In the early Caenorhabditis elegans embryo, cell polarization and cytokinesis are interrelated yet distinct processes. Here, we sought to understand a poorly understood aspect of cleavage furrow positioning. Early C. elegans embryos deficient in the cytokinetic regulator centralspindlin form furrows, due to an inhibitory activity that depends on aster positioning relative to the polar cortices. Here, we show polar relaxation is associated with depletion of cortical ECT-2, a RhoGEF, specifically at the posterior cortex. Asymmetric ECT-2 accumulation requires intact centrosomes, Aurora A (AIR-1), and myosin-dependent cortical flows. Within a localization competent ECT-2 fragment, we identified three putative phospho-acceptor sites in the PH domain of ECT-2 that render ECT-2 responsive to inhibition by AIR-1. During both polarization and cytokinesis, our results suggest that centrosomal AIR-1 breaks symmetry via ECT-2 phosphorylation; this local inhibition of ECT-2 is amplified by myosin-driven flows that generate regional ECT-2 asymmetry. Together, these mechanisms cooperate to induce polarized assembly of cortical myosin, contributing to both embryo polarization and cytokinesis.

Biophysical Models of PAR Cluster Transport by Cortical Flow in C. elegans Early Embryogenesis.

The clustering of membrane-bound proteins facilitates their transport by cortical actin flow in early Caenorhabditis elegans embryo cell polarity. PAR-3 clustering is critical for this process, yet the biophysical processes that couple protein clusters to cortical flow remain unknown. We develop a discrete, stochastic agent-based model of protein clustering and test four hypothetical models for how clusters may interact with the flow. Results show that the canonical way to assess transport characteristics from single-particle tracking data used thus far in this area, the Péclet number, is insufficient to distinguish these hypotheses and that all models can account for transport characteristics quantified by this measure. However, using this model, we demonstrate that these different cluster-cortex interactions may be distinguished using a different metric, namely the scalar projection of cluster displacement on to the flow displacement vector. Our results thus provide a testable way to use existing single-particle tracking data to test how endogenous protein clusters may interact with the cortical flow to localize during polarity establishment. To facilitate this investigation, we also develop both improved simulation and semi-analytic methodologies to quantify motion summary statistics (e.g., Péclet number and scalar projection) for these stochastic models as a function of biophysical parameters.

Modulating RhoA effectors induces transitions to oscillatory and more wavelike RhoA dynamics in Caenorhabditis elegans zygotes.

Pulsatile RhoA dynamics underlie a wide range of cell and tissue behaviors. The circuits that produce these dynamics in different cells share common architectures based on fast positive and delayed negative feedback through F-actin, but they can produce very different spatiotemporal patterns of RhoA activity. However, the underlying causes of this variation remain poorly understood. Here we asked how this variation could arise through modulation of actin network dynamics downstream of active RhoA in early Caenorhabditis elegans embryos. We find that perturbing two RhoA effectors—formin and anillin—induce transitions from nonrecurrent focal pulses to either large noisy oscillatory pulses (formin depletion) or noisy oscillatory waves (anillin depletion). In both cases these transitions could be explained by changes in local F-actin levels and depletion dynamics, leading to changes in spatial and temporal patterns of RhoA inhibition. However, the underlying mechanisms for F-actin depletion are distinct, with different dependencies on myosin II activity. Thus, modulating actomyosin network dynamics could shape the spatiotemporal dynamics of RhoA activity for different physiological or morphogenetic functions.

Computable early Caenorhabditis elegans embryo with a phase field model.

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.

PIE-1 SUMOylation promotes germline fates and piRNA-dependent silencing in C. elegans.

Germlines shape and balance heredity, integrating and regulating information from both parental and foreign sources. Insights into how germlines handle information have come from the study of factors that specify or maintain the germline fate. In early Caenorhabditis elegans embryos, the CCCH zinc finger protein PIE-1 localizes to the germline where it prevents somatic differentiation programs. Here, we show that PIE-1 also functions in the meiotic ovary where it becomes SUMOylated and engages the small ubiquitin-like modifier (SUMO)-conjugating machinery. Using whole-SUMO-proteome mass spectrometry, we identify HDAC SUMOylation as a target of PIE-1. Our analyses of genetic interactions between pie-1 and SUMO pathway mutants suggest that PIE-1 engages the SUMO machinery both to preserve the germline fate in the embryo and to promote Argonaute-mediated surveillance in the adult germline.

Roles of Actin in the Morphogenesis of the Early Caenorhabditis elegans Embryo.

The cell shape changes that ensure asymmetric cell divisions are crucial for correct development, as asymmetric divisions allow for the formation of different cell types and therefore different tissues. The first division of the Caenorhabditis elegans embryo has emerged as a powerful model for understanding asymmetric cell division. The dynamics of microtubules, polarity proteins, and the actin cytoskeleton are all key for this process. In this review, we highlight studies from the last five years revealing new insights about the role of actin dynamics in the first asymmetric cell division of the early C. elegans embryo. Recent results concerning the roles of actin and actin binding proteins in symmetry breaking, cortical flows, cortical integrity, and cleavage furrow formation are described.

PIE-1 Translation in the Germline Lineage Contributes to PIE-1 Asymmetry in the Early Caenorhabditis elegans Embryo.

In the C. elegans embryo, the germline lineage is established through successive asymmetric cell divisions that each generate a somatic and a germline daughter cell. PIE-1 is an essential maternal factor that is enriched in embryonic germline cells and is required for germline specification. We estimated the absolute concentration of PIE-1::GFP in germline cells and find that PIE-1::GFP concentration increases by roughly 4.5 fold, from 92 nM to 424 nM, between the 1 and 4-cell stages. Previous studies have shown that the preferential inheritance of PIE-1 by germline daughter cells and the degradation of PIE-1 in somatic cells are important for PIE-1 enrichment in germline cells. In this study, we provide evidence that the preferential translation of maternal PIE-1::GFP transcripts in the germline also contributes to PIE-1::GFP enrichment. Through an RNAi screen, we identified Y14 and MAG-1 (Drosophila tsunagi and mago nashi) as regulators of embryonic PIE-1::GFP levels. We show that Y14 and MAG-1 do not regulate PIE-1 degradation, segregation or synthesis in the early embryo, but do regulate the concentration of maternally-deposited PIE-1::GFP. Taken together, or findings point to an important role for translational control in the regulation of PIE-1 levels in the germline lineage.

The ARP2/3 complex prevents excessive formin activity during cytokinesis.

Cytokinesis completes cell division by constriction of an actomyosin contractile ring that separates the two daughter cells. Here we use the early Caenorhabditis elegans embryo to explore how the actin filament network in the ring and the surrounding cortex is regulated by the single cytokinesis formin CYK-1 and the ARP2/3 complex, which nucleate nonbranched and branched filaments, respectively. We show that CYK-1 and the ARP2/3 complex are the predominant F-actin nucleators responsible for generating distinct cortical F-actin architectures and that depletion of either nucleator affects the kinetics of cytokinesis. CYK-1 is critical for normal F-actin levels in the contractile ring, and acute inhibition of CYK-1 after furrow ingression slows ring constriction rate, suggesting that CYK-1 activity is required throughout ring constriction. Surprisingly, although the ARP2/3 complex does not localize in the contractile ring, depletion of the ARP2 subunit or treatment with ARP2/3 complex inhibitor delays contractile ring formation and constriction. We present evidence that the delays are due to an excess in formin-nucleated cortical F-actin, suggesting that the ARP2/3 complex negatively regulates CYK-1 activity. We conclude that the kinetics of cytokinesis are modulated by interplay between the two major actin filament nucleators.

Excitable RhoA dynamics drive pulsed contractions in the early C. elegans embryo.

Pulsed actomyosin contractility underlies diverse modes of tissue morphogenesis, but the underlying mechanisms remain poorly understood. Here, we combined quantitative imaging with genetic perturbations to identify a core mechanism for pulsed contractility in early Caenorhabditis elegans embryos. We show that pulsed accumulation of actomyosin is governed by local control of assembly and disassembly downstream of RhoA. Pulsed activation and inactivation of RhoA precede, respectively, the accumulation and disappearance of actomyosin and persist in the absence of Myosin II. We find that fast (likely indirect) autoactivation of RhoA drives pulse initiation, while delayed, F-actin-dependent accumulation of the RhoA GTPase-activating proteins RGA-3/4 provides negative feedback to terminate each pulse. A mathematical model, constrained by our data, suggests that this combination of feedbacks is tuned to generate locally excitable RhoA dynamics. We propose that excitable RhoA dynamics are a common driver for pulsed contractility that can be tuned or coupled differently to actomyosin dynamics to produce a diversity of morphogenetic outcomes.

The importance of mechanical constraints for proper polarization and psuedo-cleavage furrow generation in the early Caenorhabditis elegans embryo.

Intracellular polarization, where a cell specifies a spatial axis by segregation of specific factors, is a fundamental biological process. In the early embryo of the nematode worm Caenorhabditis elegans (C. elegans), polarization is often accompanied by deformations of the cortex, a highly contractile structure consisting of actin filaments cross-linked by the motor protein myosin (actomyosin). It has been suggested that the eggshell surrounding the early embryo plays a role in polarization although its function is not understood. Here we develop a mathematical model which couples a reaction-diffusion model of actomyosin dynamics with a phase field model of the cell cortex to implicitly track cell shape changes in the early C. elegans embryo. We investigate the potential rigidity effect of the geometric constraint imposed by the presence and size of the eggshell on polarization dynamics. Our model suggests that the geometric constraint of the eggshell is essential for proper polarization and the size of the eggshell also affects the dynamics of polarization. Therefore, we conclude that geometric constraint on a cell might affect the dynamics of a biochemical process.

Integrated Microfluidic Device for Drug Studies of Early C. Elegans Embryogenesis.

Small molecules inhibitors are powerful tools for studying multiple aspects of cell biology and stand at the forefront of drug discovery pipelines. However, in the early Caenorhabditis elegans (C. elegans) embryo, which is a powerful model system for cell and developmental biology, the use of small molecule inhibitors has been limited by the impermeability of the embryonic eggshell, the low‐throughput manual embryo isolation methods, and the lack of well‐controlled drug delivery protocols. This work reports a fully integrated microfluidic approach for studies of C. elegans early embryogenesis, including the possibility of testing small molecule inhibitors with increased throughput and versatility. The setup enables robust on‐chip extraction of embryos from gravid adult worms in a dedicated pillar array chamber by mechanical compression, followed by rapid fluidic transfer of embryos into an adjacent microtrap array. Parallel analysis of ≈100 embryos by high‐resolution time‐lapse imaging from the one‐cell stage zygote until hatching can be performed with this device. The implementation of versatile microfluidic protocols, in particular time‐controlled and reversible drug delivery to on‐chip immobilized embryos, demonstrates the potential of the device for biochemical and pharmacological assays.

Stronger net posterior cortical forces and asymmetric microtubule arrays produce simultaneous centration and rotation of the pronuclear complex in the early Caenorhabditis elegans embryo.

Positioning of microtubule-organizing centers (MTOCs) incorporates biochemical and mechanical cues for proper alignment of the mitotic spindle and cell division site. Current experimental and theoretical studies in the early Caenorhabditis elegans embryo assume remarkable changes in the origin and polarity of forces acting on the MTOCs. These changes must occur over a few minutes, between initial centration and rotation of the pronuclear complex and entry into mitosis, and the models do not replicate in vivo timing of centration and rotation. Here we propose a model that incorporates asymmetry in the microtubule arrays generated by each MTOC, which we demonstrate with in vivo measurements, and a similar asymmetric force profile to that required for posterior-directed spindle displacement during mitosis. We find that these asymmetries are capable of and important for recapitulating the simultaneous centration and rotation of the pronuclear complex observed in vivo. The combination of theoretical and experimental evidence provided here offers a unified framework for the spatial organization and forces needed for pronuclear centration, rotation, and spindle displacement in the early C. elegans embryo.

The Mitotic Spindle in the One-Cell C. elegans Embryo Is Positioned with High Precision and Stability

Precise positioning of the mitotic spindle is important for specifying the plane of cell division, which in turn determines how the cytoplasmic contents of the mother cell are partitioned into the daughter cells, and how the daughters are positioned within the tissue. During metaphase in the early Caenorhabditis elegans embryo, the spindle is aligned and centered on the anterior-posterior axis by a microtubule-dependent machinery that exerts restoring forces when the spindle is displaced from the center. To investigate the accuracy and stability of centering, we tracked the position and orientation of the mitotic spindle during the first cell division with high temporal and spatial resolution. We found that the precision is remarkably high: the cell-to-cell variation in the transverse position of the center of the spindle during metaphase, as measured by the standard deviation, was only 1.5% of the length of the short axis of the cell. Spindle position is also very stable: the standard deviation of the fluctuations in transverse spindle position during metaphase was only 0.5% of the short axis of the cell. Assuming that stability is limited by fluctuations in the number of independent motor elements such as microtubules or dyneins underlying the centering machinery, we infer that the number is ∼1000, consistent with the several thousand of astral microtubules in these cells. Astral microtubules grow out from the two spindle poles, make contact with the cell cortex, and then shrink back shortly thereafter. The high stability of centering can be accounted for quantitatively if, while making contact with the cortex, the astral microtubules buckle as they exert compressive, pushing forces. We thus propose that the large number of microtubules in the asters provides a highly precise mechanism for positioning the spindle during metaphase while assembly is completed before the onset of anaphase.

Analysis of cytokinesis by electron microscopy.

Following up on a chapter on the Correlative Light and Electron Microscopy of Early Caenorhabditis elegans Embryos in Mitosis (MCB 79, 101-119), we present an adaptation of our established protocol for the ultrastructural analysis of either permeabilized or injected embryonic systems. We prepared both drug-treated early C. elegans embryos and fluorescently labeled sea urchin embryos of Lytechinus pictus for ultrastructural studies on animal cytokinesis. Here we focus on the initial preparation steps of postmitotic embryos for high-pressure freezing and subsequent electron microscopy with an emphasis on electron tomography. The advantages and limitations of our extended protocol will be discussed.

Cdk1 phosphorylates SPAT-1/Bora to trigger PLK-1 activation and drive mitotic entry in C. elegans embryos.

The molecular mechanisms governing mitotic entry during animal development are incompletely understood. Here, we show that the mitotic kinase CDK-1 phosphorylates Suppressor of Par-Two 1 (SPAT-1)/Bora to regulate its interaction with PLK-1 and to trigger mitotic entry in early Caenorhabditis elegans embryos. Embryos expressing a SPAT-1 version that is nonphosphorylatable by CDK-1 and that is defective in PLK-1 binding in vitro present delays in mitotic entry, mimicking embryos lacking SPAT-1 or PLK-1 functions. We further show that phospho-SPAT-1 activates PLK-1 by triggering phosphorylation on its activator T loop in vitro by Aurora A. Likewise, we show that phosphorylation of human Bora by Cdk1 promotes phosphorylation of human Plk1 by Aurora A, suggesting that this mechanism is conserved in humans. Our results suggest that CDK-1 activates PLK-1 via SPAT-1 phosphorylation to promote entry into mitosis. We propose the existence of a positive feedback loop that connects Cdk1 and Plk1 activation to ensure a robust control of mitotic entry and cell division timing.

The RNA binding protein MEX-3 retains asymmetric activity in the early Caenorhabditis elegans embryo in the absence of asymmetric protein localization

The RNA binding protein MEX-3 is required to restrict translation of pal-1, the Caenorhabditis elegans caudal homolog, to the posterior of the early embryo. MEX-3 is present uniformly throughout the newly fertilized embryo, but becomes depleted in the posterior by the 4-cell stage. This MEX-3 patterning requires the CCCH zinc-finger protein MEX-5, the RNA Recognition Motif protein SPN-4, and the kinase PAR-4. Genetic and biochemical evidence suggests that MEX-5 binds to MEX-3 in the anterior of the embryo, protecting MEX-3 from degradation and allowing it to bind the pal-1 3′UTR and repress translation. MEX-3 that is not bound to MEX-5 becomes inactivated by par-4, then targeted for spn-4 dependent degradation. After the 4-cell stage, residual MEX-3 is degraded in somatic cells, and only persists in the germline precursors.To better understand regulation of mex-3, GFP was fused to MEX-3 or regions of MEX-3 and expressed in developing oocytes. GFP::MEX-3 expressed in this manner can replace endogenous MEX-3, but surprisingly is not asymmetrically localized at the 4-cell stage. These results indicate that GFP::MEX-3 retains asymmetric activity even in the absence of asymmetric protein localization. Neither the mex-3 3′UTR nor protein degradation at the 4-cell stage is strictly required. A region of MEX-3 containing a glutamine-rich region and potential ubiquitination and phosphorylation sites is sufficient for soma-germline asymmetry. Results from mex-5/6 and spn-4(RNAi) suggest two pathways for MEX-3 degradation, an early spn-4 dependent pathway and a later spn-4 independent pathway. These results indicate that mex-3 activity is regulated at multiple levels, leading to rapid and robust regulation in the quickly developing early embryo.