Early Embryos Of Xenopus Laevis Research Articles

Cell cycle oscillations driven by two interlinked bistable switches.

Regular transitions between interphase and mitosis during the cell cycle are driven by changes in the activity of the enzymatic protein complex cyclin B with cyclin-dependent kinase 1 (Cdk1). At the most basic level, this cell cycle oscillator is driven by negative feedback: active cyclin B-Cdk1 activates the anaphase-promoting complex/cyclosome, which triggers the degradation of cyclin B. Such cell cycle oscillations occur fast and periodically in the early embryos of the frog Xenopus laevis, where several positive-feedback loops leading to bistable switches in parts of the regulatory network have been experimentally identified. Here, we build cell cycle oscillator models to show how single and multiple bistable switches in parts of the underlying regulatory network change the properties of the oscillations and how they can confer robustness to the oscillator. We present a detailed bifurcation analysis of these models.

Active inference, morphogenesis, and computational psychiatry.

Active inference is a leading theory in neuroscience that provides a simple and neuro-biologically plausible account of how action and perception are coupled in producing (Bayes) optimal behavior; and has been recently used to explain a variety of psychopathological conditions. In parallel, morphogenesis has been described as the behavior of a (non-neural) cellular collective intelligence solving problems in anatomical morphospace. In this article, we establish a link between the domains of cell biology and neuroscience, by analyzing disorders of morphogenesis as disorders of (active) inference. The aim of this article is three-fold. We want to: (i) reveal a connection between disorders of morphogenesis and disorders of active inference as apparent in psychopathological conditions; (ii) show how disorders of morphogenesis can be simulated using active inference; (iii) suggest that active inference can shed light on developmental defects or aberrant morphogenetic processes, seen as disorders of information processing, and perhaps suggesting novel intervention and repair strategies. We present four simulations illustrating application of these ideas to cellular behavior during morphogenesis. Three of the simulations show that the same forms of aberrant active inference (e.g., deficits of sensory attenuation and low sensory precision) that have been used to explain psychopathological conditions (e.g., schizophrenia and autism) also produce familiar disorders of development and morphogenesis when implemented at the level of the collective behavior of a group of cells. The fourth simulation involves two cells with too high precision, in which we show that the reduction of concentration signaling and sensitivity to the signals of other cells treats the development defect. Finally, we present the results of an experimental test of one of the model's predictions in early Xenopus laevis embryos: thioridazine (a dopamine antagonist that may reduce sensory precision in biological systems) induced developmental (anatomical) defects as predicted. The use of conceptual and empirical tools from neuroscience to understand the morphogenetic behavior of pre-neural agents offers the possibility of new approaches in regenerative medicine and evolutionary developmental biology.

Studying the DNA damage response in embryonic systems.

Maintenance and surveillance of genome integrity is crucial during the very early steps of embryonic development, since de novo mutations generated during this stage can be propagated in differentiated adult cells and may lead to predisposition to diseases including cancer. Surprisingly, early embryos are characterized by a relaxed control of genome integrity, reminiscent of that observed in cancer cells. How embryos manage to produce healthy adult individuals in such conditions remains still unclear. Here, we describe protocols and methods to study and analyze the DNA damage response and genome integrity in two embryonic experimental systems, early Xenopus laevis embryos and mouse embryonic stem cells. We describe methods to study gene functions in the DNA damage response by mRNA microinjection in Xenopus embryos generated by in vitro fertilization, mutagenesis and developmental regulation of the DNA damage response. We also describe methods to analyze the DNA damage response in mESCs, including synchronization experiments that allow studying the DNA damage response at different cell cycle stages. Analysis of genome integrity in these systems may also help to shed light on the molecular mechanisms that preserve genome integrity and become dysregulated in cancer cells.

A Generalizable Optogenetic Strategy to Regulate Receptor Tyrosine Kinases during Vertebrate Embryonic Development

Ligand-independent activation of receptor tyrosine kinases (RTKs) allows for dissecting out the receptor-specific signaling outcomes from the pleiotropic effects of the ligands. In this regard, RTK intracellular domains (ICD) are of interest due to their ability to recapitulate signaling activity in a ligand-independent manner when fused to chemical or optical dimerizing domains. A common strategy for synthetic activation of RTKs involves membrane tethering of dimerizer–RTK ICD fusions. Depending on the intrinsic signaling capacity, however, this approach could entail undesirable baseline signaling activity in the absence of stimulus, thereby diminishing the system's sensitivity. Here, we observed toxicity in early Xenopus laevis embryos when using such a conventional optogenetic design for the fibroblast growth factor receptor (FGFR). To surpass this challenge, we developed a cytoplasm-to-membrane translocation approach, where FGFR ICD is recruited from the cytoplasm to the plasma membrane by light, followed by its subsequent activation via homo-association. This strategy results in the optical activation of FGFR with low background activity and high sensitivity, which allows for the light-mediated formation of ectopic tail-like structures in developing X. laevis embryos. We further generalized this strategy by developing optogenetic platforms to control three neurotrophic tropomyosin receptor kinases, TrkA, TrkB, and TrkC. We envision that these ligand-independent optogenetic RTKs will provide useful toolsets for the delineation of signaling sub-circuits in developing vertebrate embryos.

Natural size variation among embryos leads to the corresponding scaling in gene expression

Xenopus laevis frogs from laboratory stocks normally lay eggs exhibiting extensive size variability. We find that these initial size differences subsequently affect the size of the embryos prior to the onset of growth, and the size of tadpoles during the growth period. Even though these tadpoles differ in size, their tissues, organs, and structures always seem to be properly proportioned, i.e. they display static allometry. Initial axial patterning events in Xenopus occur in a spherical embryo, allowing easy documentation of their size-dependent features. We examined the size distribution of early Xenopus laevis embryos and measured diameters that differed by about 38% with a median of about 1.43 ​mm. This range of embryo sizes corresponds to about a 1.9-fold difference in surface area and a 2.6-fold difference in volume. We examined the relationship between embryo size and gene expression and observed a significant correlation between diameter and RNA content during gastrula stages. In addition, we investigated the expression levels of genes that pattern the mesoderm, induce the nervous system and mediate the progression of ectodermal cells to neural precursors in large and small embryos. We found that most of these factors were expressed at levels that scaled with the different embryo sizes and total embryo RNA content. In agreement with the changes in transcript levels, the expression domains in larger embryos increased proportionally with the increase in surface area, maintaining their relative expression domain size in relation to the total size of the embryo. Thus, our study identified a mechanism for adapting gene expression domains to embryo size by adjusting the transcript levels of the genes regulating mesoderm induction and patterning. In the neural plate, besides the scaling of the expression domains, we observed similar cell sizes and cell densities in small and large embryos suggesting that additional cell divisions took place in large embryos to compensate for the increased size. Our results show in detail the size variability among Xenopus laevis embryos and the transcriptional adaptation to scale gene expression with size. The observations further support the involvement of BMP/ADMP signaling in the scaling process.

Altering the levels of nuclear import factors in early Xenopus laevis embryos affects later development.

More than just a container for DNA, the nuclear envelope carries out a wide variety of critical and highly regulated cellular functions. One of these functions is nuclear import, and in this study we investigate how altering the levels of nuclear transport factors impacts developmental progression and organismal size. During early Xenopus laevis embryogenesis, the timing of a key developmental event, the midblastula transition (MBT), is sensitive to nuclear import factor levels. How might altering nuclear import factors and MBT timing in the early embryo affect downstream development of the organism? We microinjected X. laevis two-cell embryos with mRNA to increase levels of importin α or NTF2, resulting in differential amounts of nuclear import factors in the two halves of the embryo. Compared to controls, these embryos exhibited delayed gastrulation, curved neural plates, and bent tadpoles with different sized eyes. Furthermore, embryos microinjected with NTF2 developed into smaller froglets compared to control microinjected embryos. We propose that altering nuclear import factors and nuclear size affects MBT timing, cell size, and cell number, subsequently disrupting later development. Thus, altering nuclear import factors early in development can affect function and size at the organismal level.

Desynchronizing Embryonic Cell Division Waves Reveals the Robustness of Xenopus laevis Development

The early Xenopus laevis embryo is replete with dynamic spatial waves. One such wave, the cell division wave, emerges from the collective cell division timing of first tens and later hundreds of cells throughout the embryo. Here, we show that cell division waves do not propagate between neighboring cells and do not rely on cell-to-cell coupling to maintain their division timing. Instead, intrinsic variation in division period autonomously and gradually builds these striking patterns of cell division. Disrupting this pattern of division by placing embryos in a temperature gradient resulted in highly asynchronous entry to the midblastula transition and misexpression of the mesodermal marker Xbra. Remarkably, this gene expression defect is corrected during involution, resulting in delayed yet normal Xbra expression and viable embryos. This implies the existence of a previously unknown mechanism for normalizing mesodermal gene expression during involution.

Actomyosin-generated tension on cadherin is similar between dividing and non-dividing epithelial cells in early Xenopus laevis embryos

Epithelia represent a unique situation where polarized cells must maintain sufficiently strong cell-cell contacts to guarantee the epithelial integrity indispensable for barrier functions. Nevertheless, epithelia must also keep sufficient plasticity which is crucial during development and morphogenesis. Adherens junctions and mechanical forces produced by the actomyosin cytoskeleton are major players for epithelial integrity maintenance and plasticity regulations. To understand how the epithelium is able to meet such a challenge, it is indispensable to determine how cellular junctions and mechanical forces acting at adherens junctions are regulated. Here, we investigate the tensile forces acting on adherens junctions via cadherin during cell division in the Xenopus embryos epithelium. Using the recently developed E-cadherin FRET tension sensor and a fastFLIM prototype microscope, we were able to measure mechanical forces applied on cadherin at cell-cell junctions. We have shown that the Xenopus epithelium is under tension, approximately 3 pN which remains stable, indicating that tensile forces acting on cadherin at the adherens junction are at equilibrium. Unexpectedly, mechanical tension across cadherin was similar between dividing and non-dividing epithelial cells.

The Nedd4 binding protein 3 is required for anterior neural development in Xenopus laevis

The Fezzin family member Nedd4-binding protein 3 (N4BP3) is known to regulate axonal and dendritic branching. Here, we show that n4bp3 is expressed in the neural tissue of the early Xenopus laevis embryo including the eye, the brain and neural crest cells. Knockdown of N4bp3 in the Xenopus anterior neural tissue results in severe developmental impairment of the eye, the brain and neural crest derived cranial cartilage structures. Moreover, we demonstrate that N4bp3 depletion leads to a significant reduction of both eye and brain specific marker genes and reduced neural crest cell migration. Finally, we demonstrate an impact of N4bp3 deficiency on cell apoptosis and proliferation. Our studies indicate that N4bp3 is required for early anterior neural development of vertebrates. This is in line with a study implicating that genetic disruption of N4BP3 in humans might be related to neurodevelopmental disease.

Asymmetries in Cell Division, Cell Size, and Furrowing in the Xenopus laevis Embryo.

Asymmetric cell divisions produce two daughter cells with distinct fate. During embryogenesis, this mechanism is fundamental to build tissues and organs because it generates cell diversity. In adults, it remains crucial to maintain stem cells. The enthusiasm for asymmetric cell division is not only motivated by the beauty of the mechanism and the fundamental questions it raises, but has also very pragmatic reasons. Indeed, misregulation of asymmetric cell divisions is believed to have dramatic consequences potentially leading to pathogenesis such as cancers. In diverse model organisms, asymmetric cell divisions result in two daughter cells, which differ not only by their fate but also in size. This is the case for the early Xenopus laevis embryo, in which the two first embryonic divisions are perpendicular to each other and generate two pairs of blastomeres, which usually differ in size: one pair of blastomeres is smaller than the other. Small blastomeres will produce embryonic dorsal structures, whereas the larger pair will evolve into ventral structures. Here, we present a speculative model on the origin of the asymmetry of this cell division in the Xenopus embryo. We also discuss the apparently coincident asymmetric distribution of cell fate determinants and cell-size asymmetry of the 4-cell stage embryo. Finally, we discuss the asymmetric furrowing during epithelial cell cytokinesis occurring later during Xenopus laevis embryo development.

Dynein light intermediate chains maintain spindle bipolarity by functioning in centriole cohesion.

Cytoplasmic dynein 1 (dynein) is a minus end-directed microtubule motor protein with many cellular functions, including during cell division. The role of the light intermediate chains (LICs; DYNC1LI1 and 2) within the complex is poorly understood. In this paper, we have used small interfering RNAs or morpholino oligonucleotides to deplete the LICs in human cell lines and Xenopus laevis early embryos to dissect the LICs' role in cell division. We show that although dynein lacking LICs drives microtubule gliding at normal rates, the LICs are required for the formation and maintenance of a bipolar spindle. Multipolar spindles with poles that contain single centrioles were formed in cells lacking LICs, indicating that they are needed for maintaining centrosome integrity. The formation of multipolar spindles via centrosome splitting after LIC depletion could be rescued by inhibiting Eg5. This suggests a novel role for the dynein complex, counteracted by Eg5, in the maintenance of centriole cohesion during mitosis.

Changes in oscillatory dynamics in the cell cycle of early Xenopus laevis embryos.

Author SummaryThe early embryonic cell cycles, which initiate cell division, mark the beginning of the life of an organism. Across different phyla, these cycles have a characteristic temporal pattern, with the first cycle being long and the subsequent cycles shorter, leading to rapid increase in cell numbers. Here we have made use of the Xenopus laevis embryos to study the mechanism and significance of this temporal transition. In X. laevis embryos, the cell cycles are driven by oscillations in the activity of the cyclin B–Cdk1 complex, which regulates cell cycle progression by protein phosphorylation. We quantified the oscillatory dynamics of key regulators in the first few embryonic cell cycles, and developed an experimentally parameterized mathematical model of the oscillations. We found that a change in the balance between the Cdk1-activating phosphatase Cdc25 and the Cdk1-inhibiting kinases Wee1 and Myt1 is critical for this transition. Tuning this balance converts the cyclin B–Cdk1 oscillator from generating spiky oscillations with delayed activation, to smooth-varying oscillations with a shorter period. Moreover, we found that it is crucial for the first embryonic cell cycle to be sufficiently long, as shortening it with drugs dramatically decreases embryo viability. Our work shows how X. laevis embryos modulate their cell cycle oscillator dynamics to meet two developmental requirements: a sufficiently long first cell cycle and rapid progression of the subsequent cycles.

Scaling of dorsal‐ventral patterning in the Xenopus laevis embryo

Scaling of pattern with size has been described and studied for over a century, yet its molecular basis is understood in only a few cases. In a recent, elegant study, Inomata and colleagues proposed a new model explaining how bone morphogenic protein (BMP) activity gradient scales with embryo size in the early Xenopus laevis embryo. We discuss their results in conjunction with an alternative model we proposed previously. The expansion-repression mechanism (ExR) provides a conceptual framework unifying both mechanisms. Results of Inomata and colleagues implicate the chordin-stabilizing protein sizzled as the expander molecule enabling scaling, while we attributed this role to the BMP ligand Admp. The two expanders may work in concert, as suggested by the mathematical model of Inomata et al. We discuss approaches for differentiating the contribution of sizzled and Admp to pattern scaling.

Control of DNA Replication by the Nucleus/Cytoplasm Ratio in Xenopus

The nucleus/cytoplasm (N/C) ratio controls S phase dynamics in many biological systems, most notably the abrupt remodeling of the cell cycle that occurs at the midblastula transition in early Xenopus laevis embryos. After an initial series of rapid cleavage cycles consisting only of S and M phases, a critical N/C ratio is reached, which causes a sharp increase in the length of S phase as the cell cycle is reconfigured to resemble somatic cell cycles. How the N/C ratio determines the length of S phase has been a longstanding problem in developmental biology. Using Xenopus egg extracts, we show that DNA replication at high N/C ratio is restricted by one or more limiting substances. We report here that the protein phosphatase PP2A, in conjunction with its B55α regulatory subunit, becomes limiting for replication origin firing at high N/C ratio, and this in turn leads to reduced origin activation and an increase in the time required to complete S phase. Increasing the levels of PP2A catalytic subunit or B55α experimentally restores rapid DNA synthesis at high N/C ratio. Inversely, reduction of PP2A or B55α levels sharply extends S phase even in low N/C extracts. These results identify PP2A-B55α as a link between DNA replication and N/C ratio in egg extracts and suggest a mechanism that may influence the onset of the midblastula transition in vivo.

Preparation and analysis of biological activity of the secreted proteins of the Noggin family

We have developed methods for producing recombinant proteins of Noggin family (Noggin1 and Noggin2 of the Xenopus laevis frog) that can interact with BMP factors of TGF-beta superfamily. The genetic constructs which allow one to effectively obtain Noggin1 and Noggin2 from synthetic mRNA microinjected into Xenopus laevis early embryos, as well as in the prokaryotic expression system, were generated. The obtained proteins contain three Myc-tag epitopes on their N-terminus. This allow one to compare the expression levels of Noggin1 and Noggin 2 constructs, to purify them on the affine immunosorbent and to show the activity of Noggin proteins by analyzing their ability to bind BMP4 factor TGF-beta surperfamily by co-immunoprecipitation.

Developmental regulation of histone H2A deposition via serine-1 phosphorylation

H2A/H4 serine 1 phosphoryation (S1ph) is hypothesized to participate in many aspects of chromatin metabolism, including DNA replication, repair, recombination, mitosis, and regulation of gene expression. However, few specific functions for this ubiquitous histone modification have been demonstrated. In early Xenopus laevis embryos, development is tightly regulated to prevent premature zygotic transcription post-fertilization and to activate zygotic gene expression in a timely manner simultaneous with the mid-blastula transition (MBT). Rapid histone deposition occurs in Xenopus embryos and in sperm pronuclei assembled in cell-free Xenopus egg extract, concomitant with sperm decondensation and DNA replication. In vivo we observed that H2AS1ph appeared upon oocyte germinal vesicle breakdown (GVBD) while H4S1ph only appeared post-MBT. This is the first study to demonstrate a biological distinction between H2A and H4 S1 phosphorylation. H2A S1ph abundance in chromatin was not dependent upon DNA replication nor did it exhibit any cell-cycle changes in chromatin in cycling cell-free extract. However H4S1ph was observed on pronuclei assembled in cell-free extract in a DNA concentration-dependent fashion. The appearance of H4S1P highly correlates with the increased nuclear-to-cytoplasmic ratio found at the beginning of maternal gene expression at the MBT. Addition of wildtype H2A (H2Awt), H2AS1A (unphosphorylatable), and H2AS1E (phosphomimetic) mutant histone H2A/H2B dimers to cell-free extract showed that H2AS1A had a slower rate of chromatin incorporation than either H2Awt or H2AS1E. In cell-free extract H2Awt and H2AS1E, but not the unphosphorylated H2AS1A, interact with the histone storage chaperone Nucleoplasmin. All three interact with Nap1. Finally, overexpression of H2AS1A in Xenopusembryos results in more than 50% embryonic lethality. We conclude that H2A serine 1 phosphorylation is necessary for proper entrance into the histone chaperone pathway in the early embryo.

Use of the luciferase reporter constructs for investigation of the capacity of Noggin2 protein to inhibit cell signaling pathways in Xenopus laevis embryos

Noggin (Noggin1) protein inhibits Smad1-dependent TGF-beta signaling pathway by extracellular binding of BMP proteins. Recently we found two previously unknown representatives of Noggin family - Noggin2 and Noggin4. Here we investigate by using the luciferase reporter constructs the ability of Noggin2 to inhibit BMP-, Activin/Nodal- and Wnt-signaling pathways in the early Xenopus laevis embryos. The effectiveness of this inhibitory activity of Noggin2 is comparing with that of well-known inhibitors of the indicated pathways - the secreted proteins Cerberus and Follistatin. Besides revealing of novel properties of Noggin2, our findings demonstrate that luciferase-reporter assay is as a useful tool for signaling pathways investigation in the model of Xenopus embryos.

Cortical localization of Maternal Embryonic Leucine zipper Kinase (MELK) implicated in cytokinesis in early Xenopus embryos

MELK has been implicated in a large variety of functions. Because its level is elevated in cancer tissues and it is involved in cell proliferation, MELK is considered as a potential therapeutic target for cancers. In a recent, study we have shown that MELK is involved in cytokinesis in early Xenopus laevis embryos. MELK dynamically accumulates at the cell cortex including a narrow band corresponding to the presumptive division furrow shortly before cytokinesis onset. MELK co-localizes and interacts with anillin an important regulator of cytokinesis. In addition, MELK overexpression interferes with accumulation at the cleavage furrow of activated Rho GTPase another crucial regulator of cytokinesis. Interestingly, our study also revealed that a transition implying a change in the direction of asymmetric furrow ingression occurs during early development. After this transition, MELK, as well as other proteins involved in cytokinesis, do not localize anymore as a band at the equatorial cortex but still localizes at the cell cortex. Our results indicate that cortical localization is an important feature of MELK in X. laevis embryos.

Cortical localization of maternal embryonic leucine zipper kinase (MELK) implicated in cytokinesis in early xenopus embryos.

MELK has been implicated in a large variety of functions. Because its level is elevated in cancer tissues and it is involved in cell proliferation, MELK is considered as a potential therapeutic target for cancers. In a recent, study we have shown that MELK is involved in cytokinesis in early Xenopus laevis embryos. MELK dynamically accumulates at the cell cortex including a narrow band corresponding to the presumptive division furrow shortly before cytokinesis onset. MELK co-localizes and interacts with anillin an important regulator of cytokinesis. In addition, MELK overexpression interferes with accumulation at the cleavage furrow of activated Rho GTPase another crucial regulator of cytokinesis. Interestingly, our study also revealed that a transition implying a change in the direction of asymmetric furrow ingression occurs during early development. After this transition, MELK, as well as other proteins involved in cytokinesis, do not localize anymore as a band at the equatorial cortex but still localizes at the cell cortex. Our results indicate that cortical localization is an important feature of MELK in X. laevis embryos.

Irreversible effects of retinoic acid pulse onXenopusjaw morphogenesis: new insight into cranial neural crest specificationb

Jaws are formed by cephalic neural crest (CNCCs) and mesodermal cells migrating to the first pharyngeal arch (PA1). A complex signaling network involving different PA1 components then establishes the jaw morphogenetic program. To gather insight on this developmental process, in this study, we analyze the teratogenic effects of brief (1-15 min) pulses of low doses of retinoic acid (RA: 0.25-2 µM) or RA agonists administered to early Xenopus laevis (X.l.) embryos. We show that these brief pulses of RA cause permanent craniofacial defects specifically when treatments are performed during a 6-hr window (developmental stages NF15-NF23) that covers the period of CNCCs maintenance, migration, and specification. Earlier or later treatments have no effect. Similar treatments performed at slightly different developmental stages within this temporal window give rise to different spectra of malformations. The RA-dependent teratogenic effects observed in Xenopus can be partially rescued by folinic acid. We provide evidence suggesting that in Xenopus, as in the mouse, RA causes craniofacial malformations by perturbing signaling to CNCCs. Differently from the mouse, where RA affects CNCCs only at the end of their migration, in Xenopus, RA has an effect on CNCCs during all the period ranging from their exit from the neural tube until their arrival in the PA1. Our findings provide a conceptual framework to understand the origin of individual facial features and the evolution of different craniofacial morphotypes.