Cortical Rotation Research Articles

A maternal dorsoventral prepattern revealed by an asymmetric distribution of ventralizing molecules before fertilization in Xenopus laevis.

The establishment of the embryonic dorsoventral axis in Xenopus occurs when the radial symmetry around the egg's animal-vegetal axis is broken to give rise to the typical symmetry of Bilaterians. We have previously shown that the Notch1 protein is ventrally enriched during early embryogenesis in Xenopus laevis and zebrafish and exerts ventralizing activity through β-Catenin destabilization and the positive regulation of ventral center genes in X. laevis. These findings led us to further investigate when these asymmetries arise. In this work, we show that the asymmetrical distribution of Notch1 protein and mRNA precedes cortical rotation and even fertilization in X. laevis. Moreover, we found that in unfertilized eggs transcripts encoded by the ventralizing gene bmp4 are also asymmetrically distributed in the animal hemisphere and notch1 transcripts accumulate consistently on the same side of the eccentric maturation point. Strikingly, a Notch1 asymmetry orthogonal to the animal-vegetal axis appears during X. laevis oogenesis. Thus, we show for the first time a maternal bias in the distribution of molecules that are later involved in ventral patterning during embryonic axialization, strongly supporting the hypothesis of a dorsoventral prepattern or intrinsic bilaterality of Xenopus eggs before fertilization.

The midbody component Prc1-like is required for microtubule reorganization during cytokinesis and dorsal determinant segregation in the early zebrafish embryo.

We show that the zebrafish maternal-effect mutation too much information (tmi) corresponds to zebrafish prc1-like (prc1l), which encodes a member of the MAP65/Ase1/PRC1 family of microtubule-associated proteins. Embryos from tmi homozygous mutant mothers display cytokinesis defects in meiotic and mitotic divisions in the early embryo, indicating that Prc1l has a role in midbody formation during cell division at the egg-to-embryo transition. Unexpectedly, maternal Prc1l function is also essential for the reorganization of vegetal pole microtubules required for the segregation of dorsal determinants. Whereas Prc1 is widely regarded to crosslink microtubules in an antiparallel conformation, our studies provide evidence for an additional function of Prc1l in the bundling of parallel microtubules in the vegetal cortex of the early embryo during cortical rotation and prior to mitotic cycling. These findings highlight common yet distinct aspects of microtubule reorganization that occur during the egg-to-embryo transition, driven by maternal product for the midbody component Prc1l and required for embryonic cell division and pattern formation.

Simulating Cortical Rotation, Axis Induction, and Experimental Embryology in Amphibian Embryos Using Clay Models

Simulating Cortical Rotation, Axis Induction, and Experimental Embryology in Amphibian Embryos Using Clay Models

Maternal Wnt11b regulates cortical rotation during Xenopus axis formation: analysis of maternal-effect wnt11b mutants.

ABSTRACTAsymmetric signalling centres in the early embryo are essential for axis formation in vertebrates. These regions (e.g. amphibian dorsal morula, mammalian anterior visceral endoderm) require stabilised nuclear β-catenin, but the role of localised Wnt ligand signalling activity in their establishment remains unclear. In Xenopus, dorsal β-catenin is initiated by vegetal microtubule-mediated symmetry breaking in the fertilised egg, known as ‘cortical rotation’. Localised wnt11b mRNA and ligand-independent activators of β-catenin have been implicated in dorsal β-catenin activation, but the extent to which each contributes to axis formation in this paradigm remains unclear. Here, we describe a CRISPR-mediated maternal-effect mutation in Xenopus laevis wnt11b.L. We find that wnt11b is maternally required for robust dorsal axis formation and for timely gastrulation, and zygotically for left-right asymmetry. Importantly, we show that vegetal microtubule assembly and cortical rotation are reduced in wnt11b mutant eggs. In addition, we show that activated Wnt coreceptor Lrp6 and Dishevelled lack behaviour consistent with roles in early β-catenin stabilisation, and that neither is regulated by Wnt11b. This work thus implicates Wnt11b in the distribution of putative dorsal determinants rather than in comprising the determinants themselves. This article has an associated ‘The people behind the papers’ interview.

The people behind the papers - Douglas Houston and Marko Horb.

Over the years, conflicting data have meant that the role of Wnt in symmetry breaking and axis formation was unclear. Now, a new paper published in Development finds that maternal Wnt11b is required for robust cortical rotation inXenopus laevis, which, in turn, is required for axis induction. To find out more about this story, we caught up with two of the authors, Douglas Houston, Professor at The University of Iowa, and Marko Horb, Director of the National Xenopus Resource at the Marine Biological Laboratory in Chicago.

Lysosomes are required for early dorsal signaling in the Xenopus embryo

Lysosomes are the digestive center of the cell and play important roles in human diseases, including cancer. Previous work has suggested that late endosomes, also known as multivesicular bodies (MVBs), and lysosomes are essential for canonical Wnt pathway signaling. Sequestration of Glycogen Synthase 3 (GSK3) and of β‐catenin destruction complex components in MVBs is required for sustained canonical Wnt signaling. Little is known about the role of lysosomes during early development. In the Xenopus egg, a Wnt-like cytoplasmic determinant signal initiates formation of the body axis following a cortical rotation triggered by sperm entry. Here we report that cathepsin D was activated in lysosomes specifically on the dorsal marginal zone of the embryo at the 64-cell stage, long before zygotic transcription starts. Expansion of the MVB compartment with low-dose hydroxychloroquine (HCQ) greatly potentiated the dorsalizing effects of the Wnt agonist lithium chloride (LiCl) in embryos, and this effect required macropinocytosis. Formation of the dorsal axis required lysosomes, as indicated by brief treatments with the vacuolar ATPase (V-ATPase) inhibitors Bafilomycin A1 or Concanamycin A at the 32-cell stage. Inhibiting the MVB-forming machinery with a dominant-negative point mutation in Vacuolar Protein Sorting 4 (Vps4-EQ) interfered with the endogenous dorsal axis. The Wnt-like activity of the dorsal cytoplasmic determinant Huluwa (Hwa), and that of microinjected xWnt8 messenger RNA, also required lysosome acidification and the MVB-forming machinery. We conclude that lysosome function is required for early dorsal axis development in Xenopus. The results highlight the intertwining between membrane trafficking, lysosomes, and vertebrate axis formation.

Septins and a formin have distinct functions in anaphase chiral cortical rotation in the Caenorhabditis elegans zygote.

Many cells and tissues exhibit chirality that stems from the chirality of proteins and polymers. In the Caenorhabditis elegans zygote, actomyosin contractility drives chiral rotation of the entire cortex circumferentially around the division plane during anaphase. How contractility is translated to cell-scale chirality, and what dictates handedness, are unknown. Septins are candidate contributors to cell-scale chirality because they anchor and cross-link the actomyosin cytoskeleton. We report that septins are required for anaphase cortical rotation. In contrast, the formin CYK-1, which we found to be enriched in the posterior in early anaphase, is not required for cortical rotation but contributes to its chirality. Simultaneous loss of septin and CYK-1 function led to abnormal and often reversed cortical rotation. Our results suggest that anaphase contractility leads to chiral rotation by releasing torsional stress generated during formin-based polymerization, which is polarized along the cell anterior–posterior axis and which accumulates due to actomyosin network connectivity. Our findings shed light on the molecular and physical bases for cellular chirality in the C. elegans zygote. We also identify conditions in which chiral rotation fails but animals are developmentally viable, opening avenues for future work on the relationship between early embryonic cellular chirality and animal body plan.

Axis Specification in Zebrafish Is Robust to Cell Mixing and Reveals a Regulation of Pattern Formation by Morphogenesis

SummaryA fundamental question in developmental biology is how the early embryo establishes the spatial coordinate system that is later important for the organization of the embryonic body plan. Although we know a lot about the signaling and gene-regulatory networks required for this process, much less is understood about how these can operate to pattern tissues in the context of the extensive cell movements that drive gastrulation. In zebrafish, germ layer specification depends on the inheritance of maternal mRNAs [1, 2, 3], cortical rotation to generate a dorsal pole of β-catenin activity [4, 5, 6, 7, 8], and the release of Nodal signals from the yolk syncytial layer (YSL) [9, 10, 11, 12]. To determine whether germ layer specification is robust to altered cell-to-cell positioning, we separated embryonic cells from the yolk and allowed them to develop as spherical aggregates. These aggregates break symmetry autonomously to form elongated structures with an anterior-posterior pattern. Both forced reaggregation and endogenous cell mixing reveals how robust early axis specification is to spatial disruption of maternal pre-patterning. During these movements, a pole of Nodal signaling emerges that is required for explant elongation via the planar cell polarity (PCP) pathway. Blocking of PCP-dependent elongation disrupts the shaping of opposing poles of BMP and Wnt/TCF activity and the anterior-posterior patterning of neural tissue. These results lead us to suggest that embryo elongation plays a causal role in timing the exposure of cells to changes in BMP and Wnt signal activity during zebrafish gastrulation.Video

Non-centrosomal microtubule structures regulated by egg activation signaling contribute to cytoplasmic and cortical reorganization in the ascidian egg

In the first ascidian cell cycle, cytoplasmic and cortical reorganization is required for distributing maternal factors to their appropriate positions, resulting in the formation of the embryonic axis. This cytoplasmic reorganization is considered to depend on the cortical microfilament network in the first phase and on the sperm astral microtubule (MT) in the second phase. Recently, we described three novel MT structures: a deeply extended MT meshwork (DEM) in the entire subcortical region of the unfertilized egg, transiently accumulated MT fragments (TAF) in the vegetal pole, and a cortical MT array in the posterior vegetal cortex (CAMP). Particularly, our previous study showed CAMP to contribute to the movement of myoplasm. In addition, it is very similar to the parallel MT array, which appears during cortical rotation in Xenopus eggs. However, how these MT structures are organized is still unclear. Here, we investigated the relationship between the egg activation pathway and MT structures during the first ascidian cell cycle. First, we carefully analyzed cell cycle progression through meiosis I and II and the first mitosis, and successfully established a standard time table of cell cycle events. Using this time table as a reference, we precisely described the behavior of novel MT structures and revealed that it was closely correlated with cell cycle events. Moreover, pharmacological experiments supported the relationship between these MT structures and the signal transduction mechanisms that begin after fertilization, including Ca2+ signaling, MPF signaling, and MEK/MAPK signaling. Especially, CAMP formation was directed by activities of cyclin-dependent kinases. As these MT structures are conserved, at least, within chordate group, we emphasize the importance of understanding the controlling mechanisms of MT dynamics, which is important for embryonic axis determination in the ascidian egg.

A set of simple cell processes is sufficient to model spiral cleavage.

During cleavage, different cellular processes cause the zygote to become partitioned into a set of cells with a specific spatial arrangement. These processes include the orientation of cell division according to: an animal-vegetal gradient; the main axis (Hertwig's rule) of the cell; and the contact areas between cells or the perpendicularity between consecutive cell divisions (Sachs' rule). Cell adhesion and cortical rotation have also been proposed to be involved in spiral cleavage. We use a computational model of cell and tissue biomechanics to account for the different existing hypotheses about how the specific spatial arrangement of cells in spiral cleavage arises during development. Cell polarization by an animal-vegetal gradient, a bias to perpendicularity between consecutive cell divisions (Sachs' rule), cortical rotation and cell adhesion, when combined, reproduce the spiral cleavage, whereas other combinations of processes cannot. Specifically, cortical rotation is necessary at the 8-cell stage to direct all micromeres in the same direction. By varying the relative strength of these processes, we reproduce the spatial arrangement of cells in the blastulae of seven different invertebrate species.

Specification of anteroposterior axis by combinatorial signaling during Xenopus development.

The specification of anteroposterior (AP) axis is a fundamental and complex patterning process that sets up the embryonic polarity and shapes a multicellular organism. This process involves the integration of distinct signaling pathways to coordinate temporal-spatial gene expression and morphogenetic movements. In the frog Xenopus, extensive embryological and molecular studies have provided major advance in understanding the mechanism implicated in AP patterning. Following fertilization, cortical rotation leads to the transport of maternal determinants to the dorsal region and creates the primary dorsoventral (DV) asymmetry. The activation of maternal Wnt/ß-catenin signaling and a high Nodal signal induces the formation of the Nieuwkoop center in the dorsal-vegetal cells, which then triggers the formation of the Spemann organizer in the overlying dorsal marginal zone. It is now well established that the Spemann organizer plays a central role in building the vertebrate body axes because it provides patterning information for both DV and AP polarities. The antagonistic interactions between signals secreted in the Spemann organizer and the opposite ventral region pattern the mesoderm along the DV axis, and this DV information is translated into AP positional values during gastrulation. The formation of anterior neural tissue requires simultaneous inhibition of zygotic Wnt and bone morphogenetic protein (BMP) signals, while an endogenous gradient of Wnt, fibroblast growth factors (FGFs), retinoic acid (RA) signaling, and collinearly expressed Hox genes patterns the trunk and posterior regions. Collectively, DV asymmetry is mostly coupled to AP polarity, and cell-cell interactions mediated essentially by the same regulatory networks operate in DV and AP patterning. For further resources related to this article, please visit the WIREs website.

The dynamics of plus end polarization and microtubule assembly during Xenopus cortical rotation

The self-organization of dorsally-directed microtubules during cortical rotation in the Xenopus egg is essential for dorsal axis formation. The mechanisms controlling this process have been problematic to analyze, owing to difficulties in visualizing microtubules in living egg. Also, the order of events occurring at the onset of cortical rotation have not been satisfactorily visualized in vivo and have been inferred from staged fixed samples. To address these issues, we have characterized the dynamics of total microtubule and plus end behavior continuously throughout cortical rotation, as well as in oocytes and unfertilized eggs. Here, we show that the nascent microtubule network forms in the cortex but associates with the deep cytoplasm at the start of rotation. Importantly, plus ends remain cortical and become increasingly more numerous and active prior to rotation, with dorsal polarization occurring rapidly after the onset of rotation. Additionally, we show that vegetally localized Trim36 is required to attenuate dynamic plus end growth, suggesting that vegetal factors are needed to locally coordinate growth in the cortex.

Calcium signaling and meiotic exit at fertilization in Xenopus egg.

Calcium is a universal messenger that mediates egg activation at fertilization in all sexually reproducing species studied. However, signaling pathways leading to calcium generation and the mechanisms of calcium-induced exit from meiotic arrest vary substantially among species. Here, we review the pathways of calcium signaling and the mechanisms of meiotic exit at fertilization in the eggs of the established developmental model, African clawed frog, Xenopus laevis. We also discuss calcium involvement in the early fertilization-induced events in Xenopus egg, such as membrane depolarization, the increase in intracellular pH, cortical granule exocytosis, cortical contraction, contraction wave, cortical rotation, reformation of the nuclear envelope, sperm chromatin decondensation and sister chromatid segregation.

Hecate/Grip2a acts to reorganize the cytoskeleton in the symmetry-breaking event of embryonic axis induction.

Maternal homozygosity for three independent mutant hecate alleles results in embryos with reduced expression of dorsal organizer genes and defects in the formation of dorsoanterior structures. A positional cloning approach identified all hecate mutations as stop codons affecting the same gene, revealing that hecate encodes the Glutamate receptor interacting protein 2a (Grip2a), a protein containing multiple PDZ domains known to interact with membrane-associated factors including components of the Wnt signaling pathway. We find that grip2a mRNA is localized to the vegetal pole of the oocyte and early embryo, and that during egg activation this mRNA shifts to an off-center vegetal position corresponding to the previously proposed teleost cortical rotation. hecate mutants show defects in the alignment and bundling of microtubules at the vegetal cortex, which result in defects in the asymmetric movement of wnt8a mRNA as well as anchoring of the kinesin-associated cargo adaptor Syntabulin. We also find that, although short-range shifts in vegetal signals are affected in hecate mutant embryos, these mutants exhibit normal long-range, animally directed translocation of cortically injected dorsal beads that occurs in lateral regions of the yolk cortex. Furthermore, we show that such animally-directed movement along the lateral cortex is not restricted to a single arc corresponding to the prospective dorsal region, but occur in multiple meridional arcs even in opposite regions of the embryo. Together, our results reveal a role for Grip2a function in the reorganization and bundling of microtubules at the vegetal cortex to mediate a symmetry-breaking short-range shift corresponding to the teleost cortical rotation. The slight asymmetry achieved by this directed process is subsequently amplified by a general cortical animally-directed transport mechanism that is neither dependent on hecate function nor restricted to the prospective dorsal axis.

Timing and mechanism of the initial cue establishing handed left–right asymmetry in Caenorhabditis elegans embryos.

By the six-cell stage, embryos of Caenorhabditis elegans are morphologically L–R asymmetric with an invariant handedness that persists throughout development. We used intracellular markers to ask whether breaking of L–R symmetry could be observed at earlier stages. Observation of two- to three-cell embryos carrying intracellular markers indicated that L–R symmetry is broken concomitantly with establishment of D–V axis polarity during division of the anterior AB cell. The AB cleavage furrow initiates asymmetrically and always from the left, suggesting L–R differences in the AB cell cortex. An invariantly handed cortical rotation observed earlier during first cleavage implies that the one-cell embryo has an intrinsic chirality. We propose that L–R differences in the cortex could result from mechanical forces on asymmetric components of a chiral cortical network during the off-axis elongation of the AB-cell spindle prior to AB cleavage.

Simulation of the effects of microtubules in the cortical rotation of amphibian embryos in normal and zero gravity

This paper reports the results of computer modeling of microtubules that end up in the cortical region of a one-cell amphibian embryo, prior to the first cell division. Microtubules are modeled as initially randomly oriented semi-flexible rods, represented by several lines of point-masses interacting with one another like masses on springs with longitudinal and transverse stiffness. They are also considered to be space-filling rods floating in a viscous fluid (cytoplasm) experiencing drag forces and buoyancy from the fluid under a variable gravity field to test gravitational effects. Their randomly distributed interactions with the surrounding spherical container (the cell membrane) have a statistical nonzero average that creates a torque causing a rotational displacement between the cytoplasm and the rigid cortex. The simulation has been done for zero and normal gravity and it validates the observation that cortical rotation occurs in microgravity as well as on Earth. The speed of rotation depends on gravity, but is still substantial in microgravity.

A mean field Ising model for cortical rotation in amphibian one-cell stage embryos

We propose a new physical mechanism of cortical rotation generation in one-cell embryos of amphibians based on a phase transition in the ensemble of microtubules localized to the cortical region of the cell interior. Microtubules, protein polymers formed from tubulin heterodimers, are highly negatively charged, which results in strong electrostatic interactions over tens of nanometers, even in the presence of counterions that partially screen electrostatic interactions. A simplified model that offers a plausible representation of these effects is based on the Ising Hamiltonian, which has been robustly applied to explain a wide range of order–disorder transitions in physics, chemistry and other sciences. An Ising model phase transition, especially with the supercooperative flow alignment effect of global rotation of the cortex, provides an alternative to models of cortical rotation based on microtubule polymerization or motor molecules. Insofar as there is any reality to the concept that microtubules are involved in consciousness, we propose that cortical rotation in the one-cell embryo is a better place to look for the purported microtubule entanglement or coherence properties than the adult brain.

Cortical rotation and messenger RNA localization in Xenopus axis formation

In Xenopus eggs, fertilization initiates a rotational movement of the cortex relative to the cytoplasm, resulting in the transport of critical determinants to the future dorsal side of the embryo. Cortical rotation is mediated by microtubules, resulting in activation of the Wnt/β-catenin signaling pathway and expression of organizer genes on the dorsal side of the blastula. Similar cytoplasmic localizations resulting in β-catenin activation occur in many chordate embryos, suggesting a deeply conserved mechanism for patterning early embryos. This review summarizes the experimental evidence for the molecular basis of this model, focusing on recent maternal loss-of-function studies that shed light on two main unanswered questions: (1) what regulates microtubule assembly during cortical rotation and (2) how is Wnt/β-catenin signaling activated dorsally? In addition, as these processes depend on vegetally localized molecules in the oocyte, the mechanisms of RNA localization and novel roles for localized RNAs in axis formation are discussed. The work reviewed here provides a beginning framework for understanding the coupling of asymmetry in oogenesis with the establishment of asymmetry in the embryo.

Roles of localized mRNAs in lipid droplet function during cortical rotation in Xenopus

Roles of localized mRNAs in lipid droplet function during cortical rotation in Xenopus

Enzymes, Embryos, and Ancestors

In the 1950s, cellular regulatory mechanisms were newly recognized; with Arthur Pardee I investigated the initial enzyme of pyrimidine biosynthesis, which he discovered is controlled by feedback inhibition. The protein proved unusual in having separate but interacting sites for substrates and regulators. Howard Schachman and I dissociated the protein into different subunits, one binding regulators and one substrates. The enzyme became an early prime example of allostery. In developmental biology I studied the egg of the frog, Xenopus laevis, characterizing early processes of axis formation. My excellent students and I described cortical rotation, a 30° movement of the egg's cortex over tracks of parallel microtubules anchored to the underlying cytoplasmic core, and we perturbed it to alter Spemann's organizer and effect spectacular phenotypes. The entire sequence of events has been elucidated by others at the molecular level, making Xenopus a prime example of vertebrate axis formation. Marc Kirschner, Christopher Lowe, and I then compared hemichordate (half-chordate) and chordate early development. Despite anatomical-physiological differences, these groups share numerous steps of axis formation, ones that were probably already in use in their pre-Cambrian ancestor. I've thoroughly enjoyed exploring these areas during a 50-year period of great advances in biological sciences by the worldwide research community.