Mouse neural tube organoids self-organize floorplate through BMP-mediated cluster competition.

During neural tube (NT) development, the notochord induces an organizer, the floorplate, which secretes Sonic Hedgehog (SHH) to pattern neural progenitors. Conversely, NT organoids (NTOs) from embryonic stem cells (ESCs) spontaneously form floorplates without the notochord, demonstrating that stem cells can self-organize without embryonic inducers. Here, we investigated floorplate self-organization in clonal mouse NTOs. Expression of the floorplate marker FOXA2 was initially spatially scattered before resolving into multiple clusters, which underwent competition and sorting, resulting in a stable "winning" floorplate. We identified that BMP signaling governed long-range cluster competition. FOXA2+ clusters expressed BMP4, suppressing FOXA2 in receiving cells while simultaneously expressing the BMP-inhibitor NOGGIN, promoting cluster persistence. Noggin mutation perturbed floorplate formation in NTOs and in the NT in vivo at mid/hindbrain regions, demonstrating how the floorplate can form autonomously without the notochord. Identifying the pathways governing organizer self-organization is critical for harnessing the developmental plasticity of stem cells in tissue engineering.

Topological morphogenesis of neuroepithelial organoids.

Animal organs exhibit complex topologies involving cavities and tubular networks, which underlie their form and function1-3. However, how topology emerges during the development of organ shape, or morphogenesis, remains elusive. Here we combine tissue reconstitution and quantitative microscopy to show that tissue topology and shape is governed by two distinct modes of topological transitions4,5. One mode involves the fusion of two separate epithelia and the other involves the fusion of two ends of the same epithelium. The morphological space is captured by a single control parameter that can be traced back to the relative rates of the two epithelial fusion modes. Finally, we identify a pharmacologically accessible pathway that regulates the frequency of two modes of epithelial fusion, and demonstrate the control of organoid topology and shape. The physical principles uncovered here provide fundamental insights into the self-organization of complex tissues6.

Spatial variation of microtubule depolymerization in large asters.

Microtubule plus-end depolymerization rate is a potentially important target of physiological regulation, but it has been challenging to measure, so its role in spatial organization is poorly understood. Here we apply a method for tracking plus ends based on time difference imaging to measure depolymerization rates in large interphase asters growing in Xenopus egg extract. We observed strong spatial regulation of depolymerization rates, which were higher in the aster interior compared with the periphery, and much less regulation of polymerization or catastrophe rates. We interpret these data in terms of a limiting component model, where aster growth results in lower levels of soluble tubulin and microtubule-associated proteins (MAPs) in the interior cytosol compared with that at the periphery. The steady-state polymer fraction of tubulin was ∼30%, so tubulin is not strongly depleted in the aster interior. We propose that the limiting component for microtubule assembly is a MAP that inhibits depolymerization, and that egg asters are tuned to low microtubule density.

Spindle Scaling Is Governed by Cell Boundary Regulation of Microtubule Nucleation.

Cellular organelles such as the mitotic spindle adjust their size to the dimensions of the cell. It is widely understood that spindle scaling is governed by regulation of microtubule polymerization. Here, we use quantitative microscopy in living zebrafish embryos and Xenopus egg extracts in combination with theory to show that microtubule polymerization dynamics are insufficient to scale spindles and only contribute below a critical cell size. In contrast, microtubule nucleation governs spindle scaling for all cell sizes. We show that this hierarchical regulation arises from the partitioning of a nucleation inhibitor to the cell membrane. Our results reveal that cells differentially regulate microtubule number and length using distinct geometric cues to maintain a functional spindle architecture over a large range of cell sizes.

How to tune spindle size relative to cell size?

Cells need to regulate the size and shape of their organelles for proper function. For example, the mitotic spindle adapts its size to changes in cell size over several orders of magnitude, but we lack a mechanistic understanding of how this is achieved. Here, we review our current knowledge of how small and large spindles assemble and ask which microtubule-based biophysical processes (nucleation, polymerization dynamics, transport) may be responsible for spindle size regulation. Finally, we review possible cell-scale mechanisms that put spindle size under the regulation of cell size.

Reconstitution of a Patterned Neural Tube from Single Mouse Embryonic Stem Cells.

The recapitulation of tissue development and patterning in three-dimensional (3D) culture is an important dimension of stem cell research. Here, we describe a 3D culture protocol in which single mouse ES cells embedded in Matrigel under neural induction conditions clonally form a lumen containing, oval-shaped epithelial structure within 3 days. By Day 7 an apicobasally polarized neuroepithelium with uniformly dorsal cell identity forms. Treatment with retinoic acid at Day 2 results in posteriorization and self-organization of dorsal-ventral neural tube patterning. Neural tube organoid growth is also supported by pure laminin gels as well as poly(ethylene glycol) (PEG)-based artificial extracellular matrix hydrogels, which can be fine-tuned for key microenvironment characteristics. The rapid generation of a simple, patterned tissue in well-defined culture conditions makes the neural tube organoid a tractable model for studying neural stem cell self-organization.

Physical basis of large microtubule aster growth.

Microtubule asters - radial arrays of microtubules organized by centrosomes - play a fundamental role in the spatial coordination of animal cells. The standard model of aster growth assumes a fixed number of microtubules originating from the centrosomes. However, aster morphology in this model does not scale with cell size, and we recently found evidence for non-centrosomal microtubule nucleation. Here, we combine autocatalytic nucleation and polymerization dynamics to develop a biophysical model of aster growth. Our model predicts that asters expand as traveling waves and recapitulates all major aspects of aster growth. With increasing nucleation rate, the model predicts an explosive transition from stationary to growing asters with a discontinuous jump of the aster velocity to a nonzero value. Experiments in frog egg extract confirm the main theoretical predictions. Our results suggest that asters observed in large fish and amphibian eggs are a meshwork of short, unstable microtubules maintained by autocatalytic nucleation and provide a paradigm for the assembly of robust and evolvable polymer networks.

The Nuclear Proteome of a Vertebrate.

The composition of the nucleoplasm determines the behavior of key processes such as transcription, yet there is still no reliable and quantitative resource of nuclear proteins. Furthermore, it is still unclear how the distinct nuclear and cytoplasmic compositions are maintained. To describe the nuclear proteome quantitatively, we isolated the large nuclei of frog oocytes via microdissection and measured the nucleocytoplasmic partitioning of ∼9,000 proteins by mass spectrometry. Most proteins localize entirely to either nucleus or cytoplasm; only ∼17% partition equally. A protein's native size in a complex, but not polypeptide molecular weight, is predictive of localization: partitioned proteins exhibit native sizes larger than ∼100 kDa, whereas natively smaller proteins are equidistributed. To evaluate the role of nuclear export in maintaining localization, we inhibited Exportin 1. This resulted in the expected re-localization of proteins toward the nucleus, but only 3% of the proteome was affected. Thus, complex assembly and passive retention, rather than continuous active transport, is the dominant mechanism for the maintenance of nuclear and cytoplasmic proteomes.

Size Scaling of Microtubule Assemblies in Early Xenopus Embryos.

The first 12 cleavage divisions in Xenopus embryos provide a natural experiment in size scaling, as cell radius decreases ∼16-fold with little change in biochemistry. Analyzing both natural cleavage and egg extract partitioned into droplets revealed that mitotic spindle size scales with cell size, with an upper limit in very large cells. We discuss spindle-size scaling in the small- and large-cell regimes with a focus on the "limiting-component" hypotheses. Zygotes and early blastomeres show a scaling mismatch between spindle and cell size. This problem is solved, we argue, by interphase asters that act to position the spindle and transport chromosomes to the center of daughter cells. These tasks are executed by the spindle in smaller cells. We end by discussing possible mechanisms that limit mitotic aster size and promote interphase aster growth to cell-spanning dimensions.

Microtubule nucleation remote from centrosomes may explain how asters span large cells.

A major challenge in cell biology is to understand how nanometer-sized molecules can organize micrometer-sized cells in space and time. One solution in many animal cells is a radial array of microtubules called an aster, which is nucleated by a central organizing center and spans the entire cytoplasm. Frog (here Xenopus laevis) embryos are more than 1 mm in diameter and divide with a defined geometry every 30 min. Like smaller cells, they are organized by asters, which grow, interact, and move to precisely position the cleavage planes. It has been unclear whether asters grow to fill the enormous egg by the same mechanism used in smaller somatic cells, or whether special mechanisms are required. We addressed this question by imaging growing asters in a cell-free system derived from eggs, where asters grew to hundreds of microns in diameter. By tracking marks on the lattice, we found that microtubules could slide outward, but this was not essential for rapid aster growth. Polymer treadmilling did not occur. By measuring the number and positions of microtubule ends over time, we found that most microtubules were nucleated away from the centrosome and that interphase egg cytoplasm supported spontaneous nucleation after a time lag. We propose that aster growth is initiated by centrosomes but that asters grow by propagating a wave of microtubule nucleation stimulated by the presence of preexisting microtubules.

Spatial organization of cytokinesis signaling reconstituted in a cell-free system.

During animal cell division, the cleavage furrow is positioned by microtubules that signal to the actin cortex at the cell midplane. We developed a cell-free system to recapitulate cytokinesis signaling using cytoplasmic extract from Xenopus eggs. Microtubules grew out as asters from artificial centrosomes and met to organize antiparallel overlap zones. These zones blocked the interpenetration of neighboring asters and recruited cytokinesis midzone proteins, including the chromosomal passenger complex (CPC) and centralspindlin. The CPC was transported to overlap zones, which required two motor proteins, Kif4A and a Kif20A paralog. Using supported lipid bilayers to mimic the plasma membrane, we observed the recruitment of cleavage furrow markers, including an active RhoA reporter, at microtubule overlaps. This system opens further approaches to understanding the biophysics of cytokinesis signaling.

Organization of early frog embryos by chemical waves emanating from centrosomes.

The large cells in early vertebrate development face an extreme physical challenge in organizing their cytoplasm. For example, amphibian embryos have to divide cytoplasm that spans hundreds of micrometres every 30 min according to a precise geometry, a remarkable accomplishment given the extreme difference between molecular and cellular scales in this system. How do the biochemical reactions occurring at the molecular scale lead to this emergent behaviour of the cell as a whole? Based on recent findings, we propose that the centrosome plays a crucial role by initiating two autocatalytic reactions that travel across the large cytoplasm as chemical waves. Waves of mitotic entry and exit propagate out from centrosomes using the Cdk1 oscillator to coordinate the timing of cell division. Waves of microtubule-stimulated microtubule nucleation propagate out to assemble large asters that position spindles for the following mitosis and establish cleavage plane geometry. By initiating these chemical waves, the centrosome rapidly organizes the large cytoplasm during the short embryonic cell cycle, which would be impossible using more conventional mechanisms such as diffusion or nucleation by structural templating. Large embryo cells provide valuable insights to how cells control chemical waves, which may be a general principle for cytoplasmic organization.

Xenopus egg cytoplasm with intact actin.

We report optimized methods for preparing Xenopus egg extracts without cytochalasin D, that we term "actin-intact egg extract." These are undiluted egg cytoplasm that contains abundant organelles, and glycogen which supplies energy, and represents the least perturbed cell-free cytoplasm preparation we know of. We used this system to probe cell cycle regulation of actin and myosin-II dynamics (Field et al., 2011), and to reconstitute the large, interphase asters that organize early Xenopus embryos (Mitchison et al., 2012; Wühr, Tan, Parker, Detrich, & Mitchison, 2010). Actin-intact Xenopus egg extracts are useful for analysis of actin dynamics, and interaction of actin with other cytoplasmic systems, in a cell-free system that closely mimics egg physiology, and more generally for probing the biochemistry and biophysics of the egg, zygote, and early embryo. Detailed protocols are provided along with assays used to check cell cycle state and tips for handling and storing undiluted egg extracts.

Glycogen-supplemented mitotic cytosol for analyzing Xenopus egg microtubule organization.

Undiluted cytoplasmic extract prepared from unfertilized Xenopus laevis eggs by low-speed centrifugation (CSF extracts) is useful for reconstitution of egg microtubule dynamics and meiosis-II spindle organization, but it suffers limitations for biochemical analysis due to abundant particulates. Here, we describe preparation and the use of fully clarified, undiluted mitotic cytosol derived from CSF extract. Addition of glycogen improves the ability of this cytosol to reconstitute microtubule organization, in part through improved energy metabolism. Using fully clarified, glycogen-supplemented mitotic cytosol, we reconstituted (i) stimulation of microtubule polymerization by Ran.GTP (Groen, Coughlin, & Mitchison, 2011; Ohba, Nakamura, Nishitani, & Nishimoto, 1999) and (ii) self-organization of highly regular bipolar arrays of taxol-stabilized microtubules that we termed "pineapples" (Mitchison, Nguyen, Coughlin, & Groen, 2013). Both systems will be useful for biochemical dissection of spindle assembly mechanisms. We also describe reliable small-scale methods for preparing fluorescent antibody probes that can be used for live imaging in egg extract systems as well as standard immunofluorescence.

Context-dependent transcriptional interpretation of mitogen activated protein kinase signaling in the Drosophila embryo.

Terminal regions of the Drosophila embryo are patterned by the localized activation of Mitogen Activated Protein Kinase (MAPK), which induces zygotic genes through relief of their repression by transcriptional repressor Capicua. The levels of MAPK activation at the anterior and posterior termini are close to each other, but the expression patterns of MAPK-target genes, such as zerknüllt (zen) and tailless (tll), display strong anterior-posterior (AP) asymmetry. This region-specific response to MAPK activation provides a clear example of context-dependent interpretation of inductive signaling, a common developmental effect that remains poorly understood. In the past, the AP asymmetry of zen expression was attributed to a mechanism that depends on MAPK substrate competition. We present data suggesting that the asymmetric expression of tll is generated by a different mechanism, based on feedforward control and multiple enhancers of the tll gene. A simple mathematical model of this mechanism correctly predicts how the wild-type expression pattern of tll changes in mutants affecting the anterior, dorsoventral, and terminal patterning systems and some of their direct targets.

Branching microtubule nucleation in Xenopus egg extracts mediated by augmin and TPX2.

The microtubules that comprise mitotic spindles in animal cells are nucleated at centrosomes and by spindle assembly factors that are activated in the vicinity of chromatin. Indirect evidence has suggested that microtubules also might be nucleated from pre-existing microtubules throughout the spindle, but this process has not been observed directly. Here, we demonstrate microtubule nucleation from the sides of existing microtubules in meiotic Xenopus egg extracts. Daughter microtubules grow at a low branch angle and with the same polarity as mother filaments. Branching microtubule nucleation requires γ-tubulin and augmin and is stimulated by factors previously implicated in chromatin-stimulated nucleation, guanosine triphosphate(GTP)-bound Ran and its effector, TPX2. Because of the rapid amplification of microtubule numbers and the preservation of microtubule polarity, microtubule-dependent microtubule nucleation is well suited for spindle assembly and maintenance.

Growth, interaction, and positioning of microtubule asters in extremely large vertebrate embryo cells.

Ray Rappaport spent many years studying microtubule asters, and how they induce cleavage furrows. Here, we review recent progress on aster structure and dynamics in zygotes and early blastomeres of Xenopus laevis and Zebrafish, where cells are extremely large. Mitotic and interphase asters differ markedly in size, and only interphase asters span the cell. Growth of interphase asters occurs by a mechanism that allows microtubule density at the aster periphery to remain approximately constant as radius increases. We discuss models for aster growth, and favor a branching nucleation process. Neighboring asters that grow into each other interact to block further growth at the shared boundary. We compare the morphology of interaction zones formed between pairs of asters that grow out from the poles of the same mitotic spindle (sister asters) and between pairs not related by mitosis (non-sister asters) that meet following polyspermic fertilization. We argue growing asters recognize each other by interaction between antiparallel microtubules at the mutual boundary, and discuss models for molecular organization of interaction zones. Finally, we discuss models for how asters, and the centrosomes within them, are positioned by dynein-mediated pulling forces so as to generate stereotyped cleavage patterns. Studying these problems in extremely large cells is starting to reveal how general principles of cell organization scale with cell size.

Pronuclear migration: no attachment? No union, but a futile cycle!

How do pronuclei migrate towards each other? The zebrafish futile cycle gene is shown to encode a maternally expressed membrane protein required for nuclear attachment and migration along the sperm aster.