Cytoplasmic forces functionally reorganize nuclear condensates in oocytes.

Cells remodel their cytoplasm with force-generating cytoskeletal motors. Their activity generates random forces that stir the cytoplasm, agitating and displacing membrane-bound organelles like the nucleus in somatic and germ cells. These forces are transmitted inside the nucleus, yet their consequences on liquid-like biomolecular condensates residing in the nucleus remain unexplored. Here, we probe experimentally and computationally diverse nuclear condensates, that include nuclear speckles, Cajal bodies, and nucleoli, during cytoplasmic remodeling of female germ cells named oocytes. We discover that growing mammalian oocytes deploy cytoplasmic forces to timely impose multiscale reorganization of nuclear condensates for the success of meiotic divisions. These cytoplasmic forces accelerate nuclear condensate collision-coalescence and molecular kinetics within condensates. Disrupting the forces decelerates nuclear condensate reorganization on both scales, which correlates with compromised condensate-associated mRNA processing and hindered oocyte divisions that drive female fertility. We establish that cytoplasmic forces can reorganize nuclear condensates in an evolutionary conserved fashion in insects. Our work implies that cells evolved a mechanism, based on cytoplasmic force tuning, to functionally regulate a broad range of nuclear condensates across scales. This finding opens new perspectives when studying condensate-associated pathologies like cancer, neurodegeneration and viral infections.

Morphogenetic forces planar polarize LGN/Pins in the embryonic head during Drosophila gastrulation.

Spindle orientation is often achieved by a complex of Partner of Inscuteable (Pins)/LGN, Mushroom Body Defect (Mud)/Nuclear Mitotic Apparatus (NuMa), Gαi, and Dynein, which interacts with astral microtubules to rotate the spindle. Cortical Pins/LGN recruitment serves as a critical step in this process. Here, we identify Pins-mediated planar cell polarized divisions in several of the mitotic domains of the early Drosophila embryo. We found that neither planar cell polarity pathways nor planar polarized myosin localization determined division orientation; instead, our findings strongly suggest that Pins planar polarity and force generated from mesoderm invagination are important. Disrupting Pins polarity via overexpression of a myristoylated version of Pins caused randomized division angles. We found that disrupting forces through chemical inhibitors, depletion of an adherens junction protein, or blocking mesoderm invagination disrupted Pins planar polarity and spindle orientation. Furthermore, directional ablations that separated mesoderm from mitotic domains disrupted spindle orientation, suggesting that forces transmitted from mesoderm to mitotic domains can polarize Pins and orient division during gastrulation. To our knowledge, this is the first in vivo example where mechanical force has been shown to polarize Pins to mediate division orientation.

Collective Cell Sorting Requires Contractile Cortical Waves in Germline Cells.

Encapsulation of germline cells by layers of somatic cells forms the basic unit of female reproduction called primordial follicles in mammals and egg chambers in Drosophila. How germline and somatic tissues are coordinated for the morphogenesis of each separated unit remains poorly understood. Here, using improved live imaging of Drosophila ovaries, we uncovered periodic actomyosin waves at the cortex of germ cells. These contractile waves are associated with pressure release blebs, which project from germ cells into somatic cells. We demonstrate that these cortical activities, together with cadherin-based adhesion, are required to sort each germline cyst as one collective unit. Genetic perturbations of cortical contractility, bleb protrusion, or adhesion between germline and somatic cells induced encapsulation defects resulting from failures to encapsulate any germ cells, or the inclusion of too many germ cells per egg chamber, or even the mechanical split of germline cysts. Live-imaging experiments revealed that reducing contractility or adhesion in the germline reduced the stiffness of germline cysts and their proper anchoring to the somatic cells. Germline cysts can then be squeezed and passively pushed by constricting surrounding somatic cells, resulting in cyst splitting and cyst collisions during encapsulation. Increasing germline cysts activity or blocking somatic cell constriction movements can reveal active forward migration of germline cysts. Our results show that germ cells play an active role in physical coupling with somatic cells to produce the female gamete.

Myosin 2-Induced Mitotic Rounding Enables Columnar Epithelial Cells to Interpret Cortical Spindle Positioning Cues.

During epithelial cell proliferation, planar alignment of the mitotic spindle allows the daughter cells to stay within the epithelium. Previous work has identified cortical cues that regulate spindle orientation and the division axis [1, 2]. One such cue is cortical Pins (LGN in vertebrates) [3-6], which recruits the conserved Mud/NuMA protein and the dynein/dynactin complex to the cortex. The dynein/dynactin motor complex pulls astral microtubules to orient the spindle. Cortical Pins can therefore dictate the division axis. In addition to cortical cues, cell shape can also serve as a division orientation cue [7-9]. Here, we investigated the interplay between cortical cues and cell shape in a proliferating tissue. We analyzed division orientation in the first mitotic divisions of the early Drosophila embryo, where groups of epithelial cells synchronously divide. Using chemical inhibitors, knockdowns, and mutants with known deficits in motor activity, we showed that the myosin 2 motor is required to orient cell division in the plane of a columnar epithelium. Disrupting myosin activity caused the division axis to orient perpendicular to the epithelial plane. This effect was independent of Pins cortical localization, which became uncoupled from spindle orientation. Instead, myosin motor activity was required for the formation of the actomyosin cortex and for cell rounding upon mitotic entry. We propose that mitotic cell rounding in columnar epithelia allows cells to properly interpret cortical cues that orient the spindle. In the absence of mitotic rounding, geometric cues imposed by tight cell packing prevail and cells divide along their long apical-basal axis.

Actomyosin meshwork mechanosensing enables tissue shape to orient cell force.

Sculpting organism shape requires that cells produce forces with proper directionality. Thus, it is critical to understand how cells orient the cytoskeleton to produce forces that deform tissues. During Drosophila gastrulation, actomyosin contraction in ventral cells generates a long, narrow epithelial furrow, termed the ventral furrow, in which actomyosin fibres and tension are directed along the length of the furrow. Using a combination of genetic and mechanical perturbations that alter tissue shape, we demonstrate that geometrical and mechanical constraints act as cues to orient the cytoskeleton and tension during ventral furrow formation. We developed an in silico model of two-dimensional actomyosin meshwork contraction, demonstrating that actomyosin meshworks exhibit an inherent force orienting mechanism in response to mechanical constraints. Together, our in vivo and in silico data provide a framework for understanding how cells orient force generation, establishing a role for geometrical and mechanical patterning of force production in tissues.

Actomyosin-based tissue folding requires a multicellular myosin gradient.

Tissue folding promotes three-dimensional (3D) form during development. In many cases, folding is associated with myosin accumulation at the apical surface of epithelial cells, as seen in the vertebrate neural tube and the Drosophila ventral furrow. This type of folding is characterized by constriction of apical cell surfaces, and the resulting cell shape change is thought to cause tissue folding. Here, we use quantitative microscopy to measure the pattern of transcription, signaling, myosin activation and cell shape in the Drosophila mesoderm. We found that cells within the ventral domain accumulate different amounts of active apical non-muscle myosin 2 depending on the distance from the ventral midline. This gradient in active myosin depends on a newly quantified gradient in upstream signaling proteins. A 3D continuum model of the embryo with induced contractility demonstrates that contractility gradients, but not contractility per se, promote changes to surface curvature and folding. As predicted by the model, experimental broadening of the myosin domain in vivo disrupts tissue curvature where myosin is uniform. Our data argue that apical contractility gradients are important for tissue folding.

Stable Force Balance between Epithelial Cells Arises from F-Actin Turnover.

The propagation of force in epithelial tissues requires that the contractile cytoskeletal machinery be stably connected between cells through E-cadherin-containing adherens junctions. In many epithelial tissues, the cells' contractile network is positioned at a distance from the junction. However, the mechanism or mechanisms that connect the contractile networks to the adherens junctions, and thus mechanically connect neighboring cells, are poorly understood. Here, we identified the role for F-actin turnover in regulating the contractile cytoskeletal network's attachment to adherens junctions. Perturbing F-actin turnover via gene depletion or acute drug treatments that slow F-actin turnover destabilized the attachment between the contractile actomyosin network and adherens junctions. Our work identifies a critical role for F-actin turnover in connecting actomyosin to intercellular junctions, defining a dynamic process required for the stability of force balance across intercellular contacts in tissues.

Mechanical force sensing in tissues.

Tissue size, shape, and organization reflect individual cell behaviors such as proliferation, shape change, and movement. Evidence suggests that mechanical signals operate in tandem with biochemical cues to properly coordinate cell behavior and pattern tissues. The objective of this chapter is to present recent evidence demonstrating that forces transmitted between cells act as signals that coordinate cell behavior across tissues. We first briefly summarize molecular and cellular mechanisms by which forces are sensed by cells with an emphasis on forces generated and transmitted by cytoskeletal networks. We then discuss evidence for these mechanisms operating in multicellular contexts to coordinate complex cell and tissue behaviors that occur during embryonic development: specifically growth and morphogenesis.

Functional analysis of the NHR2 domain indicates that oligomerization of Neuralized regulates ubiquitination and endocytosis of Delta during Notch signaling.

The Notch pathway plays an integral role in development by regulating cell fate in a wide variety of multicellular organisms. A critical step in the activation of Notch signaling is the endocytosis of the Notch ligands Delta and Serrate. Ligand endocytosis is regulated by one of two E3 ubiquitin ligases, Neuralized (Neur) or Mind bomb. Neur is comprised of a C-terminal RING domain, which is required for Delta ubiquitination, and two Neur homology repeat (NHR) domains. We have previously shown that the NHR1 domain is required for Delta trafficking. Here we show that the NHR1 domain also affects the binding and internalization of Serrate. Furthermore, we show that the NHR2 domain is required for Neur function and that a point mutation in the NHR2 domain (Gly430) abolishes Neur ubiquitination activity and affects ligand internalization. Finally, we provide evidence that Neur can form oligomers in both cultured cells and fly tissues, which regulate Neur activity and, by extension, ligand internalization.

Regulation of epithelial polarity by the E3 ubiquitin ligase Neuralized and the Bearded inhibitors in Drosophila.

Understanding how epithelial polarity is established and regulated during tissue morphogenesis is a major issue. Here, we identify a regulatory mechanism important for mesoderm invagination, germ-band extension and transepithelial migration in the Drosophila melanogaster embryo. This mechanism involves the inhibition of the conserved E3 ubiquitin ligase Neuralized by proteins of the Bearded family. First, Bearded mutant embryos exhibited a loss of epithelial polarity associated with an early loss of the apical domain. Bearded regulated epithelial polarity by antagonizing neuralized. Second, repression of Bearded gene expression by Snail was required for the Snail-dependent disassembly of adherens junctions in the mesoderm. Third, neuralized was strictly required to promote the downregulation of the apical domain in the midgut epithelium and to facilitate the transepithelial migration of primordial germ cells across this epithelium. This function of Neuralized was independent of its known role in Notch signalling. Thus, Neuralized has two distinct functions in epithelial cell polarity and Notch signalling.

Temporal-spatial changes in Sonic Hedgehog expression and signaling reveal different potentials of ventral mesencephalic progenitors to populate distinct ventral midbrain nuclei.

BACKGROUND: The ventral midbrain contains a diverse array of neurons, including dopaminergic neurons of the ventral tegmental area (VTA) and substantia nigra (SN) and neurons of the red nucleus (RN). Dopaminergic and RN neurons have been shown to arise from ventral mesencephalic precursors that express Sonic Hedgehog (Shh). However, Shh expression, which is initially confined to the mesencephalic ventral midline, expands laterally and is then downregulated in the ventral midline. In contrast, expression of the Hedgehog target gene Gli1 initiates in the ventral midline prior to Shh expression, but after the onset of Shh expression it is expressed in precursors lateral to Shh-positive cells. Given these dynamic gene expression patterns, Shh and Gli1 expression could delineate different progenitor populations at distinct embryonic time points. RESULTS: We employed genetic inducible fate mapping (GIFM) to investigate whether precursors that express Shh (Shh-GIFM) or transduce Shh signaling (Gli1-GIFM) at different time points give rise to different ventral midbrain cell types. We find that precursors restricted to the ventral midline are labeled at embryonic day (E)7.5 with Gli1-GIFM, and with Shh-GIFM at E8.5. These precursors give rise to all subtypes of midbrain dopaminergic neurons and the anterior RN. A broader domain of progenitors that includes the ventral midline is marked with Gli1-GIFM at E8.5 and with Shh-GIFM at E9.5; these fate-mapped cells also contribute to all midbrain dopaminergic subtypes and to the entire RN. In contrast, a lateral progenitor domain that is labeled with Gli1-GIFM at E9.5 and with Shh-GIFM at E11.5 has a markedly reduced potential to give rise to the RN and to SN dopaminergic neurons, and preferentially gives rise to the ventral-medial VTA. In addition, cells derived from Shh- and Gli1-expressing progenitors located outside of the ventral midline give rise to astrocytes. CONCLUSIONS: We define a ventral midbrain precursor map based on the timing of Gli1 and Shh expression, and suggest that the diversity of midbrain dopaminergic neurons is at least partially determined during their precursor stage when their medial-lateral position, differential gene expression and the time when they leave the ventricular zone influence their fate decisions.

Genome engineering-based analysis of Bearded family genes reveals both functional redundancy and a nonessential function in lateral inhibition in Drosophila.

Lateral inhibition mediated by Notch receptor signaling regulates the determination of sensory organ precursor cells (SOPs) in Drosophila. The selection of SOPs from proneural cluster cells appears to rely on a negative feedback loop linking activation of the Notch receptor to downregulation of its ligand Delta within each cell. The molecular basis of this regulatory feedback mechanism is not known. Here, we have tested the role of the Bearded (Brd) family genes in this process. The Drosophila genome encodes eight Brd family members that interact with the E3 ubiquitin ligase Neuralized (Neur) and act as inhibitors of Neur-mediated Delta signaling. Genome engineering technologies were used to create specific deletions of all eight Brd family genes. We find that the Brd family genes malpha, m4, and m6 encoded by the Enhancer of split Complex (E(spl)-C) are dispensable for Drosophila development and that deletion of the five Brd family genes encoded by the Brd Complex only reduces viability. However, deletion of all Brd family genes results in embryonic lethality. Additionally, the malpha, m4, and m6 genes act redundantly with the other five Brd family genes to spatially restrict Notch activation in stage 5 embryos. These data reveal that the Brd family genes have an essential but redundant activity. While the activity of all eight Brd genes appears to be dispensable for SOP determination, clone border studies indicate that both the relative activity levels of Neur and Brd family members influence competition for the SOP fate during lateral inhibition. We propose that inhibition of Neur-Delta interaction by Brd family members is part of the feedback loop that underlies lateral inhibition in Drosophila.