The force and effect of cell proliferation

EMBO J 32: 2790–2803 doi:10.1038/emboj.2013.197; published online September102013The spatiotemporal control of cell divisions is a key factor in epithelial morphogenesis and patterning. Mao et al (2013) now describe how differential rates of proliferation within the Drosophila wing disc epithelium give rise to anisotropic tissue tension in peripheral/proximal regions of the disc. Such global tissue tension anisotropy in turn determines the orientation of cell divisions by controlling epithelial cell elongation.Oriented cell divisions play important roles in the establishment of the animal body plan by both influencing tissue morphogenesis and generating cellular diversity. Generally, the direction of the cell division plane is determined by the orientation of the mitotic spindle prior to cytokinesis. The observation that the mitotic spindle in most animal cell types aligns with the cell''s longest axis has led to the formulation of the ‘long-axis-rule'', postulating that cell shape anisotropy is the main determinant of spindle orientation (Minc et al, 2011). However, cell shape anisotropy is unlikely to be the only determinant since many cell types round up during mitosis, thereby losing their shape anisotropy and others do not follow the long-axis-rule at all. In such cases, division orientation is determined by the polarizing activity of biochemical signals originating from the environment (reviewed in Morin and Bellaïche, 2011). In addition, externally applied forces have also been suggested to control division orientation of single cells in culture independently from their effect on cell shape (Fink et al, 2011).Epithelial growth implies that cells divide parallel to the tissue plane with both daughter cells remaining integrated within the tissue. Although it has been recognized that defects in apico-basal polarity lead to spindle misalignment and disruption of epithelial architecture, the molecular mechanisms underlying this regulation are still unknown. Recent work in the Drosophila wing disc epithelium uncovered that the junctional proteins Scribbled and Discs large 1 (Dlg1) are required for proper spindle alignment parallel to the tissue plane (Nakajima et al, 2013). Similarly, in the Drosophila follicular epithelium, spindle orientation is dependent on the lateral localization of Dlg1, independently of its role in apico-basal polarity (Bergstralh et al, 2013). While such mechanisms ensure that cells divide parallel to the epithelial plane, other mechanisms must still be present to determine the orientation of the mitotic spindle within this plane.In the Drosophila wing disc epithelium, symmetric cell divisions preferentially align with the proximal-distal (PD) axis, thus elongating the organ along this axis (Baena-López et al, 2005). This preferential cell division orientation is determined by the Fat-Dachsous pathway, which promotes accumulation of the atypical myosin Dachs at PD cellular junctions. The polarized activity of Dachs in turn drives cell elongation along the PD axis, leading to a preferential orientation of the mitotic spindle along this axis (Mao et al, 2011). In this issue of The EMBO Journal, Mao et al (2013) report that while mitotic cells located in central regions of the wing disc indeed elongate and divide along the PD axis, cells located in the periphery (proximal edge) elongate and divide orthogonally to the PD axis (Figure 1). These results suggested some type of global planar tissue polarization in proximal regions of the wing disc overriding the local effects of Dachs on spindle orientation. By using laser ablation to reveal tissue tension, the authors showed that in peripheral/proximal regions of the wing disc, junctions oriented orthogonal to the PD axis (PD junctions) are under higher tension than junctions oriented along this axis (lateral junctions; Figure 1). This led them to hypothesize that anisotropic tissue tension might control division orientation of proximal wing cells. Through a combination of elegant genetic experiments and theoretical modelling, the authors then demonstrated that this global tension anisotropy in the proximal wing disc arises from higher cell division rates observed in central versus proximal regions of the wing disc. Furthermore, this apparent tension anisotropy causes concentric elongation of proximal wing disc cells orienting their mitotic spindle orthogonal to the PD axis (Figure 1).Open in a separate windowFigure 1Differential rates of cell division between distal (green) and proximal (red) regions of the Drosophila wing disc epithelium (1) give rise to global patterns of tension anisotropy within the tissue (2). This tension anisotropy promotes cell elongation along the main axis of tension, thereby controlling the orientation of cell division via cell shape anisotropies in proximal regions of the wing disc (3); D, distal; P, proximal.Collectively, these results demonstrate that differential proliferation rates within a tissue can generate global tension anisotropies, which promote cell shape changes that again influence cell division orientation. Further dissection of the mechanisms by which tissue tension controls cell division orientation will clarify if anisotropic tension controls division orientation solely through cell elongation, or if additional mechanosensing mechanisms exist that more directly convey tissue tension information to the mitotic spindle. It might also be worth exploring whether cell divisions along the main axis of tension within the wing disc affect global tension anisotropy, and whether the formation of anisotropic tension around areas of cell proliferation affects the rate of cell division therein. Such interplay between tissue tension anisotropy and cell division orientation/rate will likely be critical for maintaining physiological degrees of tissue tension and growth.In general, the work by Mao et al (2013) provides compelling evidence for a functional link between tissue tension and cell division orientation in a physiological relevant context, paving the way for future studies addressing the reciprocal relationship between these two aspects in tissue morphogenesis.     

Spatiotemporally modulated Vestigial gradient by Wingless signaling adaptively regulates cell division for precise wing size control

In animal development, the growth of a tissue or organ is timely arrested when it reaches the stereotyped correct size. How this is robustly controlled remains poorly understood. The prevalent viewpoint, which is that morphogen gradients, due to their organizing roles in development, are directly responsible for growth arrest, cannot explain a number of observations. Recent findings from studies of the Drosophila wing have revealed that the interpretation of the Wingless gradient requires signaling-induced self-inhibition and that cell proliferation is controlled by graded vestigial expression. These findings highlight a growth control mechanism that involves Wingless regulated vestigial expression, but a question is whether they can quantitatively explain the observed preciseness and robustness of wing size control. Quantitative and systematic investigation into Wingless signaling using a mathematical model has elucidated two points. First, negative regulation of the Vestigial gradient by Wingless signaling makes vestigial expression precise and robust. Second, weak Wingless signaling in a primarily small wing pouch causes a short and steep Vestigial gradient, which stimulates more cell divisions and leads to a significant expansion of the wing pouch; however, strong Wingless signaling in a primarily large wing pouch causes a long and smooth Vestigial gradient, which stimulates fewer cell divisions and results in a slight expansion of the wing pouch. These results substantially decipher an inherent mechanism of tissue and organ size control. Our model explains, and is supported by, a number of experimental observations.     

Establishment of global patterns of planar polarity during growth of the Drosophila wing epithelium

Epithelial tissues develop planar polarity that is reflected in the global alignment of hairs and cilia with respect to the tissue axes. The planar cell polarity (PCP) proteins form asymmetric and polarized domains across epithelial junctions that are aligned locally between cells and orient these external structures. Although feedback mechanisms can polarize PCP proteins intracellularly and locally align polarity between cells, how global PCP patterns are specified is not understood. It has been proposed that the graded distribution of a biasing factor could guide long-range PCP. However, we recently identified epithelial morphogenesis as a mechanism that can reorganize global PCP patterns; in the Drosophila pupal wing, oriented cell divisions and rearrangements reorient PCP from a margin-oriented pattern to one that points distally. Here, we use quantitative image analysis to study how PCP patterns first emerge in the wing. PCP appears during larval growth and is spatially oriented through the activities of three organizer regions that control disc growth and patterning. Flattening morphogen gradients emanating from these regions does not reduce intracellular polarity but distorts growth and alters specific features of the PCP pattern. Thus, PCP may be guided by morphogenesis rather than morphogen gradients.     

Epithelial polarity and spindle orientation: intersecting pathways

During asymmetric stem cell divisions, the mitotic spindle must be correctly oriented and positioned with respect to the axis of cell polarity to ensure that cell fate determinants are appropriately segregated into only one daughter cell. By contrast, epithelial cells divide symmetrically and orient their mitotic spindles perpendicular to the main apical–basal polarity axis, so that both daughter cells remain within the epithelium. Work in the past 20 years has defined a core ternary complex consisting of Pins, Mud and Gαi that participates in spindle orientation in both asymmetric and symmetric divisions. As additional factors that interact with this complex continue to be identified, a theme has emerged: there is substantial overlap between the mechanisms that orient the spindle and those that establish and maintain apical–basal polarity in epithelial cells. In this review, we examine several factors implicated in both processes, namely Canoe, Bazooka, aPKC and Discs large, and consider the implications of this work on how the spindle is oriented during epithelial cell divisions.     

Cell rearrangement and cell division during the tissue level morphogenesis of evaginating Drosophila imaginal discs

The evagination of Drosophila imaginal discs is a classic system for studying tissue level morphogenesis. Evagination involves a dramatic change in morphology and published data argue that this is mediated by cell shape changes. We have reexamined the evagination of both the leg and wing discs and find that the process involves cell rearrangement and that cell divisions take place during the process. The number of cells across the width of the ptc domain in the wing and the omb domain in the leg decreased as the tissue extended during evagination and we observed cell rearrangement to be common during this period. In addition, almost half of the cells in the region of the leg examined divided between 4 and 8 h after white prepupae formation. Interestingly, these divisions were not typically oriented parallel to the axis of elongation. Our observations show that disc evagination involves multiple cellular behaviors, as is the case for many other morphogenetic processes.     

STEM FORMATION FROM A SUCCULENT LEAF: ITS BEARING ON THEORIES OF AXIATION

The radial symmetry of shoots and roots arises from a center of symmetry within the apical meristem. When a lateral axis forms at a distance from the tip, a new center of radial symmetry must arise. We have studied the biophysics of this kind of transformation in the epidermal layer of the succulent Graptopetalum where a stem “regenerates” from organized leaf tissue. Study of the epidermal cell pattern (with scanning electron microscopy) shows that reorganization involves neither a cellular pre-pattern blocked out by oriented cell divisions nor a callus-like stage where cell files, expansion direction, and primary cell wall cellulose orientation are randomized throughout. Rather, developmental events are a function of initial position. In regions of geometrical compatibility between parent axis and prospective lateral, there is little or no modification of files, expansion, or cellulose. In regions requiring 90° changes in orientation, cellulose orientation (studied with polarized light) conforms to the new symmetry first. This is followed later by changes in the surface growth pattern and in the cell division pattern. The early establishment of a circumferential cellulose pattern in the epidermal layer could account for both the cylindrical shape of the new axis and the subsequent rearrangement of directional growth and cell file pattern.     

Cell division and morphogenesis in angiosperm embryogenesis

Angiosperm embryogenesis generates the basic body organization of flowering plants. The underlying processes of pattern formation, which establishes the diversity of position-dependent cell fates, and morphogenesis, which brings about the shape of the embryo, may not only involve intercellular communication and controlled cell expansion but also non-random cell divisions. Genetic analysis ofArabidopsisembryogenesis which displays a large invariant pattern of cell divisions suggests that unequal cell divisions segregate cell fates and are thus involved in pattern formation whereas other oriented cell divisions and differential mitotic rates reflect patterning and rather play a role in morphogenesis.     

Defective proventriculus is required for pattern formation along the proximodistal axis,cell proliferation and formation of veins in the Drosophila wing

Many genes have been identified that are required for the establishment of the dorsoventral (DV) and anteroposterior (AP) axes of the Drosophila wing. By contrast, little is known about the genes and mechanisms that pattern the proximodistal (PD) axis. Vestigial (Vg) is instrumental in patterning this axis, but the genes that mediate its effects and the mechanisms that operate during PD patterning are not known. We show that the gene defective proventriculus (dve) is required for a region of the PD axis encompassing the distal region of the proximal wing (PW) and a small part of the adjacent wing pouch. Loss-of-function of dve results in the deletion of this region and, consequently, shortening of the PD axis. dve expression is activated by Vg in a non-autonomous manner, and is repressed at the DV boundary through the combined activity of Nubbin and Wg. Besides its role in the establishment of the distal part of the PW, dve is also required for the formation of the wing veins 2 and 5, and the proliferation of wing pouch cells, especially in regions anterior to wing vein 3 and posterior to wing vein 4. The study of the regulation of dve expression provides information about the strategies employed to subdivide and pattern the PD axis, and reveals the importance of vg during this process.     

A default mechanism of spindle orientation based on cell shape is sufficient to generate cell fate diversity in polarised Xenopus blastomeres

The process of oriented divisions of polarised cells is a recurrent mechanism of cell fate diversification in development. It is commonly assumed that a specialised mechanism of spindle alignment into the axis of polarity is a prerequisite for such systems to generate cell fate diversity. Oriented divisions also take place in the frog blastula, where orientation of the spindle into the apicobasal axis of polarised blastomeres generates inner and outer cells with different fates. Here, we show that, in this system, the spindle orients according to the shape of the cells, a mechanism often thought to be a default. We show that in the embryo, fatedifferentiative, perpendicular divisions correlate with a perpendicular long axis and a small apical surface, but the long axis rather then the size of the apical domain defines the division orientation. Mitotic spindles in rounded, yet polarised, isolated Xenopus blastula cells orient randomly, but align into an experimentally introduced long axis when cells are deformed early in the cell cycle. Unlike other systems of oriented divisions, the spindle aligns at prophase, rotation behaviour is rare and restricted to small angle adjustments. Disruption of astral microtubules leads to misalignment of the spindle. These results show that a mechanism of spindle orientation that depends on cell shape rather than cortical polarity can nevertheless generate cell fate diversity from a population of polarised cells.     

Mechanical Control of Organ Size in the Development of the Drosophila Wing Disc

Control of cessation of growth in developing organs has recently been proposed to be influenced by mechanical forces acting on the tissue due to its growth. In particular, it was proposed that stretching of the tissue leads to an increase in cell proliferation. Using the model system of the Drosophila wing imaginal disc, we directly stretch the tissue finding a significant increase in cell proliferation, thus confirming this hypothesis. In addition, we characterize the growth over the entire growth period of the wing disc finding a correlation between the apical cell area and cell proliferation rate.PACS numbers: 87.19.lx, 87.18.Nq, 87.80.Ek, 87.17.Ee, 87.85.Xd     

Multiple levels of regulation specify the polarity of an asymmetric cell division in C. elegans

Wnt signaling systems play important roles in the generation of cell and tissue polarity during development. We describe a Wnt signaling system that acts in a new way to orient the polarity of an epidermal cell division in C. elegans. In this system, the EGL-20/Wnt signal acts in a permissive fashion to polarize the asymmetric division of a cell called V5. EGL-20 regulates this polarization by counteracting lateral signals from neighboring cells that would otherwise reverse the polarity of the V5 cell division. Our findings indicate that this lateral signaling pathway also involves Wnt pathway components. Overexpression of EGL-20 disrupts both the asymmetry and polarity of lateral epidermal cell divisions all along the anteroposterior (A/P) body axis. Together our findings suggest that multiple, inter-related Wnt signaling systems may act together to polarize asymmetric cell divisions in this tissue.     

Formation of the embryonic-abembryonic axis of the mouse blastocyst: relationships between orientation of early cleavage divisions and pattern of symmetric/asymmetric divisions

Setting aside pluripotent cells that give rise to the future body is a central cell fate decision in mammalian development. It requires that some blastomeres divide asymmetrically to direct cells to the inside of the embryo. Despite its importance, it is unknown whether the decision to divide symmetrically versus asymmetrically shows any spatial or temporal pattern, whether it is lineage-dependent or occurs at random, or whether it influences the orientation of the embryonic-abembryonic axis. To address these questions, we developed time-lapse microscopy to enable a complete 3D analysis of the origins, fates and divisions of all cells from the 2- to 32-cell blastocyst stage. This showed how in the majority of embryos, individual blastomeres give rise to distinct blastocyst regions. Tracking the division orientation of all cells revealed a spatial and temporal relationship between symmetric and asymmetric divisions and how this contributes to the generation of inside and outside cells and thus embryo patterning. We found that the blastocyst cavity, defining the abembryonic pole, forms where symmetric divisions predominate. Tracking cell ancestry indicated that the pattern of symmetric/asymmetric divisions of a blastomere can be influenced by its origin in relation to the animal-vegetal axis of the zygote. Thus, it appears that the orientation of the embryonic-abembryonic axis is anticipated by earlier cell division patterns. Together, our results suggest that two steps influence the allocation of cells to the blastocyst. The first step, involving orientation of 2- to 4-cell divisions along the animal-vegetal axis, can affect the second step, the establishment of inside and outside cell populations by asymmetric 8- to 32-cell divisions.     

Morphogen control of wing growth through the Fat signaling pathway

Organ growth is influenced by organ patterning, but the molecular mechanisms that link patterning to growth have remained unclear. We show that the Dpp morphogen gradient in the Drosophila wing influences growth by modulating the activity of the Fat signaling pathway. Dpp signaling regulates the expression and localization of Fat pathway components, and Fat signaling through Dachs is required for the effect of the Dpp gradient on cell proliferation. Juxtaposition of cells that express different levels of the Fat pathway regulators four-jointed and dachsous stimulates expression of Fat/Hippo pathway target genes and cell proliferation, consistent with the hypothesis that the graded expression of these genes contributes to wing growth. Moreover, uniform expression of four-jointed and dachsous in the wing inhibits cell proliferation. These observations identify Fat as a signaling pathway that links the morphogen-mediated establishment of gradients of positional values across developing organs to the regulation of organ growth.     

Effects of static or dynamic mechanical stresses on osteoblast phenotype expression in three‐dimensional contractile collagen gels

Studies performed at tissular (three‐dimensional, 3‐D) or cellular (two‐dimensional, 2‐D) levels showed that the loading pattern plays a crucial role in the osteoblastic physiology. In this study, we attempted to investigate the response of a 3‐D osteoblastic culture submitted to either no external stress or static or dynamic stresses. Rat osteosarcoma cells (ROS 17/2.8) were embedded within collagen type I lattices and studied for 3 weeks. Entrapment and proliferation of cells within the hydrated collagen gel resulted in the generation of contractile forces, which led to contraction of the collagen gel. We used this ability to evaluate the influence of three modes of mechanical stresses on the cell proliferation and differentiation: (1) the freely retracted gels (FRG) were floating in the medium, (2) the tense gels (TG) were stretched statically and isometrically, with contraction prevented in the longitudinal axis, and (3) the dynamic gels (DG) were floating gels submitted to periodic stresses (50 or 25 rpm frequency). Gels showed maximum contraction at day 12 in 50 rpm DG, followed by 25 rpm DG, then FRG (88%, 81%, 70%, respectively) and at day 16 in TG (33%). The proliferation rate was greater in TG than in FRG (+52%) but remained low in both DGs. Gel dimensions were related to the collagen concentration and on a minor extent to cell number. Cells in DG appeared rounder and larger than in other conditions. In TG, cells were elongated and oriented primarily along the tension axis. Scanning electron microscopy (SEM) showed that tension exerted by cells in TG led to reorientation of collagen fibers which, in turn, determined the spatial orientation and morphology of the cells. Transmission electron microscopy (TEM) performed at maximum proliferation showed a vast majority of cells with a distended well‐developed RER filled with granular material and numerous mitochondria. Alkaline phosphatase activity peaked close to the proliferation peak in FRG, whereas in TG, a biphasic curve was observed with a small peak at day 4 and the main peak at day 16. In DG, this activity was lower than in the two other conditions. A similar time course was observed for alkaline phosphatase gene expression as assessed by Northern blots. Regardless of the conditions, osteocalcin level showed a triphasic pattern: a first increase at day 2, followed by a decrease from day 4 to 14, and a second increase above initial values at day 18. Microanalysis‐x indicated that mineralization occurred after 14 days and TEM showed crystals within the matrix. We showed that static and dynamic mechanical stresses, in concert with 3‐D collagen matrices, played a significant role on the phenotypic modulation of osteoblast‐like cells. This experimental model provided a tool to investigate the significance and the mechanisms of mechanical activity of the 3‐D cultured osteoblast‐like cells. J. Cell. Biochem. 76:217–230, 1999. © 1999 Wiley‐Liss, Inc.     

Determination of mechanical stress distribution in Drosophila wing discs using photoelasticity

Morphogenesis, the process by which all complex biological structures are formed, is driven by an intricate interplay between genes, growth, as well as intra- and intercellular forces. While the expression of different genes changes the mechanical properties and shapes of cells, growth exerts forces in response to which tissues, organs and more complex structures are shaped. This is exemplified by a number of recent findings for instance in meristem formation in Arabidopsis and tracheal tube formation in Drosophila. However, growth not only generates forces, mechanical forces can also have an effect on growth rates, as is seen in mammalian tissues or bone growth. In fact, mechanical forces can influence the expression levels of patterning genes, allowing control of morphogenesis via mechanical feedback. In order to study the connections between mechanical stress, growth control and morphogenesis, information about the distribution of stress in a tissue is invaluable. Here, we applied stress-birefringence to the wing imaginal disc of Drosophila melanogaster, a commonly used model system for organ growth and patterning, in order to assess the stress distribution present in this tissue. For this purpose, stress-related differences in retardance are measured using a custom-built optical set-up. Applying this method, we found that the stresses are inhomogeneously distributed in the wing disc, with maximum compression in the centre of the wing pouch. This compression increases with wing disc size, showing that mechanical forces vary with the age of the tissue. These results are discussed in light of recent models proposing mechanical regulation of wing disc growth.     

Enzyme distribution patterns in the imaginal wing disc ofDrosophila melanogaster and other diptera

Summary Analysis of the development of the aldehyde oxidase (AO) pattern in the wing pouch ofD. melanogaster showed that the extension of areas with AO activity occurs in steps. This indicates that the activation of this enzyme is regulated in groups of cells. It is proposed to use the term territory for such a cell group. In the wing pouches ofD. melanogaster, D. simulans andMusca, corresponding parts of the disc become AO positive at comparable developmental stages. This indicates that AO becomes active in individual territories in a specific sequence.Borderlines of the distribution pattern of different enzymes in the wing pouch ofDrosophila and other dipteran species are in agreement with those found for the development of the AO pattern or are complementary to them. This indicates the existence of a common set of territories in the wing pouches of all higher diptera. Borderlines of patterns, as caused by different genetic constitution, are also in accord with this set of territories. The borderlines of some territories coincide with the compartmental A/P or D/V boundary. The results support the idea that both the location of compartmental boundaries and that of borderlines of enzyme territories are determined by a single mechanism.The distribution and the shape of the territories in the wing pouch is best explained by the reaction-diffusion model proposed by Meinhardt (1980), which involves three different gradients.     

Action of fat, four-jointed, dachsous and dachs in distal-to-proximal wing signaling

In the Drosophila wing, distal cells signal to proximal cells to induce the expression of Wingless, but the basis for this distal-to-proximal signaling is unknown. Here, we show that three genes that act together during the establishment of tissue polarity, fat, four-jointed and dachsous, also influence the expression of Wingless in the proximal wing. fat is required cell autonomously by proximal wing cells to repress Wingless expression, and misexpression of Wingless contributes to proximal wing overgrowth in fat mutant discs. Four-jointed and Dachsous can influence Wingless expression and Fat localization non-autonomously, consistent with the suggestion that they influence signaling to Fat-expressing cells. We also identify dachs as a gene that is genetically required downstream of fat, both for its effects on imaginal disc growth and for the expression of Wingless in the proximal wing. Our observations provide important support for the emerging view that Four-jointed, Dachsous and Fat function in an intercellular signaling pathway, identify a normal role for these proteins in signaling interactions that regulate growth and patterning of the proximal wing, and identify Dachs as a candidate downstream effector of a Fat signaling pathway.     

Division orientation: disentangling shape and mechanical forces

Oriented cell divisions are essential for the generation of cell diversity and for tissue shaping during morphogenesis. Cells in tissues are mechanically linked to their neighbors, upon which they impose, and from which they experience, physical force. Recent work in multiple systems has revealed that tissue-level physical forces can influence the orientation of cell division. A long-standing question is whether forces are communicated to the spindle orienting machinery via cell shape or directly via mechanosensing intracellular machinery. In this article, we review the current evidence from diverse model systems that show spindles are oriented by tissue-level physical forces and evaluate current models and molecular mechanisms proposed to explain how the spindle orientation machinery responds to extrinsic force.