Creative processes during vertebrate organ morphogenesis: Biophysical self-organization at the supracellular scale.

Here, we review recent developments in the literature that provide insight into self-organization at supracellular scales in vertebrate organ morphogenesis. We briefly present a historical and conceptual analysis of the term "self-organization." Based on this analysis, we suggest that self-organizing processes, at their root, possess a form of causal relationship, reciprocal causality, that is markedly distinct from linear causal chains. We survey the extent to which reciprocal causality can be used to interpret or clarify supracellular studies in development and disease. Finally, we explore how reciprocal causality can exist across length-scales, identifying situations where multiple scales require simultaneous analysis.

Morphogens enable interacting supracellular phases that generate organ architecture.

During vertebrate organogenesis, increases in morphological complexity are tightly coupled to morphogen expression. In this work, we studied how morphogens influence self-organizing processes at the collective or "supra"-cellular scale in avian skin. We made physical measurements across length scales, which revealed morphogen-enabled material property differences that were amplified at supracellular scales in comparison to cellular scales. At the supracellular scale, we found that fibroblast growth factor (FGF) promoted "solidification" of tissues, whereas bone morphogenetic protein (BMP) promoted fluidity and enhanced mechanical activity. Together, these effects created basement membrane-less compartments within mesenchymal tissue that were mechanically primed to drive avian skin tissue budding. Understanding this multiscale process requires the ability to distinguish between proximal effects of morphogens that occur at the cellular scale and their functional effects, which emerge at the supracellular scale.

Differences in cell shape, motility, and growth reflect chromosomal number variations that can be visualized with live-cell ChReporters.

Chromosome numbers often change dynamically in tumors and cultured cells, which complicates therapy as well as understanding genotype-mechanotype relationships. Here we use a live-cell "ChReporter" method to identify cells with a single chromosomal loss in efforts to better understand differences in cell shape, motility, and growth. We focus on a standard cancer line and first show clonal populations that retain the ChReporter exhibit large differences in cell and nuclear morphology as well as motility. Phenotype metrics follow simple rules, including migratory persistence scaling with speed, and cytoskeletal differences are evident from drug responses, imaging, and single-cell RNA sequencing. However, mechanotype-genotype relationships between fluorescent ChReporter-positive clones proved complex and motivated comparisons of clones that differ only in loss or retention of a Chromosome-5 ChReporter. When lost, fluorescence-null cells show low expression of Chromosome-5 genes, including a key tumor suppressor APC that regulates microtubules and proliferation. Colonies are compact, nuclei are rounded, and cells proliferate more, with drug results implicating APC, and patient survival data indicating an association in multiple tumor-types. Visual identification of genotype with ChReporters can thus help clarify mechanotype and mechano-evolution.

Confinement plus myosin-II suppression maximizes heritable loss of chromosomes, as revealed by live-cell ChReporters.

The mechanical environment of a cell can have many effects, but whether it impacts the DNA sequence of a cell has remained unexamined. To investigate this, we developed a live-cell method to measure changes in chromosome numbers. We edited constitutive genes with GFP or RFP tags on single alleles and discovered that cells that lose Chromosome reporters (ChReporters) become non-fluorescent. We applied our new tools to confined mitosis and to inhibition of the putative tumor suppressor myosin-II. We quantified compression of mitotic chromatin in vivo and demonstrated that similar compression in vitro resulted in cell death, but also rare and heritable ChReptorter loss. Myosin-II suppression rescued lethal multipolar divisions and maximized ChReporter loss during three-dimensional (3D) compression and two-dimensional (2D) lateral confinement, but not in standard 2D culture. ChReporter loss was associated with chromosome mis-segregation, rather than just the number of divisions, and loss in vitro and in mice was selected against in subsequent 2D cultures. Inhibition of the spindle assembly checkpoint (SAC) caused ChReporter loss in 2D culture, as expected, but not during 3D compression, suggesting a SAC perturbation. Thus, ChReporters enable diverse studies of viable genetic changes, and show that confinement and myosin-II affect DNA sequence and mechano-evolution.

Reciprocal cell-ECM dynamics generate supracellular fluidity underlying spontaneous follicle patterning.

During vertebrate embryogenesis, cell collectives engage in coordinated behavior to form tissue structures of increasing complexity. In the avian skin, assembly into follicles depends on intrinsic mechanical forces of the dermis, but how cell mechanics initiate pattern formation is not known. Here, we reconstitute the initiation of follicle patterning ex vivo using only freshly dissociated avian dermal cells and collagen. We find that contractile cells physically rearrange the extracellular matrix (ECM) and that ECM rearrangement further aligns cells. This exchange transforms a mechanically unlinked collective of dermal cells into a continuum, with coherent, long-range order. Combining theory with experiment, we show that this ordered cell-ECM layer behaves as an active contractile fluid that spontaneously forms regular patterns. Our study illustrates a role for mesenchymal dynamics in generating cell-level ordering and tissue-level patterning through a fluid instability-processes that may be at play across morphological symmetry-breaking contexts.

Gaussian curvature dilutes the nuclear lamina, favoring nuclear rupture, especially at high strain rate.

Nuclear rupture has long been associated with deficits or defects in lamins, with recent results also indicating a role for actomyosin stress, but key physical determinants of rupture remain unclear. Here, lamin-B filaments stably interact with the nuclear membrane at sites of low Gaussian curvature yet dilute at high curvature to favor rupture, whereas lamin-A depletion requires high strain-rates. Live-cell imaging of lamin-B1 gene-edited cancer cells is complemented by fixed-cell imaging of rupture in: iPS-derived progeria patients cells, cells within beating chick embryo hearts, and cancer cells with multi-site rupture after migration through small pores. Data fit a model of stiff filaments that detach from a curved surface.Rupture is modestly suppressed by inhibiting myosin-II and by hypotonic stress, which slow the strain-rates. Lamin-A dilution and rupture probability indeed increase above a threshold rate of nuclear pulling. Curvature-sensing mechanisms of proteins at plasma membranes, including Piezo1, might thus apply at nuclear membranes.Summary statement: High nuclear curvature drives lamina dilution and nuclear envelope rupture even when myosin stress is inhibited. Stiff filaments generally dilute from sites of high Gaussian curvature, providing mathematical fits of experiments.

Nuclear Mechanics and Cancer Cell Migration.

As a cancer cell invades adjacent tissue, penetrates a basement membrane barrier, or squeezes into a blood capillary, its nucleus can be greatly constricted. Here, we examine: (1) the passive and active deformation of the nucleus during 3D migration; (2) the nuclear structures-namely, the lamina and chromatin-that govern nuclear deformability; (3) the effect of large nuclear deformation on DNA and nuclear factors; and (4) the downstream consequences of mechanically stressing the nucleus. We focus especially on recent studies showing that constricted migration causes nuclear envelope rupture and excess DNA damage, leading to cell cycle suppression, possibly cell death, and ultimately it seems to heritable genomic variation. We first review the latest understanding of nuclear dynamics during cell migration, and then explore the functional effects of nuclear deformation, especially in relation to genome integrity and potentially cancerous mutations.

Nuclear failure, DNA damage, and cell cycle disruption after migration through small pores: a brief review.

In many contexts of development, regeneration, or disease such as cancer, a cell squeezes through a dense tissue or a basement membrane, constricting its nucleus. Here, we describe how the severity of nuclear deformation depends on a nucleus' mechanical properties that are mostly determined by the density of chromatin and by the nuclear lamina. We explain how constriction-induced nuclear deformation affects nuclear contents by causing (i) local density changes in chromatin and (ii) rupture of the nuclear lamina and envelope. Both processes mislocalize diffusible nuclear factors including key DNA repair and regulatory proteins. Importantly, these effects of constricted migration are accompanied by excess DNA damage, marked by phosphorylated histone γH2AX in fixed cells. Rupture has a number of downstream consequences that include a delayed cell cycle-consistent with a damage checkpoint-and modulation of differentiation, both of which are expected to affect migration-dependent processes ranging from wound healing to tumorigenic invasion.

Pulling the Roof Down on Anchored Nuclei.

How a smooth epithelium becomes topographically patterned in development remains incompletely understood. In this issue of Developmental Cell,Ambrosini et al. (2019) investigate how dying cells specifically indent the apical surface, finding that apical actomyosin cables contract against the apoptotic nucleus, which itself is anchored basally to the extracellular matrix.

Rescue of DNA damage after constricted migration reveals a mechano-regulated threshold for cell cycle.

Migration through 3D constrictions can cause nuclear rupture and mislocalization of nuclear proteins, but damage to DNA remains uncertain, as does any effect on cell cycle. Here, myosin II inhibition rescues rupture and partially rescues the DNA damage marker γH2AX, but an apparent block in cell cycle appears unaffected. Co-overexpression of multiple DNA repair factors or antioxidant inhibition of break formation also exert partial effects, independently of rupture. Combined treatments completely rescue cell cycle suppression by DNA damage, revealing a sigmoidal dependence of cell cycle on excess DNA damage. Migration through custom-etched pores yields the same damage threshold, with ∼4-µm pores causing intermediate levels of both damage and cell cycle suppression. High curvature imposed rapidly by pores or probes or else by small micronuclei consistently associates nuclear rupture with dilution of stiff lamin-B filaments, loss of repair factors, and entry from cytoplasm of chromatin-binding cGAS (cyclic GMP-AMP synthase). The cell cycle block caused by constricted migration is nonetheless reversible, with a potential for DNA misrepair and genome variation.

Constricted migration modulates stem cell differentiation.

Tissue regeneration at an injured site depends on proliferation, migration, and differentiation of resident stem or progenitor cells, but solid tissues are often sufficiently dense and constricting that nuclei are highly stressed by migration. In this study, constricted migration of myoblastic cell types and mesenchymal stem cells (MSCs) increases nuclear rupture, increases DNA damage, and modulates differentiation. Fewer myoblasts fuse into regenerating muscle in vivo after constricted migration in vitro, and myodifferentiation in vitro is likewise suppressed. Myosin II inhibition rescues rupture and DNA damage, implicating nuclear forces, while mitosis and the cell cycle are suppressed by constricted migration, consistent with a checkpoint. Although perturbed proliferation fails to explain defective differentiation, nuclear rupture mislocalizes differentiation-relevant MyoD and KU80 (a DNA repair factor), with nuclear entry of the DNA-binding factor cGAS. Human MSCs exhibit similar damage, but osteogenesis increases-which is relevant to bone and to calcified fibrotic tissues, including diseased muscle. Tissue repair can thus be modulated up or down by the curvature of pores through which stem cells squeeze.

Nuclear rupture at sites of high curvature compromises retention of DNA repair factors.

The nucleus is physically linked to the cytoskeleton, adhesions, and extracellular matrix-all of which sustain forces, but their relationships to DNA damage are obscure. We show that nuclear rupture with cytoplasmic mislocalization of multiple DNA repair factors correlates with high nuclear curvature imposed by an external probe or by cell attachment to either aligned collagen fibers or stiff matrix. Mislocalization is greatly enhanced by lamin A depletion, requires hours for nuclear reentry, and correlates with an increase in pan-nucleoplasmic foci of the DNA damage marker γH2AX. Excess DNA damage is rescued in ruptured nuclei by cooverexpression of multiple DNA repair factors as well as by soft matrix or inhibition of actomyosin tension. Increased contractility has the opposite effect, and stiff tumors with low lamin A indeed exhibit increased nuclear curvature, more frequent nuclear rupture, and excess DNA damage. Additional stresses likely play a role, but the data suggest high curvature promotes nuclear rupture, which compromises retention of DNA repair factors and favors sustained damage.

Constricted migration increases DNA damage and independently represses cell cycle.

Cell migration through dense tissues or small capillaries can elongate the nucleus and even damage it, and any impact on cell cycle has the potential to affect various processes including carcinogenesis. Here, nuclear rupture and DNA damage increase with constricted migration in different phases of cell cycle-which we show is partially repressed. We study several cancer lines that are contact inhibited or not and that exhibit diverse frequencies of nuclear lamina rupture after migration through small pores. DNA repair factors invariably mislocalize after migration, and an excess of DNA damage is evident as pan--nucleoplasmic foci of phosphoactivated ATM and γH2AX. Foci counts are suppressed in late cell cycle as expected of mitotic checkpoints, and migration of contact-inhibited cells through large pores into sparse microenvironments leads also as expected to cell-cycle reentry and no effect on a basal level of damage foci. Constricting pores delay such reentry while excess foci occur independent of cell-cycle phase. Knockdown of repair factors increases DNA damage independent of cell cycle, consistent with effects of constricted migration. Because such migration causes DNA damage and impedes proliferation, it illustrates a cancer cell fate choice of "go or grow."

Nuclear mechanosensing.

Structural links from the nucleus to the cytoskeleton and to the extracellular environment play a role in direct mechanosensing by nuclear factors. Here, we highlight recent studies that illustrate nuclear mechanosensation processes ranging from DNA repair and nuclear protein phospho-modulation to chromatin reorganization, lipase activation by dilation, and reversible rupture with the release of nuclear factors. Recent progresses demonstrate that these mechanosensing processes lead to modulation of gene expression such as those involved in the regulation of cytoskeletal programs and introduce copy number variations. The nuclear lamina protein lamin A has a recurring role, and various biophysical analyses prove helpful in clarifying mechanisms. The various recent observations provide further motivation to understand the regulation of nuclear mechanosensing pathways in both physiological and pathological contexts.

Genome variation across cancers scales with tissue stiffness - an invasion-mutation mechanism and implications for immune cell infiltration.

Many different types of soft and solid tumors have now been sequenced, and meta-analyses suggest that genomic variation across tumors scales with the stiffness of the tumors' tissues of origin. The opinion expressed here is based on a review of current genomics data, and it considers multiple 'mechanogenomics' mechanisms to potentially explain this scaling of mutation rate with tissue stiffness. Since stiff solid tissues have higher density of fibrous collagen matrix, which should decrease tissue porosity, cancer cell proliferation could be affected and so could invasion into stiff tissues as the nucleus is squeezed sufficiently to enhance DNA damage. Diversification of a cancer genome after constricted migration is now clear. Understanding genome changes that give rise to neo-antigens is important to selection as well as to the development of immunotherapies, and we discuss engineered monocytes/macrophages as particularly relevant to understanding infiltration into solid tumors.

SIRPA-Inhibited, Marrow-Derived Macrophages Engorge, Accumulate, and Differentiate in Antibody-Targeted Regression of Solid Tumors.

Marrow-derived macrophages are highly phagocytic, but whether they can also traffic into solid tumors and engulf cancer cells is questionable, given the well-known limitations of tumor-associated macrophages (TAMs). Here, SIRPα on macrophages from mouse and human marrow was inhibited to block recognition of its ligand, the "marker of self" CD47 on all other cells. These macrophages were then systemically injected into mice with fluorescent human tumors that had been antibody targeted. Within days, the tumors regressed, and single-cell fluorescence analyses showed that the more the macrophages engulfed, the more they accumulated within regressing tumors. Human-marrow-derived macrophages engorged on the human tumors, while TAMs were minimally phagocytic, even toward CD47-knockdown tumors. Past studies had opsonized tumors in situ with antibody and/or relied on mouse TAMs but had not injected SIRPα-inhibited cells; also, unlike past injections of anti-CD47, blood parameters remained normal and safe. Consistent with tumor-selective engorge-and-accumulate processes in vivo, phagocytosis in vitro inhibited macrophage migration through micropores that mimic features of dense 3D tissue. Accumulation of SIRPα-inhibited macrophages in tumors favored tumor regression for 1-2 weeks, but donor macrophages quickly differentiated toward non-phagocytic, high-SIRPα TAMs. Analyses of macrophages on soft (like marrow) or stiff (like solid tumors) collagenous gels demonstrated a stiffness-driven, retinoic-acid-modulated upregulation of SIRPα and the mechanosensitive nuclear marker lamin-A. Mechanosensitive differentiation was similarly evident in vivo and likely limited the anti-tumor effects, as confirmed by re-initiation of tumor regression by fresh injections of SIRPα-inhibited macrophages. Macrophage motility, phagocytosis, and differentiation in vivo are thus coupled.

Elastic-Fluid Model for DNA Damage and Mutation from Nuclear Fluid Segregation Due to Cell Migration.

When cells migrate through constricting pores, they incur DNA damage and develop genomic variation. Experiments show that this damage is not due to DNA breakage from mechanical stress on chromatin in the deformed nucleus. Here we propose a model for a mechanism by which nuclear deformation can lead to DNA damage. We treat the nucleus as an elastic-fluid system with an elastic component (chromatin) and fluid component that can be squeezed out when the nucleus is deformed. We couple the elastic-fluid model to the kinetics of DNA breakage and repair by assuming that the local volume fraction of the elastic component controls the rate of damage per unit volume due to naturally occurring DNA breaks, whereas the volume fraction of the fluid component controls the rate of repair of DNA breaks per unit volume by repair factors, which are soluble in the fluid. By comparing our results to a number of experiments on controlled migration through pores, we show that squeeze-out of the fluid, and hence of the mobile repair factors, is sufficient to account for the extent of DNA damage and genomic variation observed experimentally. We also use our model for migration through a cylindrical pore to estimate the variation with tissue stiffness of the mutation rate in tumors.

As a Nucleus Enters a Small Pore, Chromatin Stretches and Maintains Integrity, Even with DNA Breaks.

As a cell pushes or pulls its nucleus through a small constriction, the chromatin must distort and somehow maintain genomic stability despite ever-present double-strand breaks in the DNA. Here we visualize within a living cell the pore-size dependent deformation of a specific locus engineered into chromosome-1 and cleaved. An mCherry-tagged nuclease targets the submicron locus, causing DNA cleavage and recruiting repair factors such as GFP-53BP1 to a large region around the locus. Aspiration of a cell and its nucleus into a micropipette shows that chromatin aligns and stretches parallel to the pore. Extension is largest in small pores, increasing >10-fold but remaining 30-fold shorter than the DNA contour length in the locus. Brochard and de Gennes' blob model for tube geometry fits the data, with a simple modification for chromatin crowding. Continuity of the highly extended, cleaved chromatin is also maintained, consistent with folding and cross bridging of the DNA. Surprisingly, extensional integrity is unaffected by an inhibitor of the DNA repair scaffold.

DNA Damage Follows Repair Factor Depletion and Portends Genome Variation in Cancer Cells after Pore Migration.

Migration through micron-size constrictions has been seen to rupture the nucleus, release nuclear-localized GFP, and cause localized accumulations of ectopic 53BP1-a DNA repair protein. Here, constricted migration of two human cancer cell types and primary mesenchymal stem cells (MSCs) increases DNA breaks throughout the nucleoplasm as assessed by endogenous damage markers and by electrophoretic "comet" measurements. Migration also causes multiple DNA repair proteins to segregate away from DNA, with cytoplasmic mis-localization sustained for many hours as is relevant to delayed repair. Partial knockdown of repair factors that also regulate chromosome copy numbers is seen to increase DNA breaks in U2OS osteosarcoma cells without affecting migration and with nucleoplasmic patterns of damage similar to constricted migration. Such depletion also causes aberrant levels of DNA. Migration-induced nuclear damage is nonetheless reversible for wild-type and sub-cloned U2OS cells, except for lasting genomic differences between stable clones as revealed by DNA arrays and sequencing. Gains and losses of hundreds of megabases in many chromosomes are typical of the changes and heterogeneity in bone cancer. Phenotypic differences that arise from constricted migration of U2OS clones are further illustrated by a clone with a highly elongated and stable MSC-like shape that depends on microtubule assembly downstream of the transcription factor GATA4. Such changes are consistent with reversion to a more stem-like state upstream of cancerous osteoblastic cells. Migration-induced genomic instability can thus associate with heritable changes.

Nuclear constriction segregates mobile nuclear proteins away from chromatin.

As a cell squeezes its nucleus through adjacent tissue, penetrates a basement membrane, or enters a small blood capillary, chromatin density and nuclear factors could in principle be physically perturbed. Here, in cancer cell migration through rigid micropores and in passive pulling into micropipettes, local compaction of chromatin is observed coincident with depletion of mobile factors. Heterochromatin/euchromatin was previously estimated from molecular mobility measurements to occupy a volume fraction f of roughly two-thirds of the nuclear volume, but based on the relative intensity of DNA and histones in several cancer cell lines drawn into narrow constrictions, f can easily increase locally to nearly 100%. By contrast, mobile proteins in the nucleus, including a dozen that function as DNA repair proteins (e.g., BRCA1, 53BP1) or nucleases (e.g., Cas9, FokI), are depleted within the constriction, approaching 0%. Such losses-compounded by the occasional rupture of the nuclear envelope-can have important functional consequences. Studies of a nuclease that targets a locus in chromosome-1 indeed show that constricted migration delays DNA damage.