Search

The development of functional eggs and sperm are critical processes in mammalian development as they ensure successful reproduction and species propagation. While past studies have identified important genes that regulate these processes, the roles of luminal flow and fluid stress in reproductive biology remain less well understood. Here, we discuss recent evidence that support the diverse functions of luminal fluid in oogenesis, spermatogenesis and embryogenesis. We also review emerging techniques that allow for precise quantification and perturbation of tissue hydraulics in female and male reproductive systems, and propose new questions and approaches in this field. We hope this review will provide a useful resource to inspire future research in tissue hydraulics in reproductive biology and diseases.

In Brief Spermatozoa are released from Sertoli cells and flow in the seminiferous tubule to the rete testis. Our results suggest that the luminal flow in the tubules is repeatedly reversed and that this physical force helps spermatozoa release from the Sertoli cells. Abstract Spermatozoa released from Sertoli cells must be transported to the epididymis. However, the mechanism of the luminal flow in seminiferous tubules has remained unclear to date. Therefore, in this study, we investigated luminal flow and movements in the seminiferous tubules by three-dimensional analysis and in vivo imaging. Serial 5-μm-thick mouse testicular sections at 50-µm-intervals were prepared and stained by Periodic Acid-Schiff-hematoxylin. After three-dimensional reconstruction of the seminiferous tubules, the localization of the released spermatozoa and the stages observed in the sections were recorded in each reconstructed tubule. Luminal movements in the seminiferous tubules were observed by in vivo imaging using a fluorescent-reporter mouse and two-photon excitation microscopy system. Spermatozoa without contact to the seminiferous epithelium were not accumulated toward the rete testis. Additionally, such spermatozoa were found on their way not only to the most proximal rete testis but also a more distant rete testis from any stage VIII seminiferous epithelia. In vivo imaging demonstrated that the direction of the flagella of spermatozoa attached to the seminiferous epithelium was repeatedly reversed. The epithelium at the inner curve of the seminiferous tubule was shaken more actively and had fewer spermatozoa attached compared with the epithelium at the outer curve. Our results hence suggest that the luminal flow in the seminiferous tubules is repeatedly reversed and that this physical force helps spermatozoa to be released from Sertoli cells.

Living tissues are characterized by an intrinsically mechano-chemical interplay of active physical forces and complex biochemical signalling pathways. Either feature alone can give rise to complex emergent phenomena, for example mechanically driven glassy dynamics and rigidity transitions, or chemically driven reaction-diffusion instabilities. An important question is how to quantitatively assess the contribution of these different cues to the large-scale dynamics of biological materials. We address this in MDCK monolayers, considering both mechanochemical feedbacks between ERK signalling activity and cellular density as well as a mechanically active tissue rheology via a self-propelled vertex model. We show that the relative strength of active migration forces to mechanochemical couplings controls a transition from uniform active glass to periodic spatiotemporal waves. We parameterize the model from published experimental datasets on MDCK monolayers, and use it to make new predictions on the correlation functions of cellular dynamics and the dynamics of topological defects associated with the oscillatory phase of cells. Interestingly, MDCK monolayers are best described by an intermediary parameter region in which both mechanochemical couplings and noisy active propulsion have a strong influence on the dynamics. Finally, we study how tissue rheology and ERK waves feedback on one another, and uncover a mechanism via which tissue fluidity can be controlled by mechano-chemical waves both at the local and global levels.

Male penis is required to become erect during copulation. In the upper (dorsal) part of penis, the erectile tissue termed corpus cavernosum (CC) plays fundamental roles for erection by regulating the inner blood flow. When blood flows into the CC, the microvascular complex termed sinusoidal space is reported to expand during erection. A novel in vitro explant system to analyze the dynamic erectile responses during contraction/relaxation is established. The current data show regulatory contraction/relaxation processes induced by phenylephrine (PE) and nitric oxide (NO) donor mimicking dynamic erectile responses by in vitro CC explants. Two-photon excitation microscopy (TPEM) observation shows the synchronous movement of sinusoidal space and the entire CC. By taking advantages of the CC explant system, tadalafil (Cialis) was shown to increase sinusoidal relaxation. Histopathological changes have been generally reported associating with erection in several pathological conditions. Various stressed statuses have been suggested to occur in the erectile responses by previous studies. The current CC explant model enables to analyze such conditions through directly manipulating CC in the repeated contraction/relaxation processes. Expression of oxidative stress marker and contraction-related genes, Hypoxia-inducible factor 1-alpha (Hif1a), glutathione peroxidase 1 (Gpx1), Ras homolog family member A (RhoA), and Rho-associated protein kinase (Rock), was significantly increased in such repeated contraction/relaxation. Altogether, it is suggested that the system is valuable for analyzing structural changes and physiological responses to several regulators in the field of penile medicine.

Cell extrusion is a universal mode of cell removal from tissues, and it plays an important role in regulating cell numbers and eliminating unwanted cells, such as apoptotic, unfit, or cancerous. During this process, cells delaminate from the cell layer, however, the underlying mechanisms remain to be elucidated. Here, we report a conserved execution mechanism of cell extrusion. We found extracellular vesicle (EV) formation in extruding cells at a site opposite to the extrusion direction. Particularly, we found that a lipid-scramblase‒mediated local exposure of phosphatidylserine is responsible for EV formation and is crucial for executing cell extrusion, while inhibition of this process disrupted prompt cell delamination and tissue homeostasis. Furthermore, we revealed that the EV formation is governed by the mechanism in microvesicle formation, while the EVs have some hallmarks of apoptotic body. Finally, we illustrated the role of EV formation as promoting the neighboring cells’ invasion resulting in the execution of cell extrusion by experimental and mathematical modeling analysis. Taken together, this study provides the insights that membrane dynamics plays a crucial role behind the cell exit from the tissue by connecting the actions of extruding cell and the neighboring cells.

Longitudinal bone growth is achieved by a tightly controlled process termed endochondral bone formation. C-type natriuretic peptide (CNP) stimulates endochondral bone formation through binding to its specific receptor, guanylyl cyclase (GC)-B. However, CNP/GC-B signaling dynamics in different stages of endochondral bone formation have not been fully clarified, especially in terms of the interaction between the cyclic guanine monophosphate (cGMP) and cyclic adenosine monophosphate (cAMP) pathways. Here, we demonstrated that CNP activates the cAMP/protein kinase A (PKA) pathway and that this activation contributed to the elongation of the hypertrophic zone in the growth plate. Cells of the chondrogenic line ATDC5 were transfected with Förster resonance energy transfer (FRET)–based cGMP and PKA biosensors. Dual-FRET imaging revealed that CNP increased intracellular cGMP levels and PKA activities in chondrocytes. Further, CNP-induced PKA activation was enhanced following differentiation of ATDC5 cells. Live imaging of the fetal growth plate of transgenic mice, expressing a FRET biosensor for PKA, PKAchu mice, showed that CNP predominantly activates the PKA in the hypertrophic chondrocytes. Additionally, histological analysis of the growth plate of PKAchu mice demonstrated that CNP increased the length of the growth plate, but coadministration of a PKA inhibitor, H89, inhibited the growth-promoting effect of CNP only in the hypertrophic zone. In summary, we revealed that CNP-induced cGMP elevation activated the cAMP/PKA pathway, and clarified that this PKA activation contributed to the bone growth–promoting effect of CNP in hypertrophic chondrocytes. These results provide insights regarding the cross-talk between cGMP and cAMP signaling in endochondral bone formation and in the physiological role of the CNP/GC-B system.

Upon the initiation of collective cell migration, the cells at the free edge are specified as leader cells; however, the mechanism underlying the leader cell specification remains elusive. Here, we show that lamellipodial extension after the release from mechanical confinement causes sustained extracellular signal-regulated kinase (ERK) activation and underlies the leader cell specification. Live-imaging of Madin-Darby canine kidney (MDCK) cells and mouse epidermis through the use of Förster resonance energy transfer (FRET)-based biosensors showed that leader cells exhibit sustained ERK activation in a hepatocyte growth factor (HGF)-dependent manner. Meanwhile, follower cells exhibit oscillatory ERK activation waves in an epidermal growth factor (EGF) signaling-dependent manner. Lamellipodial extension at the free edge increases the cellular sensitivity to HGF. The HGF-dependent ERK activation, in turn, promotes lamellipodial extension, thereby forming a positive feedback loop between cell extension and ERK activation and specifying the cells at the free edge as the leader cells. Our findings show that the integration of physical and biochemical cues underlies the leader cell specification during collective cell migration., 細胞の集まりからリーダーが生まれる仕組み --出る杭はより出る--. 京都大学プレスリリース. 2022-09-29.

Intricate branching patterns emerge in internal organs because of the repetitive presence of simple deformations in epithelial tissues. During murine lung development, epithelial cells in distal tips of a single tube require fibroblast growth factor (FGF) signals generated by their surrounding mesenchyme to form repetitive tip bifurcations. However, it remains unknown how the cells employ FGF signaling to convert their behaviors to achieve the recursive branching processes. Here we show a self-sustained epithelial regulatory system during the murine lung branching morphogenesis, mediated by extracellular signal-regulated kinase (ERK), which acts as a downstream driver of FGF signaling. We found that tissue-scale curvature regulated ERK activity in the lung epithelium using two-photon live cell imaging and mechanical perturbations. ERK was activated specifically in epithelial tissues with a positive curvature, regardless of whether the change in curvature was attributable to morphogenesis or artificial perturbations. Moreover, ERK activation accelerated actin polymerization specifically at the apical side of cells, and mechanically contributed to the extension of the apical membrane, leading to a decrease in epithelial tissue curvature. These results indicate the existence of negative feedback loop between tissue curvature and ERK activity beyond scale. We confirmed that this regulation was sufficient to generate the recursive branching processes by a mathematical model. Taken together, we propose that ERK mediates the curvature feedback loop underlying the process of branching morphogenesis in developing lungs.

A notable example of spiral architecture in organs is the mammalian cochlear duct, where the morphology is critical for hearing function. Genetic studies have revealed necessary signaling molecules, but it remains unclear how cellular dynamics generate elongating, bending, and coiling of the cochlear duct. Here, we show that extracellular signal-regulated kinase (ERK) activation waves control collective cell migration during the murine cochlear duct development using deep tissue live-cell imaging, Förster resonance energy transfer (FRET)-based quantitation, and mathematical modeling. Long-term FRET imaging reveals that helical ERK activation propagates from the apex duct tip concomitant with the reverse multicellular flow on the lateral side of the developing cochlear duct, resulting in advection-based duct elongation. Moreover, model simulations, together with experiments, explain that the oscillatory wave trains of ERK activity and the cell flow are generated by mechanochemical feedback. Our findings propose a regulatory mechanism to coordinate the multicellular behaviors underlying the duct elongation during development., うずまき管の伸⻑を司る分子活性と細胞群の波を発見 --綱引きによる細胞群の流れと臓器の成長--. 京都大学プレスリリース. 2021-03-09.

A notable example of spiral architecture in organs is the mammalian cochlear duct, where the duct morphology is critical for hearing function. Molecular genetics has revealed the necessary signaling molecules for the formation of spirals in organs, but it remains unclear how cellular dynamics generate bending and coiling of the cochlear duct during development. Here we show two modes of multicellular dynamics underlying the morphogenetic process by combining deep tissue live-cell imaging, Förster resonance energy transfer (FRET)-based quantitation, and mathematical modeling. First, surgical separation of the cochlear duct revealed that bending forces reside primarily in the medial side of the duct. In the medial pseudostratified epithelium, we found that nuclei stall at the luminal side during interkinetic nuclear migration, which would cause the extension of the luminal side, thereby bending the duct. Second, long-term organ-scale FRET imaging of extracellular signal-regulated kinase (ERK) activity showed that helical ERK activation waves propagate from the duct tip concomitant with the reverse multicellular flow in the lateral side of the duct, resulting in advection-based duct elongation. We propose an interplay of distinct multicellular behaviors underpinning spiral morphogenesis in the developing cochlear duct.

Tracheal cartilage provides architectural integrity to the respiratory airway, and defects in this structure during embryonic development cause severe congenital anomalies. Previous genetic studies have revealed genes that are critical for the development of tracheal cartilage. However, it is still unclear how crosstalk between these proteins regulates tracheal cartilage formation. Here we show a core regulatory network underlying murine tracheal chondrogenesis from embryonic day (E) 12.5 to E15.5, by combining volumetric imaging of fluorescence reporters, inhibitor assays, and mathematical modeling. We focused on SRY-box transcription factor 9 (Sox9) and extracellular signal-regulated kinase (Erk) in the tracheal mesenchyme, and observed a synchronous, inverted U-shaped temporal change in both Sox9 expression and Erk activity with a peak at E14.5, whereas the expression level of downstream cartilage matrix genes, such as collagen II alpha 1 (Col2a1) and aggrecan (Agc1), monotonically increased. Inhibitor assays revealed that the Erk signaling pathway functions as an inhibitory regulator of tracheal cartilage differentiation during this period. These results suggest that expression of the cartilage matrix genes is controlled by an incoherent feedforward loop via Sox9 and Erk, which is supported by a mathematical model. Furthermore, the modeling analysis suggests that a Sox9-Erk incoherent feedforward regulation augment the robustness against the variation of upstream factors. The present study provides a better understanding of the regulatory network underlying the tracheal development and will be helpful for efficient induction of tracheal organoids.

Collective cell migration offers a rich field of study for non-equilibrium physics and cellular biology, revealing phenomena such as glassy dynamics [1], pattern formation [2] and active turbulence [3]. However, how mechanical and chemical signaling are integrated at the cellular level to give rise to such collective behaviors remains unclear. We address this by focusing on the highly conserved phenomenon of spatio-temporal waves of density [2, 4–8] and ERK/MAPK activation [9–11], which appear both in vitro and in vivo during collective cell migration and wound healing. First, we propose a biophysical theory, backed by mechanical and optogenetic perturbation experiments, showing that patterns can be quantitatively explained by a mechano-chemical coupling between three-dimensional active cellular tensions and the mechano-sensitive ERK/MAPK pathway. Next, we demonstrate how this biophysical mechanism can robustly induce migration in a desired orientation, and we determine a theoretically optimal pattern for inducing efficient collective migration fitting well with experimentally observed dynamics. We thereby provide a bridge between the biophysical origin of spatio-temporal instabilities and the design principles of robust and efficient long-ranged migration.

Collective cell migration offers a rich field of study for non-equilibrium physics and cellular biology, revealing phenomena such as glassy dynamics, pattern formation and active turbulence. However, how mechanical and chemical signalling are integrated at the cellular level to give rise to such collective behaviours remains unclear. We address this by focusing on the highly conserved phenomenon of spatiotemporal waves of density and extracellular signal-regulated kinase (ERK) activation, which appear both in vitro and in vivo during collective cell migration and wound healing. First, we propose a biophysical theory, backed by mechanical and optogenetic perturbation experiments, showing that patterns can be quantitatively explained by a mechanochemical coupling between active cellular tensions and the mechanosensitive ERK pathway. Next, we demonstrate how this biophysical mechanism can robustly induce long-ranged order and migration in a desired orientation, and we determine the theoretically optimal wavelength and period for inducing maximal migration towards free edges, which fits well with experimentally observed dynamics. We thereby provide a bridge between the biophysical origin of spatiotemporal instabilities and the design principles of robust and efficient long-ranged migration. Spatiotemporal waves appear during collective cell migration and are affected by mechanical forces and biochemical signalling. Here the authors develop a biophysical model that can quantitatively account for complex mechanochemical patterns, and predict how they can be used for optimal collective migration.

During collective migration of epithelial cells, the migration direction is aligned over a tissue-scale expanse. Although the collective cell migration is known to be directed by mechanical forces transmitted via cell-cell junctions, it remains elusive how the intercellular force transmission is coordinated with intracellular biochemical signaling to achieve collective movements. Here, we show that intercellular coupling of extracellular signal-regulated kinase (ERK)-mediated mechanochemical feedback yields long-distance transmission of guidance cues. Mechanical stretch activates ERK through epidermal growth factor receptor (EGFR) activation, and ERK activation triggers cell contraction. The contraction of the activated cell pulls neighboring cells, evoking another round of ERK activation and contraction in the neighbors. Furthermore, anisotropic contraction based on front-rear polarization guarantees unidirectional propagation of ERK activation, and in turn, the ERK activation waves direct multicellular alignment of the polarity, leading to long-range ordered migration. Our findings reveal that mechanical forces mediate intercellular signaling underlying sustained transmission of guidance cues for collective cell migration., 分子活性の波が細胞集団に伝わる制御機構を解明 --細胞同士の綱引きが情報を遠くに伝える--. 京都大学プレスリリース. 2020-06-04., Cells communicate by doing the 'wave'. 京都大学プレスリリース. 2020-07-22.

In vitro tubulogenesis has been employed as an experimental model system to study tissue morphogenesis of internal organs. It has been previously shown that Madin-Darby canine kidney (MDCK) cells form tubes in the presence of hepatocyte growth factor in 3D cultures. Although these cells are expected to form tube structures in some microenvironments independent of chemical stimulation, little is known about the cellular mechanisms in organizing such an anisotropic multicellular structure. Here, we report 3D culture conditions that induce MDCK tubulogenesis without growth factor stimulation. We found that the cells spontaneously form elongated tube structures through aggregation processes in a specific range of both constituent cell number and scaffold gel concentration, while they form spherical aggregates in other conditions. We then examined cellular activities affecting tubulogenesis and showed that cell proliferation is not required for the tube elongation. Furthermore, we revealed that cells in the tube tips generate traction forces and pull the surrounding scaffold gel to migrate, resulting in the tube elongation. Our results suggest that the constituent cells during the aggregation process interact each other via mechanical forces transmitted in the scaffold gel, leading to the spontaneous tube formation.

Mathematical modeling is an essential approach for the understanding of complex multicellular behaviors in tis- sue morphogenesis. Here, we review the cellular Potts model (CPM; also known as the Glazier-Graner-Hoge- weg model), an effective computational modeling framework. We discuss its usability for modeling complex developmental phenomena by examining four fundamental examples of tissue morphogenesis: (i) cell sorting, (ii) cyst formation, (iii) tube morphogenesis in kidney development, and (iv) blood vessel formation. The review provides an introduction for biologists for starting simulation analysis using the CPM framework.

OBJECTIVES Lung transplantation is the only effective therapy for patients with end-stage lung disease but an organ shortage crisis necessitates the development of alternative therapies. Recent studies have highlighted the potential of foetal tissue transplantation to facilitate the regeneration of vital organs such as liver that have been damaged by lethal diseases. Herein, with the aim of restoring pulmonary function, we hypothesized that allogenic foetal lung tissue implantation would attenuate severe respiratory failure. METHODS Lung tissue from the foetuses of pregnant green fluorescent protein-C57BL/6 mice at 13.5 days of gestation was injected into the left lungs of recipient mice. Severe lung injury was induced by paraquat, and we analysed the survival rate and pathohistological findings after 1 month. RESULTS The survival rate of the therapy group was 39%, which was significantly higher than the vehicle group at 5.9% (P = 0.034). Immunochemical staining showed that positive cytoplasmic stained cells with anti-interleukin-10 antibody were identified in the gland-like structure of embryonic day 13.5 foetal lung. At 4 weeks after orthotopic implantation, haematoxylin and eosin staining showed reduced lung inflammatory cells, reduced lung oedema and increased active cell proliferation of foetal lung cells. Lung injury score showed that the airway septal thickening revealed statistically significant differences between vehicle and foetal lung therapy (P CONCLUSIONS Immature foetal lungs improved the survival rate of mice with paraquat-induced severe lung injury, establishing the need for systematic follow-up studies. The anti-inflammatory cytokine in the tissue from embryonic day 13.5 foetal lung might suppress severe lung injury.

During cochlear development, hair cells (HCs) and supporting cells differentiate in the prosensory domain to form the organ of Corti, but how one row of inner HCs (IHCs) and three rows of outer HCs (OHCs) are organized is not well understood. Here, we investigated the process of HC induction by monitoring Atoh1 expression in cochlear explants of Atoh1-EGFP knock-in mouse embryos and showed that only the cells that express Atoh1 over a certain threshold are selected for HC fate determination. HC induction initially occurs at the medial edge of the prosensory domain to form IHCs and subsequently at the lateral edge to form OHCs, while Hedgehog signaling maintains a space between IHCs and OHCs, leading to formation of the tunnel of Corti. These results reveal dynamic Atoh1 expression in HC fate control and suggest that multi-directional signals regulate OHC induction, thereby organizing the prototype of the organ of Corti.

Size control in biological tissues involves multicellular communication via mechanical forces during development. Although fundamental cellular behaviours in response to mechanical stimuli underlie size maintenance during morphogenetic processes, the mechanisms underpinning the cellular mechano-response system that maintains size along an axis of a polarized tissue remain elusive. Here, we show how the diameter of an epithelial tube is maintained during murine epididymal development by combining quantitative imaging, mechanical perturbation and mathematical modelling. We found that epithelial cells counteract compressive forces caused by cell division exclusively along the circumferential axis of the tube to produce polarized contractile forces, eventually leading to an oriented cell rearrangement. Moreover, a mathematical model that includes the polarized mechano-responsive regime explains how the diameter of proliferating tubes is maintained. Our findings pave the way for an improved understanding of the cellular response to mechanical forces that involves collective multicellular behaviours for organizing diverse tissue morphologies., Summary: Polarized cellular constriction responding to mechanical stress controls the diameter of a developing epithelial tube during murine epididymal development.

Cellular behaviors responding to mechanical forces control the size of multicellular tissues as demonstrated in isotropic size maintenance of developing tissues. However, how mechanoresponse systems work to maintain anisotropic tissue size including tube radial size remains unknown. Here we reveal the system underlying radial size maintenance of the murine epididymal tubule by combining quantitative imaging, mathematical modeling, and mechanical perturbations. We found that an oriented cell intercalation making the tubule radial size smaller counteracts a cell tension reduction due to neighbor cell division along the tubule circumferential axis. Moreover, we demonstrated that the tubule cells enhance actomyosin constriction driving the cell intercalation in response to mechanical forces anisotropically applied on the cells. Our results suggest that epididymal tubule cells have endogenous systems for responding as active cell movement to mechanical forces exclusively along the circumferential axis, and the anisotropic cellular mechanoresponse spontaneously controls the tubule radial size.

Purpose Lung transplantation is an effective therapy for end-stage patients with chronic respiratory failure due to various benign lung diseases. The organ shortage crisis is a serious problem, so it is required to develop alternative therapies. There have been reports of organ organoids ameliorating organ function but there have been few reports of lung organoids being capable of restoring respiratory function. We therefore hypothesized that immature mouse fetal lungs would ameliorate severe respiratory failure and connect the airways between recipient and donor lungs. Methods Lungs from the fetuses of pregnant GFP-C57BL/6 mice at 13.5 days gestation were injected into the left lungs of recipient mice. Severe lung injury was induced by paraquat, and we analyzed survival rate, pulmonary function, PaO2/FiO2, lung cytokine levels and pathohistological findings with light-sheet microscopy after 1 month. Results At 4 weeks after orthotopic implantation histopathological findings showed that there was no airway connection between recipient lung and immature fetal lungs of mice. However, hematoxylin and eosin staining showed reduced lung inflammatory cells, reduced lung edema, and active cell proliferation of fetal lung cells. Maximum airway pressures of the sham, vehicle and therapy groups (n=3, 1 and 6 respectively) were 8.91±1.47, 9.75, and 9.21±1.22 mmHg after one month. The airway compliance of the sham, vehicle and therapy groups were 0.041±0.017, 0.036 and 0.037±0.008 mmHg. The arterial partial blood pressures in the left ventricle of the sham, vehicle and therapy groups under FiO2 1.0 were 449±13.4, 516 and 427±28.4 mmHg. The survival rate of the therapy group increased from 5.9% to 39%, compared with that of the vehicle group (p=0.0338 by log-rank test). There was no difference of lung cytokine levels between the vehicle and therapy groups. The low number of mice and the high standard deviation affected the results of lung cytokine levels. The level of cytokines of some treated mice showed severe lung injury, however, other mice showed decreased cytokine levels. Conclusion Immature fetal lungs did not self-organize airway structures, however, they improved the survival rate of mice with paraquat-induced severe lung injury.

Summary In all animals, collective cell movement is an essential process in many events, including wound healing and embryonic development. However, our understanding of what characterizes the emergence of multicellular collective behavior is still far from complete. In this article we showed the fundamental cellular processes that drive collective cell movement by means of integrated approaches, including precise quantification measurements and mathematical modeling of measured data. First, we observed the dependence of the collective behaviors of cultured human skin cells on Ca2+ concentrations. When the culturing area confined by a PDMS sheet was suddenly expanded by removing the sheet, the group of cells moved to the expanded area with higher collectivity at higher Ca2+ concentrations. Next, we quantitatively measured cellular responses to the Ca2+ treatments, such as cell growth, cell division, and the strength of intercellular adhesion. Using a femtosecond-laser-based assay, an original method for estimating intercellular adhesion, we found that the strength of intercellular adhesion has an approximately 13-fold range in our treatments. Incorporating the quantitative data into a mathematical model, we then confirmed that the model well reproduced the multicellular behaviors we observed, demonstrating that the strength of intercellular adhesion sufficiently determines the generation of collective cell movement. Finally, we performed extensive numerical experiments, and the results suggested that the emergence of collective cell movement is derived by an optimal balance between the strength of intercellular adhesion and the intensity of cell migration.

Tissue pattern formation during development is a reproducible morphogenetic process organized by a series of kinetic cellular activities, leading to the building of functional and stable organs. Recent studies focusing on mechanical aspects have revealed physical mechanisms on how the cellular activities contribute to the formation of reproducible tissue patterns; however, the understanding for what factors achieve the reproducibility of such patterning and how it occurs is far from complete. Here, I focus on a tube pattern formation during murine epididymal development, and show that two factors influencing physical design for the patterning, the proliferative zone within the tubule and the viscosity of tissues surrounding to the tubule, control the reproducibility of epididymal tubule pattern, using a mathematical model based on experimental data. Extensive numerical simulation of the simple mathematical model revealed that a spatially localized proliferative zone within the tubule, observed in experiments, results in more reproducible tubule pattern. Moreover, I found that the viscosity of tissues surrounding to the tubule imposes a trade-off regarding pattern reproducibility and spatial accuracy relating to the region where the tubule pattern is formed. This indicates an existence of optimality in material properties of tissues for the robust patterning of epididymal tubule. The results obtained by numerical analysis based on experimental observations provide a general insight on how physical design realizes robust tissue pattern formation.

Extracellular signal-regulated kinase (ERK) is a key factor in the widely used signaling cascade of phosphorylation-dephosphorylation cycles and plays pivotal roles in many aspects of biological processes. Experimental studies in yeast and in Drosophila embryo have suggested that the phosphorylation and spatial localization of ERK are influenced by the level of its downstream substrates. However, the mechanism, through which these substrates control properties of ERK signaling, has been unclear. I propose a mass-action kinetic model of ERK cycle with its substrate, and demonstrate that the substrate can modulate the ERK activity by directly interacting with ERK. The model shows that the addition of substrate controls the level of ERK phosphorylation positively or negatively, depending on the balance between dissociation constants of ERK-substrate interaction and properties of ERK cyclic signaling in the absence of the substrate. In addition, by considering cellular compartments, cytosol and nucleus, the substrate can lead to nuclear accumulation of ERK, suggesting that the substrate can act as a nuclear anchor of ERK. The model gives a possible mechanism that can account for substrate-mediated modulation of ERK signaling.

Whole-tissue quantification at single-cell resolution has become an inevitable approach for further quantitative understanding of morphogenesis in organ development. The feasibility of the approach has been dramatically increased by recent technological improvements in optical tissue clearing and microscopy. However, the series of procedures required for this approach to lead to successful whole-tissue quantification is far from developed. To provide the appropriate procedure, we here show tips for each critical step of the entire process, including fixation for immunofluorescence, optical clearing, and digital image processing, using developing murine internal organs such as epididymis, kidney, and lung as an example. Through comparison of fixative solutions and of clearing methods, we found optimal conditions to achieve clearer deep-tissue imaging of specific immunolabeled targets and explain what methods result in vivid volume imaging. In addition, we demonstrated that three-dimensional digital image processing after optical clearing produces objective quantitative data for the whole-tissue analysis, focusing on the spatial distribution of mitotic cells in the epididymal tubule. The procedure for the whole-tissue quantification shown in this article should contribute to systematic measurements of cellular processes in developing organs, accelerating the further understanding of morphogenesis at the single cell level.