Tissue hydraulics in reproduction.

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.

Live imaging approach of dynamic multicellular responses in ERK signaling during vertebrate tissue development.

The chemical and mechanical responses of cells via the exchange of information during growth and development result in the formation of biological tissues. Information processing within the cells through the signaling pathways and networks inherent to the constituent cells has been well-studied. However, the cell signaling mechanisms responsible for generating dynamic multicellular responses in developing tissues remain unclear. Here, I review the dynamic multicellular response systems during the development and growth of vertebrate tissues based on the extracellular signal-regulated kinase (ERK) pathway. First, an overview of the function of the ERK signaling network in cells is provided, followed by descriptions of biosensors essential for live imaging of the quantification of ERK activity in tissues. Then adducing four examples, I highlight the contribution of live imaging techniques for studying the involvement of spatio-temporal patterns of ERK activity change in tissue development and growth. In addition, theoretical implications of ERK signaling are also discussed from the viewpoint of dynamic systems. This review might help in understanding ERK-mediated dynamic multicellular responses and tissue morphogenesis.

Polarized cellular mechano-response system for maintaining radial size in developing epithelial tubes.

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.

Three-dimensional live imaging of Atoh1 reveals the dynamics of hair cell induction and organization in the developing cochlea.

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.

Three-dimensional analysis and in vivo imaging for sperm release and transport in the murine seminiferous tubule.

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. 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.

Development of Surgical and Visualization Procedures to Analyze Vasculatures by Mouse Tail Edema Model.

BACKGROUND: Because of the high frequency of chronic edema formation in the current "aged" society, analyses and detailed observation of post-surgical edema are getting more required. Post-surgical examination of the dynamic vasculature including L.V. (Lymphatic Vasculature) to monitor edema formation has not been efficiently performed. Hence, procedures for investigating such vasculature are essential. By inserting transparent sheet into the cutaneous layer of mouse tails as a novel surgery model (the Tail Edema by Silicone sheet mediated Transparency protocol; TEST), the novel procedures are introduced and analyzed by series of histological analyses including video-based L.V. observation and 3D histological reconstruction of vasculatures in mouse tails. RESULTS: The dynamic generation of post-surgical main and fine (neo) L.V. connective structure during the edematous recovery process was visualized by series of studies with a novel surgery model. Snapshot images taken from live binocular image recording for TEST samples suggested the presence of main and elongating fine (neo) L.V. structure. After the ligation of L.V., the enlargement of main L.V. was confirmed. In the case of light sheet fluorescence microscopy (LSFM) observation, such L.V. connections were also suggested by using transparent 3D samples. Finally, the generation of neo blood vessels particularly in the region adjacent to the silicone sheet and the operated boundary region was suggested in 3D reconstruction images. However, direct detection of elongating fine (neo) L.V. was not suitable for analysis by such LSFM and 3D reconstruction procedures. Thus, such methods utilizing fixed tissues are appropriate for general observation for the operated region including of L.V. CONCLUSIONS: The current surgical procedures and analysis on the post-surgical status are the first case to observe vasculatures in vivo with a transparent sheet. Systematic analyses including the FITC-dextran mediated snap shot images observation suggest the elongation of fine (neo) lymphatic vasculature. Post-surgical analyses including LSFM and 3D histological structural reconstruction, are suitable to reveal the fixed structures of blood and lymphatic vessels formation.

Dynamic erectile responses of a novel penile organ model utilizing TPEM†.

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.

Cellular Potts modeling of complex multicellular behaviors in tissue morphogenesis.

Mathematical modeling is an essential approach for the understanding of complex multicellular behaviors in tissue morphogenesis. Here, we review the cellular Potts model (CPM; also known as the Glazier-Graner-Hogeweg 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.

In vitro tubulogenesis of Madin-Darby canine kidney (MDCK) spheroids occurs depending on constituent cell number and scaffold gel concentration.

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 study on robust tissue pattern formation in growing epididymal tubule.

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.

ERK-mediated curvature feedback regulates branching morphogenesis in lung epithelial tissue.

Intricate branching patterns emerge in internal organs due to the recurrent occurrence of simple deformations in epithelial tissues. During murine lung development, epithelial cells in distal tips of the single tube require fibroblast growth factor (FGF) signals emanating from 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 mechano-chemical regulatory system underlying lung branching morphogenesis, orchestrated by extracellular signal-regulated kinase (ERK) 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 activation occurs specifically in epithelial tissues exhibiting positive curvature, regardless of whether the change in curvature was attributable to morphogenesis or perturbations. Moreover, ERK activation accelerates actin polymerization preferentially at the apical side of cells, mechanically contributing to the extension of the apical membrane, culminating in a reduction of epithelial tissue curvature. These results indicate the existence of a negative feedback loop between tissue curvature and ERK activity that transcends spatial scales. Our mathematical model confirms that this regulatory mechanism is sufficient to generate the recursive branching processes. Taken together, we propose that ERK orchestrates a curvature feedback loop pivotal to the self-organized patterning of tissues.

Tug-of-war via ERK signaling pathway for tissue organization - ERK activation to force generation.

Extracellular signal-regulated kinase (ERK) plays a crucial role in regulating collective cell behaviors observed in diverse biological phenomena. Emerging studies have shed light on the involvement of the ERK signaling pathway in the reception and generation of mechanical forces, thereby governing local mechanical interactions within multicellular tissues. Although limited in number, studies have provided insights into how ERK-mediated mechanical interactions contribute to multicellular organization. Here we explore the impact of ERK-mediated mechanical interactions on tissue morphogenesis, cell extrusion in homeostasis, and their interplay with the physical microenvironments of the extracellular matrix. We conclude that the coupling system of ERK activity with mechanical forces offers a promising avenue to unravel the emergent collective dynamics underlying tissue organization.

Collective cell dynamics and luminal fluid flow in the epididymis: A mechanobiological perspective.

BACKGROUND: The mammalian epididymis is a specialized duct system that serves a critical role in sperm maturation and storage. Its distinctive, highly coiled tissue morphology provides a unique opportunity to investigate the link between form and function in reproductive biology. Although recent genetic studies have identified key genes and signaling pathways involved in the development and physiological functions of the epididymis, there has been limited discussion about the underlying dynamic and mechanical processes that govern these phenomena. AIMS: In this review, we aim to address this gap by examining two key aspects of the epididymis across its developmental and physiological phases. RESULTS AND DISCUSSION: First, we discuss how the complex morphology of the Wolffian/epididymal duct emerges through collective cell dynamics, including duct elongation, cell proliferation, and arrangement during embryonic development. Second, we highlight dynamic aspects of luminal fluid flow in the epididymis, essential for regulating the microenvironment for sperm maturation and motility, and discuss how this phenomenon emerges and interplays with epididymal epithelial cells. CONCLUSION: This review not only aims to summarize current knowledge but also to provide a starting point for further exploration of mechanobiological aspects related to the cellular and extracellular fluid dynamics in the epididymis.

Stretching the limits of extracellular signal-related kinase (ERK) signaling - Cell mechanosensing to ERK activation.

Extracellular signal-regulated kinase (ERK) has been recognized as a critical regulator in various physiological and pathological processes. Extensive research has elucidated the signaling mechanisms governing ERK activation via biochemical regulations with upstream molecules, particularly receptor tyrosine kinases (RTKs). However, recent advances have highlighted the role of mechanical forces in activating the RTK-ERK signaling pathways, thereby opening new avenues of research into mechanochemical interplay in multicellular tissues. Here, we review the force-induced ERK activation in cells and propose possible mechanosensing mechanisms underlying the mechanoresponsive ERK activation. We conclude that mechanical forces are not merely passive factors shaping cells and tissues but also active regulators of cellular signaling pathways controlling collective cell behaviors.

Apoptotic extracellular vesicle formation via local phosphatidylserine exposure drives efficient cell extrusion.

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. However, the underlying mechanisms of cell delamination from the cell layer are unclear. Here, we report a conserved execution mechanism of apoptotic cell extrusion. We found extracellular vesicle (EV) formation in extruding mammalian and Drosophila cells at a site opposite to the extrusion direction. Lipid-scramblase-mediated local exposure of phosphatidylserine is responsible for EV formation and is crucial for executing cell extrusion. Inhibition of this process disrupts prompt cell delamination and tissue homeostasis. Although the EV has hallmarks of an apoptotic body, its formation is governed by the mechanism of microvesicle formation. Experimental and mathematical modeling analysis illustrated that EV formation promotes neighboring cells' invasion. This study showed that membrane dynamics play a crucial role in cell exit by connecting the actions of the extruding cell and neighboring cells.

Mathematical study of the role of Delta/Notch lateral inhibition during primary branching of Drosophila trachea development.

A wide range of cellular developmental processes employ intercellular signaling via the Delta/Notch lateral inhibitory pathway to achieve stable spatial patterning. Recent genetic experiments have shown the importance of Delta/Notch lateral inhibition for regulating the number of tip cells in the tracheal primary branching of Drosophila. To examine the role of Delta/Notch regulation in the tip-cell selection, we analyzed a mathematical model of a simple lateral inhibitory system having input signals. Mathematical and numerical analyses revealed that the lateral inhibition did not amplify the signal difference between neighboring cells over the parameter ranges in which the spatial pattern of tip selection was realized. We also show that the number of tip cells becomes less affected by a fluctuation of the input gradient signal as the lateral inhibition becomes stronger. In addition, we demonstrate that the lateral inhibitory regulation enhances the robustness of the tip-cell selection compared with a system regulated by self-inhibition, an alternative means of inhibitory regulation. These results suggest that the lateral inhibition promotes the robustness of tip-cell selection in the tracheal development of Drosophila.

C-type Natriuretic Peptide-induced PKA Activation Promotes Endochondral Bone Formation in Hypertrophic Chondrocytes.

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.

Functional visualization of NK cell-mediated killing of metastatic single tumor cells.

Natural killer (NK) cells lyse invading tumor cells to limit metastatic growth in the lung, but how some cancers evade this host protective mechanism to establish a growing lesion is unknown. Here, we have combined ultra-sensitive bioluminescence imaging with intravital two-photon microscopy involving genetically encoded biosensors to examine this question. NK cells eliminated disseminated tumor cells from the lung within 24 hr of arrival, but not thereafter. Intravital dynamic imaging revealed that 50% of NK-tumor cell encounters lead to tumor cell death in the first 4 hr after tumor cell arrival, but after 24 hr of arrival, nearly 100% of the interactions result in the survival of the tumor cell. During this 24-hr period, the probability of ERK activation in NK cells upon encountering the tumor cells was decreased from 68% to 8%, which correlated with the loss of the activating ligand CD155/PVR/Necl5 from the tumor cell surface. Thus, by quantitatively visualizing, the NK-tumor cell interaction at the early stage of metastasis, we have revealed the crucial parameters of NK cell immune surveillance in the lung.

A feedback loop between lamellipodial extension and HGF-ERK signaling specifies leader cells during collective cell migration.

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.

A visualization system for erectile vascular dynamics.

Erection is an essential process which requires the male penis for copulation. This copulatory process depends on the vascular dynamic regulation of the penis. The corpus cavernosum (CC) in the upper (dorsal) part of the penis plays a major role in regulating blood flow inside the penis. When the CC is filled with blood, the sinusoids, including micro-vessels, dilate during erection. The CC is an androgen-dependent organ, and various genital abnormalities including erectile dysfunction (ED) are widely known. Previous studies have shown that androgen deprivation by castration results in significantly decreased smooth muscles of the CC. Experimental works in erectile biology have previously measured intracavernosal penile pressure and mechanical tension. Such reports analyze limited features without assessing the dynamic aspects of the erectile process. In the current study, we established a novel explant system enabling direct visual imaging of the sinusoidal lumen to evaluate the dynamic movement of the cavernous space. To analyze the alternation of sinusoidal spaces, micro-dissected CC explants by patent blue dye injection were incubated and examined for their structural alternations during relaxation/contraction. The dynamic process of relaxation/contraction was analyzed with various external factors administered to the CC. The system enabled the imaging of relaxation/contraction of the lumens of the sinusoids and the collagen-containing tissues. Histological analysis on the explant system also showed the relaxation/contraction. Thus, the system mimics the regulatory process of dynamic relaxation/contraction in the erectile response. The current system also enabled evaluating the erectile pathophysiology. In the current study, the lumen of sinusoids relaxed/contracted in castrated mice similarly with normal mice. These results suggested that the dynamic erectile relaxation/contraction process was similarly retained in castrated mice. However, the system also revealed decreased duration time of erection in castrated mice. The current study is expected to promote further understanding of the pathophysiology of ED, which will be useful for new treatments in the future. Hence, the current system provides unique information to investigate the novel regulations of erectile function, which can provide tools for analyzing the pathology of ED.