descSPIM: an affordable and easy-to-build light-sheet microscope optimized for tissue clearing techniques.

Despite widespread adoption of tissue clearing techniques in recent years, poor access to suitable light-sheet fluorescence microscopes remains a major obstacle for biomedical end-users. Here, we present descSPIM (desktop-equipped SPIM for cleared specimens), a low-cost ($20,000-50,000), low-expertise (one-day installation by a non-expert), yet practical do-it-yourself light-sheet microscope as a solution for this bottleneck. Even the most fundamental configuration of descSPIM enables multi-color imaging of whole mouse brains and a cancer cell line-derived xenograft tumor mass for the visualization of neurocircuitry, assessment of drug distribution, and pathological examination by false-colored hematoxylin and eosin staining in a three-dimensional manner. Academically open-sourced ( https://github.com/dbsb-juntendo/descSPIM ), descSPIM allows routine three-dimensional imaging of cleared samples in minutes. Thus, the dissemination of descSPIM will accelerate biomedical discoveries driven by tissue clearing technologies.

Endothelial cells regulate alveolar morphogenesis by constructing basement membranes acting as a scaffold for myofibroblasts.

Alveologenesis is a spatially coordinated morphogenetic event, during which alveolar myofibroblasts surround the terminal sacs constructed by epithelial cells and endothelial cells (ECs), then contract to form secondary septa to generate alveoli in the lungs. Recent studies have demonstrated the important role of alveolar ECs in this morphogenetic event. However, the mechanisms underlying EC-mediated alveologenesis remain unknown. Herein, we show that ECs regulate alveologenesis by constructing basement membranes (BMs) acting as a scaffold for myofibroblasts to induce septa formation through activating mechanical signaling. Rap1, a small GTPase of the Ras superfamily, is known to stimulate integrin-mediated cell adhesions. EC-specific Rap1-deficient (Rap1iECKO) mice exhibit impaired septa formation and hypo-alveolarization due to the decreased mechanical signaling in myofibroblasts. In Rap1iECKO mice, ECs fail to stimulate integrin ß1 to recruit Collagen type IV (Col-4) into BMs required for myofibroblast-mediated septa formation. Consistently, EC-specific integrin ß1-deficient mice show hypo-alveolarization, defective mechanical signaling in myofibroblasts, and disorganized BMs. These data demonstrate that alveolar ECs promote integrin ß1-mediated Col-4 recruitment in a Rap1-dependent manner, thereby constructing BMs acting as a scaffold for myofibroblasts to induce mechanical signal-mediated alveologenesis. Thus, this study unveils a mechanism of organ morphogenesis mediated by ECs through intrinsic functions.

Rap1 small GTPase is essential for maintaining pulmonary endothelial barrier function in mice.

Vascular permeability is dynamically but tightly controlled by vascular endothelial (VE)-cadherin-mediated endothelial cell-cell junctions to maintain homeostasis. Thus, impairments of VE-cadherin-mediated cell adhesions lead to hyperpermeability, promoting the development and progression of various disease processes. Notably, the lungs are a highly vulnerable organ wherein pulmonary inflammation and infection result in vascular leakage. Herein, we showed that Rap1, a small GTPase, plays an essential role for maintaining pulmonary endothelial barrier function in mice. Endothelial cell-specific Rap1a/Rap1b double knockout mice exhibited severe pulmonary edema. They also showed vascular leakage in the hearts, but not in the brains. En face analyses of the pulmonary arteries and 3D-immunofluorescence analyses of the lungs revealed that Rap1 potentiates VE-cadherin-mediated endothelial cell-cell junctions through dynamic actin cytoskeleton reorganization. Rap1 inhibits formation of cytoplasmic actin bundles perpendicularly binding VE-cadherin adhesions through inhibition of a Rho-ROCK pathway-induced activation of cytoplasmic nonmuscle myosin II (NM-II). Simultaneously, Rap1 induces junctional NM-II activation to create circumferential actin bundles, which anchor and stabilize VE-cadherin at cell-cell junctions. We also showed that the mice carrying only one allele of either Rap1a or Rap1b out of the two Rap1 genes are more vulnerable to lipopolysaccharide (LPS)-induced pulmonary vascular leakage than wild-type mice, while activation of Rap1 by administration of 007, an activator for Epac, attenuates LPS-induced increase in pulmonary endothelial permeability in wild-type mice. Thus, we demonstrate that Rap1 plays an essential role for maintaining pulmonary endothelial barrier functions under physiological conditions and provides protection against inflammation-induced pulmonary vascular leakage.

Coronary artery established through amniote evolution.

Coronary arteries are a critical part of the vascular system and provide nourishment to the heart. In humans, even minor defects in coronary arteries can be lethal, emphasizing their importance for survival. However, some teleosts survive without coronary arteries, suggesting that there may have been some evolutionary changes in the morphology and function of coronary arteries in the tetrapod lineage. Here, we propose that the true ventricular coronary arteries were newly established during amniote evolution through remodeling of the ancestral coronary vasculature. In mouse (Mus musculus) and Japanese quail (Coturnix japonica) embryos, the coronary arteries unique to amniotes are established by the reconstitution of transient vascular plexuses: aortic subepicardial vessels (ASVs) in the outflow tract and the primitive coronary plexus on the ventricle. In contrast, amphibians (Hyla japonica, Lithobates catesbeianus, Xenopus laevis, and Cynops pyrrhogaster) retain the ASV-like vasculature as truncal coronary arteries throughout their lives and have no primitive coronary plexus. The anatomy and development of zebrafish (Danio rerio) and chondrichthyans suggest that their hypobranchial arteries are ASV-like structures serving as the root of the coronary vasculature throughout their lives. Thus, the ventricular coronary artery of adult amniotes is a novel structure that has acquired a new remodeling process, while the ASVs, which occur transiently during embryonic development, are remnants of the ancestral coronary vessels. This evolutionary change may be related to the modification of branchial arteries, indicating considerable morphological changes underlying the physiological transition during amniote evolution.

Coronary arteries are tasked with supplying the heart with oxygenated blood and nutrients. Any blockage or developmental problem in these blood vessels can have severe and sometimes lethal consequences. Due to their importance for health, researchers have extensively studied how coronary arteries form in humans and mice; a more limited range of studies have also looked at their equivalent in zebrafish. However, little is known about these structures develop in animals such as birds, amphibians, or other groups of fish. This makes it difficult to retrace the evolutionary processes that have given rise to the coronary arteries we are familiar with in mammals. To address this knowledge gap, Mizukami et al. set out to compare blood vessel development around the heart of mammals, birds, amphibians, and fish. To do this, they performed detailed anatomical studies of blood vessel structure at different stages of development in mice as well as quail, frogs and newts, zebrafish and sharks. In both mice and quail, small arterial subepicardial vessels (or ASVs) emerged early in development around the heart; these subsequently reorganised and remodelled themselves to give rise to the 'true' coronary arteries characteristic of the mature heart. Frogs and newts also developed similar ASV-like structures; however, unlike their mammalian and bird equivalents, these vessels did not reorganise, instead being retained into adulthood. In fish, blood vessel development resembled that of amphibians, suggesting that the coronary artery-like structures seen in some fish are an 'ancestral' form of ASVs, rather than the equivalent of the mature coronary arteries in mammals and birds. This work sheds light on the evolutionary processes shaping essential structures in the heart. In the future, Mizukami et al. hope that this knowledge will help develop a greater range of experimental animal models for studying heart disease and potential treatments.

Novel regulatory mechanisms underlying angiogenesis during wound healing revealed by fluorescence-based live-imaging in zebrafish.

Angiogenesis is a dynamic morphogenetic process that refers to the growth of new blood vessels from the pre-existing vessels and is critical for tissue repair during wound healing. In adult normal tissues, quiescent endothelial cells and pericytes maintain vascular integrity, whereas angiogenesis is immediately induced upon tissue injury, thereby forming neovascular networks to maintain homeostasis. However, impaired angiogenesis results in development of chronic and non-healing wounds in various diseases such as diabetes and peripheral artery diseases. Zebrafish are a vertebrate model organism widely used for studying many medical and life science fields. Indeed, the molecular and cellular mechanisms underlying regulation of wound angiogenesis have recently been studied by performing fluorescence-based live-imaging of adult zebrafish. In this review, we describe how endothelial cells and pericytes establish neovascular networks during wound angiogenesis and also introduce a novel role of blood flow-driven intraluminal pressure in regulating angiogenesis during wound healing.

Blood Flow Regulates Glomerular Capillary Formation in Zebrafish Pronephros.

Background: The renal glomerulus is a tuft of capillaries in Bowman's capsule and functions as a blood-filtration unit in the kidney. The unique glomerular capillary tuft structure is relatively conserved through vertebrate species. However, the morphogenetic mechanism governing glomerular capillary tuft formation remains elusive. Methods: To clarify how glomerular capillaries develop, we analyzed glomerular capillary formation in the zebrafish pronephros by exploiting fluorescence-based bio-imaging technology. Results: During glomerular capillary formation in the zebrafish pronephros, endothelial cells initially sprouted from the dorsal aorta and formed the capillaries surrounding the bilateral glomerular primordia in response to podocyte progenitor-derived vascular endothelial growth factor-A. After formation, blood flow immediately occurred in the glomerular primordia-associated capillaries, while in the absence of blood flow, they were transformed into sheet-like structures enveloping the glomerular primordia. Subsequently, blood flow induced formation of Bowman's space at the lateral sides of the bilateral glomerular primordia. Concomitantly, podocyte progenitors enveloped their surrounding capillaries while moving toward and coalescing at the midline. These capillaries then underwent extensive expansion and remodeling to establish a functional glomerular capillary tuft. However, stopping blood flow inhibited the remodeling of bilateral glomerular primordia, which therefore remained unvascularized but covered by the vascular sheets. Conclusions: We delineated the morphogenetic processes governing glomerular capillary tuft formation in the zebrafish pronephros and demonstrated crucial roles of blood flow in its formation. Blood flow maintains tubular structures of the capillaries surrounding the glomerular primordia and promotes glomerular incorporation of these vessels by inducing the remodeling of glomerular primordia.

KCNJ8/ABCC9-containing K-ATP channel modulates brain vascular smooth muscle development and neurovascular coupling.

Loss- or gain-of-function mutations in ATP-sensitive potassium channel (K-ATP)-encoding genes, KCNJ8 and ABCC9, cause human central nervous system disorders with unknown pathogenesis. Here, using mice, zebrafish, and cell culture models, we investigated cellular and molecular causes of brain dysfunctions derived from altered K-ATP channel function. We show that genetic/chemical inhibition or activation of KCNJ8/ABCC9-containing K-ATP channel function leads to brain-selective suppression or promotion of arterial/arteriolar vascular smooth muscle cell (VSMC) differentiation, respectively. We further show that brain VSMCs develop from KCNJ8/ABCC9-containing K-ATP channel-expressing mural cell progenitor and that K-ATP channel cell autonomously regulates VSMC differentiation through modulation of intracellular Ca2+ oscillation via voltage-dependent calcium channels. Consistent with defective VSMC development, Kcnj8 knockout mice showed deficiency in vasoconstrictive capacity and neuronal-evoked vasodilation leading to local hyperemia. Our results demonstrate a role for KCNJ8/ABCC9-containing K-ATP channels in the differentiation of brain VSMC, which in turn is necessary for fine-tuning of cerebral blood flow.

Mechanical loading of intraluminal pressure mediates wound angiogenesis by regulating the TOCA family of F-BAR proteins.

Angiogenesis is regulated in coordinated fashion by chemical and mechanical cues acting on endothelial cells (ECs). However, the mechanobiological mechanisms of angiogenesis remain unknown. Herein, we demonstrate a crucial role of blood flow-driven intraluminal pressure (IP) in regulating wound angiogenesis. During wound angiogenesis, blood flow-driven IP loading inhibits elongation of injured blood vessels located at sites upstream from blood flow, while downstream injured vessels actively elongate. In downstream injured vessels, F-BAR proteins, TOCA1 and CIP4, localize at leading edge of ECs to promote N-WASP-dependent Arp2/3 complex-mediated actin polymerization and front-rear polarization for vessel elongation. In contrast, IP loading expands upstream injured vessels and stretches ECs, preventing leading edge localization of TOCA1 and CIP4 to inhibit directed EC migration and vessel elongation. These data indicate that the TOCA family of F-BAR proteins are key actin regulatory proteins required for directed EC migration and sense mechanical cell stretching to regulate wound angiogenesis.

Proper migration of lymphatic endothelial cells requires survival and guidance cues from arterial mural cells.

The migration of lymphatic endothelial cells (LECs) is key for the development of the complex and vast lymphatic vascular network that pervades most tissues in an organism. In zebrafish, arterial intersegmental vessels together with chemokines have been shown to promote lymphatic cell migration from the horizontal myoseptum (HM). We observed that emergence of mural cells around the intersegmental arteries coincides with lymphatic departure from HM which raised the possibility that arterial mural cells promote LEC migration. Our live imaging and cell ablation experiments revealed that LECs migrate slower and fail to establish the lymphatic vascular network in the absence of arterial mural cells. We determined that mural cells are a source for the C-X-C motif chemokine 12 (Cxcl12a and Cxcl12b), vascular endothelial growth factor C (Vegfc) and collagen and calcium-binding EGF domain-containing protein 1 (Ccbe1). We showed that chemokine and growth factor signalling function cooperatively to induce robust LEC migration. Specifically, Vegfc-Vegfr3 signalling, but not chemokines, induces extracellular signal-regulated kinase (ERK) activation in LECs, and has an additional pro-survival role in LECs during the migration. Together, the identification of mural cells as a source for signals that guide LEC migration and survival will be important in the future design for rebuilding lymphatic vessels in disease contexts.

Lysine demethylase 2B regulates angiogenesis via Jumonji C dependent suppression of angiogenic transcription factors.

Vascular endothelial growth factor (VEGF) signaling plays a central role in vascular development and maintenance of vascular homeostasis. In endothelial cells (ECs), VEGF activates the gene expression of angiogenic transcription factors (TFs), followed by induction of downstream angiogenic responsive genes. Recent findings support that histone modification dynamics contribute to the transcriptional control of genes that are important for EC functions. Lysine demethylase 2B (KDM2B) demethylates histone H3K4me3 and H3K36me2/3 and mediates the monoubiquitination of histone H2AK119. KDM2B functions as a transcriptional repressor in somatic cell reprogramming and tumor development. However, the role of KDM2B in VEGF signaling remains to be elucidated. Here, we show that KDM2B knockdown enhances VEGF-induced angiogenesis in cultured human ECs via increased migration and proliferation. In contrast, ectopic expression of KDM2B inhibits angiogenesis. The function of KDM2B may depend on its catalytic Jumonji C domain. Genome-wide analysis further reveals that KDM2B selectively controls the transcription of VEGF-induced angiogenic TFs that are associated with increased H3K4me3/H3K36me3 and decreased H2AK119ub. These findings suggest an essential role of KDM2B in VEGF signaling in ECs. As dysregulation of VEGF signaling in ECs is involved in various diseases, including cancer, KDM2B may be a potential therapeutic target in VEGF-mediated vasculopathic diseases.

Protocol for whole-mount X-gal staining combined with tissue clearing in embryo and adult mouse using CUBIC.

Here we describe an optimized protocol for X-gal staining of tissue clearing embryo and adult mouse using CUBIC. The activity of LacZ knock-in reflecting endogenous expression of genes of interest in the whole body can be visualized by X-gal staining. This protocol is suitable for examining the developmental stage-specific expression of genes of interest spatially and temporally. For complete details on the use and execution of this protocol, please refer to Watanabe-Takano et al. (2021).

An ATX-LPA6-Gα13-ROCK axis shapes and maintains caudal vein plexus in zebrafish.

Lysophosphatidic acid (LPA) is a potential regulator of vascular formation derived from blood. In this study, we utilized zebrafish as a model organism to monitor the blood vessel formation in detail. Zebrafish mutant of ATX, an LPA-producing enzyme, had a defect in the caudal vein plexus (CVP). Pharmacological inhibition of ATX resulted in a fusion of the delicate vessels in the CVP to form large sac-like vessels. Mutant embryos of LPA6 receptor and downstream Gα13 showed the same phenotype. Administration of OMPT, a stable LPA-analog, induced rapid CVP constriction, which was attenuated significantly in the LPA6 mutant. We also found that blood flow-induced CVP formation was dependent on ATX. The present study demonstrated that the ATX-LPA6 axis acts cooperatively with blood flow and contributes to the formation and maintenance of the CVP by generating contractive force in endothelial cells.

Zebrafish Vascular Mural Cell Biology: Recent Advances, Development, and Functions.

Recruitment of mural cells to the vascular wall is essential for forming the vasculature as well as maintaining proper vascular functions. In recent years, zebrafish genetic tools for mural cell biology have improved substantially. Fluorescently labeled zebrafish mural cell reporter lines enable us to study, with higher spatiotemporal resolution than ever, the processes of mural cell development from their progenitors. Furthermore, recent phenotypic analysis of platelet-derived growth factor beta mutant zebrafish revealed well-conserved organotypic mural cell development and functions in vertebrates with the unique features of zebrafish. However, comprehensive reviews of zebrafish mural cells are lacking. Therefore, herein, we highlight recent advances in zebrafish mural cell tools. We also summarize the fundamental features of zebrafish mural cell development, especially at early stages, and functions.

Rap1 Small GTPase Regulates Vascular Endothelial-Cadherin-Mediated Endothelial Cell-Cell Junctions and Vascular Permeability.

The vascular permeability of the endothelium is finely controlled by vascular endothelial (VE)-cadherin-mediated endothelial cell-cell junctions. In the majority of normal adult tissues, endothelial cells in blood vessels maintain vascular permeability at a relatively low level, while in response to inflammation, they limit vascular barrier function to induce plasma leakage and extravasation of immune cells as a defense mechanism. Thus, the dynamic but also simultaneously tight regulation of vascular permeability by endothelial cells is responsible for maintaining homeostasis and, as such, impairments of its underlying mechanisms result in hyperpermeability, leading to the development and progression of various diseases including coronavirus disease 2019 (COVID-19), a newly emerging infectious disease. Recently, increasing numbers of studies have been unveiling the important role of Rap1, a small guanosine 5'-triphosphatase (GTPase) belonging to the Ras superfamily, in the regulation of vascular permeability. Rap1 enhances VE-cadherin-mediated endothelial cell-cell junctions to potentiate vascular barrier functions via dynamic reorganization of the actin cytoskeleton. Importantly, Rap1 signaling activation reportedly improves vascular barrier function in animal models of various diseases associated with vascular hyperpermeability, suggesting that Rap1 might be an ideal target for drugs intended to prevent vascular barrier dysfunction. Here, we describe recent progress in understanding the mechanisms by which Rap1 potentiates VE-cadherin-mediated endothelial cell-cell adhesions and vascular barrier function. We also discuss how alterations in Rap1 signaling are related to vascular barrier dysfunction in diseases such as acute pulmonary injury and malignancies. In addition, we examine the possibility of Rap1 signaling as a target of drugs for treating diseases associated with vascular hyperpermeability.

Mechanical load regulates bone growth via periosteal Osteocrin.

Mechanical stimuli including loading after birth promote bone growth. However, little is known about how mechanical force triggers biochemical signals to regulate bone growth. Here, we identified a periosteal-osteoblast-derived secretory peptide, Osteocrin (OSTN), as a mechanotransducer involved in load-induced long bone growth. OSTN produced by periosteal osteoblasts regulates growth plate growth by enhancing C-type natriuretic peptide (CNP)-dependent proliferation and maturation of chondrocytes, leading to elongation of long bones. Additionally, OSTN cooperates with CNP to regulate bone formation. CNP stimulates osteogenic differentiation of periosteal osteoprogenitors to induce bone formation. OSTN binds to natriuretic peptide receptor 3 (NPR3) in periosteal osteoprogenitors, thereby preventing NPR3-mediated clearance of CNP and consequently facilitating CNP-signal-mediated bone growth. Importantly, physiological loading induces Ostn expression in periosteal osteoblasts by suppressing Forkhead box protein O1 (FoxO1) transcription factor. Thus, this study reveals a crucial role of OSTN as a mechanotransducer converting mechanical loading to CNP-dependent bone formation.

Conserved and context-dependent roles for pdgfrb signaling during zebrafish vascular mural cell development.

Platelet derived growth factor beta and its receptor, Pdgfrb, play essential roles in the development of vascular mural cells, including pericytes and vascular smooth muscle cells. To determine if this role was conserved in zebrafish, we analyzed pdgfb and pdgfrb mutant lines. Similar to mouse, pdgfb and pdgfrb mutant zebrafish lack brain pericytes and exhibit anatomically selective loss of vascular smooth muscle coverage. Despite these defects, pdgfrb mutant zebrafish did not otherwise exhibit circulatory defects at larval stages. However, beginning at juvenile stages, we observed severe cranial hemorrhage and vessel dilation associated with loss of pericytes and vascular smooth muscle cells in pdgfrb mutants. Similar to mouse, pdgfrb mutant zebrafish also displayed structural defects in the glomerulus, but normal development of hepatic stellate cells. We also noted defective mural cell investment on coronary vessels with concomitant defects in their development. Together, our studies support a conserved requirement for Pdgfrb signaling in mural cells. In addition, these zebrafish mutants provide an important model for definitive investigation of mural cells during early embryonic stages without confounding secondary effects from circulatory defects.

A New Model for Specific Visualization of Skin Graft Neoangiogenesis Using Flt1-tdsRed BAC Transgenic Mice.

BACKGROUND: Neovascularization plays a critical role in skin graft survival. Up to date, the lack of specificity to solely track the newly sprouting blood vessels has remained a limiting factor in skin graft transplantation models. Therefore, the authors developed a new model by using Flt1-tdsRed BAC transgenic mice. Flt1 is a vascular endothelial growth factor receptor expressed by sprouting endothelial cells mediating neoangiogenesis. The authors determined whether this model reliably visualizes neovascularization by quantifying tdsRed fluorescence in the graft over 14 days. METHODS: Cross-transplantation of two full-thickness 1 × 1-cm dorsal skin grafts was performed between 6- to 8-week-old male Flt1 mice and KSN/Slc nude mice (n = 5). The percentage of graft area occupied by tdsRed fluorescence in the central and lateral areas of the graft on days 3, 5, 9, and 14 was determined using confocal-laser scanning microscopy. RESULTS: Flt1+ endothelial cells migrating from the transgenic wound bed into the nude graft were first visible in the reticular dermis of the graft center on day 3 (0.5 ± 0.1; p < 0.05). Peak neovascularization was observed on day 9 in the lateral and central parts, increasing by 2- to 4-fold (4.6 ± 0.8 and 4.2 ± 0.9; p < 0.001). Notably, some limited neoangiogenesis was displayed within the Flt grafts on nude mice, particularly in the center. No neovascularization was observed from the wound margins. CONCLUSION: The ability of the Flt1-tdsRed transgenic mouse model to efficiently identify the origin of the skin-graft vasculature and visualize graft neovascularization over time suggests its potential utility for developing techniques that promote graft neovascularization.

Protocol for analysis of integrin-mediated cell adhesion of lateral plate mesoderm cells isolated from zebrafish embryos.

Lateral plate mesoderm (LPM) cells differentiate into various cell types including endothelial and hematopoietic cells. In zebrafish embryos, LPM cells migrate toward the midline along the ventral surfaces of somites during which their cell fate specification depends upon efficient integrin-mediated cell adhesion and migration. Herein, we present a protocol for analysis of integrin-mediated cell adhesion of LPM cells isolated from zebrafish embryos. This allows the study of the molecular mechanisms underlying integrin activation required for LPM cell fate specification. For complete details on the use and execution of this protocol, please refer to Rho et al. (2019).

RhoJ integrates attractive and repulsive cues in directional migration of endothelial cells.

During angiogenesis, VEGF acts as an attractive cue for endothelial cells (ECs), while Sema3E mediates repulsive cues. Here, we show that the small GTPase RhoJ integrates these opposing signals in directional EC migration. In the GTP-bound state, RhoJ interacts with the cytoplasmic domain of PlexinD1. Upon Sema3E stimulation, RhoJ released from PlexinD1 induces cell contraction. PlexinD1-bound RhoJ further facilitates Sema3E-induced PlexinD1-VEGFR2 association, VEGFR2 transphosphorylation at Y1214, and p38 MAPK activation, leading to reverse EC migration. Upon VEGF stimulation, RhoJ is required for the formation of the holoreceptor complex comprising VEGFR2, PlexinD1, and neuropilin-1, thereby preventing degradation of internalized VEGFR2, prolonging downstream signal transductions via PLCγ, Erk, and Akt, and promoting forward EC migration. After conversion to the GDP-bound state, RhoJ shifts from PlexinD1 to VEGFR2, which then terminates the VEGFR2 signals. RhoJ deficiency in ECs efficiently suppressed aberrant angiogenesis in ischemic retina. These findings suggest that distinct Rho GTPases may act as context-dependent integrators of chemotactic cues in directional cell migration and may serve as candidate therapeutic targets to manipulate cell motility in disease or tissue regeneration.