Lymphoangiocrine signals promote cardiac growth and repair.

Recent studies have suggested that lymphatics help to restore heart function after cardiac injury1-6. Here we report that lymphatics promote cardiac growth, repair and cardioprotection in mice. We show that a lymphoangiocrine signal produced by lymphatic endothelial cells (LECs) controls the proliferation and survival of cardiomyocytes during heart development, improves neonatal cardiac regeneration and is cardioprotective after myocardial infarction. Embryos that lack LECs develop smaller hearts as a consequence of reduced cardiomyocyte proliferation and increased cardiomyocyte apoptosis. Culturing primary mouse cardiomyocytes in LEC-conditioned medium increases cardiomyocyte proliferation and survival, which indicates that LECs produce lymphoangiocrine signals that control cardiomyocyte homeostasis. Characterization of the LEC secretome identified the extracellular protein reelin (RELN) as a key component of this process. Moreover, we report that LEC-specific Reln-null mouse embryos develop smaller hearts, that RELN is required for efficient heart repair and function after neonatal myocardial infarction, and that cardiac delivery of RELN using collagen patches improves heart function in adult mice after myocardial infarction by a cardioprotective effect. These results highlight a lymphoangiocrine role of LECs during cardiac development and injury response, and identify RELN as an important mediator of this function.

Characterization of Atherosclerotic Mice Reveals a Sex-Dependent Susceptibility to Plaque Calcification but No Major Changes in the Lymphatics in the Arterial Wall.

Lymphatics participate in reverse cholesterol transport, and their presence in the arterial wall of the great vessels and prior experimental results suggest their possible role in the development of atherosclerosis. The aim of this study was to characterize the lymphatic vasculature of the arterial wall in atherosclerosis. Tissue sections and tissue-cleared aortas of wild-type mice unveiled significant differences in the density of the arterial lymphatic network throughout the arterial tree. Male and female Ldlr-/- and ApoE-/- mice on a Western diet showed sex-dependent differences in plaque formation and calcification. Female mice on a Western diet developed more calcification of atherosclerotic plaques than males. The lymphatic vessels within the aortic wall of these mice showed no major changes regarding the number of lymphatic junctions and end points or the lymphatic area. However, female mice on a Western diet showed moderate dilation of lymphatic vessels in the abdominal aorta and exhibited indications of increased peripheral lymphatic function, findings that require further studies to understand the role of lymphatics in the arterial wall during the development of atherosclerosis.

Visualization of Organ-Specific Lymphatic Growth: An Efficient Approach to Labeling Molecular Markers in Cleared Tissues.

Organ-specific lymphatics are essential for the maintenance of healthy organ function and lymphatic dysfunction can lead to the development of various diseases. However, the precise role of those lymphatic structures remains unknown, mainly due to inefficient visualization techniques. Here, we present an efficient approach to visualizing organ-specific lymphatic growth. We used a modified CUBIC protocol to clear mouse organs and combined it with whole-mount immunostaining to visualize lymphatic structures. We acquired images using upright, stereo and confocal microscopy and quantified them with AngioTool, a tool for the quantification of vascular networks. Using our approach, we then characterized the organ-specific lymphatic vasculature of the Flt4kd/+ mouse model, showing symptoms of lymphatic dysfunction. Our approach enabled us to visualize the lymphatic vasculature of organs and to analyze and quantify structural changes. We detected morphologically altered lymphatic vessels in all investigated organs of Flt4kd/+ mice, including the lungs, small intestine, heart and uterus, but no lymphatic structures in the skin. Quantifications showed that these mice have fewer and dilated lymphatic vessels in the small intestine and the lungs. Our results demonstrate that our approach can be used to investigate the importance of organ-specific lymphatics under both physiological and pathophysiological conditions.

Foliate Lymphoid Aggregates as Novel Forms of Serous Lymphocyte Entry Sites of Peritoneal B Cells and High-Grade B Cell Lymphomas.

The cellular homeostasis of lymphoid tissues is determined by the continuous interactions of mobile hematopoietic cells within specialized microenvironments created by sessile stromal cells. In contrast to the lymph nodes and mucosal lymphoid tissues with well-defined entry and exit routes, the movement of leukocytes in the peritoneal cavity is largely unknown. In this study, we report that, in addition to the omental milky spots and fat-associated lymphoid clusters, in mice, the serous surface of the mesenteric adipose streaks contains lymphocyte-rich organoids comprised of a highly compacted leaf-like part connected to the adipose tissue that can also efficiently bind B cells and high-grade B cell lymphoma (diffuse large B cell lymphoma) cells. Denoted as foliate lymphoid aggregates (FLAgs), these structures show incomplete T/B segregation and a partially differentiated stromal architecture. LYVE-1-positive macrophages covering FLAgs efficiently bind i.p. injected normal B cells as well as different types of diffuse large B cell lymphoma cells. Within FLAgs, the lymphocytes compartmentalize according to their chemokine receptor pattern and subsequently migrate toward the mesenteric lymph nodes via the mesenteric lymphatic capillaries. The blood supply of FLAgs includes short vascular segments displaying peripheral lymph node addressin, and the extravasation of lymphocytes to the omental and mesenteric adipose tissues is partly mediated by L-selectin. The appearance of i.p. injected cells in mesenteric lymph nodes suggests that the mesentery-associated lymphatics may also collect leukocytes from the fat-associated lymphoid clusters and FLAgs, thus combining the mucosal and serous exit of mobile leukocytes and increasing the range of drainage sites for the peritoneal expansion of lymphoid malignancies.

Podoplanin-rich stromal networks induce dendritic cell motility via activation of the C-type lectin receptor CLEC-2.

To initiate adaptive immunity, dendritic cells (DCs) move from parenchymal tissues to lymphoid organs by migrating along stromal scaffolds that display the glycoprotein podoplanin (PDPN). PDPN is expressed by lymphatic endothelial and fibroblastic reticular cells and promotes blood-lymph separation during development by activating the C-type lectin receptor, CLEC-2, on platelets. Here, we describe a role for CLEC-2 in the morphodynamic behavior and motility of DCs. CLEC-2 deficiency in DCs impaired their entry into lymphatics and trafficking to and within lymph nodes, thereby reducing T cell priming. CLEC-2 engagement of PDPN was necessary for DCs to spread and migrate along stromal surfaces and sufficient to induce membrane protrusions. CLEC-2 activation triggered cell spreading via downregulation of RhoA activity and myosin light-chain phosphorylation and triggered F-actin-rich protrusions via Vav signaling and Rac1 activation. Thus, activation of CLEC-2 by PDPN rearranges the actin cytoskeleton in DCs to promote efficient motility along stromal surfaces.

Mechanosensation and Mechanotransduction by Lymphatic Endothelial Cells Act as Important Regulators of Lymphatic Development and Function.

Our understanding of the function and development of the lymphatic system is expanding rapidly due to the identification of specific molecular markers and the availability of novel genetic approaches. In connection, it has been demonstrated that mechanical forces contribute to the endothelial cell fate commitment and play a critical role in influencing lymphatic endothelial cell shape and alignment by promoting sprouting, development, maturation of the lymphatic network, and coordinating lymphatic valve morphogenesis and the stabilization of lymphatic valves. However, the mechanosignaling and mechanotransduction pathways involved in these processes are poorly understood. Here, we provide an overview of the impact of mechanical forces on lymphatics and summarize the current understanding of the molecular mechanisms involved in the mechanosensation and mechanotransduction by lymphatic endothelial cells. We also discuss how these mechanosensitive pathways affect endothelial cell fate and regulate lymphatic development and function. A better understanding of these mechanisms may provide a deeper insight into the pathophysiology of various diseases associated with impaired lymphatic function, such as lymphedema and may eventually lead to the discovery of novel therapeutic targets for these conditions.

Pyrophosphate therapy prevents trauma-induced calcification in the mouse model of neurogenic heterotopic ossification.

Trauma-induced calcification is the pathological consequence of complex injuries which often affect the central nervous system and other parts of the body simultaneously. We demonstrated by an animal model recapitulating the calcification of the above condition that adrenaline transmits the stress signal of brain injury to the calcifying tissues. We have also found that although the level of plasma pyrophosphate, the endogenous inhibitor of calcification, was normal in calcifying animals, it could not counteract the acute calcification. However, externally added pyrophosphate inhibited calcification even when it was administered after the complex injuries. Our finding suggests a potentially powerful clinical intervention of calcification triggered by polytrauma injuries which has no effective treatment.

The secreted lymphangiogenic factor CCBE1 is essential for fetal liver erythropoiesis.

The secreted protein CCBE1 is required for lymphatic vessel growth in fish and mice, and mutations in the CCBE1 gene cause Hennekam syndrome, a primary human lymphedema. Here we show that loss of CCBE1 also confers severe anemia in midgestation mouse embryos due to defective definitive erythropoiesis. Fetal liver erythroid precursors of Ccbe1 null mice exhibit reduced proliferation and increased apoptosis. Colony-forming assays and hematopoietic reconstitution studies suggest that CCBE1 promotes fetal liver erythropoiesis cell nonautonomously. Consistent with these findings, Ccbe1(lacZ) reporter expression is not detected in hematopoietic cells and conditional deletion of Ccbe1 in hematopoietic cells does not confer anemia. The expression of the erythropoietic factors erythropoietin and stem cell factor is preserved in CCBE1 null embryos, but erythroblastic island (EBI) formation is reduced due to abnormal macrophage function. In contrast to the profound effects on fetal liver erythropoiesis, postnatal deletion of Ccbe1 does not confer anemia, even under conditions of erythropoietic stress, and EBI formation is normal in the bone marrow of adult CCBE1 knockout mice. Our findings reveal that CCBE1 plays an essential role in regulating the fetal liver erythropoietic environment and suggest that EBI formation is regulated differently in the fetal liver and bone marrow.

Phosphoinositide 3-OH kinase regulates integrin-dependent processes in neutrophils by signaling through its effector ARAP3.

ARAP3, a GTPase activating protein for Rho and Arf family GTPases, is one of many phosphoinositide 3-OH kinase (PI3K) effectors. In this study, we investigate the regulatory input of PI3K upstream of ARAP3 by analyzing neutrophils from an ARAP3 pleckstrin homology (PH) domain point mutation knock-in mouse (R302, 303A), in which ARAP3 is uncoupled from activation by PI3K. ARAP3 PH domain point mutant neutrophils are characterized by disturbed responses linked to stimulation by either integrin ligands or immobilized immune complexes. These cells exhibit increased ß2 integrin inside-out signaling (binding affinity and avidity), and our work suggests the disturbed responses to immobilized immune complexes are secondary to this. In vitro, neutrophil chemotaxis is affected in the mutant. In vivo, ARAP3 PH domain point mutant bone marrow chimeras exhibit reduced neutrophil recruitment to the peritoneum on induction of sterile peritonitis and also reduced inflammation in a model for rheumatoid arthritis. The current work suggests a dramatic regulatory input of PI3K into the regulation of ß2 integrin activity, and processes dependent on this, by signaling through its effector ARAP3.

CCBE1 regulates the development and prevents the age-dependent regression of meningeal lymphatics.

Recent studies have described the importance of lymphatics in numerous organ-specific physiological and pathological processes. The role of meningeal lymphatics in various neurological and cerebrovascular diseases has been suggested. It has also been shown that these structures develop postnatally and are altered by aging and that the vascular endothelial growth factor C (VEGFC)/ vascular endothelial growth factor receptor 3 (VEGFR3) signaling plays an essential role in the development and maintenance of them. However, the molecular mechanisms governing the development and maintenance of meningeal lymphatics are still poorly characterized. Recent in vitro cell culture-based experiments, and in vivo studies in zebrafish and mouse skin suggest that collagen and calcium binding EGF domains 1 (CCBE1) is involved in the processing of VEGFC. However, the organ-specific role of CCBE1 in developmental lymphangiogenesis and maintenance of lymphatics remains unclear. Here, we aimed to investigate the organ-specific functions of CCBE1 in developmental lymphangiogenesis and maintenance of meningeal lymphatics during aging. We demonstrate that inducible deletion of CCBE1 leads to impaired postnatal development of the meningeal lymphatics and decreased macromolecule drainage to deep cervical lymph nodes. The structural integrity and density of meningeal lymphatics are gradually altered during aging. Furthermore, the meningeal lymphatic structures in adults showed regression after inducible CCBE1 deletion. Collectively, our results indicate the importance of CCBE1-dependent mechanisms not only in the development, but also in the prevention of the age-related regression of meningeal lymphatics. Therefore, targeting CCBE1 may be a good therapeutic strategy to prevent age-related degeneration of meningeal lymphatics.

Lymphatic-Dependent Modulation of the Sensitization and Elicitation Phases of Contact Hypersensitivity.

Allergic contact dermatitis is a common inflammatory skin disease comprising 2 phases. During sensitization, immune cells are activated by exposure to various allergens, whereas repeated antigen exposure induces local inflammation during elicitation. In this study, we utilized mouse models lacking lymphatics in different skin regions to characterize the role of lymphatics separately in the 2 phases, using contact hypersensitivity as a model of human allergic inflammatory skin diseases. Lymphatic-deficient mice exhibited no major difference to single antigen exposure compared to controls. However, mice lacking lymphatics in both phases displayed reduced inflammation after repeated antigen exposure. Similarly, diminished immune response was observed in mice lacking lymphatics only in sensitization, whereas the absence of lymphatics only in the elicitation phase resulted in a more pronounced inflammatory immune response. This exaggerated inflammation is driven by neutrophils impacting regulatory T cell number. Collectively, our results demonstrate that skin lymphatics play an important but distinct role in the 2 phases of contact hypersensitivity. During sensitization, lymphatics contribute to the development of the antigen-specific immunization, whereas in elicitation, they moderate the inflammatory response and leukocyte infiltration in a neutrophil-dependent manner. These findings underscore the need for novel therapeutic strategies targeting the lymphatics in the context of allergic skin diseases.

NETosis: an emerging therapeutic target in renal diseases.

Introduction: Neutrophil extracellular traps (NETs) are web-like structures composed of nuclear and granular components. The primary role of NETS is to prevent the dissemination of microbes and facilitate their elimination. However, this process is accompanied by collateral proinflammatory adverse effects when the NET release becomes uncontrollable, or clearance is impaired. Although NET-induced organ damage is conducted primarily and indirectly via immune complexes and the subsequent release of cytokines, their direct effects on cells are also remarkable. NETosis plays a critical pathogenic role in several renal disorders, such as the early phase of acute tubular necrosis, anti-neutrophil cytoplasmic antibody-mediated renal vasculitis, lupus nephritis, thrombotic microangiopathies, anti-glomerular basement membrane disease, and diabetic nephropathy. Their substantial contribution in the course of these disorders makes them a desirable target in the therapeutic armamentarium. This article gives an in-depth review of the heterogeneous pathogenesis and physiological regulations of NETosis and its pivotal role in renal diseases. Based on the pathogenesis, the article also outlines the current therapeutic options and possible molecular targets in the treatment of NET-related renal disorders. Methods: We carried out thorough literature research published in PubMed and Google Scholar, including a comprehensive review and analysis of the classification, pathomechanisms, and a broad spectrum of NET-related kidney disorders. Conclusions: NETosis plays a pivotal role in certain renal diseases. It initiates and maintains inflammatory and autoimmune disorders, thus making it a desirable target for improving patient and renal outcomes. Better understanding and clinical translation of the pathogenesis are crucial aspects to treatment, for improving patient, and renal outcomes.

The selective inhibition of the Syk tyrosine kinase ameliorates experimental autoimmune arthritis.

Autoimmune arthritis - such as rheumatoid arthritis - affect a significant proportion of the population, which can cause everyday joint pain, decreased mobility and reduced quality of life. Despite having more and more therapeutic options available, there are still a lot of patients who cannot reach remission or low disease activity by current therapies. This causes an urgent need for the development of new treatment options. The Syk tyrosine kinase plays an essential role in B cell receptor, Fc receptor and integrin signaling. It has been shown that the hematopoietic cell-specific deletion of Syk resulted in a complete protection against autoantibody-induced experimental arthritis. This prompted us to test the effect of entospletinib, a second generation, Syk-selective inhibitor, which has a tolerable safety profile according to hematological clinical trials, in experimental autoimmune arthritis. We found that entospletinib dose-dependently decreased the macroscopic signs of joint inflammation, while it did not affect the health status of the animals. In line with these findings, local neutrophil accumulation and cytokine levels were reduced compared to the vehicle-treated group, while macrophage accumulation and synovial fibroblast numbers were not significantly altered. Meanwhile, entospletinib dose-dependently decreased the cell responses of immune complex- or integrin ligand-activated neutrophils. Overall, we found that selective Syk inhibition by entospletinib reduced the activity of autoantibody-induced experimental arthritis, which seems to be based mainly on the effect of the inhibitor on neutrophil functions. Our data raise the possibility that entospletinib could be a good drug candidate in the treatment of human autoimmune arthritis.

Absence of Nkx2-3 induces ectopic lymphatic endothelial differentiation associated with impaired extramedullary stress hematopoiesis in the spleen.

The red and white pulps as two main parts of the spleen are arranged around distinct types of vasculature, and perform significantly different functions in both humans and mice. Previous observations indicated a profound alteration of the local vessel specialization in mice lacking Nkx2-3 homeodomain transcription factor, including contradictory results suggesting presence of an ectopic lymphatic vascular structure. Furthermore, how the absence of Nkx2-3 and the consequential changes in endothelial components affect the extramedullary hematopoietic activity restricted to the splenic red pulp is unknown. In this work, we investigated the role of Nkx2-3 homeodomain transcription factor as a major morphogenic determinant for vascular specification, and its effect in the extramedullary hematopoiesis following acute blood loss and pharmacological stimulation of megakaryocyte differentiation after treatment with thrombopoietin-receptor mimetic Romiplostim. We found that, in mice lacking Nkx2-3, Prox1-positive lymphatic capillaries containing gp38/CD31 double positive lymphatic endothelial cells develop, arranged into an extensive meshwork, while the Clever1-positive venous segments of red pulp blood vasculature are absent. This lymphatic endothelial shift is coupled with a severely compromised splenic erythropoiesis and a significantly reduced splenic megakaryocyte colony formation following Romiplostim treatment in mice lacking Nkx2-3. These findings indicate that the shift of microvascular patterning in the absence of Nkx2-3 includes the emergence of ectopic Prox1-positive lymphatic vessels, and that this pivoting towards lymph node-like vascular patterning is associated with an impaired reserve hematopoietic capacity of the splenic red pulp.

Phospholipase Cγ2 is required for basal but not oestrogen deficiency-induced bone resorption.

BACKGROUND: Osteoclasts play a critical role in bone resorption under basal conditions, but they also contribute to pathological bone loss during diseases including postmenopausal osteoporosis. Phospholipase Cγ2 (PLCγ2) is an important signalling molecule in diverse haematopoietic lineages. Here, we tested the role of PLCγ2 in basal and ovariectomy-induced bone resorption, as well as in in vitro osteoclast cultures using PLCγ2-deficient (PLCγ2(-/-) ) mice. MATERIALS AND METHODS: The trabecular architecture of long bone metaphyses was tested by micro-CT and histomorphometric analyses. Postmenopausal osteoporosis was modelled by surgical ovariectomy. Osteoclast development and function, gene expression and PLCγ2 phosphorylation were tested on in vitro osteoclast and macrophage cultures. RESULTS: PLCγ2(-/-) mice had significantly higher trabecular bone mass under basal conditions than wild-type mice. PLCγ2 was required for in vitro development and resorptive function of osteoclasts, but not for upregulation of osteoclast-specific gene expression. PLCγ2 was phosphorylated in a Src-family-dependent manner upon macrophage adhesion but not upon stimulation by M-CSF or RANKL. Surprisingly, ovariectomy-induced bone resorption in PLCγ2(-/-) mice was similar to, or even more robust than, that in wild-type animals. CONCLUSIONS: Our results indicate that PLCγ2 participates in bone resorption under basal conditions, likely because of its role in adhesion receptor signalling during osteoclast development. In contrast, PLCγ2 does not appear to play a major role in ovariectomy-induced bone loss. These results suggest that basal and oestrogen deficiency-induced bone resorption utilizes different signalling pathways and that PLCγ2 may not be a suitable therapeutic target in postmenopausal osteoporosis.

Ex vivo confocal Raman microspectroscopy of porcine dura mater supported by optical clearing.

The effect of tissue optical clearing (TOC) to increase the probing depth and observe in-depth structure of the ex vivo porcine dura mater was studied by confocal Raman microspectroscopy (CRM). Raman intensities were significantly increased at the depth of 250 µm for all collagen bands after treatment with glycerol. The influence of glycerol on collagen hydration was also investigated. The results indicate that the process of TOC can be divided into three main steps. The first one is a fast process of tissue dehydration accompanied by collagen shrinkage while the second relatively slow process is related to the glycerol penetration into the interfibrillar space of collagen combined with swelling of tissue. The third step is collagen dissociation caused by the high concentration of glycerol. To the best of our knowledge, this study is the first example to introduce the TOC technique in assisting CRM of ex vivo dura mater in-depth probing.

3D culturing of human pluripotent stem cells-derived endothelial cells for vascular regeneration.

Rationale: Human induced pluripotent stem cell-derived endothelial cells can be candidates for engineering therapeutic vascular grafts. Methods: Here, we studied the role of three-dimensional culture on their characteristics and function both in vitro and in vivo. Results: We found that differentiated hPSC-EC can re-populate decellularized biomatrices; they remain viable, undergo maturation and arterial/venous specification. Human PSC-EC develop antifibrotic, vasoactive and anti-inflammatory properties during recellularization. In vivo, a robust increase in perfusion was detected at the engraftment sites after subcutaneous implantation of an hPSC-EC-laden hydrogel in rats. Histology confirmed survival and formation of capillary-like structures, suggesting the incorporation of hPSC-EC into host microvasculature. In a canine model, hiPSC-EC-seeded onto decellularised vascular segments were functional as aortic grafts. Similarly, we showed the retention and maturation of hiPSC-EC and dynamic remodelling of the vessel wall with good maintenance of vascular patency. Conclusions: A combination of hPSC-EC and biomatrices may be a promising approach to repair ischemic tissues.

Genetic deficiency of Syk protects mice from autoantibody-induced arthritis.

OBJECTIVE: The Syk tyrosine kinase plays an important role in diverse functions in hematopoietic lineage cells. Although previous in vitro and pharmacologic analyses suggested Syk to be a possible player in the development of autoimmune arthritis, no in vivo genetic studies addressing that issue have yet been reported. The aim of the present study was to test whether genetic deficiency of Syk affects autoantibody-induced experimental arthritis in the K/BxN serum-transfer model. METHODS: Syk(-/-) bone marrow chimeras carrying a Syk-deficient hematopoietic system were generated by transplanting Syk(-/-) fetal liver cells into lethally irradiated wild-type recipients. After complete repopulation of the hematopoietic compartment, autoantibody-mediated arthritis was induced by injection of arthritogenic K/BxN serum. Arthritis development was monitored by macroscopic and microscopic observation of the ankle joints, micro-computed tomography of bone morphology, as well as a joint function assay. RESULTS: Genetic deficiency of Syk in the hematopoietic compartment completely blocked the development of all macroscopic and microscopic signs of arthritis. The Syk(-/-) mutation also prevented the appearance of periarticular bone erosions. Finally, Syk(-/-) bone marrow chimeras were completely protected from arthritis-induced loss of articular function. CONCLUSION: Our results indicate that Syk is critically involved in the development of all clinically relevant aspects of autoantibody-mediated K/BxN serum-transfer arthritis in experimental mice. These results provide the first in vivo genetic evidence of the role of Syk in the development of autoimmune arthritis.

Fluorescence-Based Real-Time Analysis of Osteoclast Development.

Osteoclasts are multinucleated cells of hematopoietic origin which are critically involved in physiological and pathological bone resorption. They develop from myeloid progenitors through characteristic gene expression changes and intercellular fusion. This process is directed by M-CSF and RANKL which are also able to trigger osteoclast development from bone marrow cells in vitro. Osteoclasts are conventionally visualized by histochemical staining followed by manual counting, which hinders kinetic studies and automated quantification. Here we describe two fluorescence-based assays for the real-time analysis of myeloid cell to osteoclast development (FRAMCO) in primary mouse bone marrow cell cultures. Both assays rely on red-to-green fluorescence conversion of the membrane-targeted tdTomato/membrane-targeted eGFP (mTmG) transgene by Cre recombinase driven by the osteoclast-specific cathepsin K promoter (Ctsk-Cre). In the first assay (FRAMCO1.1), osteoclast-specific gene expression triggers red-to-green color conversion of cells carrying both the Ctsk-Cre and mTmG transgenes. In the second assay (FRAMCO1.2), red-to-green fluorescence conversion is triggered by fusion of neighboring co-cultured bone marrow cells separately carrying either the Ctsk-Cre or the mTmG transgenes. The two assays were tested using a high-content confocal fluorescence imaging system, followed by automated quantification. The FRAMCO1.1 assay showed robust red-to-green fluorescence conversion of more than 50% of the culture (including mononuclear cells) within 3 days under osteoclastogenic conditions. The FRAMCO1.2 assay showed a less robust but still readily measurable red-to-green color conversion in multinuclear cells within 5 days of differentiation. The assays required both the Ctsk-Cre and the mTmG transgenes and gave no signals in parallel macrophage cultures. The proper functioning of the two assays was also confirmed at the DNA, mRNA and bulk protein level. The assay systems were validated using lisophosphatidylcholine, a previously reported inhibitor of preosteoclast fusion. Taken together, our assays allow high-throughput automated real-time analysis of two critical aspects of osteoclast development, facilitating the screening for novel drug candidates for the pharmacological control of osteoclast-mediated bone resorption.