Muscle-specific stress fibers give rise to sarcomeres in cardiomyocytes.

The sarcomere is the contractile unit within cardiomyocytes driving heart muscle contraction. We sought to test the mechanisms regulating actin and myosin filament assembly during sarcomere formation. Therefore, we developed an assay using human cardiomyocytes to monitor sarcomere assembly. We report a population of muscle stress fibers, similar to actin arcs in non-muscle cells, which are essential sarcomere precursors. We show sarcomeric actin filaments arise directly from muscle stress fibers. This requires formins (e.g., FHOD3), non-muscle myosin IIA and non-muscle myosin IIB. Furthermore, we show short cardiac myosin II filaments grow to form ~1.5 µm long filaments that then 'stitch' together to form the stack of filaments at the core of the sarcomere (i.e., the A-band). A-band assembly is dependent on the proper organization of actin filaments and, as such, is also dependent on FHOD3 and myosin IIB. We use this experimental paradigm to present evidence for a unifying model of sarcomere assembly.

BVES is required for maintenance of colonic epithelial integrity in experimental colitis by modifying intestinal permeability.

Blood vessel epicardial substance (BVES), or POPDC1, is a tight junction-associated transmembrane protein that modulates epithelial-to-mesenchymal transition (EMT) via junctional signaling pathways. There have been no in vivo studies investigating the role of BVES in colitis. We hypothesized that BVES is critical for maintaining colonic epithelial integrity. At baseline, Bves-/- mouse colons demonstrate increased crypt height, elevated proliferation, decreased apoptosis, altered intestinal lineage allocation, and dysregulation of tight junctions with functional deficits in permeability and altered intestinal immunity. Bves-/- mice inoculated with Citrobacter rodentium had greater colonic injury, increased colonic and mesenteric lymph node bacterial colonization, and altered immune responses after infection. We propose that increased bacterial colonization and translocation result in amplified immune responses and worsened injury. Similarly, dextran sodium sulfate (DSS) treatment resulted in greater histologic injury in Bves-/- mice. Two different human cell lines (Caco2 and HEK293Ts) co-cultured with enteropathogenic E. coli showed increased attaching/effacing lesions in the absence of BVES. Finally, BVES mRNA levels were reduced in human ulcerative colitis (UC) biopsy specimens. Collectively, these studies suggest that BVES plays a protective role both in ulcerative and infectious colitis and identify BVES as a critical protector of colonic mucosal integrity.

Loss of CENP-F Results in Dilated Cardiomyopathy with Severe Disruption of Cardiac Myocyte Architecture.

Centromere-binding protein F (CENP-F) is a very large and complex protein with many and varied binding partners including components of the microtubule network. Numerous CENP-F functions impacting diverse cellular behaviors have been identified. Importantly, emerging data have shown that CENP-F loss- or gain-of-function has critical effects on human development and disease. Still, it must be noted that data at the single cardiac myocyte level examining the impact of CENP-F loss-of-function on fundamental cellular behavior is missing. To address this gap in our knowledge, we analyzed basic cell structure and function in cardiac myocytes devoid of CENP-F. We found many diverse structural abnormalities including disruption of the microtubule network impacting critical characteristics of the cardiac myocyte. This is the first report linking microtubule network malfunction to cardiomyopathy. Importantly, we also present data demonstrating a direct link between a CENP-F single nucleotide polymorphism (snp) and human cardiac disease. In a proximate sense, these data examining CENP-F function explain the cellular basis underlying heart disease in this genetic model and, in a larger sense, they will hopefully provide a platform upon which the field can explore diverse cellular outcomes in wide-ranging areas of research on this critical protein.

BVES regulates c-Myc stability via PP2A and suppresses colitis-induced tumourigenesis.

OBJECTIVE: Blood vessel epicardial substance (BVES) is a tight junction-associated protein that regulates epithelial-mesenchymal states and is underexpressed in epithelial malignancy. However, the functional impact of BVES loss on tumourigenesis is unknown. Here we define the in vivo role of BVES in colitis-associated cancer (CAC), its cellular function and its relevance to patients with IBD. DESIGN: We determined BVES promoter methylation status using an Infinium HumanMethylation450 array screen of patients with UC with and without CAC. We also measured BVES mRNA levels in a tissue microarray consisting of normal colons and CAC samples. Bves-/- and wild-type mice (controls) were administered azoxymethane (AOM) and dextran sodium sulfate (DSS) to induce tumour formation. Last, we used a yeast two-hybrid screen to identify BVES interactors and performed mechanistic studies in multiple cell lines to define how BVES reduces c-Myc levels. RESULTS: BVES mRNA was reduced in tumours from patients with CAC via promoter hypermethylation. Importantly, BVES promoter hypermethylation was concurrently present in distant non-malignant-appearing mucosa. As seen in human patients, Bves was underexpressed in experimental inflammatory carcinogenesis, and Bves-/- mice had increased tumour multiplicity and degree of dysplasia after AOM/DSS administration. Molecular analysis of Bves-/- tumours revealed Wnt activation and increased c-Myc levels. Mechanistically, we identified a new signalling pathway whereby BVES interacts with PR61α, a protein phosphatase 2A regulatory subunit, to mediate c-Myc destruction. CONCLUSION: Loss of BVES promotes inflammatory tumourigenesis through dysregulation of Wnt signalling and the oncogene c-Myc. BVES promoter methylation status may serve as a CAC biomarker.

Loss of CENP-F results in distinct microtubule-related defects without chromosomal abnormalities.

Microtubule (MT)-binding centromere protein F (CENP-F) was previously shown to play a role exclusively in chromosome segregation during cellular division. Many cell models of CENP-F depletion show a lag in the cell cycle and aneuploidy. Here, using our novel genetic deletion model, we show that CENP-F also regulates a broader range of cellular functions outside of cell division. We characterized CENP-F(+/+) and CENP-F(-/-) mouse embryonic fibroblasts (MEFs) and found drastic differences in multiple cellular functions during interphase, including cell migration, focal adhesion dynamics, and primary cilia formation. We discovered that CENP-F(-/-) MEFs have severely diminished MT dynamics, which underlies the phenotypes we describe. These data, combined with recent biochemical research demonstrating the strong binding of CENP-F to the MT network, support the conclusion that CENP-F is a powerful regulator of MT dynamics during interphase and affects heterogeneous cell functions.

BVES Regulates Intestinal Stem Cell Programs and Intestinal Crypt Viability after Radiation.

Blood vessel epicardial substance (BVES/Popdc1) is a junctional-associated transmembrane protein that is underexpressed in a number of malignancies and regulates epithelial-to-mesenchymal transition. We previously identified a role for BVES in regulation of the Wnt pathway, a modulator of intestinal stem cell programs, but its role in small intestinal (SI) biology remains unexplored. We hypothesized that BVES influences intestinal stem cell programs and is critical to SI homeostasis after radiation injury. At baseline, Bves(-/-) mice demonstrated increased crypt height, as well as elevated proliferation and expression of the stem cell marker Lgr5 compared to wild-type (WT) mice. Intercross with Lgr5-EGFP reporter mice confirmed expansion of the stem cell compartment in Bves(-/-) mice. To examine stem cell function after BVES deletion, we used ex vivo 3D-enteroid cultures. Bves(-/-) enteroids demonstrated increased stemness compared to WT, when examining parameters such as plating efficiency, stem spheroid formation, and retention of peripheral cystic structures. Furthermore, we observed increased proliferation, expression of crypt-base columnar "CBC" and "+4" stem cell markers, amplified Wnt signaling, and responsiveness to Wnt activation in the Bves(-/-) enteroids. Bves expression was downregulated after radiation in WT mice. Moreover, after radiation, Bves(-/-) mice demonstrated significantly greater SI crypt viability, proliferation, and amplified Wnt signaling in comparison to WT mice. Bves(-/-) mice also demonstrated elevations in Lgr5 and Ascl2 expression, and putative damage-responsive stem cell populations marked by Bmi1 and TERT. Therefore, BVES is a key regulator of intestinal stem cell programs and mucosal homeostasis. Stem Cells 2016;34:1626-1636.

The kinetochore protein, CENPF, is mutated in human ciliopathy and microcephaly phenotypes.

BACKGROUND: Mutations in microtubule-regulating genes are associated with disorders of neuronal migration and microcephaly. Regulation of centriole length has been shown to underlie the pathogenesis of certain ciliopathy phenotypes. Using a next-generation sequencing approach, we identified mutations in a novel centriolar disease gene in a kindred with an embryonic lethal ciliopathy phenotype and in a patient with primary microcephaly. METHODS AND RESULTS: Whole exome sequencing data from a non-consanguineous Caucasian kindred exhibiting mid-gestation lethality and ciliopathic malformations revealed two novel non-synonymous variants in CENPF, a microtubule-regulating gene. All four affected fetuses showed segregation for two mutated alleles [IVS5-2A>C, predicted to abolish the consensus splice-acceptor site from exon 6; c.1744G>T, p.E582X]. In a second unrelated patient exhibiting microcephaly, we identified two CENPF mutations [c.1744G>T, p.E582X; c.8692 C>T, p.R2898X] by whole exome sequencing. We found that CENP-F colocalised with Ninein at the subdistal appendages of the mother centriole in mouse inner medullary collecting duct cells. Intraflagellar transport protein-88 (IFT-88) colocalised with CENP-F along the ciliary axonemes of renal epithelial cells in age-matched control human fetuses but did not in truncated cilia of mutant CENPF kidneys. Pairwise co-immunoprecipitation assays of mitotic and serum-starved HEKT293 cells confirmed that IFT88 precipitates with endogenous CENP-F. CONCLUSIONS: Our data identify CENPF as a new centriolar disease gene implicated in severe human ciliopathy and microcephaly related phenotypes. CENP-F has a novel putative function in ciliogenesis and cortical neurogenesis.

Heterogeneity in vascular smooth muscle cell embryonic origin in relation to adult structure, physiology, and disease.

Regional differences in vascular physiology and disease response exist throughout the vascular tree. While these differences in physiology and disease correspond to regional vascular environmental conditions, there is also compelling evidence that the embryonic origins of the smooth muscle inherent to the vessels may play a role. Here, we review what is known regarding the role of embryonic origin of vascular smooth muscle cells during vascular development. The focus of this review is to highlight the heterogeneity in the origins of vascular smooth muscle cells and the resulting regional physiologies of the vessels. Our goal is to stimulate future investigation into this area and provide a better understanding of vascular organogenesis and disease. .

Isolation and physiological analysis of mouse cardiomyocytes.

Cardiomyocytes, the workhorse cell of the heart, contain exquisitely organized cytoskeletal and contractile elements that generate the contractile force used to pump blood. Individual cardiomyocytes were first isolated over 40 years ago in order to better study the physiology and structure of heart muscle. Techniques have rapidly improved to include enzymatic digestion via coronary perfusion. More recently, analyzing the contractility and calcium flux of isolated myocytes has provided a vital tool in the cellular and sub-cellular analysis of heart failure. Echocardiography and EKGs provide information about the heart at an organ level only. Cardiomyocyte cell culture systems exist, but cells lack physiologically essential structures such as organized sarcomeres and t-tubules required for myocyte function within the heart. In the protocol presented here, cardiomyocytes are isolated via Langendorff perfusion. The heart is removed from the mouse, mounted via the aorta to a cannula, perfused with digestion enzymes, and cells are introduced to increasing calcium concentrations. Edge and sarcomere detection software is used to analyze contractility, and a calcium binding fluorescent dye is used to visualize calcium transients of electrically paced cardiomyocytes; increasing understanding of the role cellular changes play in heart dysfunction. Traditionally used to test drug effects on cardiomyocytes, we employ this system to compare myocytes from WT mice and mice with a mutation that causes dilated cardiomyopathy. This protocol is unique in its comparison of live cells from mice with known heart function and known genetics. Many experimental conditions are reliably compared, including genetic or environmental manipulation, infection, drug treatment, and more. Beyond physiologic data, isolated cardiomyocytes are easily fixed and stained for cytoskeletal elements. Isolating cardiomyocytes via perfusion is an extremely versatile method, useful in studying cellular changes that accompany or lead to heart failure in a variety of experimental conditions.

Resident progenitors, not exogenous migratory cells, generate the majority of visceral mesothelium in organogenesis.

Historically, analyses of mesothelial differentiation have focused on the heart where a highly migratory population of progenitors originating from a localized "extrinsic" source moves to and over the developing organ. This model long stood alone as the paradigm for generation of this cell type. Here, using chick/quail chimeric grafting and subsequent identification of mesothelial cell populations, we demonstrate that a different mechanism for the generation of mesothelia exists in vertebrate organogenesis. In this newly discovered model, mesothelial progenitors are intrinsic to organs of the developing digestive and respiratory systems. Additionally, we demonstrate that the early heart stands alone in its ability to recruit an entirely exogenous mesothelial cell layer during development. Thus, the newly identified "organ intrinsic" model of mesotheliogenesis appears to predominate while the long-studied cardiac model of mesothelial development may be the outlier.

Embryonic domains of the aorta derived from diverse origins exhibit distinct properties that converge into a common phenotype in the adult.

Vascular smooth muscle cells (VSMCs) are derived from distinct embryonic origins. Vessels originating from differing smooth muscle cell populations have distinct vascular and pathological properties involving calcification, atherosclerosis, and structural defects such as aneurysm and coarctation. We hypothesized that domains within a single vessel, such as the aorta, vary in phenotype based on embryonic origin. Gene profiling and myographic analyses demonstrated that embryonic ascending and descending aortic domains exhibited distinct phenotypes. In vitro analyses demonstrated that VSMCs from each region were dissimilar in terms of cytoskeletal and migratory properties, and retention of different gene expression patterns. Using the same analysis, we found that these same two domains are indistinguishable in the adult vessel. Our data demonstrate that VSMCs from different embryonic origins are functionally distinct in the embryonic mouse, but converge to assume a common phenotype in the aorta of healthy adults. These findings have fundamental implications for aortic development, function and disease progression.

The chick embryo as an expanding experimental model for cancer and cardiovascular research.

A long and productive history in biomedical research defines the chick as a model for human biology. Fundamental discoveries, including the description of directional circulation propelled by the heart and the link between oncogenes and the formation of cancer, indicate its utility in cardiac biology and cancer. Despite the more recent arrival of several vertebrate and invertebrate animal models during the last century, the chick embryo remains a commonly used model for vertebrate biology and provides a tractable biological template. With new molecular and genetic tools applied to the avian genome, the chick embryo is accelerating the discovery of normal development and elusive disease processes. Moreover, progress in imaging and chick culture technologies is advancing real-time visualization of dynamic biological events, such as tissue morphogenesis, angiogenesis, and cancer metastasis. A rich background of information, coupled with new technologies and relative ease of maintenance, suggest an expanding utility for the chick embryo in cardiac biology and cancer research.

Bves and NDRG4 regulate directional epicardial cell migration through autocrine extracellular matrix deposition.

Directional cell movement is universally required for tissue morphogenesis. Although it is known that cell/matrix interactions are essential for directional movement in heart development, the mechanisms governing these interactions require elucidation. Here we demonstrate that a novel protein/protein interaction between blood vessel epicardial substance (Bves) and N-myc downstream regulated gene 4 (NDRG4) is critical for regulation of epicardial cell directional movement, as disruption of this interaction randomizes migratory patterns. Our studies show that Bves/NDRG4 interaction is required for trafficking of internalized fibronectin through the "autocrine extracellular matrix (ECM) deposition" fibronectin recycling pathway. Of importance, we demonstrate that Bves/NDRG4-mediated fibronectin recycling is indeed essential for epicardial cell directional movement, thus linking these two cell processes. Finally, total internal reflectance fluorescence microscopy shows that Bves/NDRG4 interaction is required for fusion of recycling endosomes with the basal cell surface, providing a molecular mechanism of motility substrate delivery that regulates cell directional movement. This is the first evidence of a molecular function for Bves and NDRG4 proteins within broader subcellular trafficking paradigms. These data identify novel regulators of a critical vesicle-docking step required for autocrine ECM deposition and explain how Bves facilitates cell-microenvironment interactions in the regulation of epicardial cell-directed movement.

Autotaxin signaling governs phenotypic heterogeneity in visceral and parietal mesothelia.

Mesothelia, which cover all coelomic organs and body cavities in vertebrates, perform diverse functions in embryonic and adult life. Yet, mesothelia are traditionally viewed as simple, uniform epithelia. Here we demonstrate distinct differences between visceral and parietal mesothelia, the most basic subdivision of this tissue type, in terms of gene expression, adhesion, migration, and invasion. Gene profiling determined that autotaxin, a secreted lysophospholipase D originally discovered as a tumor cell-motility-stimulating factor, was expressed exclusively in the more motile and invasive visceral mesothelia and at abnormally high levels in mesotheliomas. Gain and loss of function studies demonstrate that autotaxin signaling is indeed a critical factor responsible for phenotypic differences within mesothelia. Furthermore, we demonstrate that known and novel small molecule inhibitors of the autotaxin signaling pathway dramatically blunt migratory and invasive behaviors of aggressive mesotheliomas. Taken together, this study reveals distinct phenotypes within the mesothelial cell lineage, demonstrates that differential autotaxin expression is the molecular underpinning for these differences, and provides a novel target and lead compounds to intervene in invasive mesotheliomas.

Omental grafting: a cell-based therapy for blood vessel repair.

Clinicians regularly transplant omental pedicles to repair a wide variety of injured tissues, but the basic mechanism underlying this efficacious procedure is not understood. One possibility that has not been addressed is the ability of omentum to directly contribute regenerative cells to injured tissues. We hypothesized that if omental progenitor cells could be mobilized to incorporate into damaged tissue, the power of this therapy would be greatly expanded. Labelled omental grafts were transplanted into a murine carotid artery injury model. Selected grafts were treated with thymosin ß4 (Tß4) prior to transplantation to investigate the effects of chemical potentiation on healing. We found treatment of grafts with Tß4-induced progenitor cells to fully integrate into the wall of injured vessels and differentiate into vascular smooth muscle. Myographic studies determined that arteries receiving Tß4-stimulated grafts were functionally indistinguishable from uninjured controls. Concurrent in vitro analyses showed that Tß4 promoted proliferation, migration and trans-differentiation of cells via AKT signalling. This study is the first to demonstrate that omentum can provide progenitor cells for repair, thus revealing a novel and naturally occurring source of vascular smooth muscle for use in cell-based therapies. Furthermore, our data show that this system can be optimized with inducing factors, highlighting a more powerful therapeutic potential than that of its current clinical application. This is a paradigm-setting concept that lays the foundation for the use of chemical genetics to enhance therapeutic outcomes in a myriad of fields.

Thymosin ß4 mobilizes mesothelial cells for blood vessel repair.

Mesothelium is the simple squamous epithelium covering all abdominal organs and the coeloms in which those organs reside. While the structural characteristics of this cell type were documented a century ago, its potential in development, disease, and wound healing is only now becoming apparent. In the embryo, mesothelia provide vasculogenic cells for the developing heart, lungs, and gut. Furthermore, adult mesothelial cells can be reactivated using thymosin ß4 and mobilized to aid in tissue repair. Despite their positive role in development and repair, mesothelia are also susceptible to adhesion and tumor formation. With knowledge that the mesothelium is an important mediator of tissue repair as well as disease, it will be important to identify other factors like thymosin ß4 that have the ability to potentiate these cells. Future use of chemical and genetic agents in conjunction with mesothelial cells will lead to enhanced therapeutic potential and mitigation of deleterious outcomes.

Comprehensive timeline of mesodermal development in the quail small intestine.

BACKGROUND: To generate the mature intestine, splanchnic mesoderm diversifies into six different tissue layers each with multiple cell types through concurrent and complex morphogenetic events. Hindering the progress of research in the field is the lack of a detailed description of the fundamental morphological changes that constitute development of the intestinal mesoderm. RESULTS: We used immunofluorescence and morphometric analyses of wild-type and Tg(tie1:H2B-eYFP) quail embryos to establish a comprehensive timeline of mesodermal development in the avian intestine. The following landmark features were analyzed from appearance of the intestinal primordium through generation of the definitive structure: radial compartment formation, basement membrane dynamics, mesothelial differentiation, mesenchymal expansion and growth patterns, smooth muscle differentiation, and maturation of the vasculature. In this way, structural relationships between mesodermal components were identified over time. CONCLUSIONS: This integrated analysis presents a roadmap for investigators and clinicians to evaluate diverse experimental data obtained at individual stages of intestinal development within the longitudinal context of intestinal morphogenesis.

Identification of a novel developmental mechanism in the generation of mesothelia.

Mesothelium is the surface layer of all coelomic organs and is crucial for the generation of their vasculature. Still, our understanding of the genesis of this essential cell type is restricted to the heart where a localized exogenous population of cells, the proepicardium, migrates to and envelops the myocardium supplying mesothelial, vascular and stromal cell lineages. Currently it is not known whether this pattern of development is specific to the heart or applies broadly to other coelomic organs. Using two independent long-term lineage-tracing studies, we demonstrate that mesothelial progenitors of the intestine are intrinsic to the gut tube anlage. Furthermore, a novel chick-quail chimera model of gut morphogenesis reveals these mesothelial progenitors are broadly distributed throughout the gut primordium and are not derived from a localized and exogenous proepicardium-like source of cells. These data demonstrate an intrinsic origin of mesothelial cells to a coelomic organ and provide a novel mechanism for the generation of mesothelial cells.

Cardiac-specific deletion of the microtubule-binding protein CENP-F causes dilated cardiomyopathy.

CENP-F is a large multifunctional protein with demonstrated regulatory roles in cell proliferation, vesicular transport and cell shape through its association with the microtubule (MT) network. Until now, analysis of CENP-F has been limited to in vitro analysis. Here, using a Cre-loxP system, we report the in vivo disruption of CENP-F gene function in murine cardiomyocytes, a cell type displaying high levels of CENP-F expression. Loss of CENP-F function in developing myocytes leads to decreased cell division, blunting of trabeculation and an initially smaller, thin-walled heart. Still, embryos are born at predicted mendelian ratios on an outbred background. After birth, hearts lacking CENP-F display disruption of their intercalated discs and loss of MT integrity particularly at the costamere; these two structures are essential for cell coupling/electrical conduction and force transduction in the heart. Inhibition of myocyte proliferation and cell coupling as well as loss of MT maintenance is consistent with previous reports of generalized CENP-F function in isolated cells. One hundred percent of these animals develop progressive dilated cardiomyopathy with heart block and scarring, and there is a 20% mortality rate. Importantly, although it has long been postulated that the MT cytoskeleton plays a role in the development of heart disease, this study is the first to reveal a direct genetic link between disruption of this network and cardiomyopathy. Finally, this study has broad implications for development and disease because CENP-F loss of function affects a diverse array of cell-type-specific activities in other organs.