The zebrafish grime mutant uncovers an evolutionarily conserved role for Tmem161b in the control of cardiac rhythm.

The establishment of cardiac function in the developing embryo is essential to ensure blood flow and, therefore, growth and survival of the animal. The molecular mechanisms controlling normal cardiac rhythm remain to be fully elucidated. From a forward genetic screen, we identified a unique mutant, grime, that displayed a specific cardiac arrhythmia phenotype. We show that loss-of-function mutations in tmem161b are responsible for the phenotype, identifying Tmem161b as a regulator of cardiac rhythm in zebrafish. To examine the evolutionary conservation of this function, we generated knockout mice for Tmem161b. Tmem161b knockout mice are neonatal lethal and cardiomyocytes exhibit arrhythmic calcium oscillations. Mechanistically, we find that Tmem161b is expressed at the cell membrane of excitable cells and live imaging shows it is required for action potential repolarization in the developing heart. Electrophysiology on isolated cardiomyocytes demonstrates that Tmem161b is essential to inhibit Ca2+ and K+ currents in cardiomyocytes. Importantly, Tmem161b haploinsufficiency leads to cardiac rhythm phenotypes, implicating it as a candidate gene in heritable cardiac arrhythmia. Overall, these data describe Tmem161b as a highly conserved regulator of cardiac rhythm that functions to modulate ion channel activity in zebrafish and mice.

Nppa and Nppb act redundantly during zebrafish cardiac development to confine AVC marker expression and reduce cardiac jelly volume.

Atrial natriuretic peptide (nppa/anf) and brain natriuretic peptide (nppb/bnp) form a gene cluster with expression in the chambers of the developing heart. Despite restricted expression, a function in cardiac development has not been demonstrated by mutant analysis. This is attributed to functional redundancy; however, their genomic location in cis has impeded formal analysis. Using genome editing, we have generated mutants for nppa and nppb, and found that single mutants were indistinguishable from wild type, whereas nppa/nppb double mutants displayed heart morphogenesis defects and pericardial oedema. Analysis of atrioventricular canal (AVC) markers show expansion of bmp4, tbx2b, has2 and versican expression into the atrium of double mutants. This expanded expression correlates with increased extracellular matrix in the atrium. Using a biosensor for hyaluronic acid to measure the cardiac jelly (cardiac extracellular matrix), we confirmed cardiac jelly expansion in nppa/nppb double mutants. Finally, bmp4 knockdown rescued the expansion of has2 expression and cardiac jelly in double mutants. This definitively shows that nppa and nppb function redundantly during cardiac development to restrict gene expression to the AVC, preventing excessive cardiac jelly synthesis in the atrial chamber.

Gene expression identifies metabolic and functional differences between intramuscular and subcutaneous adipocytes in cattle.

BACKGROUND: This study used a genome-wide screen of gene expression to better understand the metabolic and functional differences between commercially valuable intramuscular fat (IMF) and commercially wasteful subcutaneous (SC) fat depots in Bos taurus beef cattle. RESULTS: We confirmed many findings previously made at the biochemical level and made new discoveries. The fundamental lipogenic machinery, such as ACACA and FASN encoding the rate limiting Acetyl CoA carboxylase and Fatty Acid synthase were expressed at 1.6-1.8 fold lower levels in IMF, consistent with previous findings. The FA elongation pathway including the rate limiting ELOVL6 was also coordinately downregulated in IMF compared to SC as expected. A 2-fold lower expression in IMF of ACSS2 encoding Acetyl Coenzyme A synthetase is consistent with utilisation of less acetate for lipogenesis in IMF compared to SC as previously determined using radioisotope incorporation. Reduced saturation of fat in the SC depot is reflected by 2.4 fold higher expression of the SCD gene encoding the Δ9 desaturase enzyme. Surprisingly, CH25H encoding the cholesterol 25 hydroxylase enzyme was ~ 36 fold upregulated in IMF compared to SC. Moreover, its expression in whole muscle tissue appears representative of the proportional representation of bovine marbling adipocytes. This suite of observations prompted quantification of a set of oxysterols (oxidised forms of cholesterol) in the plasma of 8 cattle exhibiting varying IMF. Using Liquid Chromatography-Mass Spectrometry (LC-MS) we found the levels of several oxysterols were significantly associated with multiple marbling measurements across the musculature, but (with just one exception) no other carcass phenotypes. CONCLUSIONS: These data build on our molecular understanding of ruminant fat depot biology and suggest oxysterols represent a promising circulating biomarker for cattle marbling.

Complement C5aR1 Signaling Promotes Polarization and Proliferation of Embryonic Neural Progenitor Cells through PKCζ.

The complement system, typically associated with innate immunity, is emerging as a key controller of nonimmune systems including in development, with recent studies linking complement mutations with neurodevelopmental disease. A key effector of the complement response is the activation fragment C5a, which, through its receptor C5aR1, is a potent driver of inflammation. Surprisingly, C5aR1 is also expressed during early mammalian embryogenesis; however, no clearly defined function is ascribed to C5aR1 in development. Here we demonstrate polarized expression of C5aR1 on the apical surface of mouse embryonic neural progenitor cells in vivo and on human embryonic stem cell-derived neural progenitors. We also show that signaling of endogenous C5a during mouse embryogenesis drives proliferation of neural progenitor cells within the ventricular zone and is required for normal brain histogenesis. C5aR1 signaling in neural progenitors was dependent on atypical protein kinase C ζ, a mediator of stem cell polarity, with C5aR1 inhibition reducing proliferation and symmetric division of apical neural progenitors in human and mouse models. C5aR1 signaling was shown to promote the maintenance of cell polarity, with exogenous C5a increasing the retention of polarized rosette architecture in human neural progenitors after physical or chemical disruption. Transient inhibition of C5aR1 during neurogenesis in developing mice led to behavioral abnormalities in both sexes and MRI-detected brain microstructural alterations, in studied males, demonstrating a requirement of C5aR1 signaling for appropriate brain development. This study thus identifies a functional role for C5a-C5aR1 signaling in mammalian neurogenesis and provides mechanistic insight into recently identified complement gene mutations and brain disorders.SIGNIFICANCE STATEMENT The complement system, traditionally known as a controller of innate immunity, now stands as a multifaceted signaling family with a broad range of physiological actions. These include roles in the brain, where complement activation is associated with diseases, including epilepsy and schizophrenia. This study has explored complement regulation of neurogenesis, identifying a novel relationship between the complement activation peptide C5a and the neural progenitor proliferation underpinning formation of the mammalian brain. C5a was identified as a regulator of cell polarity, with inhibition of C5a receptors during embryogenesis leading to abnormal brain development and behavioral deficits. This work demonstrates mechanisms through which dysregulation of complement causes developmental disease and highlights the potential risk of complement inhibition for therapeutic purposes in pregnancy.

Autoantibodies against homocysteinylated protein in a mouse model of folate deficiency-induced neural tube defects.

BACKGROUND: Periconceptional supplementation with folic acid results in a significant reduction in the incidence of neural tube defects (NTDs). Nonetheless, NTDs remain a leading cause of perinatal morbidity and mortality worldwide, and the mechanism(s) by which folate exerts its protective effects are unknown. Homocysteine is an amino acid that accumulates under conditions of folate-deficiency, and is suggested as a risk factor for NTDs. One proposed mechanism of homocysteine toxicity is its accumulation into proteins in a process termed homocysteinylation. METHODS & RESULTS: Herein, we used a folate-deficient diet in pregnant mice to demonstrate that there is: (i) a significant inverse correlation between maternal serum folate levels and serum homocysteine; (ii) a significant positive correlation between serum homocysteine levels and titers of autoantibodies against homocysteinylated protein; and (iii) a significant increase in congenital malformations and NTDs in mice deficient in serum folate. Furthermore, in mice administered the folate-deplete diet before conception, supplementation with folic acid during the gestational period completely rescued the embryos from congenital defects, and resulted in homocysteinylated protein titers at term that are comparable to that of mice administered a folate-replete diet throughout both the pre- and postconception period. These results demonstrate that a low-folate diet that induces NTDs also increases protein homocysteinylation and the subsequent generation of autoantibodies against homocysteinylated proteins. CONCLUSION: These data support the hypotheses that homocysteinylation results in neo-self antigen formation under conditions of maternal folate deficiency, and that this process is reversible with folic acid supplementation.

C5a receptor signaling prevents folate deficiency-induced neural tube defects in mice.

The complement system is involved in a range of diverse developmental processes, including cell survival, growth, differentiation, and regeneration. However, little is known about the role of complement in embryogenesis. In this study, we demonstrate a novel role for the canonical complement 5a receptor (C5aR) in the development of the mammalian neural tube under conditions of maternal dietary folic acid deficiency. Specifically, we found C5aR and C5 to be expressed throughout the period of neurulation in wild-type mice and localized the expression to the cephalic regions of the developing neural tube. C5aR was also found to be expressed in the neuroepithelium of early human embryos. Ablation of the C5ar1 gene or the administration of a specific C5aR peptide antagonist to folic acid-deficient pregnant mice resulted in a high prevalence of severe anterior neural tube defect-associated congenital malformations. These findings provide a new and compelling insight into the role of the complement system during mammalian embryonic development.

Neural tube defects, folate, and immune modulation.

Periconceptional supplementation with folic acid has led to a significant worldwide reduction in the incidence of neural tube defects (NTDs). However, despite increasing awareness of the benefits of folic acid supplementation and the implementation of food fortification programs in many countries, NTDs continue to be a leading cause of perinatal morbidity and mortality worldwide. Furthermore, there exists a significant subgroup of women who appear to be resistant to the protective effects of folic acid supplementation. The following review addresses emerging clinical and experimental evidence for a role of the immune system in the etiopathogenesis of NTDs, with the aim of developing novel preventative strategies to further reduce the incidence of NTD-affected pregnancies. In particular, recent studies demonstrating novel roles and interactions between innate immune factors such as the complement cascade, neurulation, and folate metabolism are explored.

EPHA4-FC TREATMENT REDUCES ISCHEMIA/REPERFUSION-INDUCED INTESTINAL INJURY BY INHIBITING VASCULAR PERMEABILITY.

The inflammatory response is characterized by increased endothelial permeability, which permits the passage of fluid and inflammatory cells into interstitial spaces. The Eph/ephrin receptor ligand system plays a role in inflammation through a signaling cascade, which modifies Rho-GTPase activity. We hypothesized that blocking Eph/ephrin signaling using an EphA4-Fc would result in decreased inflammation and tissue injury in a model of ischemia/reperfusion (I/R) injury. Mice undergoing intestinal I/R pretreated with the EphA4-Fc had significantly reduced intestinal injury compared to mice injected with the control Fc. This reduction in I/R injury was accompanied by significantly reduced neutrophil infiltration, but did not affect intestinal inflammatory cytokine generation. Using microdialysis, we identified that intestinal I/R induced a marked increase in systemic vascular leakage, which was completely abrogated in EphA4-Fc-treated mice. Finally, we confirmed the direct role of Eph/ephrin signaling in endothelial leakage by demonstrating that EphA4-Fc inhibited tumor necrosis factor-α-induced vascular permeability in human umbilical vein endothelial cells. This study identifies that Eph/ephrin interaction induces proinflammatory signaling in vivo by inducing vascular leak and neutrophil infiltration, which results in tissue injury in intestinal I/R. Therefore, therapeutic targeting of Eph/ephrin interaction using inhibitors, such as EphA4-Fc, may be a novel method to prevent tissue injury in acute inflammation by influencing endothelial integrity and by controlling vascular leak.

Co-ordinated expression of innate immune molecules during mouse neurulation.

The innate immune system is the first line of defence against pathogens and infection. Recently, it has become apparent that many innate immune factors have roles outside of immunity and there is growing evidence that these factors play important functional roles during the development of a range of model organisms. Several studies have documented developmental expression of individual factors of the toll-like receptor and complement systems, and we recently demonstrated a key role for complement C5a receptor (C5aR1) signalling in neural tube closure in mice. Despite these emerging studies, a comprehensive expression analysis of these molecules in embryonic development is lacking. In the current study, we therefore, examined the expression of key innate immune factors in the early development period of neurulation (7.5-10.5dpc) in mice. We found that complement factor genes were differentially expressed during this period of murine development. Interestingly, the expression patterns we identified preclude activation of the classical and alternative pathways and formation of the membrane attack complex. Additionally, several other classes of innate immune molecules were expressed during the period of neurulation, including toll-like receptors (TLR-2, -3, -4 and -9), receptor for advanced glycation end-products (RAGE), and their signalling adapters (TRAF-4, TRAF-6, TAK-1 and MyD88). Taken together, this study highlights a number of innate immune factors as potential novel players in early embryonic development.

RNF14 is a regulator of mitochondrial and immune function in muscle.

BACKGROUND: Muscle development and remodelling, mitochondrial physiology and inflammation are thought to be inter-related and to have implications for metabolism in both health and disease. However, our understanding of their molecular control is incomplete. RESULTS: In this study we have confirmed that the ring finger 14 protein (RNF14), a poorly understood transcriptional regulator, influences the expression of both mitochondrial and immune-related genes. The prediction was based on a combination of network connectivity and differential connectivity in cattle (a non-model organism) and mice data sets, with a focus on skeletal muscle. They assigned similar probability to mammalian RNF14 playing a regulatory role in mitochondrial and immune gene expression. To try and resolve this apparent ambiguity we performed a genome-wide microarray expression analysis on mouse C2C12 myoblasts transiently transfected with two Rnf14 transcript variants that encode 2 naturally occurring but different RNF14 protein isoforms. The effect of both constructs was significantly different to the control samples (untransfected cells and cells transfected with an empty vector). Cluster analyses revealed that transfection with the two Rnf14 constructs yielded discrete expression signatures from each other, but in both cases a substantial set of genes annotated as encoding proteins related to immune function were perturbed. These included cytokines and interferon regulatory factors. Additionally, transfection of the longer transcript variant 1 coordinately increased the expression of 12 (of the total 13) mitochondrial proteins encoded by the mitochondrial genome, 3 of which were significant in isolated pair-wise comparisons (Mt-coxII, Mt-nd2 and mt-nd4l). This apparent additional mitochondrial function may be attributable to the RWD protein domain that is present only in the longer RNF14 isoform. CONCLUSIONS: RNF14 influences the expression of both mitochondrial and immune related genes in a skeletal muscle context, and has likely implications for the inter-relationship between bioenergetic status and inflammation.

The SNX-PX-BAR family in macropinocytosis: the regulation of macropinosome formation by SNX-PX-BAR proteins.

BACKGROUND: Macropinocytosis is an actin-driven endocytic process, whereby membrane ruffles fold back onto the plasma membrane to form large (>0.2 µm in diameter) endocytic organelles called macropinosomes. Relative to other endocytic pathways, little is known about the molecular mechanisms involved in macropinocytosis. Recently, members of the Sorting Nexin (SNX) family have been localized to the cell surface and early macropinosomes, and implicated in macropinosome formation. SNX-PX-BAR proteins form a subset of the SNX family and their lipid-binding (PX) and membrane-curvature sensing (BAR) domain architecture further implicates their functional involvement in macropinosome formation. METHODOLOGY/PRINCIPAL FINDINGS: We exploited the tractability of macropinosomes through image-based screening and systematic overexpression of SNX-PX-BAR proteins to quantitate their effect on macropinosome formation. SNX1 (40.9+/-3.19 macropinosomes), SNX5 (36.99+/-4.48 macropinosomes), SNX9 (37.55+/-2.4 macropinosomes), SNX18 (88.2+/-8 macropinosomes), SNX33 (65.25+/-6.95 macropinosomes) all exhibited statistically significant (p<0.05) increases in average macropinosome numbers per 100 transfected cells as compared to control cells (24.44+/-1.81 macropinosomes). SNX1, SNX5, SNX9, and SNX18 were also found to associate with early-stage macropinosomes within 5 minutes following organelle formation. The modulation of intracellular PI(3,4,5)P(3) levels through overexpression of PTEN or a lipid phosphatase-deficient mutant PTEN(G129E) was also observed to significantly reduce or elevate macropinosome formation respectively; coexpression of PTEN(G129E) with SNX9 or SNX18 synergistically elevated macropinosome formation to 119.4+/-7.13 and 91.4+/-6.37 macropinosomes respectively (p<0.05). CONCLUSIONS/SIGNIFICANCE: SNX1, SNX5, SNX9, SNX18, and SNX33 were all found to elevate macropinosome formation and (with the exception of SNX33) associate with early-stage macropinosomes. Moreover the effects of SNX9 and SNX18 overexpression in elevating macropinocytosis is likely to be synergistic with the increase in PI(3,4,5)P(3) levels, which is known to accumulate on the cell surface and early-stage macropinocytic cups. Together these findings represent the first systematic functional study into the impact of the SNX-PX-BAR family on macropinocytosis.

Phosphatidylinositol 3'-kinase signalling supports cell height in established epithelial monolayers.

Cell-cell interactions influence epithelial morphogenesis through an interplay between cell adhesion, trafficking and the cytoskeleton. These cellular processes are coordinated, often by cell signals found at cell-cell contacts. One such contact-based signal is the phosphatidylinositol 3'-kinase (PI3-kinase; PI3K) pathway. PI3-kinase is best understood for its role in mitogenic signalling, where it regulates cell survival, proliferation and differentiation. Its precise morphogenetic impacts in epithelia are, in contrast, less well-understood. Using phosphoinositide-specific biosensors we confirmed that E-cadherin-based cell-cell contacts are enriched in PIP(3), the principal product of PI3-kinase. We then used pharmacologic inhibitors to assess the morphogenetic impact of PI3-kinase in MDCK and MCF7 monolayers. We found that inhibiting PI3-kinase caused a reduction in epithelial cell height that was reversible upon removal of the drugs. This was not attributable to changes in E-cadherin expression or homophilic adhesion. Nor were there detectable changes in cell polarity. While Myosin II has been implicated in regulating keratinocyte height, we found no effect of PI3-kinase inhibition on apparent Myosin II activity; nor did direct inhibition of Myosin II alter epithelial height. Instead, in pursuing signalling pathways downstream of PI3-kinase we found that blocking Rac signalling, but not mTOR, reduced epithelial cell height, as did PI3-kinase inhibition. Overall, our findings suggest that PI3-kinase exerts a major morphogenetic impact in simple cultured epithelia through preservation of cell height. This is independent of potential effects on adhesion or polarity, but may occur through PI3-kinase-stimulated Rac signaling.

Evaluation of candidate markers for the peritubular myoid cell lineage in the developing mouse testis.

Despite the importance of peritubular myoid (PM) cells in the histogenesis of the fetal testis, understanding the origin and function of these cells has been hampered by the lack of suitable markers. The current study was aimed at identifying molecular markers for PM cells during the early stages of testis development in the mouse embryo. Expression of candidate marker genes was tested by section in situ hybridisation, in some instances followed by immunofluorescent detection of protein products. Collagen type-I, inhibinbetaA, caldesmon 1 and tropomyosin 1 were found to be expressed by early-stage PM cells. These markers were also expressed in subsets of interstitial cells, most likely reflecting their common embryological provenance from migrating mesonephric cells. Although not strictly specific for PM cells, these markers are likely to be useful in studying the biology of early PM cells in the fetal testis.