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.
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.
These data build on our molecular understanding of ruminant fat depot biology and suggest oxysterols represent a promising circulating biomarker for cattle marbling.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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.
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).
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.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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, inhibin{szligbeta}A, 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.
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
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.
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.
(
) and
(
) 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
has impeded formal analysis. Using genome-editing, we generated mutants for
and
and found single mutants indistinguishable from wildtype whereas
/
double mutants display heart morphogenesis defects and pericardial oedema. Analysis of atrioventricular canal (AVC) markers show expansion of
,
and
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 confirm cardiac jelly expansion in
/
double mutants. Finally,
knockdown rescues the expansion of
expression and cardiac jelly in double mutants. This definitively shows that
and
function redundantly during cardiac development to restrict gene expression to the AVC, preventing excessive cardiac jelly synthesis in the atrial chamber.
•Expression of innate immune molecules in the developing mouse occurs prior to the formation of a functional cellular immune system.•Expression of the central complement components (C3, C5) and ...complement regulators (CD55, CD59, fH) occurs during neurulation.•The alternative and classical pathways are not functional due to the lack of expression of initiating factors.•The membrane attack complex is unlikely to be formed during neurulation due to lack of detectable C9 expression.•Toll-like receptors and adaptor proteins are also expressed during neurulation, whereas formyl peptide receptors are absent.
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.
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,
that displayed a specific cardiac arrhythmia phenotype. We show that loss-of-function mutations in
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 Ca
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.
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