During development, the ventricular conduction system (VCS) arises from the trabecular or spongy myocardium. VCS and trabecular myocytes proliferate at a significantly slower rate than compact zone myocardial cells, establishing a transmural cell cycle gradient. The molecular determinants of VCS/trabecular myocyte cell cycle arrest are not known. Given the importance of pocket proteins (Rb, p107 and p130) in mediating G0/G1 arrest in many cell types, we examined the role of this gene family in regulating cell cycle exit of the trabecular myocardium and ventricular conduction system. Using a combinatorial knockout strategy, we found that graded loss of pocket proteins results in a spectrum of heart and lung defects. p107/p130 double knockout (dKO) hearts manifest dysregulated proliferation within the compact myocardium and trabecular bases, while the remaining trabecular region cell cycle exits normally. Consequently, dKO hearts exhibit defective cardiac compaction, septal hyperplasia and biventricular outflow tract obstruction, while the VCS appears relatively normal. Loss of all three pocket proteins (3KO) is necessary to completely disrupt the transmural cell cycle gradient. 3KO hearts exhibit massive overgrowth of the trabecular myocardium and ventricular conduction system, which leads to fetal heart failure and death. Hearts carrying a single pocket protein allele are able to maintain the transmural cell cycle gradient. These results demonstrate the exquisite sensitivity of trabecular and conduction myocytes to pocket protein function during ventricular chamber development.
Cardiac conduction system; Cardiogenesis; Cell cycle; Chamber development; Pocket proteins; Trabecular myocardium
A significant fraction of mice deficient in either glial cell-derived neurotrophic factor (GDNF) or its co-receptors (Gfrα1, Ret), undergoes ureteric bud (UB) outgrowth leading to the formation of a rudimentary kidney. Previous studies using the isolated Wolffian duct (WD) culture indicate that activation of fibroblast growth factor (FGF) receptor signaling, together with suppression of BMP/Activin signaling, is critical for GDNF-independent WD budding (Maeshima et al., 2007). By expression analysis of embryonic kidney from Ret(−/−) mice, we found the upregulation of several FGFs, including FGF7. To examine the intracellular pathways, we then analyzed GDNF-dependent and GDNF-independent budding in the isolated WD culture. In both conditions, Akt activation was found to be important; however, whereas this occurred through PI3-kinase in GDNF-dependent budding, in the case of GDNF-independent budding, Akt activation was apparently via a PI3-kinase independent mechanism. Jnk signaling and the AP-1 transcription factor complex were also implicated in GDNF-independent budding. FosB, a binding partner of c-Jun in the formation of AP-1, was the most highly upregulated gene in the ret knockout kidney (in which budding had still occurred), and we found that its siRNA-mediated knockdown in isolated WDs also blocked GDNF-independent budding. Taken together with the finding that inhibition of Jnk signaling does not block Akt activation/phosphorylation in GDNF-independent budding, the data support necessary roles for both FosB/Jun/AP-1 signaling and PI3-kinase-independent activation of Akt in GDNF-independent budding. A model is proposed for signaling events that involve Akt and JNK working to regulate GDNF-independent WD budding.
Ureteric bud; Kidney development; Wolffian duct; AKT; Jun-Fos; JNK signaling; ret; Fibroblast growth factor
Gastrin-releasing peptide (GRP), a neuropeptide initially isolated from porcine stomach, shares sequence similarity with bombesin. GRP and its receptors are present in the brains and peripheral tissues of several species of teleost fish, but little is known about the ventilatory and cardiovascular effects of this peptide in these vertebrates. The goal of this study was to compare the central and peripheral actions of picomolar doses of trout GRP on ventilatory and cardiovascular variables in the unanesthetized rainbow trout. Compared to vehicle, intracerebroventricular (ICV) injection of GRP (1–50 pmol) significantly elevated the ventilation rate (ƒV) and the ventilation amplitude (VAMP), and consequently the total ventilation (VTOT). The maximum hyperventilatory effect of GRP (VTOT: +225%), observed at a dose of 50 pmol, was mostly due to its stimulatory action on VAMP (+170%) rather than ƒV (+20%). In addition, ICV GRP (50 pmol) produced a significant increase in mean dorsal aortic blood pressure (PDA) (+35%) and in heart rate (ƒH) (+25%). Intra-arterial injections of GRP (5–100 pmol) were without sustained effect on the ventilatory variables but produced sporadic and transient increases in ventilatory movement at doses of 50 and 100 pmol. At these doses, GRP elevated PDA by +20% but only the 50 pmol dose significantly increased HR (+15%). In conclusion, our study suggests that endogenous GRP within the brain of the trout may act as a potent neurotransmitter and/or neuromodulator in the regulation of cardio-ventilatory functions. In the periphery, endogenous GRP may act as locally-acting and/or circulating neurohormone with an involvement in vasoregulatory mechanisms.
Central GRP; Intracerebroventricular injection; Hyperventilation; Hypertension; Teleost
Myeloperoxidase (MPO) is a heme-containing enzyme released from activated leukocytes into the extracellular space during inflammation. Its main function is the production of hypohalous acids that are potent oxidants. MPO can also modulate cell signaling and inflammatory responses independently of its enzymatic activity. Because MPO is regarded as an important risk factor for cardiovascular diseases associated with increased platelet activity, we studied the effects of MPO on human platelet functional properties. Laser scanning confocal microscopy was used to reveal carbohydrate-independent MPO binding to human platelet membrane. Adding MPO to platelets did not activate their aggregation under basal conditions (without agonist). In contrast, MPO augmented agonist-induced platelet aggregation, which was not prevented by MPO enzymatic activity inhibitors. It was found that exposure of platelets to MPO leads to actin cytoskeleton reorganization and an increase in their elasticity. Furthermore, MPO evoked a rise in cytosolic Ca2+ through enhancement of store-operated Ca2+ entry (SOCE). Together, these findings indicate that MPO is not a direct agonist but rather a mediator that binds to human platelets, induces actin cytoskeleton reorganization and affects the mechanical stiffness of human platelets, resulting in potentiating SOCE and agonist-induced human platelet aggregation. Therefore, an increased activity of platelets in vascular disease can, at least partly, be provided by MPO elevated concentrations.
Myeloperoxidase; Platelets; Actin cytoskeleton; Aggregation; SOCE
Cell cycle events are driven by Cyclin dependent kinases (CDKs) and by their counter-acting phosphatases. Activation of the Cdk1:Cyclin B complex during mitotic entry is controlled by the Wee1/Myt1 inhibitory kinases and by Cdc25 activatory phosphatase, which are themselves regulated by Cdk1:Cyclin B within two positive circuits. Impairing these two feedbacks with chemical inhibitors induces a transient entry into M phase referred to as mitotic collapse. The pathology of mitotic collapse reveals that the positive circuits play a significant role in maintaining the M phase state. To better understand the function of these feedback loops during G2/M transition, we propose a simple model for mitotic entry in mammalian cells including spatial control over Greatwall kinase phosphorylation. After parameter calibration, the model is able to recapture the complex and non-intuitive molecular dynamics reported by Potapova et al. (Potapova et al., 2011). Moreover, it predicts the temporal patterns of other mitotic regulators which have not yet been experimentally tested and suggests a general design principle of cell cycle control: latching switches buffer the cellular stresses which accompany cell cycle processes to ensure that the transitions are smooth and robust.
G2/M transition; Mitotic entry; Mitotic collapse; Greatwall kinase; Spatial control of mitosis; Cell cycle
It is strongly suspected that potassium (K+) channels are involved in various aspects of prostate cancer development, such as cell growth. However, the molecular nature of those K+ channels implicated in prostate cancer cell proliferation and the mechanisms through which they control proliferation are still unknown. This study uses pharmacological, biophysical and molecular approaches to show that the main voltage-dependent K+ current in prostate cancer LNCaP cells is carried by large-conductance BK channels. Indeed, most of the voltage-dependent current was inhibited by inhibitors of BK channels (paxillin and iberiotoxin) and by siRNA targeting BK channels. In addition, we reveal that BK channels constitute the main K+ channel family involved in setting the resting membrane potential in LNCaP cells at around −40 mV. This consequently promotes a constitutive calcium entry through T-type Cav3.2 calcium channels. We demonstrate, using single-channel recording, confocal imaging and co-immunoprecipitation approaches, that both channels form macromolecular complexes. Finally, using flow cytometry cell cycle measurements, cell survival assays and Ki67 immunofluorescent staining, we show that both BK and Cav3.2 channels participate in the proliferation of prostate cancer cells.
BK channels; KCa1.1; Cav3.2; CACNA1H; T-type calcium channels; Proliferation; Prostate; Cancer cell growth
This study examined the effects of testosterone (T) on the contractile properties of two sexually dimorphic forelimb muscles and one non-dimorphic muscle in male bullfrogs (Rana catesbeiana, Shaw 1802). The dimorphic muscles in castrated males with testosterone replacement (T+) achieved higher forces and lower fatigability than did castrated males without replaced testosterone (T0 males), but the magnitude of the differences was low and many of the pair-wise comparisons of each muscle property were not statistically significant. However, when taken as a whole, the means of seven contractile properties varied in the directions expected of masculine values in T+ animals in the sexually dimorphic muscles. Moreover, these data, compared with previous data on male and female bullfrogs, show that values for T+ males are similar to normal males and are significantly different from females. The T0 males tended to be intermediate in character between T+ males and females, generally retaining masculine values. This suggests that the exposure of young males to T in their first breeding season produces a masculinizing effect on the sexually dimorphic muscles that is not reversed between breeding seasons when T levels are low. The relatively minor differences in contractile properties between T+ and T0 males may indicate that as circulating T levels rise during breeding season in normal males, contractile properties can be enhanced rapidly to maximal functional levels for breeding success.
Forelimb muscles; Sexual dimorphism; Testosterone; Bullfrog
The otx2 gene encodes a transcription factor (OTX2) essential in the formation of the brain and sensory systems. Specifically, OTX2-positive cells are associated with axons in the olfactory system of mice and otx2 is upregulated in odour-exposed zebrafish, indicating a possible role in olfactory imprinting. In this study, otx2 was used as a candidate gene to investigate the molecular mechanisms of olfactory imprinting to settlement cues in the coral reef anemonefish, Amphiprion percula. The A. percula otx2 (Ap-otx2) gene was elucidated, validated, and its expression tested in settlement-stage A. percula by exposing them to behaviourally relevant olfactory settlement cues in the first 24 hours post-hatching, or daily throughout the larval phase. In-situ hybridisation revealed expression of Ap-otx2 throughout the olfactory epithelium with increased transcript staining in odour-exposed settlement-stage larval fish compared to no-odour controls, in all scenarios. This suggests that Ap-otx2 may be involved in olfactory imprinting to behaviourally relevant settlement odours in A. percula.
Olfaction; Coral reef fish; Habitat selection; Memory; Candidate gene; In-situ hybridisation
PC12-27, a PC12 clone characterized by high levels of the transcription repressor REST and by very low mTORC2 activity, had been shown to be unresponsive to NGF, possibly because of its lack of the specific TrkA receptor. The neurotrophin receptor repressed by high REST in PC12-27 cells, however, is shown now to be not TrkA, which is normal, but p75NTR, whose expression is inhibited at the transcriptional level. When treated with NGF, the PC12-27 cells lacking p75NTR exhibited a defective TrkA autophosphorylation restricted, however, to the TrkA(Y490) site, and an impairment of the PI3K signaling cascade. This defect was sustained in part by a mTORC1-dependent feed-back inhibition that in wtPC12 cells appeared marginal. Transfection of p75NTR to a level and surface distribution analogous to wtPC12 did not modify various high REST-dependent properties of PC12-27 cells such as high β-catenin, low TSC2 and high proliferation rate. In contrast, the defective PI3K signaling cascade and its associated mTORC2 activity were largely rescued together with the NGF-induced neurite outgrowth response. These changes were not due to p75NTR alone but required its cooperation with TrkA. Our results demonstrate that, in PC12, high REST induces alterations of NGF signaling which, however, are indirect, dependent on the repression of p75NTR; and that the well-known potentiation by p75NTR of the TrkA signaling does not concern all the effects induced by NGF but primarily the PI3K cascade and its associated mTORC2, a complex known to play an important role in neural cell differentiation.
REST; PC12-27; ERK and PI3K cascades; mTORC2; mTORC1-dependent feed-back inhibition
The concept of contact inhibition of locomotion (CIL) describes the ability of a cell to change the direction of its movement after contact with another cell. It has been shown to be responsible for physiological and developmental processes such as wound healing, macrophage dispersion and neural crest cell migration; whereas its loss facilitates cancer cell invasion and metastatic dissemination. Different assays have been developed to analyze CIL in tissue culture models. However, these methods have several caveats. Collisions happen at low frequency between freely migrating cells and the orientation of the cells at the time of contact is not predictable. Moreover, the computational analysis required by these assays is often complicated and it retains a certain degree of discretion. Here, we show that confinement of neural crest cell migration on a single dimension by using a micropatterned substrate allows standardized and predictable cell–cell collision. CIL can thus easily be quantified by direct measurement of simple cellular parameters such as the distance between nuclei after collision. We tested some of the signaling pathways previously identified as involved in CIL, such as small GTPases and non-canonical Wnt signaling, using this new method for CIL analysis. The restricted directionality of migration of cells in lines is a powerful strategy to obtain higher predictability and higher efficiency of the CIL response upon cell–cell collisions.
Contact inhibition of locomotion; Neural crest; Micropatterned fibronectin substrates
The importance of the blood- and lymph vessels in the transport of essential fluids, gases, macromolecules and cells in vertebrates warrants optimal insight into the regulatory mechanisms underlying their development. Mouse and zebrafish models of lymphatic development are instrumental for gene discovery and gene characterization but are challenging for certain aspects, e.g. no direct accessibility of embryonic stages, or non-straightforward visualization of early lymphatic sprouting, respectively. We previously demonstrated that the Xenopus tadpole is a valuable model to study the processes of lymphatic development. However, a fluorescent Xenopus reporter directly visualizing the lymph vessels was lacking. Here, we created transgenic Tg(Flk1:eGFP) Xenopus laevis reporter lines expressing green fluorescent protein (GFP) in blood- and lymph vessels driven by the Flk1 (VEGFR-2) promoter. We also established a high-resolution fluorescent dye labeling technique selectively and persistently visualizing lymphatic endothelial cells, even in conditions of impaired lymph vessel formation or drainage function upon silencing of lymphangiogenic factors. Next, we applied the model to dynamically document blood and lymphatic sprouting and patterning of the initially avascular tadpole fin. Furthermore, quantifiable models of spontaneous or induced lymphatic sprouting into the tadpole fin were developed for dynamic analysis of loss-of-function and gain-of-function phenotypes using pharmacologic or genetic manipulation. Together with angiography and lymphangiography to assess functionality, Tg(Flk1:eGFP) reporter tadpoles readily allowed detailed lymphatic phenotyping of live tadpoles by fluorescence microscopy. The Tg(Flk1:eGFP) tadpoles represent a versatile model for functional lymph/angiogenomics and drug screening.
Xenopus; Lymphangiogenesis; Imaging
Mature osteoblasts are the cells responsible for bone formation and are derived from precursor osteoblasts. However, the mechanisms that control this differentiation are poorly understood. In fact, unlike the majority of organs in the body, which are composed of “soft” tissue from which cells can easily be isolated and studied, the “hard” mineralized tissue of bone has made it difficult to study the function of bone cells. Here, we established an in vitro model that mimics this differentiation under physiological conditions. We obtained mature osteoblasts and characterized them on the basis of the following parameters: the strong expression of osteoblastic markers, such as Runx2 and Col-I; the achievement of specific dimensions (the cell volume increases 26-fold compared to the osteoblast precursors); and the production of an abundant extracellular matrix also called osteoid. We demonstrated that the differentiation of osteoblast precursors into mature osteoblasts requires the continuous activation of Bone Morphogenetic Protein (BMP) receptors, which we established with the immobilization of a BMP-2mimetic peptide on a synthetic matrix mimicking in vivo microenvironment. Importantly, we demonstrated that the organization of the F-actin network and acetylated microtubules of the cells were modified during the differentiation process. We showed that the perturbation of the F-actin cytoskeleton organization abolished the differentiation process. In addition, we demonstrated that expression of the Runx2 gene is required for this differentiation. These findings demonstrate the retro-regulation of cytoplasmic and genic components due to the continuous induction of BMP-2 and also provide more detailed insights into the correct signaling of BMPs for cell differentiation in bone tissue.
Mature osteoblasts; BMP-2; Runx2; Actin cytoskeleton; Super-resolution optical profilometry
Cancer patients are known to be highly susceptible to Pseudomonas aeruginosa (Pa) infection, but it remains unknown whether alterations at the tumor cell level can contribute to infection. This study explored how cellular changes associated with tumor metastasis influence Pa infection using highly metastatic MTLn3 cells and non-metastatic MTC cells as cell culture models. MTLn3 cells were found to be more sensitive to Pa infection than MTC cells based on increased translocation of the type III secretion effector, ExoS, into MTLn3 cells. Subsequent studies found that higher levels of ExoS translocation into MTLn3 cells related to Pa entry and secretion of ExoS within MTLn3 cells, rather than conventional ExoS translocation by external Pa. ExoS includes both Rho GTPase activating protein (GAP) and ADP-ribosyltransferase (ADPRT) enzyme activities, and differences in MTLn3 and MTC cell responsiveness to ExoS were found to relate to the targeting of ExoS-GAP activity to Rho GTPases. MTLn3 cell migration is mediated by RhoA activation at the leading edge, and inhibition of RhoA activity decreased ExoS translocation into MTLn3 cells to levels similar to those of MTC cells. The ability of Pa to be internalized and transfer ExoS more efficiently in association with Rho activation during tumor metastasis confirms that alterations in cell migration that occur in conjunction with tumor metastasis contribute to Pa infection in cancer patients. This study also raises the possibility that Pa might serve as a biological tool for dissecting or detecting cellular alterations associated with tumor metastasis.
Tumor metastasis; MTC and MTLn3 cells; Pseudomonas aeruginosa; Rho GTPase; Cell migration
Development of epithelial cell polarity is a highly dynamic process, and often established by the sequential recruitment of conserved protein complexes, such as the Par or the Crumbs (Crb) complex. However, detailed insights into the refinement of polarity and the formation of the complexes are still lacking. Here, we established fluorescently tagged Lin7c, a core member of the Crb complex, as an ideal tool to follow development of polarity in zebrafish epithelia. We find that in gastrula stages, RFP-Lin7c is found in the cytosol of the enveloping layer, while Pard3-GFP is already polarized at this stage. During development of the retinal epithelium, RFP-Lin7c localization is refined from being cytosolic at 14 hours post fertilization (hpf) to almost entirely apical in cells of the eye cup at 28 hpf. This apical Lin7c localization depends on the Crb complex members Oko meduzy and Nagie oko. Thus, fluorescently tagged Lin7c can be used in a broad range of epithelia to follow polarity maturation in vivo and specifically to elucidate the sequence of events determining Crb complex-mediated polarity.
Polarity; Neuroepithelia; Retina; Crumbs; Zebrafish
Cullin-RING ubiquitin ligases (CRLs) mediate the ubiquitination of numerous protein substrates and target them for proteasomal degradation. The function of CRLs is under tight regulation by Cullin-binding proteins. It has been reported that the Spliceosome-associated protein 130 (SAP130/SF3b-3) binds to several Cullin proteins, yet it remains unknown whether SAP130 plays any role in regulating the function of CRLs. Here, we report that SAP130 overexpression reduces the binding of adaptor protein Skp1 and substrate receptor Skp2 to Cul1, whereas it has no effect on CAND1 binding to Cul1. Overexpression of SAP130 decreases the degradation rate of p27, a protein substrate of the SCFSkp2 ligase. Interestingly, silencing of SAP130 also inhibits the degradation of p27, suggesting a dual role for SAP130 in the regulation of SCF activity. We hypothesized that the regulatory role of SAP130 could extend to other CRLs; however, overexpression of SAP130 is unable to affect the protein stability of the Cul2 and Cul3 substrates, HIF-1 and NRF-2. SAP130 binds to Cul1, Cul2 and Cul4 with similar affinity, and it binds to Cul3 more strongly. SAP130 localizes in both the nucleus and the cytoplasm. Hence, the inability of SAP130 to regulate Cul2 and Cul3 CRLs cannot be explained by low binding affinity of SAP130 to these cullins or by subcellular sequestration of SAP130. We propose a novel role for SAP130 in the regulation of SCF, whereby SAP130 physically competes with the adaptor protein/F-box protein for Cul1 binding and interferes with the assembly of a functional SCF ligase.
Cullin-RING ligase 1; SAP130; p27
Pex3 is an evolutionarily conserved type III peroxisomal membrane protein required for peroxisome formation. It is inserted into the ER membrane and sorted via an ER subdomain (the peroxisomal ER, or pER) to peroxisomes. By constructing chimeras between Pex3 and the type III ER membrane protein Sec66, we have been able to separate the signals that mediate insertion of Pex3 into the ER from those that mediate sorting within the ER to the pER subdomain. The N-terminal 17-amino acid segment of Pex3 contains two signals that are each sufficient for sorting to the pER: a chimeric protein containing the N-terminal domain of Pex3 fused to the transmembrane and cytoplasmic segments of Sec66 sorts to the pER in wild type cells, and does not colocalise with peroxisomes. Subsequent transport to existing peroxisomes requires the Pex3 transmembrane segment. When expressed in Drosophila S2R+ cells, ScPex3 targeting to peroxisomes is dependent on the intra-ER sorting signals in the N-terminal segment. The N-terminal segments of both human and Drosophila Pex3 contain intra-ER sorting information and can replace that of ScPex3. Our analysis has uncovered the signals within Pex3 required for the various steps of its transport to peroxisomes. Our generation of versions of Pex3 that are blocked at each stage along its transport pathway provides a tool to dissect the mechanism, as well as the molecular machinery required at each step of the pathway.
Peroxisome; Peroxin; Pex3; Peroxisomal ER; Intra-ER sorting
Kallmann's syndrome is caused by the failure of olfactory axons and gonadotropin-releasing hormone (GnRH) neurons to enter the embryonic forebrain, resulting in anosmia and sterility. Sox10 mutations have been associated with Kallmann's syndrome phenotypes, but their effect on olfactory system development is unknown. We recently showed that Sox10 is expressed by neural crest-derived olfactory ensheathing cells (OECs). Here, we demonstrate that in homozygous Sox10lacZ/lacZ mouse embryos, OEC differentiation is disrupted; olfactory axons accumulate in the ventromedial olfactory nerve layer and fewer olfactory receptor neurons express the maturation marker OMP (most likely owing to the failure of axonal targeting). Furthermore, GnRH neurons clump together in the periphery and a smaller proportion enters the forebrain. Our data suggest that human Sox10 mutations cause Kallmann's syndrome by disrupting the differentiation of OECs, which promote embryonic olfactory axon targeting and hence olfactory receptor neuron maturation, and GnRH neuron migration to the forebrain.
Sox10; Olfactory ensheathing glia; GnRH neurons; Kallmann's syndrome
Transcription factor Nrf2 and its repressor Keap1 regulate a network of cytoprotective genes involving more than 1% of the genome, their best known targets being drug-metabolizing and antioxidant genes. Here we demonstrate a novel role for this pathway in directly regulating mitochondrial bioenergetics in murine neurons and embryonic fibroblasts. Loss of Nrf2 leads to mitochondrial depolarisation, decreased ATP levels and impaired respiration, whereas genetic activation of Nrf2 increases the mitochondrial membrane potential and ATP levels, the rate of respiration and the efficiency of oxidative phosphorylation. We further show that Nrf2-deficient cells have increased production of ATP in glycolysis, which is then used by the F1Fo-ATPase for maintenance of the mitochondrial membrane potential. While the levels and in vitro activities of the respiratory complexes are unaffected by Nrf2 deletion, their activities in isolated mitochondria and intact live cells are substantially impaired. In addition, the rate of regeneration of NADH after inhibition of respiration is much slower in Nrf2-knockout cells than in their wild-type counterparts. Taken together, these results show that Nrf2 directly regulates cellular energy metabolism through modulating the availability of substrates for mitochondrial respiration. Our findings highlight the importance of efficient energy metabolism in Nrf2-mediated cytoprotection.
Nrf2; Keap1; Energy metabolism; Oxidative phosphorylation; Mitochondria
Although approximately 50% of Down Syndrome (DS) patients have heart abnormalities, they exhibit an overprotection against cardiac abnormalities related with the connective tissue, for example a lower risk of coronary artery disease. A recent study reported a case of a person affected by DS who carried mutations in FBN1, the gene causative for a connective tissue disorder called Marfan Syndrome (MFS). The fact that the person did not have any cardiac alterations suggested compensation effects due to DS. This observation is supported by a previous DS meta-analysis at the molecular level where we have found an overall upregulation of FBN1 (which is usually downregulated in MFS). Additionally, that result was cross-validated with independent expression data from DS heart tissue. The aim of this work is to elucidate the role of FBN1 in DS and to establish a molecular link to MFS and MFS-related syndromes using a computational approach. To reach that, we conducted different analytical approaches over two DS studies (our previous meta-analysis and independent expression data from DS heart tissue) and revealed expression alterations in the FBN1 interaction network, in FBN1 co-expressed genes and FBN1-related pathways. After merging the significant results from different datasets with a Bayesian approach, we prioritized 85 genes that were able to distinguish control from DS cases. We further found evidence for several of these genes (47%), such as FBN1, DCN, and COL1A2, being dysregulated in MFS and MFS-related diseases. Consequently, we further encourage the scientific community to take into account FBN1 and its related network for the study of DS cardiovascular characteristics.
Down Syndrome; Marfan Syndrome; Cardiovascular; Heart; Bioinformatics
The liver, gall bladder, and ventral pancreas are formed from the posterior region of the ventral foregut. After hepatic induction, Sox17+/Pdx1+ pancreatobiliary common progenitor cells differentiate into Sox17+/Pdx1− gall bladder progenitors and Sox17−/Pdx1+ ventral pancreatic progenitors, but the cell-extrinsic signals that regulate this differentiation process are unknown. This study shows that the septum transversum mesenchyme (STM) grows in the posterior direction after E8.5, becoming adjacent to the presumptive gall bladder region, to induce gall bladder development. In this induction process, STM-derived BMP4 induces differentiation from common progenitor cells adjacent to the STM into gall bladder progenitor cells, by maintaining Sox17 expression and suppressing Pdx1 expression. Furthermore, the STM suppresses ectopic activation of the liver program in the posterior region of the ventral foregut following hepatic induction through an Fgf10/Fgfr2b/Sox9 signaling pathway. Thus, the STM plays pivotal roles in gall bladder development by both inductive and suppressive effects.
Septum transversum mesenchyme; Liver; Gall bladder; Ventral pancreas; Mouse
The lipid mediator sphingosine-1-phosphate (S1P) is a regulator of cardiac development in zebrafish, as disruption of its receptor s1pr2 or transporter spns2 causes migration defects in cardiac progenitors. To examine the genetic interaction of S1P signaling and the cell adhesion molecule fibronectin, we have established a fn;spns2 double mutant. Cardiac migration defects in fn;spns2 mutants were more severe than those in fn or spns2 mutants. We further found that the lower jaw morphology was disorganized in the fn;spns2 mutant, while it had a slightly shortened anterior–posterior distance in the ventral pharyngeal arch in fn and spns2 mutants relative to wild type. Knockdown of fn in the s1pr2 mutant, but not in the s1pr1 mutant, resulted in severe defects in cardiac migration and ventral pharyngeal arch arrangement. Further, in the background of the fn mutant, knockdown of endothelin receptor A (ednra), which was downregulated in the spns2 mutant, caused pharyngeal defects resembling those in the fn;spns2 mutant. These results strongly suggest that Spns2-S1PR2 signaling and fibronectin cooperatively regulate both cardiac and lower jaw development in zebrafish.
Sphingosine-1-phosphate; spns2; fibronectin; Jaw; Heart
Neural stem cells (NSCs) can be obtained from a variety of sources, but not all NSCs exhibit the same characteristics. We have examined how the level of glycogen synthase kinase-3 activity regulates NSCs obtained from different sources: the mouse embryonic striatum, embryonic hippocampus, and mouse ES cells. Growth of striatal NSCs is enhanced by mild inhibition of GSK-3 but not by strong inhibition that is accompanied by Wnt/TCF transcriptional activation. In contrast, the growth of hippocampal NSCs is enhanced by both mild inhibition of GSK-3 as well as stronger inhibition. Active Wnt/TCF signaling, which occurs normally in the embryonic hippocampus, is required for growth of neural stem and progenitor cells. In the embryonic striatal germinal zone, however, TCF signaling is normally absent and its activation inhibits growth of NSCs from this region. Using a genetic model for progressive loss of GSK-3, we find that primitive ES cell-derived NSCs resemble striatal NSCs. That is, partial loss of GSK-3 alleles leads to an increase in NSCs while complete ablation of GSK-3, and activation of TCF-signaling, leads to their decline. Furthermore, expression of dominant negative TCF-4 in the GSK-3-null background was effective in blocking expression of Wnt-response genes and was also able to rescue neuronal gene expression. These results reveal that GSK-3 regulates NSCs by divergent pathways depending on the tissue of origin. The responses of these neural precursor cells may be contingent on baseline Wnt/TCF signaling occurring in a particular tissue.
Neural stem cells; Wnt signaling; ES cells; Neurosphere assay
The Hippo pathway has a central role in coordinating tissue growth and apoptosis. Mutations that compromise Hippo pathway activity cause tissue overgrowth and have been causally linked to cancer. In Drosophila, the transcriptional coactivator Yorkie mediates Hippo pathway activity to control the expression of cyclin E and Myc to promote cell proliferation, as well as the expression of bantam miRNA and DIAP1 to inhibit cell death. Here we present evidence that the Hippo pathway acts via Yorkie and p53 to control the expression of the proapoptotic gene reaper. Yorkie further mediates reaper levels post-transcriptionally through regulation of members of the miR-2 microRNA family to prevent apoptosis. These findings provide evidence that the Hippo pathway acts via several distinct routes to limit proliferation-induced apoptosis.
p53; Hippo pathway; Yorkie/YAP; reaper; MicroRNA; Apoptosis; Proliferation
Pseudoachondroplasia and multiple epiphyseal dysplasia are genetic skeletal diseases resulting from mutations in cartilage structural proteins. Electron microscopy and immunohistochemistry previously showed that the appearance of the cartilage extracellular matrix (ECM) in targeted mouse models of these diseases is disrupted; however, the precise changes in ECM organization and the pathological consequences remain unknown. Our aim was to determine the effects of matrilin-3 and COMP mutations on the composition and extractability of ECM components to inform how these detrimental changes might influence cartilage organization and degeneration.
Cartilage was sequentially extracted using increasing denaturants and the extraction profiles of specific proteins determined using SDS-PAGE/Western blotting. Furthermore, the relative composition of protein pools was determined using mass spectrometry for a non-biased semi-quantitative analysis.
Western blotting revealed changes in the extraction of matrilins, COMP and collagen IX in mutant cartilage. Mass spectrometry confirmed quantitative changes in the extraction of structural and non-structural ECM proteins, including proteins with roles in cellular processes such as protein folding and trafficking. In particular, genotype-specific differences in the extraction of collagens XII and XIV and tenascins C and X were identified; interestingly, increased expression of several of these genes has recently been implicated in susceptibility and/or progression of murine osteoarthritis.
We demonstrated that mutation of matrilin-3 and COMP caused changes in the extractability of other cartilage proteins and that proteomic analyses of Matn3 V194D, Comp T585M and Comp DelD469 mouse models revealed both common and discrete disease signatures that provide novel insight into skeletal disease mechanisms and cartilage degradation.
Cartilage; Genetic skeletal disease; Proteomics; Pseudoachondroplasia; Multiple epiphyseal dysplasia