Sprouty2 mediated tuning of signalling is essential for somite myogenesis

Background: Negative regulators of signal transduction cascades play critical roles in controlling different aspects of normal embryonic development. Sprouty2 (Spry2) negatively regulates receptor tyrosine kinases (RTK) and FGF signalling and is important in differentiation, cell migration and proliferation. In vertebrate embryos, Spry2 is expressed in paraxial mesoderm and in forming somites. Expression is maintained in the myotome until late stages of somite differentiation. However, its role and mode of action during somite myogenesis is still unclear. Results: Here, we analysed chick Spry2 expression and showed that it overlaps with that of myogenic regulatory factors MyoD and Mgn. Targeted mis-expression of Spry2 led to inhibition of myogenesis, whilst its C-terminal domain led to an increased number of myogenic cells by stimulating cell proliferation. Conclusions: Spry2 is expressed in somite myotomes and its expression overlaps with myogenic regulatory factors. Overexpression and dominant-negative interference showed that Spry2 plays a crucial role in regulating chick myogenesis by fine tuning of FGF signaling through a negative feedback loop. We also propose that mir-23, mir-27 and mir-128 could be part of the negative feedback loop mechanism. Our analysis is the first to shed some light on in vivo Spry2 function during chick somite myogenesis

Assessing the efficiency and significance of Methylated DNA Immunoprecipitation (MeDIP) assays in using in vitro methylated genomic DNA

<p>Abstract</p> <p>Background</p> <p>DNA methylation contributes to the regulation of gene expression during development and cellular differentiation. The recently developed Methylated DNA ImmunoPrecipitation (MeDIP) assay allows a comprehensive analysis of this epigenetic mark at the genomic level in normal and disease-derived cells. However, estimating the efficiency of the MeDIP technique is difficult without previous knowledge of the methylation status of a given cell population. Attempts to circumvent this problem have involved the use of <it>in vitro </it>methylated DNA in parallel to the investigated samples. Taking advantage of this stratagem, we sought to improve the sensitivity of the approach and to assess potential biases resulting from DNA amplification and hybridization procedures using MeDIP samples.</p> <p>Findings</p> <p>We performed MeDIP assays using <it>in vitro </it>methylated DNA, with or without previous DNA amplification, and hybridization to a human promoter array. We observed that CpG content at gene promoters indeed correlates strongly with the MeDIP signal obtained using <it>in vitro </it>methylated DNA, even when lowering significantly the amount of starting material. In analyzing MeDIP products that were subjected to whole genome amplification (WGA), we also revealed a strong bias against CpG-rich promoters during this amplification procedure, which may potentially affect the significance of the resulting data.</p> <p>Conclusion</p> <p>We illustrate the use of <it>in vitro </it>methylated DNA to assess the efficiency and accuracy of MeDIP procedures. We report that efficient and reproducible genome-wide data can be obtained via MeDIP experiments using relatively low amount of starting genomic DNA; and emphasize for the precaution that must be taken in data analysis when an additional DNA amplification step is required.</p

Vesicoureteral Reflux and Other Urinary Tract Malformations in Mice Compound Heterozygous for Pax2 and Emx2

Congenital anomalies of the kidney and urinary tract (CAKUT) are the most common cause of chronic kidney disease in children. This disease group includes a spectrum of urinary tract defects including vesicoureteral reflux, duplex kidneys and other developmental defects that can be found alone or in combination. To identify new regulators of CAKUT, we tested the genetic cooperativity between several key regulators of urogenital system development in mice. We found a high incidence of urinary tract anomalies in Pax2;Emx2 compound heterozygous mice that are not found in single heterozygous mice. Pax2+/−;Emx2+/− mice harbor duplex systems associated with urinary tract obstruction, bifid ureter and a high penetrance of vesicoureteral reflux. Remarkably, most compound heterozygous mice refluxed at low intravesical pressure. Early analysis of Pax2+/−;Emx2+/− embryos point to ureter budding defects as the primary cause of urinary tract anomalies. We additionally establish Pax2 as a direct regulator of Emx2 expression in the Wolffian duct. Together, these results identify a haploinsufficient genetic combination resulting in CAKUT-like phenotype, including a high sensitivity to vesicoureteral reflux. As both genes are located on human chromosome 10q, which is lost in a proportion of VUR patients, these findings may help understand VUR and CAKUT in humans

Left-Right Function of dmrt2 Genes Is Not Conserved between Zebrafish and Mouse

Background: Members of the Dmrt family, generally associated with sex determination, were shown to be involved in several other functions during embryonic development. Dmrt2 has been studied in the context of zebrafish development where, due to a duplication event, two paralog genes dmrt2a and dmrt2b are present. Both zebrafish dmrt2a/terra and dmrt2b are important to regulate left-right patterning in the lateral plate mesoderm. In addition, dmrt2a/terra is necessary for symmetric somite formation while dmrt2b regulates somite differentiation impacting on slow muscle development. One dmrt2 gene is also expressed in the mouse embryo, where it is necessary for somite differentiation but with an impact on axial skeleton development. However, nothing was known about its role during left-right patterning in the lateral plate mesoderm or in the symmetric synchronization of somite formation. Methodology/Principal Findings: Using a dmrt2 mutant mouse line, we show that this gene is not involved in symmetric somite formation and does not regulate the laterality pathway that controls left-right asymmetric organ positioning. We reveal that dmrt2a/terra is present in the zebrafish laterality organ, the Kupffer’s vesicle, while its homologue is excluded from the mouse equivalent structure, the node. On the basis of evolutionary sub-functionalization and neo-functionalization theories we discuss this absence of functional conservation. Conclusions/Significance: Our results show that the role of dmrt2 gene is not conserved during zebrafish and mous

Expression of the 60 kDa and 71 kDa heat shock proteins and presence of antibodies against the 71 kDa heat shock protein in pediatric patients with immune thrombocytopenic purpura

BACKGROUND: Immune thrombocytopenic purpura (ITP) is an autoimmune disease characterized by platelet destruction resulting from autoantibodies against platelet proteins, particularly platelet glycoprotein IIb/IIIa. Heat shock proteins (Hsp) have been shown to be major antigenic determinants in some autoimmune diseases. Antibodies to Hsps have also been reported to be associated with a number of pathological states. METHODS: Using western blot, we measured the levels of the 60 kDa heat shock protein (Hsp60) and of the inducible 71 kDa member of the Hsp70 family (Hsp71) in lymphocytes and the presence of antibodies against these hsps in plasma of 29 pediatric patients with ITP before the treatment and in 6 other patients before and after treatment. RESULTS: Interestingly only one out of 29 patients showed detectable Hsp60 in lymphocytes while this heat shock protein was detected in the 30 control children. Hsp71 levels were slightly lower in lymphocytes of patients with ITP than in controls (1567.8 ± 753.2 via 1763.2 ± 641.8 integrated optical density (IOD) units). There was a small increase of Hsp71 after recovery from ITP. The titers of plasma antibodies against Hsp60 and Hsp71 were also examined. Antibodies against Hsp71 were more common in ITP patients (15/29) than in control children (5/30). The titer of anti-Hsp71 was also higher in children patients with ITP. The prevalence of ITP children with antibodies against Hsp71 (51.7%) was as high as those with antibodies against platelet membrane glycoproteins (58.3%). CONCLUSIONS: In summary, pediatric patients with ITP showed no detectable expression of Hsp60 in lymphocytes and a high prevalence of antibody against Hsp71 in plasma. These changes add to our understanding of the pathogenesis of ITP and may be important for the diagnosis, prognosis and treatment of ITP

Plasma antibodies against heat shock protein 70 correlate with the incidence and severity of asthma in a Chinese population

BACKGROUND: The heat shock proteins (Hsps) are induced by stresses such as allergic factors and inflammatory responses in bronchi epithelial cells and therefore may be detectable in patients with asthma. However, the etiologic link between anti-Hsps and asthma (its severity and related inflammatory responses such as interleukin-4 and immunoglobulin E) has not been established. We determined whether antibodies against Hsp60 and Hsp70 were present in patients with asthma and evaluated their associations with risk and severity of asthma. METHODS: We determined the levels of anti-Hsp60 and anti-Hsp70 by immunoblot and their associations with risk and symptom severity of asthma in 95 patients with asthma and 99 matched non-symptomatic controls using multivariate logistic regression analysis. RESULTS: Compared to the controls, asthma patients were more likely to have detectable anti-Hsp60 (17.2% vs 5.1%) and anti-Hsp70 (33.7% vs 8.1%) (p ≤ 0.001). In particular, the presence of anti-Hsp70 was associated with a greater than 2 fold risk for asthma (adjusted OR = 2.21; 95% CI = 1.35~3.59). Furthermore, both anti-Hsp60 and anti-Hsp70 levels were positively correlated with symptom severity (p < 0.05) as well as interleukin-4 and immunoglobulin E (p < 0.05). Individuals with antibodies against anti-Hsp60 and anti-Hsp70 were more likely to have a family history of asthma (p < 0.001) and higher plasma concentrations of total immunoglobulin E (p = 0.001) and interleukin-4 (p < 0.05) than those without antibodies. CONCLUSIONS: These data suggest that anti-Hsp60 and especially anti-Hsp70 correlate with the attacks and severity of asthma. The underlying molecular mechanisms linking antibodies to heat shock proteins and asthma remain to be investigated

Sprouty Proteins Inhibit Receptor-mediated Activation of Phosphatidylinositol-specific Phospholipase C

PLCγ03B3 binds Spry1 and Spry2. Overexpression of Spry decreased PLCγ03B3 activity and IP3 and DAG production, whereas Spry-deficient cells yielded more IP3. Spry overexpression inhibited T-cell receptor signaling and Spry1 null T-cells hyperproliferated with TCR ligation. Through action of PLCγ03B3, Spry may influence signaling through multiple receptors

Gata3 Acts Downstream of β-Catenin Signaling to Prevent Ectopic Metanephric Kidney Induction

Metanephric kidney induction critically depends on mesenchymal–epithelial interactions in the caudal region of the nephric (or Wolffian) duct. Central to this process, GDNF secreted from the metanephric mesenchyme induces ureter budding by activating the Ret receptor expressed in the nephric duct epithelium. A failure to regulate this pathway is believed to be responsible for a large proportion of the developmental anomalies affecting the urogenital system. Here, we show that the nephric duct-specific inactivation of the transcription factor gene Gata3 leads to massive ectopic ureter budding. This results in a spectrum of urogenital malformations including kidney adysplasia, duplex systems, and hydroureter, as well as vas deferens hyperplasia and uterine agenesis. The variability of developmental defects is reminiscent of the congenital anomalies of the kidney and urinary tract (CAKUT) observed in human. We show that Gata3 inactivation causes premature nephric duct cell differentiation and loss of Ret receptor gene expression. These changes ultimately affect nephric duct epithelium homeostasis, leading to ectopic budding of interspersed cells still expressing the Ret receptor. Importantly, the formation of these ectopic buds requires both GDNF/Ret and Fgf signaling activities. We further identify Gata3 as a central mediator of β-catenin function in the nephric duct and demonstrate that the β-catenin/Gata3 pathway prevents premature cell differentiation independently of its role in regulating Ret expression. Together, these results establish a genetic cascade in which Gata3 acts downstream of β-catenin, but upstream of Ret, to prevent ectopic ureter budding and premature cell differentiation in the nephric duct

Mesenchymal Stromal Cells Primed with Paclitaxel Provide a New Approach for Cancer Therapy

BACKGROUND: Mesenchymal stromal cells may represent an ideal candidate to deliver anti-cancer drugs. In a previous study, we demonstrated that exposure of mouse bone marrow derived stromal cells to Doxorubicin led them to acquire anti-proliferative potential towards co-cultured haematopoietic stem cells (HSCs). We thus hypothesized whether freshly isolated human bone marrow Mesenchymal stem cells (hMSCs) and mature murine stromal cells (SR4987 line) primed in vitro with anti-cancer drugs and then localized near cancer cells, could inhibit proliferation. METHODS AND PRINCIPAL FINDINGS: Paclitaxel (PTX) was used to prime culture of hMSCs and SR4987. Incorporation of PTX into hMSCs was studied by using FICT-labelled-PTX and analyzed by FACS and confocal microscopy. Release of PTX in culture medium by PTX primed hMSCs (hMSCsPTX) was investigated by HPLC. Culture of Endothelial cells (ECs) and aorta ring assay were used to test the anti-angiogenic activity of hMSCsPTX and PTX primed SR4987(SR4987PTX), while anti-tumor activity was tested in vitro on the proliferation of different tumor cell lines and in vivo by co-transplanting hMSCsPTX and SR4987PTX with cancer cells in mice. Nevertheless, despite a loss of cells due to chemo-induced apoptosis, both hMSCs and SR4987 were able to rapidly incorporate PTX and could slowly release PTX in the culture medium in a time dependent manner. PTX primed cells acquired a potent anti-tumor and anti-angiogenic activity in vitro that was dose dependent, and demonstrable by using their conditioned medium or by co-culture assay. Finally, hMSCsPTX and SR4987PTX co-injected with human cancer cells (DU145 and U87MG) and mouse melanoma cells (B16) in immunodeficient and in syngenic mice significantly delayed tumor takes and reduced tumor growth. CONCLUSIONS: These data demonstrate, for the first time, that without any genetic manipulation, mesenchymal stromal cells can uptake and subsequently slowly release PTX. This may lead to potential new tools to increase efficacy of cancer therapy

Genome wide mapping reveals PDE4B as an IL-2 induced STAT5 target gene in activated human PBMCs and lymphoid cancer cells

IL-2 is the primary growth factor for promoting survival and proliferation of activated T cells that occurs following engagement of the Janus Kinase (JAK)1-3/and Signal Transducer and Activator of Transcription (STAT) 5 signaling pathway. STAT5 has two isoforms: STAT5A and STAT5B ( commonly referred to as STAT5) which, in T cells, play redundant roles transcribing cell cycle and survival genes. As such, inhibition of STAT5 by a variety of mechanisms can rapidly induce apoptosis in certain lymphoid tumor cells, suggesting that it and its target genes represent therapeutic targets to control certain lymphoid diseases. To search for these molecules we aligned IL-2 regulated genes detected by Affymetrix gene expression microarrays with the STAT5 cistrome identified by chip-on-ChIP analysis in an IL-2-dependent human leukemia cell line, Kit225. Select overlapping genes were then validated using qRT(2)PCR medium-throughput arrays in human PHA-activated PBMCs. Of 19 putative genes, one key regulator of T cell receptor signaling, PDE4B, was identified as a novel target, which was readily up-regulated at the protein level (3 h) in IL-2 stimulated, activated human PBMCs. Surprisingly, only purified CD8+ primary T-cells expressed PDE4B, but not CD4+ cells. Moreover, PDE4B was found to be highly expressed in CD4+ lymphoid cancer cells, which suggests that it may represent a physiological role unique to the CD8+ and lymphoid cancer cells and thus might represent a target for pharmaceutical intervention for certain lymphoid diseases