Lindgaardmcfadden2227

The placenta releases large quantities of extracellular vesicles (EVs) that likely facilitate communication between the embryo/fetus and the mother. We isolated EVs from second trimester human cytotrophoblasts (CTBs) by differential ultracentrifugation and characterized them using transmission electron microscopy, immunoblotting and mass spectrometry. selleck inhibitor The 100,000  g pellet was enriched for vesicles with a cup-like morphology typical of exosomes. They expressed markers specific to this vesicle type, CD9 and HRS, and the trophoblast proteins placental alkaline phosphatase and HLA-G. Global profiling by mass spectrometry showed that placental EVs were enriched for proteins that function in transport and viral processes. A cytokine array revealed that the CTB 100,000  g pellet contained a significant amount of tumor necrosis factor α (TNFα). CTB EVs increased decidual stromal cell (dESF) transcription and secretion of NF-κB targets, including IL8, as measured by qRT-PCR and cytokine array. A soluble form of the TNFα receptor inhibited the ability of CTB 100,000  g EVs to increase dESF secretion of IL8. Overall, the data suggest that CTB EVs enhance decidual cell release of inflammatory cytokines, which we theorize is an important component of successful pregnancy.Fragile X mental retardation protein (FMRP) is an RNA-binding protein abundant in the nervous system. Functional loss of FMRP leads to sensory dysfunction and severe intellectual disabilities. In the auditory system, FMRP deficiency alters neuronal function and synaptic connectivity and results in perturbed processing of sound information. Nevertheless, roles of FMRP in embryonic development of the auditory hindbrain have not been identified. Here, we developed high-specificity approaches to genetically track and manipulate throughout development of the Atoh1+ neuronal cell type, which is highly conserved in vertebrates, in the cochlear nucleus of chicken embryos. We identified distinct FMRP-containing granules in the growing axons of Atoh1+ neurons and post-migrating NM cells. FMRP downregulation induced by CRISPR/Cas9 and shRNA techniques resulted in perturbed axonal pathfinding, delay in midline crossing, excess branching of neurites, and axonal targeting errors during the period of circuit development. Together, these results provide the first in vivo identification of FMRP localization and actions in developing axons of auditory neurons, and demonstrate the importance of investigating early embryonic alterations toward understanding the pathogenesis of neurodevelopmental disorders.Homeostasis of intestinal stem cells (ISCs) is maintained by the orchestration of niche factors and intrinsic signaling networks. Here, we have found that deletion of Erk1 and Erk2 (Erk1/2) in intestinal epithelial cells at embryonic stages resulted in an unexpected increase in cell proliferation and migration, expansion of ISCs, and formation of polyp-like structures, leading to postnatal death. Deficiency of epithelial Erk1/2 results in defects in secretory cell differentiation as well as impaired mesenchymal cell proliferation and maturation. Deletion of Erk1/2 strongly activated Wnt signaling through both cell-autonomous and non-autonomous mechanisms. In epithelial cells, Erk1/2 depletion resulted in loss of feedback regulation, leading to Ras/Raf cascade activation that transactivated Akt activity to stimulate the mTor and Wnt/β-catenin pathways. Moreover, Erk1/2 deficiency reduced the levels of Indian hedgehog and the expression of downstream pathway components, including mesenchymal Bmp4 - a Wnt suppressor in intestines. Inhibition of mTor signaling by rapamycin partially rescued Erk1/2 depletion-induced intestinal defects and significantly prolonged the lifespan of mutant mice. These data demonstrate that Erk/Mapk signaling functions as a key modulator of Wnt signaling through coordination of epithelial-mesenchymal interactions during intestinal development.Central nervous system (CNS) blood vessels contain a functional blood-brain barrier (BBB) that is necessary for neuronal survival and activity. Although Wnt/β-catenin signaling is essential for BBB development, its downstream targets within the neurovasculature remain poorly understood. To identify targets of Wnt/β-catenin signaling underlying BBB maturation, we performed a microarray analysis that identified Fgfbp1 as a novel Wnt/β-catenin-regulated gene in mouse brain endothelial cells (mBECs). Fgfbp1 is expressed in the CNS endothelium and secreted into the vascular basement membrane during BBB formation. Endothelial genetic ablation of Fgfbp1 results in transient hypervascularization but delays BBB maturation in specific CNS regions, as evidenced by both upregulation of Plvap and increased tracer leakage across the neurovasculature due to reduced Wnt/β-catenin activity. In addition, collagen IV deposition in the vascular basement membrane is reduced in mutant mice, leading to defective endothelial cell-pericyte interactions. Fgfbp1 is required cell-autonomously in mBECs to concentrate Wnt ligands near cell junctions and promote maturation of their barrier properties in vitro Thus, Fgfbp1 is a crucial extracellular matrix protein during BBB maturation that regulates cell-cell interactions and Wnt/β-catenin activity.The identification of novel mechanisms to maintain embryonic stem cell (ESC) pluripotency is of crucial importance, because the currently used culture conditions are not suitable for ESCs from all species. In this study, we show that the protein kinase D (PKD) inhibitor CID755673 (CID) is able to maintain the undifferentiated state of mouse ESCs in combination with the mitogen-activated protein kinase kinase (MEK) inhibitor. The expression levels of PKD members, including PKD1, PKD2 and PKD3, were low in mouse ESCs but significantly increased under differentiation conditions. Therefore, depletion of three PKD genes was able to phenocopy PKD inhibition. Mechanistically, PKD inhibition activated PI3K/AKT signaling by increasing the level of AKT phosphorylation, and the addition of a PI3K/AKT signaling pathway inhibitor partially reduced the cellular response to PKD inhibition. Importantly, the self-renewal-promoting effect of CID was maintained in human ESCs. Simultaneous knockdown of the three human PKD isoforms enabled short-term self-renewal in human ESCs, whereas PI3K/AKT signaling inhibition eliminated this self-renewal ability downstream of the PKD inhibitor.