Project description:How organ size and form are controlled during development is a major question of biology. Blood vessels have been shown to be essential for early development of the liver and pancreas, and are fundamental to normal and pathological tissue growth. Here we report that non-nutritional signals from blood vessels surprisingly act to restrain pancreas growth. Elimination of endothelial cells increases the size of embryonic pancreatic buds. Conversely, VEGF-induced hypervascularization decreases pancreas size. The growth phenotype results from vascular restriction of pancreatic tip cell formation, lateral branching and differentiation of the pancreatic epithelium into endocrine and acinar cells. The effects are seen both in vivo and ex vivo, indicating a perfusion-independent mechanism. Thus the vasculature controls pancreas morphogenesis and growth by reducing branching and differentiation of primitive epithelial cells.
Project description:The genetic mechanisms that determine the size of the adult pancreas are not fully understood. Here we address the importance of the imprinted Igf2 gene for growth and function of the mouse pancreas. We used reporter lines to monitor Igf2 levels during late fetal and postnatal pancreatic development and demonstrate that Igf2 is mostly expressed within the mesenchyme. We manipulated Igf2 levels in vivo in the major pancreas cell types using Cre lines and found that loss of Igf2 from the developing mesenchyme results in pancreatic hypoplasia, associated with loss of acinar and beta-cell mass, postnatal whole-body growth restriction and maternal glucose intolerance during pregnancy. Overexpression of Igf2 directed to the mesenchyme results in pancreatic overgrowth. Importantly, deletion of Igf2 from the developing epithelium has no growth effects. Our findings demonstrate that a major role for Igf2 is the regulation of pancreas size, and reveal an unforeseen key function for mesenchymal IGF signalling in pancreatic growth.
Project description:How organ size and form are controlled during development is a major question of biology. Blood vessels have been shown to be essential for early development of the liver and pancreas, and are fundamental to normal and pathological tissue growth. Here we report that non-nutritional signals from blood vessels surprisingly act to restrain pancreas growth. Elimination of endothelial cells increases the size of embryonic pancreatic buds. Conversely, VEGF-induced hypervascularization decreases pancreas size. The growth phenotype results from vascular restriction of pancreatic tip cell formation, lateral branching and differentiation of the pancreatic epithelium into endocrine and acinar cells. The effects are seen both in vivo and ex vivo, indicating a perfusion-independent mechanism. Thus the vasculature controls pancreas morphogenesis and growth by reducing branching and differentiation of primitive epithelial cells. For transcriptome analysis, RNA was isolated using QIAGEN RNeasy micro Kit from pancreatic buds of Pdx1-tTA (n=3) and littermate Pdx1-tTA; TET-VEGF (n=3) e12.5 embryos, or from e12.5 wild type pancreatic buds explanted and treated with VEGFR2i (n=5) or vehicle (n=5) for 2 days. Pooled samples were hybridized to Affymetrix mouse gene 1.0 st arrays. The arrays were RMA normalized using Partek Genomic Suite 6.5. Differentially regulated genes were selected based on p-values and ratios using t-test.
Project description:Scaling up the functioning of synthetic circuits from microplates to bioreactors is far from trivial to achieve. We here test the scalability performance of a previously developed growth switch for increasing product yields in bacteria, based on external control of RNA polymerase expression. We show that, in liter-scale bioreactors operating in fed-batch mode, growth-arrested Escherichia coli cells are able to convert glucose to glycerol at an increased yield. A multi-omics quantification of the physiology of the cells shows that apart from acetate production, few metabolic side-effects occur, while a number of specific responses to growth slow-down and growth arrest are launched on the transcriptional level. These responses include the downregulation of genes involved in growth-associated processes, such as amino acid and nucleotide metabolism and translation, and the upregulation of a heat response. Interestingly, these transcriptional responses are buffered on the proteomic level, probably due to the strong decrease of the total mRNA concentration after the diminution of transcriptional activity and the absence of growth dilution of proteins. This transforms the growth-arrested cells into “bags of proteins“ with a functioning metabolism. More generally, the analysis shows that physiological characterization of bacterial cells hosting a synthetic circuit may reveal complex patterns of adaptation on different time-scales, dynamically interacting with the bioreactor environment.
Project description:Beyond forming bone, osteoblasts play pivotal roles in various biological processes, including hematopoiesis and bone metastasis. Extracellular vesicles (EVs) have recently been implicated in intercellular communication via transfer of proteins and nucleic acids between cells. Here, we focused on the proteomic characterization of non-mineralizing (NMOBs) and mineralizing (MOBs) human osteoblast (SV-HFOs) EVs and investigated their effect on human prostate cancer (PC3) cells by microscopic, proteomic and gene expression analyses. Proteomic analysis showed that 97% of the proteins were shared among NMOB and MOB EVs, and 30% were novel osteoblast-specific EV proteins. Label-free quantification demonstrated mineralization stage-dependent five-fold enrichment of 59 and 451 EV proteins in NMOBs and MOBs, respectively. Interestingly, bioinformatic analyses of the osteoblast EV proteomes and EV-regulated prostate cancer gene expression profiles showed that they converged on pathways involved in cell survival and growth. This was verified by in vitro proliferation assays where osteoblast EV uptake led to two-fold increase in PC3 cell growth compared to cell-free culture medium-derived vesicle controls. Our findings elucidate the mineralization stage-specific protein content of osteoblast-secreted EVs, show a novel way by which osteoblasts communicate with prostate cancer, and open up innovative avenues for therapeutic intervention. PC3 cells were treated with extracellular vesicles from non-mineralizing and mineralizing SV-HFOs for three different incubation times (4hrs, 24hrs, 48hr)
Project description:Beyond forming bone, osteoblasts play pivotal roles in various biological processes, including hematopoiesis and bone metastasis. Extracellular vesicles (EVs) have recently been implicated in intercellular communication via transfer of proteins and nucleic acids between cells. Here, we focused on the proteomic characterization of non-mineralizing (NMOBs) and mineralizing (MOBs) human osteoblast (SV-HFOs) EVs and investigated their effect on human prostate cancer (PC3) cells by microscopic, proteomic and gene expression analyses. Proteomic analysis showed that 97% of the proteins were shared among NMOB and MOB EVs, and 30% were novel osteoblast-specific EV proteins. Label-free quantification demonstrated mineralization stage-dependent five-fold enrichment of 59 and 451 EV proteins in NMOBs and MOBs, respectively. Interestingly, bioinformatic analyses of the osteoblast EV proteomes and EV-regulated prostate cancer gene expression profiles showed that they converged on pathways involved in cell survival and growth. This was verified by in vitro proliferation assays where osteoblast EV uptake led to two-fold increase in PC3 cell growth compared to cell-free culture medium-derived vesicle controls. Our findings elucidate the mineralization stage-specific protein content of osteoblast-secreted EVs, show a novel way by which osteoblasts communicate with prostate cancer, and open up innovative avenues for therapeutic intervention.