Gene expression across mammalian organ development

The evolution of gene expression in mammalian organ development remains largely uncharacterized. Here we report the transcriptomes of seven organs (cerebrum, cerebellum, heart, kidney, liver, ovary and testis) across developmental time points from early organogenesis to adulthood for human, macaque, mouse, rat, rabbit, opossum and chicken. Comparisons of gene expression patterns identified developmental stage correspondences across species, and differences in the timing of key events during the development of the gonads. We found that the breadth of gene expression and the extent of purifying selection gradually decrease during development, whereas the amount of positive selection and expression of new genes increase. We identified differences in the temporal trajectories of expression of individual genes across species, with brain tissues showing the smallest percentage of trajectory changes, and the liver and testis showing the largest. Our work provides a resource of developmental transcriptomes of seven organs across seven species, and comparative analyses that characterize the development and evolution of mammalian organs

Sequential Accumulation of ‘Driver’ Pathway Mutations Induces the Upregulation of Hydrogen-Sulfide-Producing Enzymes in Human Colonic Epithelial Cell Organoids

Recently, a CRISPR-Cas9 genome-editing system was developed with introduced sequential ‘driver’ mutations in the WNT, MAPK, TGF-β, TP53 and PI3K pathways into organoids derived from normal human intestinal epithelial cells. Prior studies have demonstrated that isogenic organoids harboring mutations in the tumor suppressor genes APC, SMAD4 and TP53, as well as the oncogene KRAS, assumed more proliferative and invasive properties in vitro and in vivo. A separate body of studies implicates the role of various hydrogen sulfide (H2S)-producing enzymes in the pathogenesis of colon cancer. The current study was designed to determine if the sequential mutations in the above pathway affect the expression of various H2S producing enzymes. Western blotting was used to detect the expression of the H2S-producing enzymes cystathionine β-synthase (CBS), cystathionine γ-lyase (CSE) and 3-mercaptopyruvate sulfurtransferase (3-MST), as well as several key enzymes involved in H2S degradation such as thiosulfate sulfurtransferase/rhodanese (TST), ethylmalonic encephalopathy 1 protein/persulfide dioxygenase (ETHE1) and sulfide-quinone oxidoreductase (SQR). H2S levels were detected by live-cell imaging using a fluorescent H2S probe. Bioenergetic parameters were assessed by Extracellular Flux Analysis; markers of epithelial-mesenchymal transition (EMT) were assessed by Western blotting. The results show that the consecutive mutations produced gradual upregulations in CBS expression—in particular in its truncated (45 kDa) form—as well as in CSE and 3-MST expression. In more advanced organoids, when the upregulation of H2S-producing enzymes coincided with the downregulation of the H2S-degrading enzyme SQR, increased H2S generation was also detected. This effect coincided with the upregulation of cellular bioenergetics (mitochondrial respiration and/or glycolysis) and an upregulation of the Wnt/β-catenin pathway, a key effector of EMT. Thus sequential mutations in colon epithelial cells according to the Vogelstein sequence are associated with a gradual upregulation of multiple H2S generating pathways, which, in turn, translates into functional changes in cellular bioenergetics and dedifferentiation, producing more aggressive and more invasive colon cancer phenotypes

Role of Cystathionine β-Synthase and 3-Mercaptopyruvate Sulfurtransferase in the Regulation of Proliferation, Migration, and Bioenergetics of Murine Breast Cancer Cells

Cystathionine β-synthase (CBS), CSE (cystathionine γ-lyase) and 3-mercaptopyruvate sulfurtransferase (3-MST) have emerged as three significant sources of hydrogen sulfide (H2S) in various forms of mammalian cancer. Here, we investigated the functional role of CBS’ and 3-MST’s catalytic activity in the murine breast cancer cell line EO771. The CBS/CSE inhibitor aminooxyacetic acid (AOAA) and the 3-MST inhibitor 2-[(4-hydroxy-6-methylpyrimidin-2-yl)sulfanyl]-1-(naphthalen-1-yl)ethan-1-one (HMPSNE) were used to assess the role of endogenous H2S in the modulation of breast cancer cell proliferation, migration, bioenergetics and viability in vitro. Methods included measurements of cell viability (MTT and LDH assays), cell proliferation and in vitro wound healing (IncuCyte) and cellular bioenergetics (Seahorse extracellular flux analysis). CBS and 3-MST, as well as expression were detected by Western blotting; H2S production was measured by the fluorescent dye AzMC. The results show that EO771 cells express CBS, CSE and 3-MST protein, as well as several enzymes involved in H2S degradation (SQR, TST, and ETHE1). Pharmacological inhibition of CBS or 3-MST inhibited H2S production, suppressed cellular bioenergetics and attenuated cell proliferation. Cell migration was only inhibited by the 3-MST inhibitor, but not the CBS/CSE inhibitor. Inhibition of CBS/CSE of 3-MST did not significantly affect basal cell viability; inhibition of 3-MST (but not of CBS/CSE) slightly enhanced the cytotoxic effects of oxidative stress (hydrogen peroxide challenge). From these findings, we conclude that endogenous H2S, generated by 3-MST and to a lower degree by CBS/CSE, significantly contributes to the maintenance of bioenergetics, proliferation and migration in murine breast cancer cells and may also exert a minor role as a cytoprotectant.</p

CXCR4 and CXCR7 receptors modulate invasive, but not adhesive properties of transduced NB cells <i>in vitro</i>.

<p><b>A.</b> CXCR4 and CXCR7 cell surface expression in the NB8 transduced cell lines. CXCR4, CXCR7 or a combination of both CXCL12 receptors was overexpressed in the IGR-NB8 cell line by retroviral infection. NB8pMigr cell line represents control cells transduced with the pMigr empty vector. Percent of CXCR4 (blue) and CXCR7 (green) positive transduced cells are indicated. Grey histogram: cells stained without primary antibodies; blue and green histograms: cells stained with anti-CXCR4 and anti-CXCR7, respectively. <b>B</b>. Adhesion capacities of NB8x4, NB8x7, NB8x4x7, and the mock transduced NB8pMigr control cell lines on HUVEC monolayer <i>in vitro</i>. Mean ± SD of the ratio of adherent NB cell number relative to that of NB8pMigr control cells are plotted in the graph. Five independent experiments were conducted in triplicates. <b>C.</b> Invasive capacities of transduced NB8 cells toward CXCL12 through Matrigel. Mean ratio ± SD of the number of invasive cells in presence versus in absence of CXCL12 are plotted in the bare graph. Three independent experiments were conducted in triplicates. Multiple comparisons were performed using the parametric one way Anova test with Bonferroni corrections: *p<0.05, ***p<0.0005.</p

CXCR4 and CXCR7 receptors enhance metastatic growth in the liver.

<p><b>A.</b> Pictures of one representative liver of each group at sacrifice (W6). <b>B.</b> Vimentin IHC staining of liver sections of 5 mice per group at W6. <b>C.</b> Ki67 and cleaved caspase-3 (C3) IHC of one representative hepatic metastasis at W6 (magnifications 10x and 40x). Positive control for cleaved C3 is inserted in the small insert (NB8x4 orthotopic tumor, magnification 40x).</p

CXCR7 and CXCR4 favor site-specific homing of circulating NB cells after iv injection.

<p><b>A.</b> Vimentin IHC staining of paraffin-embedded sections of orthotopic primary tumors derived from NB8pMigr, NB8x4, NB8x7, and NB8x4x7 cells (magnification 40x) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125616#pone.0125616.ref035" target="_blank">35</a>], and normal lung and liver tissues from NSG mice (magnification 10x). <b>B.</b> One representative picture of vimentin IHC staining of liver tissue sections from 4 mice per group at W2 post iv injection (magnification 4x). <b>C.</b> Graph representing individual numbers, and mean numbers of hepatic metastases per group at W2 ± SD. Tumor foci were counted under light microscope in two complete liver tissue sections per mouse (Mann Whitney test, *p<0.05, **p<0.005). <b>D.</b> Vimentin IHC staining of one representative lung tissue sections per group at W2 post injection (magnification 10x). <b>E.</b> Vimentin IHC staining of representative lung tissue sections from two mice per group at W6 post injection (magnification 20x). <b>F.</b> Graph representing numbers of vimentin positive micrometastases in lungs for each mouse, and mean numbers per group ± SD at W6 post injection. Vimentin-positive micrometastases were counted in 11 randomly selected fields (magnification 10x) of one section of paraffin-embedded lung tissue of five mice per group (Mann Whitney test, *p<0.05).</p