Project description:The existence of conventional dendritic cells (cDCs) has not yet been demonstrated outside mammals. In this paper, we identified bona fide cDCs in chicken spleen. Comparative profiling of global and of immune response gene expression, morphology, and T cell activation properties show that cDCs and macrophages (MPs) exist as distinct mononuclear phagocytes in chicken, resembling their human and mouse cell counterparts. Using computational analysis, core gene expression signatures for cDCs, MPs, T and B cells across chicken, human and mouse were established, which will facilitate the identification of these subsets in other vertebrates. Overall this study, by extending the newly uncovered cDC and MP paradigm to chicken, suggests that the generation of these two phagocyte lineages occurred before the reptile to mammal and bird transition in evolution. It opens avenues for the design of new vaccines and neutraceuticals that are mandatory for the sustained supply of poultry products in the expanding human population.
Project description:Transcription profiling by array of differential gene expression in chicken primary B cells infected ex vivo with attenuated and very virulent strains of infectious bursal disease virus (IBDV)
Project description:We reported that stable expression of constitutively active intra cellular Notch (ICN), in quail neuroretina (QNR) cells transformed by a conditional v-Src mutant (QNR/v-src cells), resulted in the suppression of their transformed properties. Acquisition of a normal phenotype coincided with a major switch in cell identity, as these undifferentiated QNR/v-src cells acquired characteristics of glial differentiation. Similar loss of transformation and gene reprogramming can be achieved in QNR/v-src cells, stably expressing the human CBF protein, RBP-Jk, whose activity was rendered ligand independent by fusion to the VP16 transactivator. These major phenotypic changed are correlated with a dominant interference with signaling effectors, regulating cell morphology and cytoskeleton organization. To understand the mechanisms by which Notch signaling activation suppressed v-Src induced cell transformation and induced differentiation, we compared the transcription profile of QNR cells transformed by a v-Src mutant encoding a temperature sensitive oncoprotein (QNR/v-src), with that of cells stably expressing ICN (QNR/v-src/ICN) or RBP-Jk-VP16 (QNR/v-src/RBP-Jk-VP16). Total RNA was extracted from QNR/v-src, QNR/v-src/ICN or QNR/v-src/RBP-Jk-VP16 cells maintained at permissive (37°C) or restrictive (41°C) temperature. cDNA from QNR/v-src cells was probed with that of QNR/v-src/ICN or QNR/v-src/RBP-Jk-VP16 at both temperature on microarrays spotted with 13,000 cDNA from chicken EST collections designed by the genomic facility of the Fred Hutchinson Cancer Research Center (Seattle). For two sets of sample, dye swap experiments were performed.
Project description:Cell transformation by the Src tyrosine kinase is characterized by extensive changes in gene expression. To describe these changes, investigators have relied extensively on the study of immortalized rodent cell lines or heterogeneous tumor samples that limit the identification of differentially expressed genes or may not represent the full spectrum of biological processes regulated during transformation. In this study, we took advantage of transformation-deficient and temperature sensitive mutants of the Rous sarcoma virus to characterize the patterns of gene expression in two types of primary cells, namely chicken embryo fibroblasts (CEF) and chicken neuro-retinal (CNR) cells. Keywords: viral transformation of primary cells, transformation, transformation deficient mutant, temperature sensitive mutant, v-Src Chicken embryo fibroblasts (CEF) were infected with the wild-type strain Schmidt-Ruppin A RSV or non-transforming strain NY315 RSV or the non-transforming control virus RCASBP(A) to assess genes involved in v-Src-dependent transformation of CEF. Chicken embryo fibroblasts (CEF) were infected with the temperature sensitve strain NY72-4 RSV and cultured either at non-permissive temperature (41.5M-KM-^ZC) or permissive temperature (37M-KM-^ZC) to assess genes involved in v-Src-dependent transformation of CEF. Chicken neuroretina cells (CNR) were infected with the temperature sensitve strain NY72-4 RSV and cultured either at non-permissive temperature (41.5M-KM-^ZC) or permissive temperature (37M-KM-^ZC) to assess genes involved in v-Src-dependent transformation of CNR and compared to CEF.
Project description:Nontransformed cells form heterotypic cadherin junctions with adjacent transformed cells to inhibit tumor cell growth and motility. Transformed cells must override this form of growth control, called contact normalization, to invade and metastasize during cancer progression. Heterocellular cadherin junctions between transformed and nontransformed cells are needed for this process. However, specific mechanisms downstream of cadherin signaling have not been clearly elucidated. Here, we utilized a b-catenin reporter construct to determine if contact normalization affects Wnt signaling in transformed cells. b-catenin driven GFP expression in Src transformed mouse embryonic cells was decreased when cultured with cadherin competent nontransformed cells compared to transformed cells cultured with themselves, but not when cultured with cadherin deficient nontransformed cells. We also utilized a layered culture system to investigate the effects of oncogenic transformation and contact normalization on gene expression and oncogenic Src kinase mediated phosphorylation events. RNA-Seq analysis found that the cadherin dependent contact normalization inhibited the expression of 22 transcripts that were induced by Src transformation, and increased the expression of 78 transcripts that were suppressed by Src transformation. Phosphoproteomic analysis of cells expressing a temperature sensitive Src kinase construct found that contact normalization decreased phosphorylation of 10 proteins on tyrosine residues that were phosphorylated within 1 hour of Src kinase activation in transformed cells. Taken together, these results indicate that cadherin dependent contact normalization inhibits Wnt signaling to regulate oncogenic kinase activity and gene expression, particularly PDPN expression, in transformed cells in order to control tumor progression.