Project description:Analysis of differential gene expression. The influence of a constitutively activated mutant Kit receptor on gene expression in fetal hematopoietic cells was analyzed. Results provide information of genes and cellular processes that are influenced by Kit signaling. Total RNA obtained from embryonic day E13.5 fetal liver of double transgenic R26-LSL-KITD816V:Vav-iCre mice compared to single transgenic controls. R26-LSL-KITD816V mice have been registered with the mouse genome database (MGI:5516508, allele named Gt(ROSA)26sorTM1(GFP-cKIT*)Hsc). Vav-iCre mice have been described by De Boer et al. in 2003.
Project description:MiRNAs have the potential to regulate cellular differentiation programs. However, miRNA-deficiency in primary hematopoietic stem cells (HSCs) results in HSC depletion in mice, leaving the question of whether miRNAs play a role in early-lineage decisions unanswered. To address this issue, we deleted Dicer1, which encodes an essential RNaseIII enzyme for miRNA biogenesis, in murine CCAAT/enhancer-binding protein alpha (C/EBPA)-positive myeloid-committed progenitors in vivo. In contrast to the results in HSCs, we found that miRNA depletion affected neither the number of myeloid progenitors nor the percentage of C/EBPA-positive progenitor cells. Analysis of gene-expression profiles from wild type and Dicer1-deficient granulocyte-macrophage progenitors (GMPs) revealed that 20 miRNA families were active in GMPs. Of the derepressed miRNA targets in Dicer1-null GMPs, 27% are normally exclusively expressed in HSCs or are specific for multi-potent progenitors and erythropoiesis, indicating an altered gene-expression landscape. Dicer1-deficient GMPs were defective in myeloid development in vitro and exhibited an increased replating capacity, indicating a regained self-renewal potential of these cells. In mice, Dicer1 deletion blocked monocytic differentiation, depleted macrophages and caused myeloid dysplasia with morphological features of Pelger-Huët anomaly. These results provide evidence for a miRNA-controlled switch for a cellular program of self-renewal and expansion towards myeloid differentiation in GMPs. To generate Cebpa-Cre;R26-LSL-Eyfp;Dicer1wt/fl/Dicer1fl/fl mice, we crossed mice that contain floxed Dicer1 alleles (Dicer1fl) with Cebpa-Cre;R26-LSL-Eyfp reporter mice 2. Fetal livers were obtained on embryonic day (E) 13.5. Routine genotyping of Dicer1; Cebpa-Cre;R26-LSL-Eyfp mice was performed by PCR assays of DNA from tail or toe biopsies. For transplantation, 6 to 8-week-old recipient mice (C57Bl/6, Jackson Laboratories) were irradiated (8.5 Gy) and tail-vein injected with fetal liver single-cell suspensions. Typically, cells from each fetal liver were transplanted into two recipient mice. Hematopoietic tissues were analyzed 6-10 weeks post transplantation. EYFP positive GMPs from the bone marrow of Dicer wt control (n=3), Dicer -/wt (n=3 and Dicer fl/fl (n=3) were sorted and analyzed for gene expression profiles.
Project description:RNAseq analysis was performed on liver of tet-YAP and tet-Myc transgenic mice induced for 2 days (R26-mice) or 4 weeks (laptTA-mice) or in liver tumor noduli.
Project description:Single-cell RNA sequencing analysis was performed on primary pancreatic ductal adenocarcinoma derrived cell line from PiKP (P48-Cre; R26-rtTa-IRES-EGFP; tetO-LSL-KrasG12D/+; Trp53L/L) murine tumors. In this study we establish that oncogenic Kras is the predominant driver of T cell paucity and myeloid infiltration in the pancreatic tumor microenvironment. Then, we use scRNA seq analysis of cultured PiKP cells with and without Kras* extinction to identify immune related genes involved in driving Kras* mediated immune supression in PDAC.
Project description:Hippo signaling is highly associated with activity in the stem cell compartment of many epithelial tissues. In this study, we examined if Hippo signaling inhibition (by inducing Yap expression) could convert differentiated cells into a progenitor like phenotype. Rosa26-lsl-YFP mice had recombination induced and cells were FACS sorted using YFP as a marker at either 1 week or 6 weeks after recombination. YFP expression is a surrogate for Yap expression (our protein of interest) and time was varied in vivo. RNA was immediately extracted after sorting and labeled to be hybridized to the array. FACS sorted Yap expressing liver cells were sorted one or six weeks after induction and compared to fetal liver samples as controls The control data from fetal liver samples have been previously published and is available in the GEO database as GSE12117 (GSM305568, GSM305569, GSM305570). The complete dataset representing: Yap Expressiong Liver samples and the fetal liver control Samples from Series GSE12117 (re-processed in this study), is linked below as a supplementary file.
Project description:C/EBPalpha is a transcription factor critically involved in myeloid development and indispensable for formation of granulocytes. To track the cellular fate of stem and progenitor (LSK) cells, which express C/EBPalpha, we developed a mouse model expressing Cre recombinase from the Cebpa promoter and an inducible EYFP allele. We show that Cebpa/EYFP+ cells represent a significant subset of LSK cells, which predominantly give rise to myeloid cells in steady state hematopoiesis. C/EBPalpha induced a robust myeloid gene expression signature and downregulated E2A-induced regulators of early lymphoid development. In addition, Cebpa/EYFP+ cells comprise a fraction of early thymic progenitors (ETP) with robust myeloid potential. However, Cebpa/EYFP+ LSK and ETP cells retained the ability to develop into erythroid and T-lymphoid lineages, respectively. These findings support an instructive, but argue against a lineage restrictive role of C/EBPalpha in multipotent hematopoietic and thymic progenitors. We performed global gene expression profiling of double-sorted Cebpa/EYFP+ and Cebpa/EYFP- LSK cells of pooled Cebpa Cre/wt R26 EYFP reporter mice to identify differentially regulated genes in Cebpa+ versus Cebpa- LSK cells. RNA was isolated from three biological replicates of Cebpa/EYFP+ LSK cells and two biological replicates of Cebpa/EYFP- LSK cells. To determine if the identified genes were truly dependent on Cebpa expression, we also performed global gene expression profilling of Cebpa/EYFP+ and Cebpa/EYFP- fetal liver LSK cells of Cebpa Cre/fl R26 EYFP mice. Induction of Cebpa/Cre expression in these mice leads to Cre-mediated recombination of the floxed wt Cebpa allele resulting in a complete Cebpa knock-out. In this case, RNA was isolated from two biological replicates of either Cebpa/EYFP+ and Cebpa/EYFP- LSK cells. In addition, we included one biological replicate of Cebpa/EYFP+ and Cebpa/EYFP- fetal liver LSK cells of Cebpa Cre/wt R26 EYFP mice to determine the correlation of differentially regulated genes in bone marrow and fetal liver LSK cells.
Project description:Liver mitochondria play a central role in metabolic adaptations to changing nutritional states, yet their dynamic regulation upon anticipated changes in the energy state has remained unaddressed. Here, we show that sensory food perception rapidly induces mitochondrial fission in the liver via protein kinase B/AKT-dependent phosphorylation of serine 131 of the Mitochondrial fission factor (MFFS131), a response mediated via activation of hypothalamic Pro-opiomelanocortin (POMC)-expressing neurons. A non-phosphorylatable MFFS131G knock-in mutation abrogates AKT-induced mitochondrial fragmentation in vitro. In vivo, MFFS131G knock-in mice display altered liver mitochondrial dynamics upon refeeding and impaired insulin stimulated suppression of gluconeogenesis. Collectively, we reveal a critical role for rapid activation of a hypothalamic/liver axis to adapt mitochondrial function to anticipated changes of nutritional state in control of hepatic glucose metabolism. R26-fl-Akt-C mice Mice carrying a conditional myristoylation tagged Akt-C transgene in the ROSA26 locus were used to activate AKT in the liver with a liver specific Cre-dependent virus. The generation of this line has been described previously (V. Kohlhaas et al, 2021) For AAV mediated liver-specific delivery of Cre, R26-fl-Akt-C or control mice were injected with a AAV8-TBG-iCre virus (VB1724, Vector Biolabs). This repository contains two experiments a) Liver of liver active Akt-CA and b) Insulin stimulation of primary heptocytes. Please note that replicate one of the hepatocyte dataset have been removed from the analysis due to the limited number of posphosites compared to others.
Project description:Cancer cachexia syndrome is observed in 80% of patients with advanced-stage cancer, and it is one of the most frequent causes of death. Severe wasting accounts for more than 80% in patients with advanced pancreatic cancer. Here we wanted to define, by using an microarray approach and the Pdx1-cre;LSL-KrasG12D;INK4a/arffl/fl, the pathways involved in muscle, liver and white adipose tissue wasting. The aim of our work was to characterize as extensively as possible the pathways activated by the pancreatic cancer-induced cachectic tissues. For this purpose, we generated and compared genome-wide expression profiles of white adipose tissue, skeletal muscle and liver, from Pdx1-cre;LSL-KrasG12D;INK4a/arffl/fl and LSL-KrasG12D;INK4a/arffl/fl mice at 10 weeks-old. Tissue samples by triplicate was obtained from liver, muscle and adipose tissues in both groups, controls and cachectic mice. Total RNA samples was processed and profiled on Affymetrix Mouse Gene 1.0 ST arrays as previously described (Cano et al, 2012)
Project description:Chemokine signaling is important for the seeding of different sites by hematopoietic stem cells during development. Serum Response Factor (SRF) controls multiple genes governing adhesion and migration, mainly by recruiting members of the Myocardin-Related Transcription Factor (MRTF) family of G-actin regulated cofactors. We used vav-iCre to inactivate MRTF-SRF signaling early during hematopoietic development. In both Srf- and Mrtf-deleted animals, hematopoiesis in fetal liver and spleen is intact, but does not become established in fetal bone marrow. Srf-null HSC/Ps (hematopoietic stem/progenitor cells) fail to effectively engraft in transplantation experiments, exhibiting normal proximal signaling responses to SDF-1, but reduced adhesiveness, F-actin assembly, and reduced motility. Srf-null HSC/Ps fail to polarise in response to SDF-1, and cannot migrate through restrictive membrane pores to SDF-1 or Scf in vitro. Mrtf-null HSC/Ps were also defective in chemotactic responses to SDF-1. MRTF-SRF signaling is thus critical for the response to chemokine signaling during hematopoietic development.