Project description:Mist1+ cells and parietal cells in mouse stomach were separatedly sorted, and RNAs were isolated. Mist1 (also known as Bhlha15) is expressed in gastric chief cells and gastric stem cells in mice. However, more specific genes for each population needs to be identified to better understand the precise biology in these cell populations. In order to address cell specific gene signature, we separately sorted Mist1+ gastric chief cells and Mist1+ gastric stem cells by FACS, and performed microarray analysis. Mist1+ gastric chief cells were sorted by using Mist1-CreERT; R26-TdTomato mouse stomach, immediately after tamoxifen administration. Mist1+ gastric stem cells were sorted by chief cell-ablated Mist1-CreERT; R26-TdTomato mouse stomach, combining with Lgr5-DTR mice. Lgr5-expressing chief cells were ablated by giving DT into these mice. As a control, acid-secreting gastric parietal cell samples were used. Mice were treated with or without Lgr5-DT ablation before sorting.
Project description:MIST1 is a bHLH transcription factor that is necessary for the maturation of gastric zymogenic cells as they differentiate from their precursor mucous neck cells. In this experiment, mucous neck cells and zymogenic cells of normal, adult C57BL/6 and MIST1 knockout mice were laser-capture microdissected in order to determine MIST1-dependent, zymogenic cell specific gene expression.
Project description:The transcription factor MIST1 is required for final maturation of secretory cells of diverse tissues, including gastric digestive-enzyme secreting zymogenic (chief) cells (ZCs). Here, we show that MIST1 directly activates RAB26, RAB3D and several other genes. Experiment Overall Design: We used microarrays to determine genes upregulated following transient MIST1 transfection. Two gastric cell lines were used: HGC-27 and AGS. Three chips were generated from each cell line: parental (untransfected), GFP+empty vector (a control for effects of transfection), and GFP+MIST1. The latter two chips were generated from cells flow sorted based on GFP fluorescence to isolate cells with high levels of transfection. Chips were analyzed by dChip to identify genes expressed in both MIST1-transfected cell populations relative to their respective controls in multiple Boolean 'AND' comparisons.
Project description:Mist1+CD24hi cells and Mist1+CD24lo cells in mouse small intestine were separatedly sorted, and RNAs were isolated. Mice were treated with irradiation, Lgr5-DT ablation, doxorubicin, or NICD expression before sorting.
Project description:MIST1 over-expression has not been analyzed in a cell type that does not express endogenous MIST1. Here, we force-express ectopic MIST1 in the parietal cells of mouse stomachs. We extracted whole corpus stomach mRNA and compared gene expression levels between MIST1-expressing mouse stomachs to controls.
Project description:The transcription factor MIST1 is required for final maturation of secretory cells of diverse tissues, including gastric digestive-enzyme secreting zymogenic (chief) cells (ZCs). Here, we show that MIST1 directly activates RAB26, RAB3D and several other genes.
Project description:Although early developmental processes involve cell fate decisions that define the body axes and establish progenitor cell pools, development does not cease once cells are specified. Instead, most cells undergo specific maturation events where changes in the cell transcriptome ensure that the proper gene products are expressed to carry out unique physiological functions. Pancreatic acinar cells mature post-natally to handle an extensive protein synthetic load, establsih organized apical-basal polarity for zymogen granule trafficking, and assemble gap-junctions to perimt efficient cell-cell communication. Despite significant progress in defining transcriptional networks that control initial acinar cell specification and differentiation decisions, little is know regarding the role of transcription factors in the specification and maintenance of maturation events. One candidate maturation effector is MIST1, a secretory cell-restricted transcription factor that has been implicated in controlling regulated exocytosis events in a number of cell types. Embryonic knock-out of MIST1 generates acinar cells that fail to establish an apical-basal organization, fail to properly localize zymogen granule and fail to communicate intra-cellularly, making the exocrine organ highly suceptible to pancreatic diseases. In an effort to identify the gene expression differences responsible for MIST1 regulating mature acinar properties. We generated a tamoxifen-inducible mouse model where MIST1 expression could be activated in vivoand performed gene expression arrays on wildtype, MIST1-null, and induced MIST1 pancreatic RNA. RNA was isolated from pancreata of 8 week old mice using the Qiagen RNeasy Midi kit. Pancreta of wildtype, MIST1-null, and MIST1-null with a tamoxifen inducible MIST1-expressing transgene were harvested 36 hours post-tamoxifen administration. Therefore, this experiment provides information on steady-state gene expression differences between wildtype and MIST1-null mice as well as immediate gene expression changes induced by MIST1 expression.
Project description:MIST1 is a bHLH transcription factor that is necessary for the maturation of gastric zymogenic cells as they differentiate from their precursor mucous neck cells. In this experiment, mucous neck cells and zymogenic cells of normal, adult C57BL/6 and MIST1 knockout mice were laser-capture microdissected in order to determine MIST1-dependent, zymogenic cell specific gene expression. Stomachs were excised immediately following sacrifice, quickly flushed with room-temperature PBS, inflated by duodenal injection of OCT compound, frozen in Cytocool II, and cut into serial 7-M-NM-<m-thick cryosections, which were mounted on Superfrost slides, fixed in 70% EtOH, rehydrated in nuclease-free water, and then incubated in Alexa Fluor 488-conjugated Griffonia simplicifolia GS-II (diluted 1:500 in nuclease-free water) for 15 min. Sections were washed in nuclease-free water and dehydrated in graded ethanol followed by xylene. ZCs were identified as corpus cells that were basal to GS-II labeling and which did not show the dark silhouettes and characteristic shape of parietal cells following xylene dehydration. Four wild-type mice and 5 Mist1M-bM-^HM-^R/M-bM-^HM-^R mice were used for dissection (PixCell II LCM apparatus [7.5-M-NM-<m spot diameter] and CapSure HS LCM caps) to generate two caps per mouse. RNA was purified by PicoPure kit, and RNA integrity was confirmed by an Agilent 2100 Bioanalyzer. All RNA from each cap was treated with DNase I and then reverse transcribed using the SuperScript III (Invitrogen) standard protocol (most cDNA syntheses started with 10 ng of total RNA). Biotinylated cRNA probes were hybridized to the GeneChips. Gene chip arrays used in these experiments were Affymetrix Mouse Gene 1.0ST arrays. Chip quality control and GeneChip to GeneChip comparisons to generate expression profiles were performed using dChip.
Project description:MIST1 over-expression has not been analyzed in a cell type that does not express endogenous MIST1. Here, we force-express ectopic MIST1 in the hepatocytes of mouse livers. We extracted mRNA and compared gene expression levels between MIST1-expressing mouse livers to controls.