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Bulk RNA sequencing on sorted blood cells from Drosophila lymph glands [PL iCC mCC bulk RNA-Seq]

ABSTRACT: We report the use of bulk mRNA sequencing on FACS-sorted blood cells to identify new and unique genetic markers for each major differentiated cell type of the Drosophila lymph gland. We used flow cytomotery in a wild-type genetic background whihc contains two fluorescent reporters that allow for the separation of 3 distinct populations. Plasmatcytes express the markers Hml, Pxn, vkg, Col4a1, and NimC1 , while crystal cells are markered by expression of hnt, lz, ProPO. mCC has higher expression of ProPO relitive to iCC, but lower expression of Hml and DsRed. We identified novel markers that are differentially expressed in each of the three major differentiated blood cell populations (PL, iCC, and mCC) that have been useful to definetely identify populations in scRNA-Seq and give information about transcript isoforms in each of the distinct populations.

ORGANISM(S): Drosophila melanogaster

PROVIDER: GSE168820 | GEO | 2021/11/03

REPOSITORIES: GEO

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Bulk RNA sequencing on sorted blood cells from Drosophila lymph glands [MZ IZ bulk RNA-seq]

Project description:We report the use of bulk mRNA sequencing on FACS-sorted blood cells to identify new and unique genetic markers for each major zone of the Drosophila lymph gland. We used flow cytomotery in a wild-type genetic background whihc contains two fluorescent reporters that allow for the separation of 4 distinct populations. Progenitors of the medullary zone (MZ) are marked by dome-meso-EGFP expression. Differentitating cels of the cortical zone (CZ) are marked by Hml-DsRed. These two sorted populations (MZ and CZ) are positive for the expected zone-specific markers (i.e. Tep4, dome, and shg for MZ and Hml, Pxn, vkg, Col4a1, and NimC1 for plasmatocytes of the CZ), however, the CZ population also expresses crystal cell markers (hnt, lz, ProPO), indicating that it is a mix of plasmatocytes and crystal cells. Intermediate progenitors of the intermediate zone (IZ), which are not well understood and do not have specific markers, but are characterized by simultaneous expression of EGFP and DsRed (double positives) were also sorted. In addtiion, there is a population of lymph gland cells that is double negative for both EGFP and DsRed fluorescence. This double negative population contains a mix of cell types including posterior signaling center (PSC) cells, MZ and CZ cells that have lost fluorescence, late crystal cells that do not express Hml (early CCs do express), and non-blood cells (i.e. dorsal vessel and pericardial cells). These double negatives were excluded from the analysis for these reasons. We analyzed transcript data of these 4 sorted populations in the early third instart larvae (90-96 hours after eggs lay at 25 degrees C). We identified novel markers that are differentially expressed in each of the three major blood cell populations (MZ, IZ, CZ) that have been useful to definetely identify populations in scRNA-Seq and give information about transcript isoforms in each of the distinct populations.

2021-11-03 | GSE168821 | GEO

Single cell RNA-sequencing on blood cells from Drosophila lymph glands [scRNA-seq]

Project description:We report the use of single cell mRNA sequencing to identify new blood cell types, subtypes, genetic markers, and important developmental pathways in the Drosophila lymph gland. We analyzed transcript data for 21,157 cells from wild-type early third instar larvae (90-93 hours after eggs lay at 25 degrees C) which contain reporter constructs that mark progenitors and differentiated cells. We find that lymph gland blood cells can be catorgorized into 9 major clusters. One cluster corrresponds to the niche cells of the posterior signaling center (PSC) that expressed the PSC markers Antennopedia and Collier. Progenitors of the medullary zone (MZ) are found in two distinct clusters (MZ1 and MZ2), both of which express the MZ markers domeless and Tep4. One cluster contains intermediate progenitors of the intermediate zone (IZ), and these express markers such as CG30090, lectin-24A, and MFS3 that we found to be IZ-specific in previous bulk RNA-seq experiments with sorted lymph gland cells. Another cluster (PL) contains plasmatocytes that express NimC1, as well as Hml, Pxn, vkg, and Col4a1. Crystal cells are found in one major population that subclusters into two distinct subclusters (CC1 and CC2) that express the markers lz, Hnt, and proPO.. Another transitory population that we dub proplasmatocytes expresses Hml, Pxn, vkg, and Col4a1, but not PL marker Nimc1 or IZ-markers. One cluster is not well understood, butis highly enriched for cell cycle- and replication stress genes. Pseudotime and Trajectory analysis results in fruther sub-division of these zones based on pseudotime states and aids in building a model of lymph gland development that will be the basis for future studies.

2021-11-03 | GSE168819 | GEO

Bulk RNA sequencing on sorted blood cells from Drosophila lymph glands [PL iCC mCC bulk RNA-Seq]

Project description:Bulk RNA sequencing on sorted blood cells from Drosophila lymph glands [PL iCC mCC bulk RNA-Seq]

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Expression from hemocytes misexpressing Idh-R195H vs. controls

Project description:Expression profile for hemocytes from hml-Gal4, UAS-2xEGFP larvae were compared to hemocytes from hml-Gal4, UAS-2xEGFP; UAS-Idh-R195H larvae We sought to determine which genes and pathways were upregulated or downregulated in hemocytes expressing Idh-R195H, the Drosophila homolog of the human cancer-associated IDH1-R132H mutant, in vivo. We sought to obtain homogeneous populations of hemocytes from each genotype. We compared larvae expressing Idh-R195H from the hml-Gal4 driver to larvae with the hml-Gal4 driver alone.

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NOTCH-YAP1/TEAD-DNMT1 axis drives hepatocyte reprogramming into intrahepatic cholangiocarcinoma [bulk RNA-Seq]

Project description:BACKGROUND AND AIM: Intrahepatic cholangiocarcinoma (ICC) is a liver tumor of increasing incidence and devastating prognosis. A critical barrier to developing an effective regimen for ICC is its intra- and inter-tumoral molecular heterogeneity, which may in part be due to its diverse cellular origin. We investigated clinical relevance and the molecular mechanisms underlying hepatocyte (HC)-driven ICC development. METHODS: Expression of ICC driver genes in human diseased livers at risk for ICC development were examined. Sleeping beauty and hydrodynamic tail vein injection based Akt-NICD/YAP1 ICC model was used to investigate pathogenetic roles of SOX9 and YAP1 in HC-driven ICC. These models and in silico studies led to identification of DNA methyltransferase-1 (DNMT1) as a YAP1 target, which was validated by both loss- and gain-of-function studies. RESULTS: Co-expression of AKT along with NICD/YAP1 in HC yielded ICC which resembled proliferative, Notch-activated, and stem cell-like subclasses of clinical ICC. NICD induced SOX9 and YAP1 in HC-driven ICC and deletion of either significantly delays ICC development. Yap1 deletion or TEAD inhibition, but not Sox9 deletion, impaired HC-to-biliary epithelial cell (BEC) reprogramming. DNMT1 was discovered as a novel downstream effector of YAP1-TEAD complex that directs HC-to-BEC/ICC fate-switch. DNMT1 loss prevented Notch/YAP1-dependent HC-driven cholangiocarcinogenesis, and DNMT1 re-expression restored ICC development following TEAD repression. Co-expression of DNMT1 with AKT was sufficient to induce tumor development including ICC. DNMT1 was detected in subset of HCs and dysplastic BECs in cholestatic human livers prone to ICC development. CONCLUSION: We identify a novel NOTCH-YAP1/TEAD-DNMT1 axis essential for of HC-to-BEC/ICC conversion, may be relevant in cholestasis-to-ICC pathogenesis in the clinic.

2023-07-25 | GSE200471 | GEO

Transcriptome of Smooth Muscle Cells, Interstitial Cells of Cajal and PDGFRα+ Cells

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Single-cell RNA-seq of primary human gastric interstitial cells of Cajal

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2022-07-04 | E-MTAB-9602 | biostudies-arrayexpress

Transcriptome of Smooth Muscle Cells, Interstitial Cells of Cajal and PDGFRα+ Cells

2015-07-31 | GSE57776 | GEO

Transcriptome-wide identification of transcripts regulated by MBNL3 in hepatocellular carcinoma cells

Project description:Understanding the roles of splicing factors and splicing events during human tumorigenesis would open new avenues for targeted therapeutics. Here we identify an oncofetal splicing factor MBNL3, which indicates poor prognosis, and promotes hepatocellular carcinoma (HCC) tumorigenesis. Genetic MBNL3 inhibition almost completely abolishes HCC tumorigenesis. Transcriptomic analysis revealed that MBNL3 induces PXN antisense transcript 1 exon 4 inclusion. The transcript lacking exon 4 binds to coding sequences of PXN mRNA, causes dissociation of translation elongation factors from PXN mRNA, thereby inhibiting PXN mRNA translation. Whereas the transcript containing exon 4 preferentially binds to 3' untranslated region of PXN mRNA, protects PXN mRNA from microRNA-24-AGO2 complex-induced degradation, thereby increasing PXN expression. Through inducing exon 4 inclusion, MBNL3 upregulates PXN expression, which mediates the pro-tumorigenic roles of MBNL3. Collectively, these data demonstrate detailed mechanistic links between oncofetal splicing factor, splicing event, and tumorigenesis, and establish splicing factors and splicing events would be potential therapeutic targets.

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