In-vivo inhibition of Fgfr2b signalling in FACS-isolated AECs from embryonic mouse lungs from E16.5 to E18.5
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ABSTRACT:
PROVIDER: PRJNA476355 | ENA |
REPOSITORIES: ENA
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Project description:Rosa26rtTA;tetOsFgfr2b (C75Bl6) induced from E16.5 until E18.5, lungs were harvest at E18.5, AECI and AECII were sorted using FACS. Mice were kept on water and food ad libitum.
2021-06-01 | GSE115878 | GEO
Project description:In this experiment we inhibited FGF signalling in E16.5 mouse lungs by using a dominant negative mouse model. We are interested in assessing the genetic regulation by FGF signalling at this stage of lung development. Specifically, we used a tetracycline controlled transcriptional activation model (Tet(o)) to manipulate FGF signalling. Genetically modified male mice that expressed a reverse tetracycline-controlled trans activator (rtTA) on the Rosa26 locus and a soluble FGFR2b on the Tet(o) promoter (Tet(o)sFgfr2b) (B6.Cg-Gt(ROSA)26Sortm1.1(rtTA,EGFP)NagyTg(tetO-Fgfr2b/Igh)1.3Jaw/sbel), were crossed with females lacking the Tet(o)sFgfr2b allele. Pregnant females were injected with doxycycline intraperitonally at E16.5. Doxycycline then induced the transcription soluble FGFR2b in the embryos, which inhibited FGF signalling by sequestering FGF ligands, preventing them from binding to the native FGFR2b receptor. We harvested embryonic lungs after 9 hrs of inhibition. We took the left lobe of each sample for RNA isolation. We set-up our matings so that 50% of the embryos in a litter were expected to contain the soluble FGFR2b. The remaining littermates will serve as the control group. We will compare the gene expression profiles of the experimental embryonic lungs with those of littermate controls.
2021-06-01 | GSE115877 | GEO
Project description:We are interested in studying the role of FGF/FGFR2b signalling on lung alveolar epithelial bipotent progenitors. Bipotent progenitors give rise to alveolar type 1 and type 2 cells (AECI and AECII, respectively). Previous research has shown that AECII cells isolated from lungs where FGF signalling has been globally inhibited show a gene signature much closer to type 1 cells. Furthermore, the relative number of AECI in experimental lungs was increased compared to control lungs. This suggests FGF signalling promotes AECII differentiation. We want to test the hypothesis that FGF signalling promotes the differentiation of AECII by signalling to bipotent progenitors. To test this hypothesis, we have used a transgenic mouse line (SpcCreERT2_Fgfr2bflox_tomatoflox), which, upon administration of tamoxifen, can be used to knock-out the FGFR2b receptor in SFTPC + cells, while labelling those cells red. We injected mice carrying embryos with and without the Fgfr2bflox allele (experimental and control, respectively) at E14.5 and at E15.5. At this timepoint, SFTPC is a marker for bipotent progenitors. Thus, by administering tamoxifen at this timepoint, bipotent progenitors were targeted to knock-out FGFR2b, and thus inhibit FGF/FGFR2b signalling to these cells. We harvested lungs at E18.5, a timepoint where the differentiation of AECI and AECII is well underway. Harvested lungs were prepared for FACS-based cell sorting. Cells were sorted by gating for total epithelial cells, then RFP + cells (representing bipotent progenitors), then AECI and AECII cells. These cells were collected, and their total RNA isolated for gene array analysis. We will then compare the AECI and AECII gene signatures of isolated cells between experimental and control samples.
2021-06-01 | GSE115879 | GEO
Project description:In-vivo inhibition of Fgfr2b signalling in FACS-isolated Sftpc-expressing cells in embryonic mouse lungs from E14.5 to E18.5
| PRJNA476354 | ENA
Project description:In-vivo inhibition of Fgfr2b signalling in embryonic whole mouse lungs at E16.5
| PRJNA476352 | ENA
Project description:FGFR2b signalling interacts with ßcatenin signalling to regulate early branching morphogenesis. Earlier, we identified a set of genes regulated in the epithelium of the developing lung by the global inhibition of FGFR2b signalling after 6 and 9 hours, in vivo. In this experiment, we used a pharmacological inhibitor of ßcatenin signalling, IQ1, to block ßcatenin signalling in isolated E12.5 wildtype lungs for 9 and 24 hrs, in vitro. We intend to assess the overlap between genes regulated in our in vivo experiment with those regulated in our in vitro, IQ1 experiment. In so doing, we hope to identify genes regulated by the FGFR2b/ßcatenin signalling pathway. Samples were obtained by isolating E12.5 lungs from C57bl/6J mice. Isolated lungs were then cultured on filters with culture medium supplied with 10 uM IQ1 (experimental) or vehicle (control), for 9 and 24 hrs. After these times, the experiments were stopped, and samples were prepared for RNA isolation.
2019-01-25 | GSE124155 | GEO
Project description:In this experiment we inhibited FGF signalling in E14.5 mouse lungs by using a dominant negative mouse model. We are interested in assessing the genetic regulation by FGF signalling at this stage of lung development. Specifically, we used a tetracycline controlled transcriptional activation model (Tet(o)) to manipulate FGF signalling. Genetically modified male mice that expressed a reverse tetracycline-controlled trans activator (rtTA) on the Rosa26 locus and a soluble FGFR2b on the Tet(o) promoter (Tet(o)sFgfr2b) (B6.Cg-Gt(ROSA)26Sortm1.1(rtTA,EGFP)NagyTg(tetO-Fgfr2b/Igh)1.3Jaw/sbel), were crossed with females lacking the Tet(o)sFgfr2b allele. Pregnant females were injected with doxycycline intraperitonally at E14.5. Doxycycline then induced the transcription soluble FGFR2b in the embryos, which inhibited FGF signalling by sequestering FGF ligands, preventing them from binding to the native FGFR2b receptor. We harvested embryonic lungs after 9 hrs of inhibition. We took the left lobe of each sample for RNA isolation. We set-up our matings so that 50% of the embryos in a litter were expected to contain the soluble FGFR2b. The remaining littermates will serve as the control group. We will compare the gene expression profiles of the experimental embryonic lungs with those of littermate controls.
2020-06-22 | GSE115876 | GEO
Project description:FGFR2b/ßcatenin signalling pathway in embryonic mouse lungs
| PRJNA510849 | ENA
Project description:We used microfluidic single cell RNA-seq on 198 individual mouse lung epithelial cells at 4 different stages throughout development to measure the transcriptional states which define the developmental and cellular hierarchy of the distal mouse lung epithelium. We classified 80 cells comprising the distal lung epithelium at E18.5 into distinct populations using an unbiased genome-wide approach that did not require a priori knowledge of the underlying cell types or prior purification of cell types. This M-bM-^@M-^\reverse tissue engineeringM-bM-^@M-^] approach confirmed the basic outlines of the conventional model of cell type diversity in the distal lung and led to the discovery of a large number of novel transcriptional regulators and cell type markers that discriminate between the different populations. Moreover, we reconstructed the steps during maturation of bipotential progenitors into both alveolar lineages based on the presence of undifferentiated, differentiated as well as differentiation intermediate cells at the single time point E18.5. Finally, we followed Sftpc-positive cells throughout their lifecycle (E14.5, E16.5, E18.5, adult) and identified 7 gene sets that differentiate between the multipotential, bipotential, mature, as well as intermediate states of the AT2 lineage. 198 single-cell transcriptomes from mouse lung epithelium were analyzed in total, two 200-cell bulk control samples as well as one no-cell control; All single cell and control samples contain 92 external RNA spike-ins; For time point E18.5, three individual experiments were performed using 3 different pregnant mice (3 biological replicates): Replicate 1 (pooled sibling lungs) yielded 20 single cell transcriptomes, replicate 2 (one single embryonic lung) yielded 34 single cell transcriptomes and replicate 3 (pooled siibling lungs) yielded 26 single cell transcriptomes; In addition, a 200-cell bulk control sample was prepared for E18.5 replicate 1 and E18.5 replicate 3 experiments; A no-cell control sample was generated for the E18.5 replicate 1 experiment; For time point E14.5, one experiment (one pregnant mouse, pooled sibling lungs) was performed yielding 45 single cell transcriptomes; For time point E16.5, one experiment (one pregnant mouse, pooled sibling lungs) was performed yielding 27 single cell transcriptomes; For the adult time point, one 107 day old mouse was used and transcriptomes of 46 single cells were obtained; All single cell samples were processed on the microfluidic platform, 200-cell-bulk and no-cell control samples were processed in microliter volumes in PCR tubes.
2014-04-08 | E-GEOD-52583 | biostudies-arrayexpress
Project description:In-vivo inhibition of Fgfr2b signalling in embryonic mouse lungs
| PRJNA476349 | ENA