Project description:Transcriptional profiling of Control and RbpjkCNULL lungs from E18.5. For microarray profiling, total RNA from E18.5 lungs was submitted for labeling and hybridization (Mouse Gene 1.0 ST Whole Genome Array, Affymetrix) to the Boston University Microarray Core facility. Clara cells (CCs) are a morphologically and operationally heterogeneous population of secretoglobin Scgb1a1-expressing secretory cells crucial for airway homeostasis and post-injury repair. Insights into the extent and origin of CC diversity have been hindered by the limited knowledge of markers of these cells and their precursors. To identify novel putative markers of CCs we characterized global changes in gene expression in embryonic lungs in which CCs were suppressed by conditional disruption of Notch signaling (Rbpjkcnull). Microarray profiling and Real Time PCR (qRT-PCR) identified eleven genes differentially downregulated in the E18.5 airways of Rbpjkcnull compared to controls, nearly half not previously known to mark CCs. Remarkably, in situ hybridization (ISH) revealed overlapping but also distinct domains of expression of these genes in E18.5 controls. Notably, Reg3g, Chad, Gabrp and Lrrc26 were selectively expressed in CCs of proximal airways and Upk3a expression was highly enriched in a CC subpopulation surrounding Neuroepithelial Bodies (NEBs). All genes expressed in the adult airways were found to be expressed in adult CCs and downregulated by selective CC ablation in a Naphthalene model of injury. Flow cytometry-based isolation of CCs from different airway regions of adult B1-EGFP reporter mice and qRT-PCR corroborated the spatial enrichment in gene expression observed by ISH. Remarkably, although, Scgb3a2, Upk3a, Cyp2f2, Cbr2, Krt15 could be detected unambiguously in airway progenitors as early as E14.5, only Scgb3a2 and Upk3a had their expression suppressed by disruption of Notch signaling, implicating both genes as the earliest markers for CC differentiation. Our study supports the idea that the diversification of the CC phenotype already arises during embryonic development and identifies candidate markers that can be used to investigate this process.

Project description:Transcriptional profiling of Control and RbpjkCNULL lungs from E18.5. For microarray profiling, total RNA from E18.5 lungs was submitted for labeling and hybridization (Mouse Gene 1.0 ST Whole Genome Array, Affymetrix) to the Boston University Microarray Core facility. Clara cells (CCs) are a morphologically and operationally heterogeneous population of secretoglobin Scgb1a1-expressing secretory cells crucial for airway homeostasis and post-injury repair. Insights into the extent and origin of CC diversity have been hindered by the limited knowledge of markers of these cells and their precursors. To identify novel putative markers of CCs we characterized global changes in gene expression in embryonic lungs in which CCs were suppressed by conditional disruption of Notch signaling (Rbpjkcnull). Microarray profiling and Real Time PCR (qRT-PCR) identified eleven genes differentially downregulated in the E18.5 airways of Rbpjkcnull compared to controls, nearly half not previously known to mark CCs. Remarkably, in situ hybridization (ISH) revealed overlapping but also distinct domains of expression of these genes in E18.5 controls. Notably, Reg3g, Chad, Gabrp and Lrrc26 were selectively expressed in CCs of proximal airways and Upk3a expression was highly enriched in a CC subpopulation surrounding Neuroepithelial Bodies (NEBs). All genes expressed in the adult airways were found to be expressed in adult CCs and downregulated by selective CC ablation in a Naphthalene model of injury. Flow cytometry-based isolation of CCs from different airway regions of adult B1-EGFP reporter mice and qRT-PCR corroborated the spatial enrichment in gene expression observed by ISH. Remarkably, although, Scgb3a2, Upk3a, Cyp2f2, Cbr2, Krt15 could be detected unambiguously in airway progenitors as early as E14.5, only Scgb3a2 and Upk3a had their expression suppressed by disruption of Notch signaling, implicating both genes as the earliest markers for CC differentiation. Our study supports the idea that the diversification of the CC phenotype already arises during embryonic development and identifies candidate markers that can be used to investigate this process. Control (n=3) versus RbpjkCNULL(n=3)

Project description:Perivascular adipose tissue (PVAT) regulates vascular function by secreting vasoactive substances. In mice, Notch signaling is activated in the PVAT during diet-induced obesity, and leads to the loss of the thermogenic phenotype and adipocyte whitening due to increased lipid accumulation. We used the Adiponectin-Cre (Adipoq-Cre) strain to activate a ligand-independent Notch1 intracellular domain transgene (N1ICD) to drive constitutive Notch signaling in the adipose tissues (N1ICD;Adipoq-Cre). We previously found that constitutive activation of Notch1 signaling in the PVAT phenocopied the effects of diet-induced obesity. To understand the downstream pathways activated by Notch signaling, we performed a proteomic analysis of the PVAT from control versus N1ICD;Adipoq-Cre mice. This comparison identified prominent changes in the protein signatures related to metabolism, adipocyte homeostasis, mitochondrial function, and ferroptosis. PVAT-derived stromal vascular fraction cells were derived from our mouse strains to study the cellular and molecular phenotypes during adipogenic induction. We found that cells with activated Notch signaling displayed decreased mitochondrial respiration despite similar levels of adipogenesis and mitochondrial number. We observed variable regulation of the proteins related to mitochondrial dynamics and ferroptosis, including PHB3, PINK1, pDRP1, and the phospholipid hydroperoxidase GPX4. Mitochondria regulate some forms of ferroptosis, which is a regulated process of cell death driven by lipid peroxidation. Accordingly, we found that Notch activation promoted lipid peroxidation and ferroptosis in PVAT-derived adipocytes. Because the PVAT phenotype is a regulator of vascular reactivity, we tested the effect of Notch activation in PVAT on vasoreactivity using wire myography. The aortae from the N1ICD;Adipoq-Cre mice had increased vasocontraction and decreased vasorelaxation in a PVAT-dependent and age-dependent manner. Our data provide support for the novel concept that increased Notch signaling in the adipose tissue leads to PVAT whitening, impaired mitochondrial function, increased ferroptosis, and loss of a protective vasodilatory signal. Our study advances our understanding of how Notch signaling in adipocytes affects mitochondrial dynamics, which impacts vascular physiology.

Dataset Information

Presenilin gene function and Notch signaling feedback regulation in the developing mouse lens.

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Presenilin gene function and Notch signaling feedback regulation in the developing mouse lens.

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