Project description:Mutations in PROP1 are the most common cause of hypopituitarism in humans; therefore, unraveling its mechanism of action is highly relevant from a therapeutic perspective. Our current understanding of the role of PROP1 in the pituitary gland is limited to the regulation of pituitary transcription factors Hesx1 and Pit1. To elucidate the comprehensive PROP1-dependent gene regulatory network, we conducted genome wide analysis of PROP1 DNA binding and effects on gene expression in mutant tissues, isolated stem cells and engineered cell lines. We determined that PROP1 is essential for maintaining proliferation of stem cells and stimulating them to undergo an epithelial to mesenchymal transition-like process necessary for cell migration and differentiation. Genomic profiling reveals that PROP1 binds to and represses claudin 23, characteristic of epithelial cells, and it activates EMT inducer genes: Zeb2, Notch2 and Gli2. Our findings identify PROP1 as a central transcriptional component of pituitary stem cell differentiation.
Project description:Mutations in PROP1 are the most common cause of hypopituitarism in humans; therefore, unraveling its mechanism of action is highly relevant from a therapeutic perspective. Our current understanding of the role of PROP1 in the pituitary gland is limited to the regulation of pituitary transcription factors Hesx1 and Pit1. To elucidate the comprehensive PROP1-dependent gene regulatory network, we conducted genome wide analysis of PROP1 DNA binding and effects on gene expression in mutant tissues, isolated stem cells and engineered cell lines. We determined that PROP1 is essential for maintaining proliferation of stem cells and stimulating them to undergo an epithelial to mesenchymal transition-like process necessary for cell migration and differentiation. Genomic profiling reveals that PROP1 binds to and represses claudin 23, characteristic of epithelial cells, and it activates EMT inducer genes: Zeb2, Notch2 and Gli2. Our findings identify PROP1 as a central transcriptional component of pituitary stem cell differentiation.
Project description:The anterior pituitary gland plays central roles in body growth, reproduction, metabolism and the stress response. In this study, we performed single-cell RNA-sequencing (scRNA-seq) of 4,113 individual cells from human fetal pituitaries. We characterized divergent developmental trajectories with distinct transitional intermediate states in five hormone-producing cell lineages. Furthermore, we characterized the cellular heterogeneity of pituitary stem cells, identifying a hybrid epithelial/mesenchymal state and an early-to-late state transition. These analyses define a single-cell resolution roadmap for human pituitary development.
Project description:Prop1 controls the expression of genes besides Pit1 that are important for cell mgration, survival and differentiation in the mouse and human pituitary gland. Microarray analysis was used to compare pituitary RNA from newborn Prop1 and Pit1 mutants and their wild type littermates.
Project description:Prop1 controls the expression of genes besides Pit1 that are important for cell mgration, survival and differentiation in the mouse and human pituitary gland. Microarray analysis was used to compare pituitary RNA from newborn Prop1 and Pit1 mutants and their wild type littermates. Total RNA was collected and pooled from Prop1df/df, Porp1+/+, Pit1dw/dw, and Pit+/+ at the age of P1. 5 pools per genotype were hybridized to the Affymetrix Mouse Genome 430 2.0 array.
Project description:Mutations in the pituitary specific transcription factor Prophet of Pit-1 (PROP1) are the most common genetic etiology of combined pituitary hormone deficiency (CPHD). CPHD is associated with short stature, attributable to growth hormone deficiency and/or thyroid stimulating hormone deficiency, as well as hypothyroidism and infertility. Pathogenic lesions impair pituitary development and differentiation of endocrine cells. We performed single-cell RNA sequencing of pituitary cells from a wild-type and a Prop1-mutant P4 female to elucidate population-specific differential gene expression. We observed a Smoc2+ve population that expressed low Sox2, which trajectory analyses suggest are a transitional cell state as stem cells differentiate into endocrine cells. We also detected ectopic expression of Sox21 in these cells in the Prop1df/df mutant. Prop1-mutant mice are known to overexpress Pou3f4, which we now show to be also enriched in this Smoc2+ve population. We sought to elucidate the role of Pou3f4 during pituitary development and to determine the contributions of Pou3f4 upregulation to pituitary disease by utilizing double-mutant mice lacking both Prop1 and Pou3f4. However, our data showed that Pou3f4 is not required for normal pituitary development and function. Double mutants further demonstrated that the upregulation of Pou3f4 was not causative for the overexpression of Sox21. These data indicate loss of Pou3f4 is not a potential cause of CPHD, and further studies may investigate the functional consequence of upregulation of Pou3f4 and Sox21, if any, in the novel Smoc2+ve cell population.
Project description:Dietary vitamin A is metabolized into bioactive retinoic acid in vivo and regulates the development of many embryonic tissues. Retinoic acid signaling is active in the oral ectoderm-derived tissues of the neuroendocrine system, but its role there has not yet been fully explored. We show here that retinoic acid signaling is active during pituitary organogenesis and dependent on the pituitary transcription factor Prop1. Prop1-mutant mice show reduced expression of the aldehyde dehydrogenase gene Aldh1a2, which metabolizes the vitamin A-intermediate retinaldehyde into retinoic acid. In order to elucidate the specific function of RA signaling during neuroendocrine development, we studied a conditional deletion of Aldh1a2 and a dominant-negative mouse model of inhibited retinoic acid signaling during pituitary organogenesis. These models partially phenocopy Prop1-mutant mice by exhibiting embryonic pituitary dysmorphology and reduced hormone expression, especially of thyroid-stimulating hormone. These findings establish the critical role of retinoic acid in embryonic pituitary stem cell progression to differentiated hormone cells and raise the question of gene-by-environment interactions as contributors to pituitary development and disease.
Project description:Epithelial-mesenchymal transition (EMT) is a process by which epithelial cells lose cell-cell contact and gain cancer malignancy such as invasion, stemness, chemoresistance and metastasis. Reverse precess, mesenchymal-epithelial transition (MET) is also important for colonization. Extracellular vesicles (EVs) secreted from cancer cells are also important for cancer malignancy. To analyze RNAs from cells and EVs during EMT and MET, RNA sequencing was performed using E-cadherin-RFP/Py2T reporter system.