Browsing by Subject "Cell Differentiation"
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Item Balancing Renewal and Differentiation of Progenitor Cells in the Developing Kidney(2017-11-30) Ramalingam, Harini; Marciano, Denise; Carroll, Thomas J.; Cleaver, Ondine; Olson, Eric N.Mammalian kidneys perform the important function of blood filtration. All the filtered wastes are concentrated into urine and excreted from the body. The kidney performs these functions through individual functional units called nephrons. While nephron number may decrease during one's lifetime (due to various kidney injuries or hypoxic events), new nephrons do not form in adults. This is most likely due to the absence of nephron precursor cells. If nephron number falls below a certain threshold, the kidneys stop functioning properly pre-disposing individuals to a myriad of medical conditions including renal failure. While dialysis is a treatment option, the survival rate of a dialysis patients is poor. Currently, kidney transplantation is the only long-term treatment possible, but sufficient numbers of transplantable kidneys are not available. Various stem cell therapies and kidney re-engineering are actively being pursued as viable treatment paths. For successful progress in those approaches, we need access to lots of progenitors. To obtain this a thorough understanding of kidney development and the various signals that play important roles in nephron endowment is imperative. Despite many decades of work invested in this field, there are still many unknowns. Regulation of renewal and differentiation of the progenitors during the process of nephron formation is the focus of my dissertation.Item BMP Signaling Regulates Germ Cell Pluripotency, Sexual Differentiation, and Cancer Susceptibility(2016-10-14) Sanchez, Angelica; Castrillon, Diego H.; Amatruda, James F.; Cobb, Melanie H.; Cleaver, OndineTesticular germ cell tumors are the most common malignancy found in young men between the ages of 14-40. While these tumors are highly curable with cisplatin based combined chemotherapies, the treatments come with very detrimental side effects and ultimately fail in up to 15% of patients. These patients have no other avenues of treatment and often times succumb to the disease. Very little is currently known about the biology of the tumors but risk factors highlight possible mechanisms of action. For example, patients with Disorders of Sex Development (DSD) have increased risk for developing malignant germ cell tumors. DSDs manifest at birth and present with atypical gonadal or anatomical sex as well as chromosomal aberrations[1, 2]. Some cases are explained by the presence of chromosomal aberrations, but the cause of many others remains unknown. Such syndromes thus highlight the potential links between germ cell pluripotency, sexual differentiation and cancer susceptibility. A recent GWAS study [3] identified association of BMP7 with testicular dysgenesis syndrome; however the molecular mechanisms behind this association remain unknown. Previously, we described the development of testicular germ cell tumors in zebrafish carrying a mutation in bmpr1bb, a BMP family receptor, and demonstrated that human GCTs have defects in BMP signaling. Here I use this model and next-generation sequencing analysis in a cross-species comparative oncology approach to identify genes and pathways with fundamental importance to the development of the human disease. I further defined the role of specific BMP ligands in mediating germ cell differentiation and identified reciprocal somatic- and germ cell BMP signaling events that regulate germ cell differentiation and maturation. Using genetic crosses to further impair BMP signaling, I found that zebrafish doubly heterozygous for mutations in bmpr1bb and bmp2b, bmp7a or smad5 have profoundly impaired gonadogenesis and altered male:female sex ratios. Affected fish also exhibit markedly abnormal gonadal differentiation, including the presence of undifferentiated gonadal tissue and the occurrence of biphenotypic gonads, as well as greatly increased germ cell tumor susceptibility. Our findings implicate defective BMP pathway signaling as a potential factor in DSDs and GCT susceptibility. Our goals are to identify biological mechanisms that govern germ cell differentiation and to understand how defects in this process cause human disease.Item Characterizing Renal Interstitial Heterogeneity and Its Role in Nephron Patterning(2020-08-01T05:00:00.000Z) England, Alicia Rachel; Marciano, Denise; Carroll, Thomas J.; Cleaver, Ondine; Petroll, W. Matthew; Varner, VictorChronic kidney disease (CKD) is a growing national health concern affecting 30 million Americans. CKD can lead to permanent loss of kidney function requiring treatment for survival, yet the only two treatment options, dialysis and transplant, are unable to meet the needs of the millions impacted. There are numerous efforts to engineer renal replacement therapy tissue yet most focus primarily on the nephron (the functional unit of the kidney) and the vasculature. Although these efforts are exciting, they face two significant obstacles. First, current progenitor cell-based technologies have only produced relatively immature tissues. Second, most efforts have overlooked the importance of renal stroma, a cell type that not only impacts the development of the renal parenchyma, but which also plays a crucial role in kidney physiology as well as having multiple endocrine functions. Using a combination of single-cell ribonucleic acid sequencing (scRNA-seq) and messenger RNA (mRNA) in situ hybridization we have found that the mouse embryonic renal stroma is a molecularly heterogeneous population of cells with different cell types occupying anatomically distinct positions that correlate with anatomically and functionally distinct regions of the adjacent parenchyma. We find that human fetal kidney interstitium shows a similar degree of heterogeneity to the mouse extending this phenomenon beyond our model system. Further, we find that beta-catenin has a cell-autonomous role in the development of a medullary subset of the interstitium and that this non-autonomously affects the development of the adjacent tissue. These data suggest stromal sub-types establish unique microenvironmental niches that provide signals which regulate the differentiation/segmentation of the nephron. We find that interstitial heterogeneity is evident at the earliest stages of renal development, and that interstitial patterning develops independent of signals from the nephron tubules. Using a novel nephrogenic zone cell differentiation assay, we find a subpopulation preferentially promotes proximal tubule differentiation and non-autonomously promotes proliferation. These data highlight a functional role of the renal interstitium in renal development that cannot be ignored in current renal regeneration efforts.Item Dynamic Behaviors of Langerhans Cells In Situ(2007-06-12) Ward, Brant Russell; Takashima, AkiraThe epithelial surfaces of the body, being constantly exposed to both innocuous and potentially harmful substances and organisms, are protected by an array of immune cells called Langerhans cells (LCs). As a bridge between the innate (non-specific) and adaptive (specific) immune responses, LCs sense injurious stimuli and contribute to tissue inflammation as well as initiate the antibody- or cell-mediated specific responses to pathogenic organisms. These events represent pivotal occurrences in the instigation of the immune response, and thus are important targets for experimental investigatation. To explore the dynamic behaviors of LCs within the epithelial tissues, LCs in living corneal and epidermal tissue were examined ex vivo and in vivo by multiphoton and confocal laser scanning microscopy. LCs at baseline exhibit a unique behavior characterized by rhythmic extension and retraction of the dendritic processes. To explore the responses of LCs to tissue injury, LCs in the cornea and epidermis were examined after pinpoint thermal or diffuse chemical injury. After these stimuli, LCs displayed more active dSEARCH and lateral movement through the tissue. These behavioral responses were dependent on the inflammatory cytokines IL-1 and TNFa, suggesting a requirement for these cytokines in LCs responses to injury. After infection of the epidermis with pathogenic bacteria in vivo, LCs displayed similar responses, showing increased dSEARCH and lateral migration. These results indicate that LCs in different tissues react to varying pathogenic stimuli with common behavioral responses and add a new dimension to our understanding of LC biology.Item EGFR and Akt Signaling in Rhabdomyosarcoma Pathogenesis(2018-07-25) Granados, Valerie Ann; Amatruda, James F.; Olson, Eric N.; Lum, Lawrence; Galindo, ReneRhabdomyosarcoma is an aggressive soft-tissue malignancy comprised microscopically of neoplastic skeletal muscle-lineage precursors that fail to exit the cell-cycle and fuse into syncytial muscle - the underlying pathogenetic mechanisms for which remain unclear. We previously identified that misregulated myoblast fusion signaling via the TANC1 adaptor molecule promotes neoplastic transformation in RMS cells. As TANC1 is not presently pharmacologically targetable, here we have turned to our Drosophila RMS-related model to identify myoblast fusion-related elements potentially targetable in RMS. Genetic modifier screening against the fly model revealed that decreased Epidermal Growth Factor Receptor (EGFR) activity, which regulates myoblast fusion programming in flies, suppresses PAX-FOXO1 (PF)-induced lethality. As EGFR is pharmacologically targetable, we demonstrate that EGFR inhibitors antagonize RMS in a ERMS-RD cell line, but that other RMS cell lines are resistant. Further interrogation finds that EGFR inhibitor-sensitive cells exhibit marked down-regulated activation of the Akt intracellular signaling transducer, but not MEK/MAPK or STAT3, suggesting that Akt promotes and/or sustains RMS. We then demonstrate that Akt pharmacologic inhibition antagonizes RMS in vitro and in vivo, including RMS cells resistant to EGFR inhibition. We additionally find that sustained Akt1 activity promotes RMS cell terminal differentiation-arrest. Together, these findings point towards Akt activity as a broad RMS underpinning and therapeutic vulnerability.Item Elucidating the Role of Cellular Architecture in the Developing Pancreas(2015-11-30) Marty Santos, Leilani Marie; MacDonald, Raymond J.; Johnson, Jane E.; Carroll, Thomas J.; Cleaver, OndineMany studies have focused on examining the intrinsic factors such as transcriptional regulators that instruct the step-wise acquisition of β-cell fate in the developing pancreas, with the intention of recapitulating the events necessary in order to generate these cells in vitro for replacement therapies. Directed differentiation protocols have improved upon transitioning from 2D to 3D cultures, indicating that the 3D microenvironment in which β cells are born is critical for the acquisition of their cell fate. However, little is known about the mechanisms through which the 3D architecture of the developing pancreas mediates cell fate specification and epithelial organization. In order to address some of the remaining gaps in the field, we proceeded to characterize the Pdx1-/- embryo, a mutant in which pancreatic cell fate and architecture had been reported to fail early in its development, to determine whether the developmental failure was related to defects in the epithelial architecture. After elucidating that Pdx1 is a transcriptional regulator of the cellular adhesion molecule E-cadherin, we then examined the effect that tissue-specific deletion of this molecule has on the developing pancreas. We determined that E-cadherin regulates both endocrine cell fate and isletogenesis, as we observe that there is a reduction in endocrine progenitors and total endocrine volume, in addition to a failure of the endocrine cells to coalesce into islets. Our findings also demonstrate that acinar cells are lost in the post-natal E-cadherinf/f;Pdx1Cre pancreas, due to an increase in cell death, suggesting that E-cadherin is capable of regulating cell survival. This body of work indicates that architectural molecules play a critical role in the regulation of cell fate specification and epithelial morphogenesis in the developing pancreas.Item FOXO is the Mediator Linking Temporal Differentiation and the Insulin Signaling Pathway(2005-12-20) Lah, Carol Joonhyun; Cameron, ScottThe timing of differentiation is crucial for the correct development of an organism, because specific pathways can be used reiteratively to differentiate cells. Until recently, the molecular mechanism behind the temporal control of differentiation has remained elusive. Bateman and McNeill (2004) revealed a novel role for the insulin/insulin-like growth factor receptor (InR) pathway in regulating the timing of differentiation in neuronal photoreceptor cells in the Drosophila compound eye. The link between the InR pathway and temporal differentiation is significant, because of the implication that external factors, e.g. nutrition, are tightly coupled to the timing of differentiation. This proposal tests the hypothesis that FOXO, a crucial component of the InR pathway, mediates the regulation of developmental timing. The aims are the following: 1. Observe if dFOXO mutants affect temporal differentiation in the Drosophila eye. 2. Perform epistasis experiments to determine if dFOXO is downstream of other insulin signaling components. 3. Analyze the downstream targets of dFOXO that may play a role in neuronal differentiation.Item Innate and Adaptive Immune Mechanisms of Pathogen-Specific T Helper 17 Cell Differentiation(2019-12-09) Gao, Yajing; Niederkorn, Jerry Y.; Wakeland, Edward K.; Pasare, Chandrashekhar; Pfeiffer, Julie K.; Reese, Tiffany A.; Fu, Yang-XinDendritic cells (DCs) are critical for the differentiation of pathogen-specific CD4 T cells. However, to what extent innate cues from DCs dictate transcriptional changes in T cells remains elusive. Here, we used DCs stimulated with distinct pathogens to prime CD4 T cells in vitro, and found that these T cells express unique transcriptional profiles dictated by the nature of the priming pathogen. More specifically, the transcriptome of in vitro pathogen-primed T helper 17 (Th17) cells resembled that of in vivo primed Th17 cells but was remarkably distinct from cytokine-polarized Th17 cells. We identified caspase-1 as one of the unique genes upregulated only in pathogen-primed Th17 cells and discovered a critical role for T-cell-intrinsic caspase-1, independent of its function in inflammasome, in optimal priming of Th17 responses. T cells lacking caspase-1 failed to induce colitis or confer protection to C. rodentium infection due to suboptimal Th17 cell differentiation in vivo. This study underlines the importance of DC-mediated priming in identifying novel regulators of T cell differentiation. DC sensing of fungal pathogens, certain commensal bacteria and extracellular enteric pathogens leads to the generation of Th17 response. However, the nature of specific innate signals that uniquely promote Th17 cells has not been fully understood. We find that sensing of C. rodentium and other enteric pathogens induces unfolded protein response (UPR) in DCs that is critical for Th17 differentiation. Mechanistically, activation of PERK-eIF2α-ATF4 arm of UPR in DCs selectively enhances the production of IL-6 and IL-23. Genetic ablation of PERK in DCs or pharmacological inhibition of eIF2α phosphorylation blunts the production of IL-23 and IL-6, leading to impairment of Th17/22 differentiation and defective protection against enteropathogenic infection. Additionally, forced induction of UPR led to Th17 cell differentiation by non-Th17-inducing pathogens. We propose that the integration of PRR signaling with other sensors of disturbed cellular homeostasis, such as pathogen-induced stress, allows the innate immune system to induce tailored adaptive immune responses.Item Mapping the Landscape of Acquired Vulnerabilities in Ovarian Cancer(2013-06-05) Shields, Benjamin Baker; Castrillon, Diego H.; White, Michael A.; Altschuler, Steven J.; Brekken, Rolf A.Recent undertakings to identify the genetic lesions associated with ovarian cancer have noted the striking diversity of mutations occurring in this disease. This genetic diversity has complicated the search for novel therapies. However, recent data has suggested that one commonality of ovarian tumors might be ablation of miRNA biogenesis. Here I conducted a broad-scale gain-of-function microRNA (miRNA) screen in 16 ovarian cancer cell lines to annotate the functional landscape present in such a chaotic genetic background. miRNAs function as multigenic perturbations allowing for interrogation of maximal gene space with few experiments. This screen identified multiple miRNAs reducing cell viability with the majority of hits being toxic in only one or two lines screened. This surprising finding reflected the commonality of altered miRNA function in ovarian tumors while also suggesting that specifics of this alteration in function are unique to each tumor. To investigate more public vulnerabilities, I focused mechanistic studies on miRNAs displaying penetrance in greater than 5 cell lines. miR-517a reduced cell viability in over 30% of the panel and also reduced tumor burden in vivo. Functional analysis of the predicted targets of miR-517a revealed that expression of this miRNA reduced protein levels of ARCN1, a member of the coatamer complex, and that knockdown of ARCN1 reduced cell viability similar to miR-517a. Another penetrant miRNA, miR-124a, reduced cell viability in 37.5% of the panel and functional analysis of this miRNA revealed it promoted a cell differentiation program. Analysis of predicted targets revealed that expression of miR-124a reduced expression the homeodomain transcription factor SIX4, resulting in increased signaling along the tumor suppressive AMPK pathway and epithelial differentiation. Furthermore, SIX4 displayed increased expression in ovarian tumors and depletion of SIX4 expression reduced tumor cell viability in vitro and in vivo. Therefore, SIX4 overexpression might function to deflect cell differentiation in tumors. Thus, the common loss of miRNA function observed in ovarian tumors might serve to maintain an undifferentiated state, and engagement of cell fate determination programs via re-expression of miRNAs can result in catastrophic consequences for cancer cell viability.Item Regulation of Adaptive and Innate Immunity by the Circadian Transcription Factor NFIL3(2014-06-13) Yu, Xiaofei; Yarovinsky, Felix; Hooper, Lora V.; Alto, Neal; Green, Carla B.; van Oers, Nicolai S. C.The day-night cycle has a profound impact on animal physiology, which has been shown to be mediated by an intracellular timing system called the circadian clock. However, little is known about whether and how the day-night cycle and the circadian clock influence host immunity. Here, I show that the circadian transcription factor NFIL3 is critical for both adaptive and innate immunity by regulating TH17 cells and innate lymphoid cells (ILCs), respectively. First, NFIL3 transcriptionally represses RORgt, the master regulator of TH17 cells, by directly binding to the Rorgt promoter and thereby suppresses TH17 cell development from naïve T helper cells. Consistent with oscillation of Nfil3 expression during the circadian cycle, Rorgt expression also oscillates in mice, with higher expression at noon and lower expression at midnight. Accordingly, naïve T helper cells show greater potential to develop into TH17 cells at noon than at midnight. Furthermore, artificially disturbing the circadian cycles of mice by manipulating their light exposure results in circadian clock-mediated disruption of TH17 homeostasis and increased susceptibility to experimentally-induced colitis. Therefore, NFIL3 regulates TH17 cell development in a circadian manner. Second, NFIL3 is essential for the development of all major types of innate lymphoid cells (ILCs) by regulating the generation of ILC progenitors in the bone marrow. One of the NFIL3-dependent progenitor populations, aLP, can differentiate into all major types of ILCs in vivo. NFIL3 controls progenitor development by activating a High Mobility Group (HMG) transcription factor Tox directly in common lymphoid progenitors (CLPs). Accordingly, restoring Tox expression in Nfil3-deficient progenitors rescues ILC development in mice. So NFIL3 regulates ILC development by activating Tox expression in bone marrow precursors. Taken together, my work demonstrates that NFIL3 is a critical regulator of host immunity and that it modulates immune functions along the circadian cycle. This study is among the first to reveal the influence of the circadian clock on host immunity and provides novel insights into the regulatory mechanisms underlying variations of immune cell development and function during the circadian cycle.Item Regulation of Transcription Through RNA Polymerase II Promoter-Proximal Pausing(2018-04-12) Tastemel, Melodi Damla; Kraus, W. Lee; Zhang, Chengcheng "Alec"; Amatruda, James F.; Banaszynski, Laura; Bai, XiaoyingCells need to respond to environmental signals in order to adapt to changing stimuli, maintain cell viability and establish fate decisions. One way the cell accomplishes this adaptation is via altering gene expression of critical genes by influencing transcription of their mRNAs. mRNA Transcription is carried out by the enzyme RNA Polymerase II (Pol II). Regulation of RNA Pol II occurs at every step of transcription, the most well studied one being transcription initiation. However, in many metazoan genes, after transcription initiates, RNA Pol II experiences a pause 20-60 nucleotides downstream of transcription start site. This is mediated by two pausing complexes negative elongation factor (NELF) and DRB-sensitivity inducing factor (DSIF). In order to pursue productive elongation, Pol II needs to be relieved from this promoter-proximal pause by recruitment of P-TEFb. Recent studies have demonstrated that regulation of transcription pausing, and elongation is necessary for cells to respond to external stimuli and for mammalian development, and that dysregulation of this network has been seen in developmental and immunological disorders, heart disease and a variety of cancers. However, what are the molecular and functional roles of Pol II pausing during normal mammalian development are yet to be elucidated. Here, using mouse embryonic stem cell differentiation as my major model system of mammalian development, I asked whether disruptions in RNA Pol II pausing would have an effect on embryonic development. I utilized CRISPR/Cas9 based genome editing to mutate pausing complex subunits nelfe (NELF) and spt5 (DSIF) in mouse embryonic stem cells. Using genomic approaches such as global run-on sequencing, I validated that promoter-proximal pausing is perturbed in these mESCs. By utilizing monolayer and three-dimensional differentiation protocols, I have observed disruption of lineage differentiation in pausing deficient mESCs. Finally, I established CRISPR/Cas9 genome editing protocols to mutate chromatin regulators and pausing factors in order to study their roles in developing organisms.Item A Role for POU3F3 in Myocyte Differentiation: Exploring New Frontier in Alveolar Rhabdomyosarcoma Development(2015-01-26) Denegre, Amelia; Granados, Valerie; Avirneni, Usha; Galindo, ReneSUMMARY: Children who are diagnosed with pediatric rhabdomyosarcoma (RMS), a mesenchymal-derived soft tissue cancer that comprises 3.5% of childhood cancers, are often delivered a bleak prognosis with little hope of a future. Despite significant advances illuminating transcription factor signaling in RMS onset and progression, research is still needed to precisely understand RMS pathogenesis on a molecular level in order to develop targeted treatment options. OBJECTIVE: The goal of this project is to explore the role of POU3F3 in myogenesis, particularly in relation to cell fusion and myocyte differentiation. METHODS: Immunofluorescence: POU3F3 knockdown cells were differentiated in 2% horse serum. On day 2 of differentiation, cells were probed with POU3F3 primary and red immunofluorescent secondary antibody, allowing for imaging of POU3F3 localization during myoblast differentiation. Western Blot: Three knock-down shPOU3F3 C2C12 cell-line constructs were tested. Western blots were performed that compared C2C12 control, POU3F3 overexpression, and POU3F3 knockdown cells. Crystal Violet: After differentiation, POU3F3 knock-down and control cell lines were stained with crystal violet stain to visualize the effect of POU3F3 knockdown on differentiation. RESULTS: Immunofluorescence: We confirmed that in knockdown C2C12 cells, POU3F3 localizes like control C2C12, in the periphery. Previous research has shown that in overexpressed POU3F3 C2C12 cell lines, POU3F3 localizes to the nucleus. The implication of the disconnect between POU3F3 location in controls and knock-downs versus overexpressed cell lines is an area that is an opportunity for further research. Western Blot: Western Blot analysis confirmed that POU3F3 knockdown was successful, and provides a platform for further POU3F3 interrogation. Crystal Violet: Crystal violet staining suggests that POU3F3 participates in a myoblast differentiation, as the control cells fuse into myotubes, while POU3F3-silenced cells do not. CONCLUSION: These initial results suggest that POU3F3 participates in muscle differentiation. Next, the Galindo lab will be probing POU3F3 function in myogenesis in greater depth, insights they will next apply to RMS.Item Roles of Ascl1 and Olig2 in the Transcriptional Regulation of Astrocytogenesis(2016-01-19) Combiths, Adam; Vue, Tou Yia; Johnson, Jane E.Ascl1 and Olig2 are transcription factors highly expressed in certain neural progenitor cells, and are known to be involved in neurogenesis and oligodendrogenesis (OL) throughout the CNS; their role in astrocytogenesis (AS) is less well explored. Recent evidence shows that Ascl1-lineage AS clones in the spinal cord (SC) are spatially restricted to either gray matter (GM) or white matter (WM), but not both, and that Olig2 may be necessary for WM astrocytogenesis in the brain. We consider the following questions: (1) Do Ascl1+ progenitors give rise to astrocytes in the brain? (2) Do astrocyte clones, in general, display the GM/WM spatial restriction seen in Ascl1-lineage astrocytes? (3a) Is Olig2 expressed by astrocytes in the SC? (3b) If it is, is this expression required for astrocytogenesis in the SC? To address (1), we used the CreERT2 system under the Ascl1 promoter to label Ascl1+ progenitor cells in the neonatal murine brain (and their progeny) with the tdTom fluorescent reporter. Adult brains were obtained and immunohistochemically (IHC) labeled for factors specific to mature AS, OL and neural lineages; AS, OL, and neurons derived from neonatal Ascl1+ progenitors were observed in every major cortical and subcortical structure, showing that neonatal Ascl1+ progenitors do give rise to AS through the brain. To address (2), we used the CreERT2-Confetti system under the promoter for hGFAP (an astrocyte-specific marker) to give sparse labeling of astrocytes in multiple colors, so that any clone (one clone representing all the progeny of a single AS-progenitor cell) will be far from and visually distinct from other clones. Adult murine SCs were obtained, sectioned, and analyzed by fluorescence microscopy. The location, morphology and clonal identity of every labeled cell was cataloged and used to construct a clonal map of AS distribution in the spinal cord from neonatal development through adulthood, revealing the presence of "mixed" (non-GM/WM-restricted) AS clones - strong evidence for the existence of a GM/WM bipotent AS progenitor cell. To address (3a), we used the CreERT2 system under hGFAP, and IHC labeled for the presence of Olig2. The presence of Olig2+;tdTom+ double positive cells (i.e., astrocytes expressing Olig2) was quantified via fluorescence microscopy. Approximately 50% of astrocytes expressed Olig2. To address (3b), we repeated the above procedure in mice with floxed Olig2 alleles, allowing conditional knockout (CKO) of Olig2 at time of induction. 50% of Olig2-CKO spinal cords showed an almost-complete lack of astrocytes, tentatively indicating a vital role of Olig2 expression in astrocytogenesis in both the GM and the WM.Item T Cell Intrinsic BCAP Links IL1R to the PI3K-mTOR Pathway and Regulates Pathogenic Th17 Differentiation(2018-07-24) Deason, Krystin Leigh; Hooper, Lora V.; Pasare, Chandrashekhar; van Oers, Nicolai S. C.; Satterthwaite, Anne B.Toll-IL-1R homology (TIR) domains are found within adaptor proteins involved in the signaling of Toll like receptors (TLRs) and Interleukin 1 receptor (IL1R) families. Previous work by our lab identified a TIR domain in the protein B cell adaptor for phosphoinositide 3-kinase (BCAP) and determined a role for BCAP in the TLR signaling pathway in myeloid cells. Due to the shared use of TIR domains by TLR and IL1R signaling pathways, I hypothesized that BCAP would also be involved in signaling downstream of the IL1 family of receptors. The IL1 cytokine family has been shown to play a major role in T cell activation, survival, and differentiation; IL1b, in particular, plays a critical role in the differentiation of Th17 lineage cells. Here, I discovered that BCAP functions downstream of IL1R in CD4 T cells and thereby regulates Th17 lineage differentiation and function. IL1b-induced PI3K-Akt-mTOR signaling is compromised in BCAP deficient T cells which leads to decreased mTOR activation, decreased glycolysis, and defective Th17 lineage commitment. Transcriptional analysis of BCAP deficient CD4 T cells revealed that BCAP is critical for the expression of genes associated with pathogenic Th17 lineage cells. Mice specifically lacking BCAP in T cells have normal development of steady state Th17 cells in vivo yet have decreased development of pathogenic Th17 diseases, such as experimental autoimmune encephalomyelitis (EAE) and T cell transfer colitis. Further, the use of a potent inhibitor of mTOR, which is downstream of BCAP activation, mimics BCAP deficiency by preventing IL1b induced differentiation of pathogenic Th17 cells. This study establishes BCAP as a critical link between IL1R and the metabolic status of activated Th17 cells and further demonstrates that BCAP is critical for the generation of pathogenic Th17 cells in vitro and in vivo.