Browsing by Subject "Notch"

The homeostatic hematopoietic stem cell compartment is comprised of quiescent long term self renewing stem cells and cycling short term stem cells with finite renewal potential. To study the molecular mechanisms governing self renewal of hematopoietic cells we must force them to enter the cell cycle and proliferate. One approach to accomplish this goal is to damage the hematopoietic compartment with ionizing radiation or cytotoxic chemotherapy. Such injuries ablate mature blood cells and drive the primitive stem cells into cycle. I have elected to use a simple model of hematopoietic damage and regeneration to study the molecular mechanisms controlling self renewal in hematopoietic stem cells. At the beginning of this project it was unclear whether the signaling pathways which homeostatically control self renewal are utilized during injury repair. In particular, there is very little understanding of the signals required for regeneration after radiation damage. We hypothesized extracellular signal transduction pathways provided by the microenvironment are critical mediators of the stem cell repair process. To address these topics and extend the previous work generated in our laboratory, I chose to pursue a candidate approach focusing on the Wnt and Notch developmental signaling pathways.

In order to examine the activation and requirement for each signaling cascade after radiation and chemotherapy damage we used a combination of loss of function and reporter mouse models. To this end, we have conducted the majority of experiments for the Wnt project in animals deficient in beta-catenin, the key transcription factor required in the pathway. Our investigations revealed the Wnt pathway is turned on within regenerating stem cells and loss of beta-catenin impairs regeneration of the stem cell compartment after both radiation and chemotherapy injury.

Using a Transgenic Notch Reporter mouse to investigate the role of Notch signaling following hematopoietic damage we determined the Notch pathway is also activated during regeneration. Furthermore, using a live imaging approach we discovered Notch activated cells change their fate choice during regeneration. To determine if Notch gain of function provides radio-protection we infected stem cells with an active form of Notch prior to radiation and then scored self renewal potential in vitro. This led us to the conclusion that Notch gain of function can provide a self renewal benefit to irradiated hematopoietic stem cells.

The mouse embryonic gonad provides an outstanding model to study the complex mechanisms involved in cell fate specification and maintenance. At the bipotential stage, both XX and XY gonads are capable of becoming testes or ovaries upon specific molecular cues. The specification of the supporting cell lineage (as either Sertoli cells in the male or granulosa cells in the female) initiates the testis or ovary program, leading to male or female fate. However, there are significant gaps in our understanding of how the somatic cells in the gonad arise, are competent to differentiate, and determine and maintain their fates. In this dissertation, we addressed these questions.

We found that NUMB (an antagonist of Notch signaling) serves as competence factor for somatic cell differentiation during early gonadogenesis. The asymmetric allocation of NUMB to the basolateral domain of actively dividing coelomic epithelial (CE) cells is indispensable to (1) maintain the totipotent stem cell-like reservoir at the CE domain, and (2) give rise to progenitor cells that can further differentiate into supporting and interstitial cell lineages. Deletion of Numb; Numbl resulted in disruption of cell polarity in the CE domain as well as a reduction of multiple differentiated cell lineages within XX and XY gonads, including supporting cells and male steroidogenic cells, which were most severely affected. We detected elevated Notch downstream signaling in the Numb; Numbl mutant gonads. Moreover, treatment of DAPT (which blocks Notch signaling) rescued the Numb; Numbl mutant phenotypes, strongly suggesting that upregulation of Notch is responsible.

Previous experiments indicate that when supporting cells commit to the male (Sertoli) fate, they must repress the alternative female (granulosa) cell fate. In another line of experiments, we investigated the hypothesis that the Polycomb repressive complex (PRC1) plays a critical role in repressing the female pathway during male gonad patterning. We found that loss of Ring1B (a component of PRC1) led to the disruption of XY gonad development specific to the posterior region of male gonads. Sry, the upstream driver of the male pathway, was not appropriately expressed in the posterior domain, which contained cells expressing female markers and, in some cases, small aggregates of undifferentiated cells. Using ChIP-Seq, we identified potential targets of PRC during male gonad development. Moreover, a key gene in the male pathway, SOX9, interacts with Ring1B, based on immunoprecipitation results, leading to the hypothesis that it may be involved in the recruitment of PRC to its target sites to execute the repression of female genes in male gonads.

Our findings provide insight into how somatic cell fate is determined and maintained during mammalian sex determination. Our results may be valuable for patients with disorders of sexual development with unidentified genetic contributions.

Rhabdomyosarcoma (RMS), the most common soft tissue sarcoma in children and adolescents, is characterized by skeletal muscle features. The Ras-driven subset, which includes the embryonal (eRMS) and pleomorphic (pRMS) histologic subtypes, is an aggressive high risk subgroup with a 5-year survival rate of <30%. Recently the YAP oncoprotein, which is ordinarily silenced by the Hippo tumor suppressor pathway, was found to be highly upregulated in RMS tumors. However, the role of YAP in the Ras-driven subset was unknown.

In patient-derived Ras-driven eRMS cell lines, we suppressed YAP genetically via shRNAs. YAP suppression decreased cell proliferation, increased myogenic differentiation, and promoted apoptosis in vitro and in vivo in subcutaneous xenografts. Pharmacologic inhibition by the YAP-TEAD inhibitor verteporfin also decreased cell proliferation and tumor growth. In a genetically defined model of Ras-driven RMS, constitutively active YAPS127A can serve as the initial oncogenic alteration whereby YAPS127A is sufficient for senescence bypass in primary skeletal muscle myoblasts, but requires expression of hTERT and oncogenic Ras for tumorigenesis in vivo. Importantly these tumors are histologically consistent with human Ras-driven RMS.

To understand the impact of YAP signaling on cell stemness, we cultured eRMS cells as 3D spheres. These spheres are enriched in stem cell genes, as well as in YAP and Notch signaling. The Notch pathway is another developmental pathway that is also highly upregulated in eRMS and contributes to tumorigenesis. Using the spheres as a model, we uncovered a bidirectional signaling circuit between YAP and Notch that regulates stemness. Active Notch signaling upregulates YAP, and YAP in turn upregulates the Notch ligands JAG1 and DLL1 and the transcription factor RBPJ. This circuit controls expression of several stem cell genes including SOX2, which is functionally required for eRMS cell stemness. Silencing this circuit for therapeutic purposes may be challenging, since the inhibition of one node (for example pharmacologic Notch blockade) can be rescued by upregulation of another (constitutive YAP expression). Instead, dual inhibition of Notch and YAP is necessary. Supporting the existence of this circuit beyond a model system, nuclear Notch and YAP protein expression are correlated in human eRMS tumors, and YAP suppression in vivo decreases both Notch signaling and SOX2 expression. In preliminary studies, we also analyzed the differential effects of the three Ras isoforms on eRMS tumorigenesis, Ras-Notch, and Ras-YAP signaling, and developed a method to culture the alveolar RMS subtype as spheres. In conclusion, the YAP oncoprotein drives Ras-driven tumorigenesis by promoting cell growth, survival, and stemness, and through signaling interactions with the Notch pathway. This study also provides rationale for combination therapies targeting YAP and Notch for the treatment of Ras-driven RMS.

Over 1.5 billion individuals are infected with intestinal helminths worldwide, with a majority of those infected living in developing nations. In developed nations intestinal helminth infections are very rare, while incidences of allergy and asthma are common. The incidence of allergic afflictions is growing rapidly every year. Both clearance of helminth infections and propagation of allergic disease are mediated by type 2 immune responses. The cytokines interleukin-4 (IL-4) and interleukin-13 (IL-13) play major roles in the propagation of type 2 immune responses. IL-4 and IL-13 are produced by a number of immune cells, within both the innate and adaptive arms of immunity, that are important in driving allergic responses.

CD4+ T follicular helper (Tfh) cells reside in the B-cell follicle and specialize in aiding the maturation of germinal center (GC) B cells. IL-4 produced by Tfh cells is required for GC B cell Immunoglobulin (Ig) class switching to type-2 isotypes, IgE and IgG1. IgE serves as a critical mediator of type-2 immune responses. CD4+ T helper 2 (Th2) cells localize to the periphery at sites of infection and damage. Th2 cells make both IL-4 and IL-13 cytokines. Th2 cells, along with multiple innate cell types, are critical for driving the peripheral hallmarks of type-2 immune responses, including mucus production and smooth muscle contractility.

Elucidating the pathways that regulate the differentiation, function, and maintenance of Tfh and Th2 cells is critically important for discovering potential therapies for allergic disease and helminth infections. Notch signaling is capable of driving Th2 IL-4 production and differentiation in vitro. However, the in vivo role for Notch signaling in Th2 populations remains unclear. The mechanisms controlling Tfh IL-4 production are largely unknown. Given that Notch signaling is required for the differentiation of Tfh cells and is known to influence cytokine production in T cells, we hypothesized that Notch signaling also plays an important role in regulating the function of Tfh cells. Nippostrongylus brasiliensis infection drives a robust type-2 immune response and allows for analysis of both Tfh and Th2 cells. Here, infection with N. brasiliensis was used to characterize whether Notch signaling is required for Th2 and Tfh differentiation and function in vivo.

Deletion of Notch receptors on T cells of infected mice results in reduced IL-4 producing Tfh, but not Th2 cells. As a result, we saw impairments in overall Tfh functionality while peripheral Th2 immunity remained intact. Notch deficient T cells had major impairments in Tfh, but not Th2, cell differentiation. Overexpression of Notch signaling in CD4+ T cells leads to increased IL-4 production by Tfh cells, but not Th2 cells. Furthermore, we identified that conventional dendritic cells (cDCs) do play a role as sources for Notch ligand early during the immune response. However, neither cDCs or follicular dendritic cells (FDCs) are essential sources of Notch ligand to drive Tfh cell differentiation.

While Notch signaling is critical for Tfh differentiation, it is not known if Notch signaling plays a continued role beyond Tfh differentiation. We used pharmacologic inhibition of Notch signaling to assess a role for Notch in Tfh maintenance. Here, we show that inhibition of Notch signaling after Tfh differentiation results in altered expression and activity of important trafficking receptors. This change was accompanied by aberrant localization of IL-4 expressing T cells in the lymph node. Additionally, late Notch inhibition resulted in an altered transcriptional program in Tfh cells. These findings suggest that Notch signaling plays a critical role in Tfh, but not Th2 driven immunity. In total, the data shown here demonstrate that Notch signaling is not only important for Tfh differentiation, but also for regulating Tfh cell fate, function, and maintenance.