Browsing by Subject "Notch"
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Item Determining the role of a small GTPase, Ral, and an endocytic factor, epsin, in Drosophila Notch signaling(2011-12) Cho, Bomsoo; Fischer, Janice AnnCell-cell communication events are crucial to determine the fate of each cell during development. Notch signaling is involved in many different contexts in determining cell fate by mediating cell-cell communication. Furthermore, regulation of the Notch transduction pathway is critical for normal cellular function, which is implicated in various diseases, including cancers. At a certain developmental time point, intrinsic or extrinsic developmental cues induce biases in ligands and Notch receptors between neighboring cells. These initial biases are further amplified by various cellular factors which eventually dictate cell fates. In Drosophila, two Notch ligands, Delta and Serrate, trigger Notch receptor activation in nearby cells by virtue of numerous regulating factors. One important question in this area is how cells become Notch signal sending or receiving cells for cell fate decisions. I show evidence about a distinct mechanism for biasing the direction of Notch signaling that depends on a small GTPase, Ral, during Drosophila photoreceptor cell development. Investigations described here indicate that Fz signaling up-regulates Ral transcription in a signal sending fate cell, the R3 precursor, and Ral represses ligand-independent activation of Notch in the R3 precursor. This event ensures R3 to become a signaler and contributes to asymmetric Notch activation in the neighboring cell, R4. Ral is a small Ras-like GTPase that regulates membrane trafficking and signaling. Here, possible Ral effector pathways that are important for Notch regulation will be proposed. To trigger Notch activation in adjacent cells, Notch ligand endocytosis by the signaling cells is necessary. Recently, it was suggested that control of membrane trafficking is important not only for ligand signaling, but also for Notch receptor activation. Furthermore, Notch receptor trafficking regulates critical cellular functions, including proliferation, which is implicated in tumors. Therefore, another important question in Notch signaling is about the role of membrane trafficking in regulation of the Notch transduction pathway. Drosophila endocytic epsin, Liquid facets [Lqf], is a key component necessary for ligand endocytosis, thereby triggering Notch activation in adjacent cells. However, its function in signal receiving cells for Notch activation has not been studied. In this dissertation, I provide evidence that epsin is also required in signal receiving cells for Notch activation in developmental contexts. Furthermore, genetic and molecular evidence suggests that epsin regulates Notch receptor trafficking via Rab5-mediated endosomal sorting pathway for Notch activation. These studies support the idea that Notch activation at the plasma membrane is not the only way to transduce Notch signaling, but the Notch receptor must enter through an epsin-mediated endocytic pathway into subcellular compartments to be activated, at least in some contexts.Item Development and validation of an analytical model for the notched pocket damper seal(Texas A&M University, 2004-09-30) Kannan Srinivas, BharathwajExperiments conducted at the Texas A&M Turbomachinery Laboratory and field applications have shown that pocket damper seals (PDS) can be used to suppress vibrations in compressors. A mathematical model is presented for the notched PDS. The notch is a prominent feature in all the PDS manufactured in recent times. The notch is provided at the exit blades of the PDS to act as a diverging clearance, which is one of the conditions for the damper seal to perform satisfactorily The model to be presented has been adapted from a theory previously developed to predict the direct stiffness and damping coefficients. The flow equations are numerically solved and a computer program is developed correspondingly. The predictions from this notched model are compared with the existing model to highlight the effect of the notch in the analysis. These predictions correlate well with the experimental results from the notched PDS. Also experimental results from testing of a two bladed PDS are compared to the code predictions thus validating the notched model. The notched model performs satisfactorily to predict the direct damping coefficients. Coastdown tests are conducted on a four bladed eight pocket PDS with a partial arc notch of large radius across the exit blades. The PDS offers positive direct damping which increases with an increase in seal inlet pressure. The low stiffness of the test rig combined with the negative stiffness of the seal made it impracticable to conduct testing above inlet pressures of 64.7 psia (4.461 bar). The existing theoretical models are compared with the experimental data collected up to 64.7 psia (4.461 bar).Item Inhibitor of differentiation 2A influences growth and differentiation of the developing vertebrate retina upstream of the notch signaling pathway(2012-08) Uribe, Rosa Anna; Gross, Jeffrey Martin; Vokes, Steven A.; Eberhart, Johann; Stein, David; Agarwala, SeemaInhibitor of differentiation (Id) family helix-loop-helix proteins regulate the proliferation, survival and differentiation of numerous cell types during development, however their function during retinal development has not been analyzed. Using loss-of-function and overexpression assays in zebrafish, I demonstrate that Id2a levels modulate retinoblast cell cycle kinetics and thereby influence neuron and glia formation in the retina. Id2a-deficient retinas possess increased numbers of cells occupying S phase, at the expense of mitotic cells, and kinetic analyses demonstrate that Id2a is required for S-phase progression and/or the transition from S to M phase. Id2a-dependent defects in retinoblast proliferation lead to microphthalmia and to an absence of nearly all differentiated inner and outer nuclear layer cell types. Overexpression of id2a has the opposite effect on retinoblast cell cycle kinetics: id2a-overexpressing retinoblasts progress from S to M phase more rapidly and they undergo mitosis more frequently, which results in macrophthalmia. Mosaic analyses reveal that Id2a function in facilitating both cell cycle progression and neuronal differentiation in the retina is non-cell-autonomous, suggesting that Id2a functions upstream of the extrinsic pathways that regulate retinogenesis. In an effort to identify which extrinsic pathways function downstream of Id2a, I discovered that Id2a function is necessary and sufficient to limit Notch pathway activity during retinogenesis. Id2a-deficient retinae possess elevated levels of Notch pathway component gene expression, while retinae overexpressing id2a possess reduced expression of Notch pathway component genes. Attenuation of Notch signaling activity by DAPT or by morpholino knockdown of Notch1a is sufficient to rescue both the proliferative and differentiation defects in Id2a-deficient retinae. In addition to regulating Notch pathway activity, through an RNA-Seq and differential gene expression analysis of Id2a-deficient retinae, I identified a number of additional intrinsic and extrinsic regulatory pathway components whose expression is regulated by Id2a. These data highlight the integral role played by Id2a in the gene regulatory network governing the transition from retinoblast proliferation to terminal differentiation during vertebrate retinogenesis.Item Inner Ear Sensory Epithelia Development and Regulation in Zebrafish(2011-10-21) Sweet, Elly MaeThe inner ear is a complex sensory organ of interconnected chambers, each with a sensory epithelium comprised of hair cells and support cells for detection of sound and motion. This dissertation focuses on the development and regulation of sensory epithelia in zebrafish and utilizes loss of function, gain of function and laser ablation techniques. Hair cells and support cells develop from an equivalence group specified by proneural genes encoding bHLH transcription factors. The vertebrate Atoh1 bHLH transciption factor is a potential candidate for this role. However, data in mouse has led some researchers to conclude it does not have a proneural activity, but, rather, is involved in later stages of hair cell differentiation. In addition, the factors regulating Atoh1 are mostly unknown. We address these issues in zebrafish and show that the zebrafish homologs atoh1a and atoh1b are required during two developmental phases, first in the preotic placode and later in the otic vesicle. They interact with the Notch pathway and are necessary and sufficient for specification of sensory epithelia. Our data confirm atoh1 genes have proneural function. We also go on to show Atoh1 works in a complex network of factors, Pax2/5/8, Sox2, Fgf and Notch. Misexpression of atoh1 alters axial patterning and leads to expanded sensory epithelia, which is enhanced by misexpression of either fgf8 or sox2. Lastly, we examine the role of sox2 in sensory epithelia development and regeneration. Sox2 has been implicated in maintainence of pluripotent stem cells as well as cell differentiation. In the inner ear, Sox2 is initially expressed in the prosensory domain and is required for its formation. Eventually, Sox2 is downregulated in hair cells and maintained in support cells; however, its later role has not been determined. We show that in the zebrafish inner ear, sox2 is expressed after sensory epithelium development has begun and, like in mouse, expression is down regulated in hair cells and maintained in support cells. Our data demonstrate a role for sox2 in maintenance of hair cells and in transdifferentation of support cells into hair cells after laser ablation. Additionally, sox2 is regulated by Aoth1a/1b, Fgf, and Notch.Item JNK2 inhibits luminal cell commitment in normal mammary glands and tumors(2013-05) Cantrell, Michael Andrew; Van Den Berg, CarlaBreast cancer is a heterogeneous disease with vastly different tumor progression kinetics and survival outcomes depending upon the differentiation state and gene expression patterns of the tumor. Effective treatments exist for patients with endocrine therapy sensitive or HER2 overexpressing tumors, but targeted treatments are not available for other tumor types. The mechanisms governing mammary tumor phenotype generation could prove critical to finding treatments. The c-Jun N-terminal kinase (JNK) pathway has recently been implicated in the inhibition of breast tumor luminal differentiation (1, 2) and JNK2, in particular, is important in mammary tumorigenesis and tumor progression (3-8). Therefore, the involvement of JNK2 in inhibition of mammary luminal cell differentiation was investigated in normal glands and tumors. Studies found that JNK2 inhibits luminal cell populations in normal mammary ducts. Additionally, JNK2 suppresses Notch activity in stem cell niche of the developing mammary gland. In vitro assays show that control over differentiation by JNK2 is due to suppression of p53-dependent Notch1 expression. Inhibition of luminal cell populations by JNK2 is also apparent in tumor cell models regardless of p53 expression. In the p53-competent Polyoma Middle T-antigen model, Notch1 expression is suppressed by JNK2. In the absence of p53, JNK2 suppresses luminal populations independent of Notch1. In this model, decreased luminal marker expression is accompanied by increased epithelial to mesenchymal transition. It was also found that JNK2-dependent epithelial to mesenchymal transition inhibits luminal populations and is driven by JNK2-dependent suppression of Brca1. JNK2 also confers resistance to estrogen signaling inhibition, and increases the metastatic ability of tumor cells in vivo. These data establish the importance of JNK2 in mammary epithelial cell differentiation in normal glands and tumors. They also suggest that JNK2 may be an effective prognostic marker or treatment target.Item Ligand-immobilized biomaterial surfaces for Notch signaling and T cell differentiation(2012-12) Kim, Myunghee Michelle; Roy, Krishnendu; Maynard, Jennifer A; Schmidt, Christine E; Suggs, Laura J; Tucker, HaleyIn vitro T cell differentiation from hematopoietic progenitor cells is a potential alternative source of T cells for adoptive therapy in treatment of cancers as well as T cell deficiencies. Presentation of Notch ligands immobilized on a surface is necessary in designing a stroma-free in vitro T cell differentiation system. Current methods for in vitro T cell differentiation have advanced greatly in the recent years, allowing development of functional T cells in vitro. However, these are limited to 2D coculture with stromal cells or culture on hard plastic surfaces with immobilized ligands, and have yet to report quantitative effects of variables such as substrate stiffness. This dissertation discusses the fabrication of 2D and 3D systems of various properties for presentation of Notch ligands for development of an efficient culture system, at the same time offering insight into the science of cell signaling and cell-material interactions. Magnetic microbeads, liposomes, as well as 2D and 3D soft hydrogel surfaces were fabricated to present Notch ligands at varying ligand densities and to study their quantitative effect on Notch signaling and T cell differentiation. The x findings of this dissertation demonstrate that substrate material plays a role in Notch signaling in combination with ligand density, and may affect downstream events of T lineage commitment. Insights gained from this research provide a new direction in the importance of culture substrate in ligand-presenting systems and allow development of new systems to support efficient generation of T cells in vitro.Item The role of auxilin and endocytosis in delta signaling(2012-05) Banks, Susan Marie-Louise; Fischer, Janice Ann; Huibregtse, Jon; Macdonald, Paul; Morgan, Jennifer; O'Halloran, TheresaNotch signaling is important for cell-cell signaling during development. Notch signaling is highly conserved across all metazoans and failure in Notch signaling is causative in many human diseases. In the Drosophila eye, activation of the Notch pathway requires Lqf (Drosophila Epsin)-dependent and Clathrin-dependent internalization of the Notch receptor ligands, Delta or Serrate, by the signal-sending cells. However, it is unclear why ligand must be internalized into the signal-sending cells to activate Notch signaling in the signal-receiving cells. Evidence suggests that in addition to Clathrin and Epsin, Auxilin is essential for signaling and is indirectly required for internalization of the Notch receptor ligand Delta. Auxilin functions in uncoating Clathrin-coated vesicles to maintain a pool of free Clathrin and Epsin in the cell. auxilin mutants were used as an entryway to identify previously unknown components of the Notch signaling pathway. An F1, FLP/FRT, EMS screen was performed and enhancers of an auxilin mutant rough eye defect were isolated. The enhancers ultimately formed one complementation group on the 2nd chromosome and fourteen complementation groups on the 3rd chromosome. Three of the 3rd chromosome complementation groups were each identified as Delta, lqf, or hsc70. A single allele was identified as faf. Delta and Epsin have known roles in signaling cells to activate Notch as described above. Hsc70 is an ATPase that functions with Auxilin to uncoat Clathrin-coated vesicles and Faf is a deubiquitinating enzyme that maintains levels of active Epsin in the cell. These results suggest I have isolated mutations in genes closely tied to Notch signaling or functioning directly with Auxilin. Mutations in two genes previously undescribed in Notch signaling in the developing Drosophila eye were also isolated from the screen and identified. The second chromosome complementation group was identified as α-adaptin. α-Adaptin is a subunit of the heterotetrameric Clathrin adaptor protein AP-2. One of the third chromosome complementation groups was identified as crumbs. Crumbs is an integral membrane protein that functions at adherens junctions and in establishing apical/basal polarity in cells. Characterizing roles for α-Adaptin and Crumbs during Notch signaling may elucidate the purpose for Delta internalization to activate Notch signaling.Item Study of Endothelial Morphogenesis in Three-Dimensional Collagen Matrices(2011-08-08) Su, Shih-ChiSprouting angiogenesis is a multi-step process consisting of basement membrane degradation, endothelial cell (EC) activation, proliferation, invasion, lumen formation, and stabilization. Such complexity reveals that the orchestration of individual genes and multiple signaling pathways are required. To better understand the mechanisms that direct the transformation of adherent ECs on the surface of collagen matrices to multicellular invading sprouts, we analyzed differential gene expression with time using an in vitro model of EC invasion driven by the combination of sphingosine-1-phosphate (S1P) and angiogenic growth factors. Gene expression changes were confirmed by real-time PCR and Western blot analyses. In addition, we have undertaken a proteomic screen to dissect downstream targets of the S1P receptors that possibly regulate EC invasion. Gene silencing or overexpression were used to examine the involvement and role of downstream targets of S1P in EC invasion into three-dimensional collagen matrices. We demonstrated that various cell adhesion molecule genes involved in adherens junction and cell-extracellular matrix (ECM) interactions were upregulated; whereas a set of genes associated with tight junctions were downregulated. Numerous genes encoding ECM proteins and proteases were induced, indicating that biosynthesis and remodeling of ECM is indispensable for sprouting angiogenesis. Knockdown of a highly upregulated gene, A Disintegrin and Metalloproteinase with Thrombospondin-type repeats-1 (ADAMTS1), decreased invasion responses, confirming a role for ADAMTS1 in mediating EC invasion. Furthermore, differential expression of multiple members of the Wnt (wingless) and Notch pathways were observed. Functional experiments indicated that inhibition and activation of the Notch signaling pathway stimulated and inhibited EC invasion responses, respectively. In addition, we identified annexin 2 as a regulator of endothelial morphogenesis. We observed that S1P triggered annexin 2 translocation from cytosol to plasma membrane and its association with vascular endothelial (VE)-cadherin. Moreover, annexin 2 depletion attenuated Akt activation, which was associated with increased phosphorylation of VE-cadherin and endothelial barrier leakage. Disrupting homotypic VE-cadherin interactions resulted in decreased Akt (but not Erk1/2) activation. Furthermore, expression of constitutively active Akt restored reduced EC invasion observed with annexin 2 and VE-cadherin knockdown. Collectively, we report that annexin 2 regulates endothelial morphogenesis through an adherens junction-mediated pathway upstream of Akt.Item The Function and Genetic Interactions of Zebrafish atoh1 and sox2: Genes Involved in Hair Cell Development and Regeneration(2010-10-12) Millimaki, Bonny ButlerThe sensory cells of the inner ear, hair cells, provide vertebrates with the ability to detect auditory stimuli and balance. In mammals, cochlear hair cells, those responsible for hearing, do not regenerate. Zebrafish hair cells do regenerate. Gaining an understanding of the role and regulation of the genes involved in the formation and regeneration of these cells may provide information important for the development of genetic therapies. We show that zebrafish atoh1 acts as the proneural gene responsible for defining the equivalence group from which hair cells form. Expression of atoh1 is dependent upon Fgf and Pax. Atoh1 induces expression of delta, resulting in activation of Notch and subsequent lateral inhibition. Another factor known to be important for hair cell formation in mice is Sox2. In zebrafish, sox2 expression is downstream of Atoh1, Notch and Fgf. Zebrafish Sox2 is not required for hair cell formation, but rather Sox2 is important for hair cell maintenance. In zebrafish, otic hair cell regeneration has not yet been characterized. We show that, following laser ablation, hair cells regenerate by way of transdifferentiation. We further show that this regeneration requires Sox2 activity. These data suggest that Sox2 acts to maintain support cell plasticity. This role is likely conserved because Sox2 is also important for stem cell plasticity in mammals. This new understanding of the role and regulation of both Atoh1 and Sox2 provides essential information that can be used to further efforts to provide genetic therapies for hair cell regeneration in mammals.