Browsing by Subject "Xenopus"
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Item Cdc6 amd replication competence in xenopus oocytes(Texas Tech University, 2002-08) Dallas, Elizabeth WhitmireThe early division cycles of an embryo following fertilization rely on the oocyte's ability to replicate DNA. During meiosis, oocytes temporarily lose their ability to replicate DNA. After a single round of pre-meiotic S-phase, oocytes enter meiosis and rapidly arrest at prophase of meiosis I. During this G2 arrest (immature oocyte), that can last months or years depending on the animal species, oocytes are unable to replicate DNA. Upon hormonal stimulation, arrested oocytes resume meiosis, reestablish DNA replication competence in meiosis I shortly after germinal vesicle breakdown (GVBD), but repress replication until fertilization. How oocytes lose and regain replication competence during meiosis are important questions underlying the production of functional gametes that will support the early development of the embryo. This study focuses on determining why immature oocytes are unable to replicate their DNA. Since the elongation machinery is known to be present and functional in the immature oocyte, we hypothesized that the ability of an oocyte to replicate its DNA is controlled at the initiation step. The initiation of DNA replication in mitotic cells requires the assembly and activation of pre-replication complexes (pre-RCs). During assembly, the Origin Recognition Complex (ORC), Cdc6, Cdtl and the Mem proteins bind to the DNA in a sequential order. Following assembly, phosphorylation by two kinases, Cdk2/Cyclin E and Cdc7/Dbf4, leads to activation of the pre-RC. We show here that immature oocytes are unable to replicate due to three defects which prevent pre-RC assembly and thus, replication ability: (1) absence of Cdc6 protein; (2) differential localization of pre-RC components; and (3) phosphorylation of the Mcm4 protein. Next we describe experiments, which illustrate that Cdc6 protein synthesis and GVBD are the only limiting events for the development of replication competence during meiosis. Since Cdc6 seems to be critical for replication competence, we also focused experiments on determining the mechanisms that regulate Cdc6 expression in the immature oocyte. It appears that Cdc6 is absent from the immature oocyte because it is not being synthesized and is also very unstable in the immature oocyte. The Cdc6 message is present in the immature oocyte, but is repressed via sequences in its 3'UTR, which are most likely cytoplasmic polyadenylation elements (CPEs).Item Characteristics of a maturation-including steroid receptor in the plasma membrane of xenopus oocytes(Texas Tech University, 1994-12) Liu, ZhimingXenopus laevis is a widely used animal in general and molecular biology research (Dawid and Sargent, 1988). This animal can be conveniently maintained under laboratory conditions and its ovarian foUicle cells are easily dissociated from its relatively large oocytes using well estabhshed methods. Thus, a considerable amount of background information on developmental and molecular biology of oocytes in this species has already been accumulated. The Xenopus oocyte was one of the first and most convincing models establishing the existence of ceU surface actions of steroids (Haukkamaa, 1987). However, our knowledge on the characteristics of the MIS receptor in the plasma membrane of Xenopus oocytes has feUed to make significant progress (Jessus and Ozon, 1993), due in part to the lack of adequate techniques for determination of receptor binding activity and for isolation and purification of the receptor. Therefore, the specific objectives of this research are to develop a direct radioreceptor assay technique for the MIS receptor of Xenopus oocyte membranes, to study the characteristics of MIS receptor binding activity and the developmental and gonadotropic regulation of MIS receptor density, and to isolate and purify the MIS receptor. In Chapter 2, the development of MIS receptor binding assay technique and characterization of the receptor binding activity is described; this chapter has been published in the joumal Biology of Reproduction (1993; 49: 980-988). In Chapter 3, the isolation and purification of a protein tentatively identified as the MIS receptor is described. In Chapter 4, an overall summary and conclusion is provided.Item Investigation of Immunoglobulin Heavy Chain Isotypes in an Ancestral Mucosal Immune Model(2012-10-19) Du, ChristinaThe importance of gut associated lymphoid tissues has been extensively reported in higher vertebrates, but less is known in lower vertebrates. In mammals immunoglobulin (Ig)A is the primary Ig of mucosal immunity. But no IgA has been identified in cold-blooded animals. In higher vertebrates, antigen must stimulate the lymphoid tissues in the intestines to elicit an IgA response, and cytokines from CD4 positive helper T cells are required for B cell switch. It is not known if this is the case in lower vertebrates, or if T cell help evolved before or after class switch recombination between functional antibody isotypes. My study will fill in these gaps in our knowledge by comparing oral antigen inoculation relative to intraperitoneal antigen inoculation in frogs (Xenopus sp.). Oral immunization is a novel approach to eliciting immune responses in Xenopus. I propose that IgX will increase with oral inoculation compared to intraperitoneal injection. This would be the first demonstration of class switch upon oral immunization to a mucosal isotype in the first vertebrates that employs higher vertebrate Ig heavy chain switch mechanism, which would shed light on the most fundamental aspects of our humoral adaptive immune system. Using a total Ig ELISA protocol, measuring total relative levels of IgM, there was no difference between the first three groups of orally immunized frogs compared to intraperitoneally immunized frogs. However, a response to serum IgX was seen in the first group. On the other hand, the refined Ag-specific ELISA protocol did present a significant increase in serum IgM response in frogs immunized systemically over orally immunized animals, but not an overall IgX response. Phylogenetic analysis suggests that, contrary to initial reports, IgA evolved from IgX. With consideration of entire constant region and individual constant domain analyses as well as synteny and function, we suggest new hypotheses of vertebrate antibody evolution to be tested as immunogenetic coverage of more species continues to expand.Item Transcriptional control of epithelial morphogenesis(2013-05) Chung, Mei-I; Wallingford, John B.How tissues and organs develop into their final shape during embryogenesis is a fascinating and long-standing question in developmental biology. Tissue morphogenesis is driven by a variety of events at the cellular level and individual cell shape change is one of the central morphogenetic engines. Thus, it is crucial to understand what signals specify the correct cell behavior in specific groups of cells during development. For my doctoral studies, I have focused on two cell shape change events, apical constriction and cilia assembly. First, we present data demonstrating that Shroom3 is essential for cell shape changes and morphogenesis in the developing vertebrate gut, where Shroom3 transcription requires the Pitx1 transcription factor. We identified a Pitx-responsive regulatory element in the genomic DNA upstream of Shroom3, and showed that Pitx proteins directly activated Shroom3 transcription in Xenopus. Moreover, we showed that ectopic expression of Pitx proteins was sufficient to induce Shroom3-dependent cytoskeletal reorganization and epithelial cell shape change. These data demonstrated new breadth to the requirements for Shroom3 in morphogenesis, and also provided a cell-biological mechanism for Pitx transcription factors action during morphogenesis. Next, we focused on understanding the transcriptional regulation of ciliogenesis. We first showed that Rfx2 transcription factor broadly controlled ciliogenesis, and by RNA- and ChIP-sequencing, we showed that Rfx2 directly regulated a wide range of genes encoding diverse ciliogenic machinery. Finally, in addition to ciliogenesis regulation, a large number of non-ciliary genes in our Rfx2 dataset led us to identify a novel role of Rfx2 in controlling insertion of multi-ciliated cells into the overlying mucociliary epithelium. Moreover, we showed here that Slit2, a target of Rfx2, was involved in multi-ciliated cell movements, possibly through mediating cortical E-cadherin level. This work allowed us to begin building a genetic network controlling multi-ciliated cells in mucociliary epithelium. Together, we showed a transcriptional regulation of apical constriction driving gut morphogenesis and a comprehensive transcriptional network that governs multi-ciliated cell development.