Life and death of the embryonic female reproductive tract

Regardless of genetic sex, amniotes develop two sets of genital ducts, the Wolffian and Müllerian ducts. Normal sexual development requires the differentiation of one duct and the regression of the other. I show that cells in the rostral most region of the coelomic epithelium (CE) are specified to a Müllerian duct fate beginning at Tail Somite Stage 19 (TS19). The Müllerian duct (MD) invaginates from the CE where it extends caudally to and reaches the Wolffian duct (WD) by TS22. Upon contact, the MD elongates to the urogenital sinus separating the WD from the CE and its formation is complete by TS34. During its elongation, the MD is associated with and dependent upon the WD and I have identified the mechanism for MD elongation. Using the Rosa26 reporter to fate map the WD, I show that the WD does not contribute cells to the MD. Using an in vitro recombinant explant culture assay I show that the entire length of the MD is derived from the CE. Furthermore, I analyzed cell proliferation and developed an in vitro assay to show that a small population of cells at the caudal tip proliferates, laying the foundation for the formation of the MD. I also show that during its formation, the MD has a distinctive mesoepithelial character. The MD in males regresses under the influence of Anti-Müllerian Hormone (AMH). Through tissue-specific gene inactivation I have identified that Acvr1 and Bmpr1a and Smad1, Smad5 and Smad8 function redundantly in transducing the AMH signal. In females the MD differentiates into an epithelial tube and eventually the female reproductive tract. However, the exact tissue into which the MD differentiates has not been determined. I therefore generated a MD specific Cre allele that will allow for the fate mapping of the MD in both females males. The MD utilizes a unique form of tubulogenesis during development and to my knowledge is the only tubule that relies upon a signal from and the presence of another distinct epithelial tube for its formation.^

A standard operation procedure and a pilot water testing for herbicides in northwest Harris County, Texas

Herbicides are used to control the growth of weeds along highways, power lines, and many other urban locations. Exposure to herbicides has been linked to adverse health outcomes. This study was initiated to pretest for the presence of herbicides in multiple water sources near intersections in a corridor in the Northwest Harris County (specifically in the Highway 6/FM 1960, North Freeway 45, US 290 and S 99 corridor). Roadside water and tap water samples were collected and analyzed for herbicides using the established Environmental Protection Agency (EPA) Method 515.4: "Determination of Chlorinated Acids in Drinking Water by Liquid-Liquid Micro-extraction, Derivatization, and Fast Gas Chromatography with Electron Capture Detection." A standard operating procedure (adapted from the US EPA Method 515.4) was developed for subsequent, larger studies of environmental fate of herbicides and non-occupational exposure risks. Preliminary testing of 16 water samples was performed to pretest the existence of trace herbicides; all concentrations that were greater than the minimum reporting limits of each analyte are reported with a 99 percent confidence. This study failed to find concentrations above the limits of detection of the method in any of the samples collected on June 15, 2008. However, this does not indicate that the waters around the NW Harris County are free of herbicides and metabolites. A larger and repeated sampling in the region would be necessary to make that claim. ^

PRODUCTION, DEGRADATION AND ACCUMULATION OF ORGANICS BY WASTE STABILIZATION POND MICROORGANISMS

The objectives of this research were: to determine the contribution of algae to commonly run water quality variables, to evaluate waste pond micoorganisms' capacity to degrade and accumulate ten EPA priority pollutants, and to determine the environmental fate of those compounds in a laboratory

LOW REACTIVE OXYGEN SPECIES AND HIGH GLYCOLYSIS IN GLIOBLASTOMA STEM CELLS:MECHANISMS AND THERAPEUTIC IMPLICATIONS

Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumor with poor prognosis due in part to drug resistance and high incidence of tumor recurrence. The drug resistant and cancer recurrence phenotype may be ascribed to the presence of glioblastoma stem cells (GSCs), which seem to reside in special stem-cell niches in vivo and require special culture conditions including certain growth factors and serum-free medium to maintain their stemness in vitro. Exposure of GSCs to fetal bovine serum (FBS) can cause their differentiation, the underlying mechanism of which remains unknown. Reactive oxygen species (ROS) play an important role in normal stem cell differentiation, but their role in affecting cancer stem cell fate remains unclear. Whether the metabolic characteristics of GSCs are different from other glioblastoma cells and can be targeted are also unknown. In this study, we used several stem-like glioblastoma cell lines derived from clinical tissues by typical neurosphere culture system or orthotopic xenografts, and showed that addition of fetal bovine serum to the medium induced an increase of ROS, leading to aberrant differentiation and decreases of stem cell markers such as CD133. We found that exposure of GSCs to serum induced their differentiation through activation of mitochondrial respiration, leading to an increase in superoxide (O2-) generation and a profound ROS stress response manifested by upregulation of oxidative stress response pathway. This increase in mitochondrial ROS led to a down-regulation of molecules including SOX2, and Olig2, and Notch1 that are important for stem cell function and an upregulation of mitochondrial superoxide dismutase SOD2 that converts O2- to H2O2. Neutralization of ROS by antioxidant N-acetyl-cysteine in the serum-treated GSCs suppressed the increase of superoxide and partially rescued the expression of SOX2, Olig2, and Notch1, and prevented the serum-induced differentiation phenotype. Additionally, GSCs showed high dependence on glycolysis for energy production. The combination of a glycolytic inhibitor 3-BrOP and a chemotherapeutic agent BCNU depleted cellular ATP and inhibited the repair of BCNU-induced DNA damage, achieving strikingly synergistic killing effects in drug resistant GSCs. This study uncovers the metabolic properties of glioblastoma stem cells and suggests that mitochondrial function and cellular redox status may profoundly affect the fates of glioblastoma stem cells via a ROS-mediated mechanism, and that the active glycolytic metabolism in cancer stem cells may provide a biochemical basis for developing novel therapeutic strategies to effectively eliminate GSCs.

Mechanism of mRNA stability control mediated by AU -rich element: Characterization of cis-elements and trans-factors

Regulation of cytoplasmic deadenylation, the first step in mRNA turnover, has direct impact on the fate of gene expression. AU-rich elements (AREs) found in the 3′ untranslated regions of many labile mRNAs are the most common RNA-destabilizing elements known in mammalian cells. Based on their sequence features and functional properties, AREs can be divided into three classes. Class I or class III ARE directs synchronous deadenylation, whereas class II ARE directs asynchronous deadenylation with the formation of poly(A)-intermediates. Through systematic mutagenesis study, we found that a cluster of five or six copies of AUUUA motifs forming various degrees of reiteration is the key feature dictating the choice between asynchronous versus synchronous deadenylation. A 20–30 nt AU-rich sequence immediately 5 ′ to this cluster of AUUUA motifs can greatly enhance its destabilizing ability and is an integral part of the AREs. These two features are the defining characteristics of class II AREs. ^ To better understand the decay mechanism of AREs, current methods have several limitations. Taking the advantage of tetracycline-regulated promoter, we developed a new transcriptional pulse strategy, Tet-system. By controlling the time and the amount of Tet addition, a pulse of RNA could be generated. Using this new system, we showed that AREs function in both growth- and density-arrested cells. The new strategy offers for the first time an opportunity to investigate control of mRNA deadenylation and decay kinetics in mammalian cells that exhibit physiologically relevant conditions. ^ As a member of heterogeneous nuclear RNA-binding protein, hnRNP D 0/AUF1 displays specific affinities for ARE sequences in vitro . But its in vivo function in ARE-mediated mRNA decay is unclear. AUF1/hnRNP D0 is composed of at least four isoforms derived by alternative RNA splicing. Each isoform exhibits different affinity for ARE sequence in vitro. Here, we examined in vivo effect of AUF1s/hnRNP D0s on degradation of ARE-containing mRNA. Our results showed that all four isoforms exhibit various RNA stabilizing effects in NIH3T3 cells, which are positively correlated with their binding affinities for ARE sequences. Further experiments indicated that AUF1/hnRNP D0 has a general role in modulating the stability of cytoplasmic mRNAs in mammalian cells. ^

Functional studies of a LIM -homeodomain transcription factor lmx1b in murine mid -hindbrain and limb development

This thesis is centered on applying molecular genetics to study pattern formation during animal development. More specifically, this thesis describes the functional studies of a LIM-homeodomain gene called lmx1b during murine embryogenesis. Lmx1b expression is restricted to the mid-hindbrain junction as well as to the dorsal mesenchyme of the limb, suggesting important functions during mid-hindbrain and limb development. To test these possibilities, lmx1b homozygous mutant mice were generated and their limb and CNS phenotypes examined. Lmx1b homozygous mutant mice exhibit a large reduction of mid-hindbrain structures, and that their limbs are symmetrical along the dorsal-ventral axis as the result of a dorsal to ventral transformation. Taken together, these studies define essential functions for lmx1b in mid-hindbrain patteming and in dorsal limb cell fate determination. However, the molecular mechanisms which accounts for these phenotypes are unknown, and whether lmx1b has same or distinctive functions during the mid-hindbrain and limb development is also unclear. ^ Recently, insight into molecular mechanisms of mid-hindbrain patterning and limb development has resulted from the identification of several factors with restricted expression patterns within these regions. These include the secreted factors wnt-1, fgf-8, wnt-7a and the transcription factors pax-2, and en-1. Targeted disruption of any of these genes in mice suggests that these genes might be involved in similar regulatory pathways. Analysis of the expression of these genes in lmx1b mutants demonstrates that lmxlb is not required for the initiation, but is required to maintain their expression at the mid-hindbrain junction. Thus, lmxlb is not required for specifying mid-hindbrain cell fates, rather, it functions to ensure the establishment or maintenance of a proper organizing center at the mid-hindbrain junction. Interestingly, lmxlb functions cell non-autonomously in chimera analysis, which indicates that lmx1b might regulate the expression of secreted factors such as wnt-1 and/or fgf-8 in the organizing center. In contrast, lmx1b functions cell autonomously in the dorsal limb to govern dorsal ventral limb development and its expression is regulated by with wnt-7a and en-1. However, single and double mutant analysis suggest that all three genes have partially overlapping functions as well as independent functions. The results point toward a complicated network of cross-talks among all three limb axes. ^

Function of GCIP (CCNDBP1), a helix -loop -helix leucine -zip protein, in cell differentiation and tumorigenesis

Cell growth and differentiation are complex and well-organized processes in which cells respond to stimuli from the environment by carrying out genetic programs. Transcription factors with helix-loop-helix (HLH) motif play critical roles in controlling the expression of genes involved in lineage commitment, cell fate determination, proliferation and tumorigenesis. This study has examined the roles of GCIP (CCNDBP1) in cell differentiation and tumorigenesis. GCIP is a recently identified HLH-leucine zipper protein without a basic region like the Id family of proteins. However, GCIP shares little sequence homology with the Id proteins and has domains with high acidic amino acids and leucine-rich regions following the HLH domain like c-Myc. Here we firstly demonstrate that GCIP is a transcription regulator related to muscle differentiation program. Overexpression of GCIP in C2C12 cells not only promotes myotube formation but also upregulates myogenic differentiation biomarkers, including MHC and myogenein. On the other hand, our finding also suggests that GCIP is a potential tumor suppressor related to cell cycle control. Expression of GCIP was significantly down-regulated in colon tumors as compared to normal colon tissues. Overexpression of GCIP in SW480 colon cancer cell line resulted in a significant inhibition on tumor cell colony formation on soft agar assays while silencing of GCIP expression by siRNA can promote cell proliferation and colony formation. In addition, results from transgenic mice specifically expressing GCIP in liver also support the idea that GCIP is involved in the early stage of hepatocarcinogenesis and decreased susceptibility to chemical hepatocarcinogenesis. ^