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Indiana University-Purdue University Indianapolis (IUPUI) / Neural crest cells (NCC) are a multipotent population of cells that form at the dorsal region of neural tube, migrate and contribute to a vast array of embryonic structures, including the majority of the head, the septum of the cardiac outflow tract (OFT), smooth muscle subpopulations, sympathetic nervous system and many other organs. Anomalous NCC morphogenesis is responsible for a wide variety of congenital defects. Importantly, several individual members of the TGFβ superfamily have been shown to play essential roles in various aspects of normal NCC development. However, it remains unclear what role Smad7, a negative regulator of TGFβ superfamily signaling, plays during development and moreover what the spatiotemporal effects are of combined suppression of TGFβ superfamily signaling during NCC formation and colonization of the developing embryo. Using a cre/loxP three-component triple transgenic system, expression of Smad7 was induced via doxycycline in the majority of pre- and post-migratory NCC lineages (via Wnt1-Cre mice). Further, expression of Smad7 was induced via doxycycline in a subset of post-migratory NCC lineages (via Periostin-Cre mice, after the NCC had reached their target organs and undergone differentiation). Induction of Smad7 within NCC significantly suppressed TGFβ superfamily signaling, as revealed via diminished phosphorylation levels of both Smad1/5/8 and Smad2/3 in vivo. This resulted in subsequent loss of NCC-derived craniofacial, pharyngeal and cardiac OFT cushion tissues. ROSA26r NCC lineage mapping demonstrated that cardiac NCC emigration and initial migration were unaffected, but subsequent colonization of the OFT was significantly reduced. At the cellular level, increased cell death was observed, but cell proliferation and NCC-derived smooth muscle differentiation were unaltered. Molecular analysis demonstrated that Smad7 induction resulted in selective increased phospho-p38 levels, which in turn resulted in the observed initiation of apoptosis in trigenic mutant embryos. Taken together, these data demonstrate that tightly regulated TGFβ superfamily signaling is essential for normal craniofacial and cardiac NCC colonization and cell survival in vivo.

Indiana University-Purdue University Indianapolis (IUPUI) / Congenital heart defects occur in approximately one percent of births every year, which makes it the most frequently occurring congenital defect in patients. The aim of this project was to use two mutant neural crest (NC) mouse models to study the mechanisms underlying congenital heart failure in utero. The first mouse model was a Pax3 systemic knockout, which was lethal by mouse gestational day 14, and had appreciably reduced numbers of migratory NC cells. The second mouse model was a Wnt1Cre-mediated NC genetic cell ablation model, which was surprisingly viable and survived to birth, despite an apparent lack of migratory NC cells. The resultant data indicated that both mouse models had similar heart structural defects including persistent truncus arteriosus, which was due to fewer or no migratory cardiac NC cells. However, in utero heart function was appreciably perturbed in Pax3 mutants when compared to that of the ablated mutant model. The loss of embryonic cardiac function in Pax3 mutants was directly attributed to a substantial decrease in the activity of the beta-adrenergic pathway. This was due to a lack of proper specification of trunk NC cells, leading to diminished levels of circulating catecholamine levels in the embryo. To definitively confirm this conclusion, poor cardiac function was successfully restored by pharmacological stimulation of the beta-adrenergic pathway via administration of isoproterenol and forskolin to pregnant dams, which led to embryonic survival of Pax3 mutants to birth. By comparison of these two mutant mouse models, perturbation in the beta-adrenergic pathway was identified as the underlying mechanism responsible for in utero heart failure and lethality in Pax3 mutant embryos. The results of this study are expected to be significant in developing future therapeutic targets for congenital heart failure in prenatal and newborn patients.

Bovine morulae (day 6: n=257) were obtained to evaluate the [effect of immunoglobulins (Ig) on early bovine embryo development in vitro. Fifty-four cows superovulations were conducted in 36 cows with follicle stimulating hormone. Embryos were collected by non-surgical procedures and morphologically evaluated and randomly assigned to culture. Embryos were cultured in Ham's F-10 containing 10% (6.4 mg/ml) steer serum (SS), 1% (.64 mg/ml) bovine gamma . globulins (GG), 1% (.64 mg/ml) bovine IgG, 1% (.64 mg/ml) bovine 1gM, 10% SS plus 1% GG, 10% SS plus 1% 1gG, or 10% SS plus 1% 1gM. Embryos were cultured to the hatched blastocyst stage or degeneration and evaluated at 12 h intervals. / Master of Science

The migration of the cranial neural crest is an essential part of cranio-facial development in every vertebrate embryo. The cranial neural crest (CNC) is a transient population of cells that forms the lateral border of the anterior neural plate. In the tailbud stage Xenopus embryo, the neural crest cells delaminate from the neural tube, and undergo a large-scale migration from the dorsal to ventral region of the embryo. The CNC travels along distinct pathways, and populates specific regions of the embryos face. Once the CNC ceases migrating, it differentiates into a variety of tissues that are essential for cranio-facial structure and function. Some of these tissues include bones, muscle, cartilage, and ganglia. The CNC receives a concert of signals from neighboring tissues during and after CNC migration as well as signals transmitted among CNC cells, which act together to determine the fate of each CNC cell. Therefore, the proper migration of the CNC is an essential part of cranio-facial development. What molecules are important for the process of CNC migration? As one might imagine, a milieu of different molecules and interactions are essential for this complicated embryological process to occur. The work presented in this dissertation will focus on the role of a cell adhesion molecule that is important for Xenopus CNC migration. Typically, the amount of cell adhesion decreases within tissues undergoing migration. This behavior is essential to allow fluidity within the tissue as it moves. However, cell adhesions are fundamental for cell migration to occur because the moving cells need a platform on which to mechanically propel themselves. These interactions can occur between the migrating cell and extracellular matrix molecules (ECM), or can happen between cells. The cranial neural crest utilizes both cell-ECM and cell-cell interactions during the process of migration. The amount of cell adhesion mediated by either of these mechanisms will depend on where the cell is located within the CNC. Cells located at the periphery of the CNC tissue, which is surrounded by a matrix of ECM, will have more cell-ECM interactions. Cells located deeper in the CNC tissue, where there is little ECM, will rely more on cell-cell interactions. The work presented in this thesis focuses on a cell-cell adhesion molecule that is part of the cadherin superfamily of molecules. With this in mind, these studies should be descriptive of the environment within the CNC, and to a less degree the environment between the CNC and the surrounding tissues. The work presented in this dissertation will focus on cadherin-11, which is a classical cadherin that is specifically expressed in the cranial neural crest during its migration. How does cadherin-11 function in the CNC during this process? The work presented here suggests that the main role of cadherin-11 in the CNC is to perform as a cell adhesion molecule. However, too much cell adhesion is inhibitory to migration. In this respect, many of the studies described in this work indicate that cadherin-11 mediated cell adhesion is tightly regulated during CNC migration. Here I show that cadherin-11 is extracellularly processed by ADAM metalloproteases, ADAM9 and ADAM13, which removes the adhesive domain of cadherin-11. This extracellular cleavage event occurs throughout CNC migration, and is likely the main mechanism that regulates cadherin-11 mediated cell adhesion. Cleavage of cadherin-11 by ADAMs does not seem to affect its ability to interact with cytoplasmic binding partners, â-catenin and p120-catenin. This observation supports the idea that the “purpose” of cadherin-11 cleavage is to regulate cell adhesion, and not to induce (cell autonomous) signaling events. Additionally, the secreted extracellular domain of cadherin-11 (EC1-3) retains biological activity. This fragment can bind to a number of cell surface molecules in tissue culture including full-length cadherin-11 and specific members of the ADAM family. This observation suggests that EC1-3 may interact with full-length cadherin-11 molecules in vivo, and inhibit cadherin-11 mediated cell adhesion during CNC migration. EC1-3 can rescue CNC migration in embryos that overexpress cadherin-11, further supporting this hypothesis. Many of the above observations have been published in my first-author paper entitled “Extracellular processing of cadherin-11 by ADAM metalloproteases is essential for Xenopus cranial neural crest migration” published in the journal Molecular Biology of the Cell in 2009. Some of the unpublished work in this dissertation further focuses on how EC1-3 effects CNC migration in an ex vivo environment. During these studies, the observation was made that overexpression of EC1-3 in a cranial neural crest explant produces abnormal directional movement. In these experiments, it appeared as though certain regions of the CNC explant were “attracting” other regions of the explant. The preliminary studies described in chapter IV are aimed at answering the question; does EC1-3 attract migrating CNC cells? Here, we generated a Matlab program in order to effectively quantify the amount of directional movement of CNC explants presented with a source of EC1-3. In addition to quantifying cell directionality, this program can also decipher between cells moving with random or directed motion, and measure the velocity of cell migration within certain coordinates. Therefore, this program should be useful other ex vivo studies that require the observation of these features. To conclude, the work presented in this dissertation suggests that the role of cadherin-11 during cranial neural crest migration is predominately based on the adhesive function. In order for CNC migration to proceed, the amount of cadherin-11 mediated cell-cell adhesion is tightly regulated throughout this process. These cell-cell interactions are likely important for “sheet” and “branch” migration where CNC cells maintain a lot of cell-cell cohesion.

京都大学 / 新制・論文博士 / 博士(医学) / 乙第13586号 / 論医博第2306号 / 新制||医||1070(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 川口 義弥, 教授 波多野 悦朗, 教授 齋藤 潤 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

Fertile White Leghorn (Gallus gallus) and Bobwhite Quail (Colinus virginianus) were subjected to 15 min exposures produced by burning solid rocket motor (SRM) fuel. Comparative mortality data were collected. Chicken eggs were further used to study the effects of exposure on water relations and blood gas parameters. Chicken embryos exposed once on day 19 or incubation demonstrated and LD50 of 204 ppm; the LD50 for quail embryos was 175 ppm. When mortality was regressed on the final exposure concentration, chicken and quail embryos exposed on days 12 and 19 showed LD50's of 127 and 86 ppm respectively, and embryos exposed on days 4, 12, and 19 had LD50's of 75 and 56 ppm. Quail embryos appeared to be more sensitive to SRM exhausts than chicken embryos, probably owing to the larger surface area to volume ration of the egg. Embryos exposed to a small daily concentration had an MLC of 117 ppm and an LD50 of approximately 200 ppm for cumulative exposure concentrations. This suggested that individual exposures were additive in effect. Eggs exposed at temperatures less than 37.5 C showed reduced lethality, while exposures at greater temperatures increased lethality. The rate of water loss from chicken eggs measured over an eight hour period increased 5 times because of a 15 min exposure. Since the increased rate of dehydration occurred during the exposure, the hydroscopic effects of exposure were extreme. When eggs lose the 18% of the initial weight normally lost from evaporation during incubation due to exposure, no more water loss was seen to occur. Blood gas analyses on 12 day embryos showed decreased pH at cumulative exposure concentrations greater than 200 ppm. Carbon monoxide in the exhausts probably increased carboxyhemoglobin, reducing buffering capacity. The acidosis was partially compensated by increased HCO3-, Exogenously derived C1-, plus increased HCO3- may shift intracellular K+, making the serum hyperkatremic. Dehydration effects further increased serum hypertonicity.

No description available.

A survey of 967 married women from Misamis Oriental, Philippines was conducted to examine pregnancy as a reproductive experience that may either be status-enhancing or status-degrading. In the study, pregnancy status was defined as a social position located within a set of relationships. The position involved ranking based on its importance to the society. Pregnancy status was said to be derived from the woman’s relationships with significant others as well as self definition. Findings from multivariate analyses indicated that four variables were significantly related to pregnancy status. These were: (1) number of live births; (2) modern role orientation; (3) woman’s educational attainment; and (4) age. Women who were younger and who have fewer children were found to have a higher regard for pregnancy as status-enhancing. Those who have less education and more traditional role orientation also reported higher pregnancy status evaluation. Although residence and socio-economic status were not highly correlated with pregnancy status, the analyses showed that respondents who came from rural areas and from lower socio-economic classes were more likely to perceive pregnancy as status-enhancing than those from urban areas and from higher socio- economic classes. Interestingly, women who have been married for more years, while controlling for their educational attainment and residence, were also found to have a higher regard for pregnancy while reporting higher number of unwanted births. However, when number of live births was considered, the negative relationship prevailed between marriage duration and pregnancy status. With such factors as number of live births, length of marriage, woman's educational attainment, and residence introduced in the final path model, the effect of pregnancy status on the woman's expressed number of unwanted births was analyzed. Findings from Linear Structural Relations (LISREL) analyses revealed that while number of live births was the most important indicator of unwanted births, pregnancy status also contributed to unwanted births as a direct and mediating factor. It is suggested that a woman’s perception of pregnancy as status-enhancing or degrading be considered as a supplementary factor in explaining fertility behavior. Since the present study is an initial effort to provide guidelines, further research is needed. / Ph. D. / incomplete_metadata

Cytokinesis is defined as the physical division of one cell into two and occurs at the end of the cell cycle. Gestation and development are defined by dividing cells; as an organism develops, cells must duplicate their genetic material, divide, and form two daughter cells. This process is fundamental to all life on our planet. Here, I present work that builds upon our understanding of cytokinesis, focusing on the differential requirements for cytokinesis in different cell types in the early C. elegans embryo, specifically, the P2 cell of the 4-cell embryo. The textbook view of cytokinesis is that all animal cells divide using the same molecular machinery. Yet, growing evidence supports both cell type-specific regulation of cytokinesis and cell type-specific consequences for cytokinesis failure. The 4-cell C. elegans embryo is a powerful model for studying cell type-specific differences in cytokinesis as the cells are already programmed to form distinct cell linages, and previously, we identified cell type-specific regulation of cytokinesis at the 4-cell stage. We weakened the contractile ring using a temperature sensitive (ts) diaphanous formin/CYK-1 mutant. Under this condition, the two anterior cells (ABa and ABp) always failed in cytokinesis, whereas the two posterior cells (EMS and P2) divided successfully at a high frequency, even without detectable F-actin in the cell division plane. Here we focus on the cell type-specific protection of cytokinesis in the P2 germ precursor cell, required to produce all gametes in the adult worm. Using a candidate-based RNAi mini-screen to identify genes required for protection of P2 cytokinesis in the formin(ts) embryos, we identified members of the CCCH Zn2+-finger protein family that are enriched in P2 and required for proper germ cell fate specification. Depletion of MEX-1, PIE-1, or POS-1 led to loss of cytokinetic protection and P2 cytokinesis failure in formin(ts) mutants, but not in control embryos. While depletion of MEX-1 affected multiple cell types, PIE-1 and POS-1 acted exclusively in the P2 cell. Further analysis revealed these germ fate regulators protect cytokinesis by preventing excessive accumulation of septin/UNC-59 and its binding partner, anillin/ANI-1, on the cell cortex in the P2 cell division plane, both negative regulators of actomyosin constriction during cytokinesis in many contexts. We further found that co-depletion of septin and PIE-1 was sufficient to both reduce anillin levels at the P2 division plane and restore cytokinetic protection of P2 in formin(ts) mutant embryos. Thus, germ fate specification protects the P2 germ precursor cell from cytokinesis failure upon damage to the actin cytoskeleton at least in part by controlling the levels of septin and anillin at the division plane.