京都大学 / 新制・課程博士 / 博士(理学) / 甲第24452号 / 理博第4951号 / 新制||理||1707(附属図書館) / 京都大学大学院理学研究科生物科学専攻 / (主査)教授 高橋 淑子, 准教授 佐藤 ゆたか, 教授 中務 真人 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DGAM

The chicken bursa of Fabricius is a primary lymphoid tissue important for B-cell development. Our long-term goal is to understand the role of bursal microenvironment in an early B-cell differentiation event initiating repertoire development through immunoglobulin gene-conversion in the chick embryo. We hypothesize that early bursal B-cell differentiation is guided by signals through cytokine receptors. Our theory is based on previous evidence for expression of the receptor tyrosine kinase superfamily members and interleukin receptors in unseparated populations of bursal B-cells and bursal tissue. Knowledge of the expressed genes that are responsible for B-cell differentiation is a prerequisite for understanding the bursal microenvironment’s function. This project uses transcriptomic analysis to examine gene expression across an early B-cell differentiation event. RNA-seq was performed with total RNA isolated from developing B-cells at embryonic day (ED) 16 and ED 19 (n=3). Approximately 90 million high quality clean reads where obtained from the cDNA libraries. The analysis revealed differentially expressed genes involved in Wnt signaling pathway, Jak-STAT pathway, metabolic pathways, tyrosine metabolism, Toll-like receptor signaling pathway, MAPK signaling pathway, and cellhesion molecules. The transcripts for surface receptors, signal transduction and transcription factors identified in this study represent gene candidates for controlling B-cell differentiation in response to bursal microenvironmental factors.

No description available.

No description available.

Os gânglios da raiz dorsal (GRD) armazenam neurônios TrkA-positivos. A percepção e transmissão de estímulos por estes neurônios dependem de uma neuritogênese adequada. Miosina (MioVa) é expressa no tecido nervoso e está presente em neuritos, corpo celular e cone de crescimento. Caracterizamos o padrão de expressão de MioVa na neuritogênese de células TrkA-positivas do GRD de galinha in vivo e in vitro. In vivo, MioVa é expressa em células que não começaram a emitir neuritos em HH25, e sua expressão persiste por toda neuritogênese. In vitro, é recrutada para o processo de re-emissão de neuritos de neurônios TrkA positivos na presença de NGF, sendo expressa em neuritos em diferentes estádios de neo-neuritogênese. Nos ensaios funcionais, observamos que a superexpressão do domínio globular de MioVa em culturas de GRD com 10 ou 100ng/ml de NGF reduz a população de células com neuritos longos e aumenta a população de células com neuritos curtos ou sem neuritos. Em conjunto, estes dados sugerem que a MioVa é importante para o estabelecimento de neuritos nociceptores. / The dorsal root ganglia (DRG) harbor the TrkA-positive neurons. The stimuli perception and transmission by these neurons depend on a proper neuritogenesis. Myosin (MyoVa) is widely expressed in nervous tissue and is present in neurites, cell body and growth cone. Here, we characterized the MyoVa expression pattern in chicken DRG TrkA-positive cells neuritogenesis, in vivo and in vitro. In vivo, at stage HH25, MyoVa was present both in cells with and without neurites and its expression persists throughout neuritogenesis. In vitro, it is recruited for the regeneration process and TrkA-positive neurons neurites re-emission in the presence of NGF, being expressed in neurites at different stages of neo-neuritogenesis. In functional assays, we observed that MyoVa globular tail overexpression in GRD cultures maintained with 10 or 100ng/ml NGF reduces the number of neurons with long neurites and increased the number of neurons with short neurites or no neurites. Taken together, these results suggest that Myosin Va is important for the establishment of nociceptor neurites.

La crête neurale (CN) est une structure multipotente transitoire de l'embryon de vertébrés. La CN céphalique (CNC), mais pas la CN troncale (CNT), fournit des tissus mésenchymateux (squelette, derme et tissus adipeux de la face). Cette capacité de la CNC est liée à l'absence d'expression des gènes de type Hox. Cependant, les cellules de la CNT possèdent des potentialités mésenchymateuses à l'état dormant, qui peuvent s'exprimer en culture. Les mécanismes moléculaires qui régulent les potentialités mésenchymateuses de la CN le long de l'axe antéro-postérieur restent incompris. Chez l'embryon d'oiseau, nous avons étudié l'influence des facteurs de transcription Hox et Six sur la formation du mésenchyme par la CN. D'une part, nos analyses in vivo et in vitro montrent que Six1 est présent dans des cellules mésenchymateuses de la CN et du mésoderme, suggérant un rôle dans le développement musculo-squelettique de la tête. D'autre part, nous avons testé l'hypothèse d'un rôle inhibiteur des facteurs Hox. Nos résultats montrent que l'expression ectopique de Hoxa2 dans les cellules de CNC en culture inhibe la production d'ostéoblastes, sans affecter celle des cellules nerveuses et mélanocytaires. Dans la CNT, nous avons trouvé que la différentiation osseuse, cartilagineuse et adipocytaire, est fortement réduite après la surexpression de Hoxa2, sans effet sur les autres phénotypes dérivés de la CN. Ces résultats suggèrent que les potentialités mésenchymateuses de la CN sont régulées, au moins en partie, par un mécanisme commun aux cellules de CNC et CNT, mettant en jeu une inhibition de l'activité du gène Hoxa2. / The neural crest (NC) is a transitory multipotent structure of the vertebrate embryo. The cephalic NC (CNC), not the trunk NC (TNC), gives rise to mesenchymal cell types (contributing to craniofacial skeleton, dermis and adipose tissue). This capacity of the CNC has been linked to the absence of Hox gene expression in the most rostral region of the embryo. However, TNC cells do have mesenchymal potentialities, although in a dormant state in vivo, but which can be disclosed after NC in vitro culture. The molecular mechanisms that regulate mesenchymal potentials of the NC cells along the rostral-caudal axis are still elusive. Here, we have used the avian embryo model to investigate the possible influence on NC mesenchymal fate, of Hox and Six transcription factor genes. On the one hand, in vivo and in vitro culture analyses show that Six1 gene is expressed in mesenchymal cell populations derived from both cranial NC and mesoderm, suggesting a role for Six1 in muscle-skeletal development in the head. On the other hand, we have tested the hypothesis of an inhibitory action of Hox genes on NC cell mesenchymal differentiation using NC in vitro cultures. In CNC cells, we found that ectopic expression of Hoxa2 strongly reduces the production of osteoblasts, while neural and melanocytic phenotypes are unaffected. In the cultured CNT cells, overexpression of Hoxa2 results in largely impaired differentiation into bone cells, chondrocytes and adipocytes, whereas other NC derivatives are unchanged. These results suggest that mesenchymal potentials of the CNC and TNC are controlled, at least in part, via a common mechanism that involves inhibition of Hoxa2 gene activity.

Developing chicken embryos have two ductus arteriosus (DA) that shunt blood away from the lungs and to the chorioallantoic membrane, the embryonic gas exchanger. In mammals, DA closure is stimulated by an increase in blood gas O2 that occurs as the animal begins to breathe with its lungs. The goal of this study was to determine the influence of O2 levels during incubation on the vascular reactivity and morphology of the O2-sensitive DA and to examine the effects of changing O2 levels during late incubation on the morphology of the DA from chicken embryos. In comparison to normoxia, hypoxia (15%) reduced venous O2 levels in day 16 and day 18 embryos and reduced aircell O2 values in day 16, day 18, and internally pipped (IP) embryos, whereas hyperoxia (30%) increased venous O2 levels and aircell O2 level in day 16, day 18, and IP embryos. In comparison to normoxia, hypoxia delayed closure of the DA, whereas hyperoxia accelerated DA closure. In comparison to the left DA from externally pipped (EP) normoxic embryos, the left DA from EP hypoxic embryos exhibited a significantly weaker contractile response to O2. The DA from day 18 hypoxic embryos exhibited a significantly weaker contractile response to norepinephrine and phenylephrine when compared with the DA from day 18 normoxic and hyperoxic embryos. The effect of incubation in hypoxia / hyperoxia during different developmental windows on the DA O2-induced contractile response was observed only in IP embryos that were incubated in normoxia for 16 days and were then moved to hyperoxia. Incubation in hypoxia / hyperoxia resulted in differences in embryo mass, yolk mass, and heart mass. There is an association between the decreased contractile response to O2 and delayed closure in the proximal portion of the DA from hypoxic embryos; as well as an increased contractile response to O2 and accelerated closure in the proximal portion of the DA from hyperoxic embryos.

Os gânglios da raiz dorsal (GRD) armazenam neurônios TrkA-positivos. A percepção e transmissão de estímulos por estes neurônios dependem de uma neuritogênese adequada. Miosina (MioVa) é expressa no tecido nervoso e está presente em neuritos, corpo celular e cone de crescimento. Caracterizamos o padrão de expressão de MioVa na neuritogênese de células TrkA-positivas do GRD de galinha in vivo e in vitro. In vivo, MioVa é expressa em células que não começaram a emitir neuritos em HH25, e sua expressão persiste por toda neuritogênese. In vitro, é recrutada para o processo de re-emissão de neuritos de neurônios TrkA positivos na presença de NGF, sendo expressa em neuritos em diferentes estádios de neo-neuritogênese. Nos ensaios funcionais, observamos que a superexpressão do domínio globular de MioVa em culturas de GRD com 10 ou 100ng/ml de NGF reduz a população de células com neuritos longos e aumenta a população de células com neuritos curtos ou sem neuritos. Em conjunto, estes dados sugerem que a MioVa é importante para o estabelecimento de neuritos nociceptores. / The dorsal root ganglia (DRG) harbor the TrkA-positive neurons. The stimuli perception and transmission by these neurons depend on a proper neuritogenesis. Myosin (MyoVa) is widely expressed in nervous tissue and is present in neurites, cell body and growth cone. Here, we characterized the MyoVa expression pattern in chicken DRG TrkA-positive cells neuritogenesis, in vivo and in vitro. In vivo, at stage HH25, MyoVa was present both in cells with and without neurites and its expression persists throughout neuritogenesis. In vitro, it is recruited for the regeneration process and TrkA-positive neurons neurites re-emission in the presence of NGF, being expressed in neurites at different stages of neo-neuritogenesis. In functional assays, we observed that MyoVa globular tail overexpression in GRD cultures maintained with 10 or 100ng/ml NGF reduces the number of neurons with long neurites and increased the number of neurons with short neurites or no neurites. Taken together, these results suggest that Myosin Va is important for the establishment of nociceptor neurites.

No description available.

Conditions that lead to reversible growth arrest (quiescence), promote the expression of a set of genes called growth arrest specific (GAS) genes. GAS genes play a crucial role in initiating and maintaining the entry into quiescence, while also activating stress responses to help the cell overcome the effects of the stressors. Gene profiling study examining the transcriptome has shown a vast number of genes that are upregulated during quiescence, among them is GEM1 (GTP binding protein overexpressed in skeletal muscle). GEM1 transcripts were elevated 18-fold in response to quiescence. GEM1 is a small monomeric GTPase from the Ras superfamily. It is involved in regulation of cytoskeleton reorganization, and inhibition of voltage gated calcium channels that ultimately prevents hormone secretion. A preliminary study determined that GEM1 is packaged into extracellular vesicles (EV). GEM1 is also reported to promote lipid accumulation and adipogenesis in goat pre-adipocytes. GEM1 is also reported to bind transcription factors that are involved in lipid homeostasis pathways. Thus, it is probable that GEM1 may play a major role in EV formation and/or release, and lipid homeostasis. This study examined the expression of GEM1 at the protein level and validates its candidacy as a GAS gene. We also created two GEM1-shRNA retroviral constructs capable of partially downregulating GEM1 expression which can serve as a molecular tool for further characterizing the function of GEM1 in quiescent CEF. / Thesis / Bachelor of Science (BSc) / GEM1 is a small monomeric GTPase, implicated in a variety of roles in eukaryotes. It plays a role in regulating adipogenesis, and hormone secretion. Most notably it regulates cytoskeleton reorganization in response to changes in calcium concentrations. Gene profiling done by Bédard Lab identified that GEM1 transcripts were highly elevated in reversible growth arrested chicken embryo fibroblasts (CEF). In this study we further explore and characterize the protein expression of GEM1 in quiescent CEF. We also design and test shRNAi retroviral constructs to downregulate GEM1 in quiescent CEF.