京都大学 / 新制・課程博士 / 博士(医学) / 甲第23471号 / 医博第4778号 / 新制||医||1053(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 山下 潤, 教授 木村 剛, 教授 湊谷 謙司 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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xv, 76 p. : ill. (some col.) Includes 4 video files. / Improved understanding of pharyngeal arch (PA) patterning and morphogenesis can reveal critical insights into the origins of craniofacial diseases, such as Fraser syndrome. PAs contain mesenchymal condensations, which give rise to most of the facial skeleton in vertebrates. Studies of Endothelin1 signaling reveal that the skeleton derived from the first two PAs are patterned into dorsal, intermediate, and ventral domains. Previous work has indicated that endothelin targets, including the Dlx genes, homeotically pattern dorsal versus ventral PA identity. I show that the Dlx gene family plays a vital role in PA intermediate-domain identity establishment. In WT fish, the PA intermediate domain is delineated by combined expression of all Dlx genes. Reduction of Dlx gene function results in loss of intermediate-domain identity. Conversely, ventral expansion of Dlx expression, seen in hand2 mutants, results in ventral expansion of intermediate-domain identity. Hence, PA intermediate-domain identity is defined by co-expression of Dlx genes. Epithelial-mesenchymal interactions play an important part in PA intermediate-domain morphogenesis. Zebrafish fras1 (epithelially expressed) and itga8 (mesenchymally expressed) mutants also show specific defects within intermediate-domain skeleton and epithelia. Facial phenotypes in fras1;itga8 double mutants look extremely similar to either single mutant, suggesting that fras1 and itga8 might participate in the same epithelial-mesenchymal interaction during PA intermediate-domain formation. Our developmental studies reveal that fras1 - and itga8 -dependent epithelial segmentation of the PA intermediate domain stabilizes developing skeletal elements. Lesions in human FRAS1 underlie many cases of Fraser syndrome, and this work provides an excellent developmental model for the craniofacial defects found in Fraser syndrome. Loss of either Dlx or fras1 function produces defects in the PA intermediate domain, yet seemingly during different developmental periods. Nonetheless, combined reduction of both Dlx and fras1 function synergistically increases skeletal defects, implying a molecular connection between early (Dlx -mediated) pattern formation and later (fras1 -mediated) pattern stabilization. Elucidation of the Dlx-fras1 interaction is an interesting topic which may unveil new molecules pertinent to Fraser syndrome. Supplemental movies highlighting skeletal and epithelial morphogenesis accompany this dissertation. / Committee in charge: Judith S. Eisen, Chairperson; Charles B. Kimmel, Advisor; John H. Postlethwait, Member; Chris Q. Doe, Member; Kennith E. Prehoda, Outside Member

Indiana University-Purdue University Indianapolis (IUPUI) / The heart is a complex organ that is composed of numerous cell types, which must integrate their programs for proper specification, differentiation, and cardiac morphogenesis. During cardiac development the basic helix-loop-helix transcription factor Hand2 is dynamically expressed within the endocardium and extra-cardiac lineages such as the epicardium, cardiac neural crest cells (cNCCs), and NCC derived components of the autonomic nervous system. To investigate Hand2 function within these populations we utilized multiple murine Hand2 Conditional Knockout (H2CKO) genetic models. These studies establish for the first time a functional requirement for Hand2 within the endocardium, as several distinct phenotypes including hypotrabeculation, tricuspid atresia, aberrant septation, and precocious coronary development are observed in endocardial H2CKOs. Molecular analyses reveal that endocardial Hand2 functions within the Notch signaling pathway to regulate expression of Nrg1, which encodes a crucial secreted growth factor. Furthermore, we demonstrate that Notch signaling regulates coronary angiogenesis via Hand2 mediated modulation of Vegf signaling. Hand2 is strongly expressed within midgestation NCC and endocardium derived cardiac cushion mesenchyme. To ascertain the function of Hand2 within these cells we employed the Periostin Cre (Postn-Cre), which marks cushion mesenchyme, a small subset of the epicardium, and components of the autonomic nervous system, to conditionally ablate Hand2. We find that Postn-Cre H2CKOs die shortly after birth despite a lack of cardiac structural defects. Gene expression analyses demonstrate that Postn-Cre ablates Hand2 from the adrenal medulla, causing downregulation of Dopamine Beta Hydroxylase (Dbh), a gene encoding a crucial catecholaminergic biosynthetic enzyme. Electrocardiograms demonstrate that 3-day postnatal Postn-Cre H2CKO pups exhibit significantly slower heart rates than control littermates. In conjunction with the aforementioned gene expression analyses, these results indicate that loss of Hand2 function within the adrenal medulla results in a catecholamine deficiency and subsequent heart failure.