제브라피쉬 초기발생에 있어서 신경세포와 혈관세포 간의 상호작용에 대한 연구 = Interactions between Motoneurons and Blood Vessel Cells in Zebrafish

다국어 초록 (Multilingual Abstract)

Blood vessels and nerves are structurally similar complex branched systems. Their guidance must be exquisitely regulated to ensure proper wiring of both networks. Recent results showed that specialized endothelial cells, resembling axonal growth cones, form the tips of growing capillaries. These endothelial tip cells guide outgrowing capillaries in response to gradients of extra cellular matrix-bound vascular endothelial growth factor. Several axon guidance molecules, including Semaphorins, Netrins, Ephrins and Slits, have also been implicated in vessel path finding and network formation. My current study focuses on the possible interaction between primary motoneurons and intersomitic vessels during early development. I established a double-transgenic zebrafish with GFP expression in blood vessel cells and RFP expression in motoneurons, enabling me to examine their development at a cellular level in live animals. With this powerful experimental animal system, I tested the involvement of midline signaling in motoneuron and vessel development. Cyclopamine, a chemical inhibitor of the Shh pathway, inhibited both motoneuron and vessel development. Specific inhibition of motoneuron development with antisense olig2 morpholino knockdown affected intersegmental vessel formation. To identify signaling guidance molecule, I analyzed expression of several candidate genes and found that slit-3 gene is down-regulated in motoneurons. These findings suggest that motoneuron has a possible interaction with vessel cells during early embryo development.

목차 (Table of Contents)

• Ⅰ. 서론 1
• Ⅱ. 재료 및 방법 14
• 1. 제브라피쉬의 사육 및 발생배의 준비 14
• 2. Whole-mount in situ hybridization 15
• 3. In vitro transcription과 anti-sense morpholino의 microinjection 17
• 4. Whole-mount immunostaining 18
• 5. Cyclopamine 처리 18
• 6. Cryostat section 19
• Ⅲ. 결과 20
• 1. Double transgenic fish 제작과 관찰 20
• 2. Midline 신호전달의 억제를 통한 운동신경세포와 혈관내피세포의 관계분석 25
• 3. Olig2 유전자 knockdown을 통한 운동신경세포와 혈관내피세포의 관계분석 29
• 4. 운동신경세포와 혈관내피세포의 관련 신호전달인자 확인 35
• Ⅳ. 고찰 41
• Ⅴ. 결론 44
• Ⅵ. 참고 문헌 44
• ABSTRACT 51