小鼠肺臟發育在過程中的細胞的增生與分化，受到間質細胞與鄰近的上皮細胞之間訊息交互作用的調控。其中sonic hedgehog (Shh)，已知可由肺臟遠端的上皮細胞分泌並且調控鄰近的間質細胞，影響間質細胞的增生與分化。然而，在肺臟發育研究中，Shh對於肺臟遠端上皮細胞的調控仍然不清楚。在此，我們使用轉殖基因鼠的動物模式，藉由具組織專一性的promoter,來誘導Smoothened在肺臟上皮細胞大量表現。我們收取15.5天與18.5天的胚胎肺臟的分析結果顯示：無論是實驗組或是對照組的小鼠胚胎，肺臟遠端上皮細胞型態皆有分化。此外，由於傳統影像技術的限制，神經與血管在肺葉中連續性的分佈有其觀察上侷限性。我們利用組織澄清試劑將組織透明化，以增加光的穿透度，並配合共軛焦顯微鏡，將三維影像技術運用在肺臟發育研究上。我們成功地以螢光染色標記神經、上皮細胞、血管，以及細胞核後，透過共軛焦顯微鏡掃描取得深層的組織影像，再以電腦軟體重製出三維的連續性的影像分析。結果顯示，我們可以獲得E15.5及E18.5胚胎肺葉中神經走向的清晰影像，並觀察到神經沿著肺臟近端上皮細胞的走向生長，血管主要則是分布在間質細胞中並圍繞著遠端上皮細胞。此技術亦可應用在小鼠肺臟腺瘤中，並獲得清晰的血管影像。此一立體影像建構技術未來將可以運用於肺臟發育以及肺癌研究。

The process of lung branching morphogenesis involves cell proliferation, differentiation and migration, which is regulated through the signals from mesenchyme and adjacent epithelium. The sonic hedgehog (Shh) is one of signaling molecules secreted from distal lung epithelium to adjacent mesenchyme, which regulates mesenchymal cells proliferation and differentiation. However, the impact of Shh signaling on distal lung epithelium development is not clear. Herein, we genetically overexpressed smoothened (Smo) in mouse lung epithelium by epithelial specific gene promoters surfactant protein C (Spc) and Shh to investigate the role of Shh signaling pathway in the distal lung epithelium development. No obvious morphological differences were observed in distal lung epithelium of smoothened overexpressed mouse embryos. However, nerve innervation and vasculogenesis in these lungs are unclear due to the limitation of traditional histology methods that provide non-stereo images of nerves and blood vessels in embryonic lungs. In the second part of my thesis study, a novel imaging method that provides global views of oriented innervations of neuronal network and epithelium branching morphogenesis was applied to developing mouse lungs. The E15.5 and E18.5 mouse embryonic lung lobes were immunofluorescence stained for neuron, epithelium, endothelium and nuclei, followed by optical clear reagent treatment. Transparent lung tissues were scanned by confocal microscopy, and reconstituted to resemble projection and three-dimensional nerve images. The image results indicated that the orientation of nerves extended along the proximal lung epithelial tubules, while the vasculature in mesenchyme surrounded distal lung epithelium in can be acquired at E15.5 and E18.5 lung lobes. This technique also can be applied to mouse lung tumors for angiogenesis study. We successfully applied a 3-D imaging technique to visualize spatial distribution of nerves, blood vessels and epithelium tubules in lung development and in lung cancer research.

Contents
摘要...............................................................................................................i
Abstract.........................................................................................................ii
Abbreviation................................................................................................iv
Chapter 1. Introduction .....................................................................................................1
1.1 Murine lung development stages.......................................................................1
1.2 The signal interaction between mesenchyme and epithelium affected the lung development......................................................................................................................2
1.3 Sonic hedgehog signal pathway.......................................................................2
1.4 Overexpressing Smo in the lung epithelium of Scx-cre/Smo mouse.............3
1.5 Aim of this study.............................................................................................4
1.6 Imaging of epithelial morphogenesis and innervation in embryonic lungs........4
1.7 Optical clear reagent FocusClearTM.............................................................5
1.8 Purpose of 3D imaging study.......................................................................5
Chapter 2. Materials and methods..................................................................................6
2.1 The list of chemicals and reagent...................................................................6
2.2 The list of antibodies and blocking serum......................................................7
2.3 The list of mouse source.................................................................................7
2.4 The animal models..........................................................................................8
2.5 Genotyping..................................................................................................... 8
2.5.1 Genotyping for Spc-rtta...........................................................................9
2.5.2 Genotyping for TetO-cre..........................................................................9
2.5.3 Genotyping for Shh................................................................................10
2.5.4 Genotyping for Smo(KI) .......................................................................10
2.5.5 Genotyping for Smo(WT) .....................................................................10
2.6 Tissue preparation............................................................................................11
2.7 Hematoxylin and Eosin staining (H&E) stain..................................................11
2.8 Immunohistochemistry, Immunofluorescence.................................................12
2.9 RNA extraction, reverse transcription and real-time PCR...............................12
2.10 Transmittance test.............................................................................................13
2.11 Tissue preparation for LSM 780 confocal scanning.........................................13
2.12 Immunofluorescence for scanning by confocal LSM 780................................14
Chapter 3. Result.............................................................................................................15
3.1 The phenotype of Scx-cre/Smo transgenic mouse embryonic lungs...............15
3.2.1 Overexpressing Smo in lung epithelium by Spc-rtta/TetO-cre system...........15
3.2.2 The phenotype of E15.5 Spc/TetO-cre/Smo and control mouse embryonic lungs...............................................................................................................................16
3.2.3 Distribution and proliferation of mouse embryonic distal lung epithelium at E15.5................................................................................................................................16
3.3.1 The phenotype of Shh-cre/Smo and Smo mouse embryonic lungs at E15.5...17
3.3.2 Identify the distribution and proliferation of Shh-cre/Smo and Smo embryonic distal lung epithelium at E15.5........................................................................................18
3.3.3 Determining Cre recombinase expression in the E14.5 embryonic lungs of Shh-cre/Smo.....................................................................................................................18
3.4.1 The traditional image technique......................................................................19
3.4.2 Transparency test in mouse embryonic lung lobes..........................................19
3.4.3 The images of whole lung lobes scanning by confocal microscopy.............20
3.4.4 Distribution of neuron and vascular in embryonic lung lobes.........................21
3.4.5 Confocal image of the lung adenocarcinoma..................................................22
Chapter 4. Discussion......................................................................................................23
References...................................................................................................................26
Figures.............................................................................................................................31
Figure 1. Breeding strategy for conditional deletion of the stop fragment in lung epithelial cell by Scx-cre.................................................................................................31
Figure 2. The H&E stain of Scx-cre/Smo transgenic embryo show the abnormal tissue pattern at E17.........................................................................................................32
Figure 3. Smo was time dependently induced by lung epithelium specific promoter, Spc. .................................................................................................................................33
Figure 4. The genotyping result of E15.5 embryos of Spc-rtta/TetO-cre/Smo X Smo..................................................................................................................................34
Figure 5. The lumen of distal lung in Spc/TetO-cre/Smo still develop and the lumen seen to larger than control.............................................................................................35
Figure 6. The distribution pattern of Sox9 in Spc/TetO-cre/Smo was no significant difference compared with control....................................................................................36
Figure 7. The proliferation of distal epithelium were determined by p-Histone H3...................................................................................................................................37
Figure 8. Breeding strategy for conditional deletion of the stop fragment in lung epithelial cell by Shh-cre.................................................................................................38
Figure 9. The morphology of mouse embryonic lung of E15.5 Smo and Shh-cre/Smo without the significant change...................................................................39
Figure 10. The histology of distal lung epithelium of E15.5 Smo and Shh-cre/Smo demonstrated the similar tissue pattern...........................................................................40
Figure 11. The proliferation of distal lung epithelium cell of e15.5 Smo and Shh-cre/Smo were not significant difference..................................................................41
Figure 12. The histology showed the distal lung epithelium development of E18.5 Shh-cre/Smo...................................................................................................................42
Figure 13. The transcription level of Shh-cre/Smo and Smo embryonic lung at E14.5 was detected by real-time PCR.............................................................................43
Figure 14. The traditional histology methods only provided a plane image...........44
Figure 15. The left lung lobes become more transparent after immersing in FocusClearTM ..................................................................................................................45
Figure 16. The high resolution projection images of nerve distribution in the E15.5 right upper lung lobe.......................................................................................................46
Figure 17. The nerve projection images of the E18.5 right upper lung lobe.........48
Figure 18. The neuron innervated along with proximal lung epithelium...............49
Figure 19. The image demonstrated that the growth direction of vascular and nerve was consistent.................................................................................................................50
Figure 20. The capillary network of proximal and distal lung at E18.5.................51
Figure 21. Reconstitute 3D images of angiogenesis of Lung adenocarcinoma derived from Spc/k-ras double transgenic mouse............................................................52

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