臺灣博碩士論文加值系統

脊索動物的中樞神經系統是由背側中空神經管構成，此構造也是脊索動物特有的構造。在脊索動物神經發育的過程中，神經管中的神經幹細胞藉由Delta/Notch信號通路產生不對稱分裂形成子細胞或繼續保留幹細胞特性。雖然脊索動物的神經發育分子機制已經被深入的研究，然而脊索動物的中樞神經系統如何演化而來仍有爭議。目前為止，關於半索動物的神經發育機制的研究較為缺乏。半索動物在演化樹上為脊索動物的旁系群，由於親緣關係和脊索動物相近，研究半索動物的神經發育可以闡明脊索動物中樞神經系統的演化過程。因此，本研究的目的在於探討間接發育型玉柱蟲Ptychodera flava的神經系統發育過程以及其中的分子機制。我利用免疫螢光染色及原位雜交技術去觀察Synaptotagmin蛋白、Ngn基因、AcSc基因、chat基因、th基因及Serotonin蛋白的分佈與基因表現位置，用以標記分化神經、神經先驅細胞及不同類型的神經。實驗結果發現，已分化神經細胞首先在原腸胚時期出現在胚胎的apical region，然後再出現在胚胎的纖毛帶上。等到tornaria larva時期，神經細胞的軸突將神經細胞之間連結得更為緊密。實驗結果又發現，在神經細胞出現之前，神經先驅基因Ngn和AcSc已經分別以salt-and-pepper的形式表現在胚胎的纖毛帶及apical region上。在Notch信號通路抑制物（DAPT）處理過的胚胎中，我觀察到神經先驅基因在胚胎apical region的表現範圍有擴大現象，並產生發育散亂的神經系統。此結果表示，Notch信號通路參與了P. flava的神經系統發育。此外，我也觀察到DAPT會影響纖毛帶及眼點的發育。而在BMP蛋白處理後的胚胎中，我發現Ngn基因和AcSc基因的表現會分別往胚胎的腹側集中或減少。以上的實驗結果顯示P. flava和脊索動物以及其他的動物一樣，都是利用Notch及BMP信號通路來調控神經系統的發育。最後，我的研究結果顯示驅動兩側對稱動物中各式神經系統的演化動力來源並非是神經發育過程的機制，而是神經系統開始發育的位置。

The central nervous system (CNS) of chordates is composed of a dorsal hollow neural tube, which represents one of the defining features of the phylum. During chordate neurogenesis, the neural stem cells located within the neural tube undergo asymmetrical cell division, and depending on the level of Delta/Notch signaling, the daughter cells become neural progenitors or remain as stem cells. Although the molecular mechanisms underlying neurogenesis of the chordate nervous system have been well studied, the evolutionary origin of the chordate CNS remains debatable. Little attention has been paid to the development of the nervous system of the hemichordates, a sister group of chordates. Because of the phylogenetic position, studies on the developmental mechanisms controlling neurogenesis in hemichordates may shed light on the evolution of the chordate nervous system. In this study, I aim to decipher the spatiotemporal neurogenesis process and its underlying molecular mechanism in Ptychodera flava, an indirect-developing hemichordate. I performed immunostaining and in situ hybridization on markers of differentiated neurons, neural progenitors and neuronal subtypes, including Synaptotagmin, Ngn, AcSc, chat, th and Serotonin. I observed that differentiated neurons appeared initially in the apical region, then in the ciliary bands at the late gastrula stage, and the neurons became hardwired at the tornaria larval stage. My results also revealed that, a few hours before the Synaptotagmin-positive neurons were observed, neural progenitor genes, Ngn and AcSc, were expressed in a salt-and-pepper pattern in the ciliary bands and the apical ectoderm, respectively. In embryos treated with DAPT, a Notch signaling inhibitor, I observed broader expression domains of the neural progenitor genes and a disorganized nervous system, indicating that P. flava neurogenesis is mediated by Notch signaling. In addition, I observed that development of the ciliary bands and the eye spots were affected by DAPT. In the BMP-treated embryos, it is known that neural patterning is affected, and I observed that expression of Ngn and AcSc was accumulated ventrally or decreased, respectively. From these results, I conclude that similar to chordates and other animals, P. flava employs Notch and BMP signaling pathways for neurogenesis and neural patterning. My study suggests that changes of where neurogenesis occurs, rather than the neurogenic process, may be the driving force for the emergence of diverse nervous systems in the bilaterian.

口試委員會審定書 i
謝辭 ii
摘要 iv
Abstract v
Table of Contents vii
List of Figures iix
List of Appendix x
Introduction 1
Background 1
Neural patterning in Bilateria 2
Neurogenesis in the Bilateria 3
Using hemichordates to investigate the evolutionary origins of the CNS of chordates 4
Overview of Ptychodera flava development 6
Previous studies of the nervous system in hemichordates 7
Aim of this study 8
Materials and Methods 9
Animal collection 9
Spawning induction, fertilization and embryo culture 9
Molecular cloning 10
DIG-labeled RNA probe preparation 10
DNP-labeled RNA probe preparation 11
Fixation of the P. flava embryos 12
Fixation of the P. flava embryos for Synaptotagmin immunostaining 12
Whole mount in situ hybridization (WMISH) 13
Double fluorescence in situ hybridization (double FISH) 14
Immunofluorescence staining 16
Treatments with DAPT 17
Results 18
Spatiotemporal development of the embryonic nervous system of P. flava 18
Expression patterns of the proneural bHLH transcription factors, Pf-neurogenin and Pf-achaete-scute, in P. flava embryos 19
Investigation of the neuronal subtypes 21
Neural progenitor genes are regulated by Notch signaling in P. flava 23
Treating DAPT before or after the late gastrula stage affects the organization of the nervous system 25
Treating DAPT before the late gastrula stage affects development of the ciliary band 26
Treating embryos with DAPT affects development of the eye 27
Overactivation of BMP signaling represses neurogenesis in P. flava 28
Discussion 31
Similar molecular mechanisms are used for neurogenesis in Metazoa 31
Larval nervous systems of indirect-developing hemichordates and sea urchins might be homologous 32
Treating DAPT before the late gastrula stage affects development of the ciliary bands 34
DAPT influences development of the eye spots 34
References 36
Figure 1. The nervous system of P. flava (A-M) Synaptotagmin immunostaining in P. flava from late gastrula (45 hpf) to tornaria larva (96 hpf) 42
Figure 2. Numbers of the neuronal cell bodies in different regions and at different stages of P. flava 43
Figure 3. Basic helix-loop-helix domain of Pf-Ngn and Pf-AcSc 44
Figure 4. Expression of Pf-Ngn and Pf-AcSc. (A-D) Expression of Pf-Ngn was detected by WMISH from 45 hpf to 72 hpf 46
Figure 5. Expression of the th and chat genes and distributions of Serotonin during P. flava embryogenesis 47
Figure 6. The timescale of neurogenesis in P. flava based on molecular markers. 48
Figure 7. Expression of Pf-delta during embryogenesis 49
Figure 8. Schemes of DAPT treating periods in P. flava 51
Figure 9. Expressions patterns of Pf-Ngn and Pf-AcSc in DAPT-treated embryos 52
Figure 10. Expression of Pf-delta in DAPT-treated embryos 54
Figure 11. Synaptotagmin immunostaining in the DAPT-treated embryos 55
Figure 12. Expression of Pf-chat and Pf-th in the DAPT-treated embryos 57
Figure 13. Serotonin immunostaining in the DAPT-treated embryos 58
Figure 14. Expression of Pf-onecut, an ciliary bands marker, in the DAPT-treated embryos 59
Figure 15. Expression of Pf-vsx and Pf-opsin in the DAPT-treated embryos 60
Figure 16. Scheme of the mBMP4 treating period in P. flava 61
Figure 17. Expression of Pf-Ngn and Pf-AcSc in mBMP4 overactivated embryos 62
Figure 18. Expression of Pf-chat and Pf-th in mBMP4 overactivated embryos 63
Appendix 1. Deuterostome phylogeny 65
Appendix 2. Development stages of P. flava 66
Appendix 3. Primers used for cloning P. flava genes 67
Appendix 4. Experimental solution recipes 68

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