蠑螈肢體再生：神經介導表現之組蛋白去乙醯化酶之角色與雙螢光嵌合體肢體再生模式之建立

Abstract

蠑螈以其在受傷後能夠重新生長大部分身體組織並恢復複雜結構的卓越能力而在脊椎動物中備受矚目。其中一個著名的例子就是牠們完全再生功能性肢體的能力。儘管在這個領域取得了重要進展，但對於控制肢體再生的分子信號的理解仍然有限。組蛋白去乙醯化酶（HDACs）在肢體再生過程中扮演著關鍵角色。本論文旨在研究HDAC1在蠑螈肢體再生中的參與情況。研究發現，在再生的早期分化階段之前，HDAC1表現呈雙相上調。使用MS-275等抑制HDAC活性的化合物會延遲幼體的肢體再生，而局部注射HDAC抑制劑則會阻礙HDAC活性、芽基組織的形成和隨後的肢體再生。HDAC1的表現在傷口表皮中更為明顯，且截肢前的去神經會阻止其表達上升和肢體再生。此外，補充神經因子有助於促進HDAC1的上調表達並增強肢體再生過程。這些發現顯示HDAC1在蠑螈肢體再生中的參與情況，並強調了神經因子在調節這一過程中的重要性。 此外，本研究探討了HDAC抑制對肢體再生轉錄反應的影響。轉錄組學分析揭示了表皮和軟組織中複雜的功能途徑。HDAC活性對阻止與組織發育和分化相關的基因過早表達至關重要。抑制HDAC1導致再生相關基因和WNT通路相關基因的過早活化。使用WNT抑制劑處理後，部分恢復HDAC1的抑制作用，改善芽基組織形成。 此外，本論文還建立紅色與綠色的轉基因蠑螈，並延續先前實驗室在肌肉接合方面的研究結果，旨在透過雙螢光移植的方式，觀察再生肢體的橫截面來檢測肌肉纖維的重新接合情況。結果顯示，不同肌肉之間的肌肉纖維接合速度不同，其中 gracilis 肌表現出外圍的重連特徵。此外，在遠端再生部位，RFP+的肌肉纖維對肌肉再生起到了貢獻作用，尤其是在小腿的腹肌表面。這個雙螢光嵌合體為瞭解蠑螈肢體再生的後期肌肉接合模式提供了新的視角。 本研究凸顯了HDAC1在蠑螈肢體再生中的重要作用。神經調控的HDAC1表達對於芽基組織的形成和成功再生至關重要。研究結果強調了在這一過程中涉及的基因表達模式和表觀遺傳修飾的複雜性。此外，肌肉纖維的重連動態為肢體再生的後期階段提供了深入的洞察。本論文推進了人們對於蠑螈肢體再生機制的理解。這些發現對於再生醫學研究具有重要意義，並可能對未來的治療方法做出貢獻。

Axolotls are renowned for their remarkable ability to regenerate various body parts, including fully functional limbs, making them stand out among vertebrates. However, our understanding of the molecular mechanisms that govern limb regeneration in axolotls remains limited. Histone deacetylases (HDACs) have been identified as crucial players in this process. This thesis aimed to investigate the role of HDAC1 in axolotl limb regeneration. The study revealed a biphasic up-regulation of HDAC1 prior to the early differentiation stage of regeneration. Inhibition of HDAC activity using the compound MS-275 caused a delay in limb regeneration in larvae, while localized injection of HDAC inhibitors hindered HDAC activity, blastema formation, and subsequent limb regeneration. Notably, HDAC1 expression was more pronounced in the wound epidermis (WE), and denervation prior to amputation prevented its elevation and subsequent limb regeneration. Furthermore, supplementation of nerve factors promoted the up-regulation of HDAC1 expression and enhanced the process of limb regeneration. These findings shed light on the involvement of HDAC1 in axolotl limb regeneration and emphasize the significance of nerve factors in regulating this process. Furthermore, this thesis delved into the transcriptional changes occurring during limb regeneration under HDAC inhibition. Transcriptome sequencing uncovered intricate functional pathways in both the epidermis and soft tissue (ST). The activity of HDACs was crucial in suppressing the premature expression of genes associated with tissue development and differentiation. Inhibiting HDAC1 resulted in premature activation of genes linked to regeneration and the WNT pathway. Interestingly, administering a WNT inhibitor partially reversed the effects of HDAC1 inhibition and enhanced blastema formation. In addition, this study established transgenic axolotls with red and green fluorescence, building upon previous research in muscle reconnection conducted in Dr. Lee’s laboratory. The aim was to observe the reconnection of muscle fibers in regenerative limbs through cross-sectional analysis using the double fluorescence transplantation method. The results revealed that the reconnection of muscle fibers varied in speed among different muscles, with the gracilis muscle exhibiting peripheral reconnection characteristics. Furthermore, in the distal regenerative regions, the RFP+ muscle fibers contributed to muscle regeneration, particularly on the ventral surface of the calf. This double fluorescence chimeric model provides a new perspective on the late-stage muscle reconnection patterns in axolotl limb regeneration. Overall, this thesis highlights the essential role of HDAC1 in axolotl limb regeneration. Nerve-mediated HDAC1 expression is crucial for blastema formation and successful regeneration. The findings underscore the intricate gene expression patterns and epigenetic modifications involved in the process. Furthermore, muscle fiber reconnection dynamics provide insights into the late stages of limb regeneration. In conclusion, this comprehensive study advances our understanding of the mechanisms underlying axolotl limb regeneration. The findings have implications for regenerative medicine research and may contribute to future therapeutic approaches.