BMP傳訊在環節動物線蚓再生過程扮演的角色之研究

Abstract

本研究的目的為觀察胚胎發育中用於腹背體軸建立的BMP傳訊途徑是否參與了線蚓個體尺度的再生過程，同時也初步描述BMP於再生過程中可能扮演的角色。首先由實驗室先前建立的轉錄組資料庫中搜尋Echy-bmp片段後將此片段cDNA選殖出來，並製作用於原位雜合反應的探針。原位雜合反應實驗結果發現不論在前端還是後端再生的過程中，Echy-bmp片段在再生第二天的表現都是最明顯的。接著再以針對BMP傳訊途徑的胞內傳訊物質的活化態p-Smad的抗體進行免疫染色實驗，發現在再生的過程中，蟲體背部區域會有明顯的p-Smad訊號。因此，BMP在再生過程不僅會被表現，且其下游訊號集中於背端區域。接下來，以小分子抑制劑DMH-1來抑制BMP的膜上受器，藉此阻礙訊息傳遞，以觀察BMP在再生中的功能。抑制的實驗結果發現，在頭部再生的過程中的第三及第四天，acetyl-alpha tubulin抗體免疫染色顯示的周邊神經有發育不良的情形，但中樞神經的縱向neuropile再生似乎沒有受到顯著影響。綜上所述，本實驗發現BMP傳訊途徑可能在線蚓再生過程中調控周邊神經發育。

This study aims to find out whether the BMP signaling pathway, which is responsible for patterning the dorsal-ventral (DV) body axis in embryonic development of many bilaterian species, is involved in the whole-body regeneration of Enchytraeus. In situ hybridization of Enchy-bmp showed that the BMP ligand is dynamically expressed during regeneration. Immunofluorescence (IF) against phospho-Smad1/5/8 (p-Smad), the activated form of the signal transducer downstream to the BMP, was used to reveal the pattern of the BMP pathway activity during regeneration. It was found that p-Smad staining is located on the dorsal side of the trunk. To identify the function of BMP signaling during regeneration, the fragment undergoing anterior regeneration was treated with the small-molecule inhibitor of the BMP receptor kinase, DMH-1, on the third day post-amputation and onward, when the blastema starts to differentiate. The efficacy of DMH-1 treatment was confirmed by p-Smad IF. IF against acetyl-alpha tubulin revealed that the organization of the peripheral nerves was disrupted in the DMH-1-treated specimen. On the other hand, the regeneration of the longitudinal neuropile in the ventral nerve cord was not affected by DMH-1 treatment. Together, these results indicate that the BMP signaling pathway may regulate the development of peripheral nerve in anterior regeneration.