尋找在線蟲中參與計畫性細胞凋亡之新的調節子

Abstract

計畫性細胞凋亡是多細胞生物發育重要的過程， 而egl-1、ced-9、ced-4、ced-3這四個基因已經被發現是線蟲的計畫性細胞凋亡關鍵調節因子。當細胞決定死亡時，EGL-1會與CED-9結合，使得CED-4可以離開CED-9而活化CED-3，然而這些基因之調控的機制並不清楚。線蟲凋亡的細胞會產生不對稱分裂，所產生的兩個子細胞，較大的細胞會繼續分裂及分化，而較小的細胞則進行計畫性細胞凋亡。為了瞭解哪些基因影響計畫性細胞凋亡，所以我們實驗室設計了一組利用帶grp-1基因突變為背景之線蟲，篩選尾巴異常的蟲之方法。當grp-1或是與促進細胞凋亡的基因單獨突變時，線蟲只會有低比例的尾巴型態異常，然而當grp-1與促進細胞凋亡的基因同時突變時，則會使線蟲產生高比例的尾巴型態異常。藉由這樣的策略，已篩選出了一百多個線蟲突變株，其中三個為grp-1;tp27, grp-1;tp24, 以及 grp-1;tp172。這三個突變株有40~65%的尾巴型態異常，藉由DAPI染色，我發現這三個突變線蟲，在尾巴部分有多出的核，另外在胚胎1.5折時期，它們也有較少的細胞屍體。除此之外，grp-1;tp27 及grp-1;tp24有相當高的比例會有多的AVM或PVM細胞。除了藉由正向遺傳學的策略，我也利用了反向遺傳學的策略去尋找可能參與在計畫性細胞死亡的基因。之前兩篇文獻利用全基因組RNAi篩選的方式找出了在果蠅中可能參與於細胞凋亡的基因，參考兩篇文獻所指出的基因，我挑選了其中15個基因進行RNAi篩選實驗。藉由第一階段尾巴型態異常篩選，以及第二階段多出的Pn.aap細胞比例之篩選，我找出了四個候選基因--- K08F11.5, F10G8.9, C52D10.12, 及nkb-1。

Programmed cell death is an important process during the development of multicellular organisms. In C. elegans, four important genes, egl-1, ced-9, ced-4, ced-3, have been shown involved in the core pathway to execute the programmed cell death. When the cell is decided to die, EGL-1 (BH3-contain protein) binds to CED-9/Bcl-2. Then, CED-4/Apaf-1 leaves from CED-9 and activate CED-3/caspase. In C. elegans, apoptotic cells are resulted from asymmetric cell division that give rise of two cells with different cell sizes and fates. The larger cell keeps dividing and differentiation, whereas the smaller one undergoes programmed cell death. However, how these genes are expressed and regulated remains largely unknown. To find new genes affecting the processes of programmed cell death, lab members performed a grp-1 enhancer screen. The screen was based on the observation that single mutation in grp-1 or any of the proapoptotic genes, egl-1, ced-4 and ced-3, resulted in a low penetrant of tail defect. But double mutations of grp-1 and any of the proapoptotic genes caused a high penetrance tail defect, which is likely attributed to an additional tail hypodermal cell. In this screen, three mutant strains grp-1;tp24, grp-1;tp27, and grp-1;tp172 were isolated. I found that these strains had moderate penetrance of tail defect. By staining with DAPI, I observed an extra nucleus in the tail region in these mutant worms. Three mutant strains are recessive and shows decreased number of cell corpses at the 1.5 fold embryo stage. In addition, grp-1;tp24 and grp-1;tp27 have higher percentage of the extra AVM and PVM phenotype than grp-1. These results show that these three mutants might involve in programmed cell death. In addition to forward genetic approaches, I also applied reverse genetic strategies to search for candidate genes that regulate programmed cell death. Recently two studies applied genome-wide RNA interference screen to identify regulators of apoptosis in Drosophila. According to these two studies 15 genes were chosen as my candidates. The grp-1 dependent tail defect analysis and subsequent analysis of extra surviving Pn.app cells, four genes, K08F11.5, F10G8.9, C52D10.12, and nkb-1, were found as candidates that might be involved in the programmed cell death.