分析BLMP-1調控線蟲尾突細胞凋亡的功能

Abstract

計畫性細胞凋亡在生物發育及維持生理恆定扮演著重要的角色。在線蟲中，當egl-1/BH-3-only gene的轉錄作用啟動，會促進大部分的體細胞活化凋亡的訊息傳遞鏈進一步死亡。EGL-1表現量的提升進而使得CED-4從CED-4-CED-9複合物中釋放，活化了下游的CED-3，最終導致細胞死亡。儘管如此，依然存在著不同的機制來開啟特定細胞的凋亡核心路徑。一對坐落在尾部的尾突細胞於bean stage前出生，隨著時間的發育，兩顆尾突細胞進行質融合，產生一顆雙核的細胞，接著分化出由微管構成的支架結構，朝尾部延伸並且協助尾巴型態的正常發育，約略於3-fold至3.2-fold stage開始執行細胞凋亡，而在約3.2-fold至3.6-fold stage產生了尾突細胞的細胞屍體。相較於大部分出生後30分鐘內即會進行細胞凋亡的體細胞，對於尾突細胞在特定時間點啟動細胞凋亡的機制與主要的調控因子依然存在著許多未知。實驗室先前的研究發現blmp-1會影響某些胚胎時期的細胞凋亡，其中包含了尾突細胞。已知blmp-1屬於zinc finger transcriptional repressor，首先我們利用blmp-1::gfp的質體表現於ced-3(n717)突變株中。從3-fold stage至胚胎晚期，blmp-1開始表現在尾突細胞內，這意謂著blmp-1表現在尾突細胞中的時間點被嚴密地調控，同時blmp-1的表現對於細胞凋亡非常地重要。為了進一步釐清blmp-1與細胞凋亡之核心路徑中ced-3、ced-4和ced-9的調控關係，首先，我將CED-3專一性地過量表現於尾突細胞中，發現可以讓blmp-1突變株中不正常存活的尾突細胞進行凋亡，因此我們認為blmp-1導入凋亡的核心路徑，其作用位置可能是ced-3的上游或者平行於ced-3。有趣的是，將全長CED-4在高濃度情性下表現在wild type中會造成尾突細胞的不正常存活，之後會再更進一步地解開此非預期內的結果。接著，我觀察到，ced-9失去功能的情形下，可以部分的rescue blmp-1所造成的尾突細胞凋亡缺失，這意謂著blmp-1需要透過ced-9的功能來促進尾突細胞的凋亡。另一方面， Dr. Shai Shaham實驗室發現dre-1可能藉由負向調控ced-9的轉譯表現，最終促進尾突細胞的死亡。已知，dre-1是SCF E3 ubiquitin ligase 複合體中的 F-box protein，它的功能主要是協助辨識受質以利蛋白質降解的進行。而我藉由轉殖基因的方法，觀察到dre-1從bean至2-fold stage只會表現於尾突細胞中，其臨近的下皮細胞無表現，令人意外的是，dre-1突然在3-fold stage消失了，約略3.2-fold至3.6-fold stage又再度開始表現於尾突細胞內。這暗示著dre-1對於啟動尾突細胞凋亡時間點的調控也是相當地重要。有趣的是，將blmp-1過量表現於dre-1 (RNAi)突變株中，可以rescue因為dre-1表現量降低造成得尾突細胞不正常存活率。這樣的結果暗指blmp-1可能平行於dre-1或者作用在其下游的位置，最終促進尾突細胞的凋亡。綜合以上所述，我們觀察了blmp-1與dre-1在尾突細胞內時間與空間上的表現情形 ，並且指出在基因層次上，blmp-1與核心路徑中的ced-3以及ced-9之間的關係。

Programmed cell death (PCD) plays an important role in animal development. In Caenorhabditis elegans, transcriptional up-regulation of egl-1/BH-3-only gene promotes most somatic cell deaths. The increased level of EGL-1 induces the release of the proapoptotic CED-4(Apaf-1) from the CED-4-CED-9 complex and hence activating the downstream CED-3 caspase. However, some cell deaths, such as tail-spike cell death, use alternative mechanisms to switch on the core PCD pathway, because egl-1 is not absolutely required for these deaths. Two tail-spike cells are generated in the tail region before the bean stage. They fuse to become a binuclear cell at the comma stage. The binuclear cell differentiates and extends a long, thin, microtubule-based spike to help tail elongation during later embryogenesis. The binuclear cell undergoes apoptosis at about the 3.2-fold to 3.6-fold stage, when tail morphogenesis is complete. The temporal regulation of tail-spike cell death is poorly understood. We found that blmp-1, which encodes a zinc finger transcriptional repressor, is required for the death of some embryonic cells, including that of tail-spike cell. Using the blmp-1 transcriptional GFP reporter in the ced-3(n717) mutant, the expression of blmp-1 was detected in the tail-spike cell from 3-fold to late embryogenesis stage. This result suggests that blmp-1 is transcriptional up-regulated prior to tail-spike cell death. We next examined the relationship of blmp-1 with the key PCD genes ced-3, ced-4 and ced-9. First, over-expression of ced-3 specifically in the tail-spike cell rescues the PCD defect in the blmp-1(s71) mutant, suggesting that blmp-1 acts upstream of, or in parallel to, ced-3 to promote tail-spike cell death. Intriguingly, over-expression of high dosage of the ced-4 full-length cDNA results in abnormal tail-spike cell survival in the wild type. Further experiment is maded to unravel this unexpected result. Second, loss of ced-9 partially suppresses the tail-spike cell death phenotype of the blmp-1(s71) mutant, suggesting that blmp-1 acts upstream of, or in parallel to ced-9. Dr. Shai Shaham lab found that dre-1, which encodes an F-box protein of an SCF (Skp1-Cullin-F-box protein) E3 ubiquitin ligase mediating protein degradation, promotes tail-spike cell death. Using transgenic approaches, we observed the expression of dre-1 disappeared suddenly in tail-spike cell at the 3-fold stage, but it reappeared at about the 3.2-fold to 3.6-fold stage, suggesting that dre-1 expression is also critical for timely tail-spike cell death. Interestingly, over-expression of blmp-1 can rescue the tail-spike cell death defect in the dre-1(RNAi) mutant, indicating blmp-1 may act downstream of, or in parallel to dre-1. Therefore, we have characterized the temporal expression pattern of blmp-1 and dre-1,and positioned blmp-1 genetically to core PCD genes ced-3 and ced-9.