澤蛭神經外胚層背腹體軸發育過程中PCSK表現與BMP前驅蛋白調控之初探

Abstract

BMP訊息路徑在動物胚胎體軸發育上扮演重要角色，目前已知其訊息傳遞的範圍，受前驅蛋白酶對BMP前驅蛋白的切割方式所影響。在脊椎動物和果蠅上，BMP4╱Dpp均以擴散形成濃度梯度的形式調控胚胎背腹體軸建立，但在澤蛭系統中，BMP5-8才是調控神經外胚層背腹體軸的關鍵BMP訊息分子，其作用方式也不同於前兩者，必須透過細胞間的直接接觸產生作用。先前研究得知，澤蛭不同細胞系譜中的BMP訊息傳遞範圍也有差異。我們可以分析BMP5-8前驅蛋白之切割處理來進一步瞭解水蛭胚胎體軸建立的上游分子機制。本研究分析澤蛭中十二個前驅蛋白轉換酶（proprotein convertase, PCs）與已知的前驅蛋白轉換酶家族之親緣關係，並以反轉錄PCR和RNA原位雜交染色法，確立澤蛭中十二個前驅蛋白轉換酶的表現。為測試前驅蛋白轉換酶的活性，嘗試以細胞表面聯結蛋白水解標示法（cell surface-linked indicator of proteolysis assay, CLIP），以及異位表現切位定點突變的受標定BMP5-8兩種方法分析，結果發現切位突變後的BMP，並不影響BMP在建立神經外胚層背腹體軸的功能。

Bone morphogenetic protein (BMP) morphogen is known to play a vital role in body axis patterning during embryogenesis. Unlike BMP4/Dpp functions as a morphogen patterning embryo dorsoventral (DV) axis in vertebrate and fly, BMP5-8 in leech, the key BMP signaling ligand specifies DV patterning, acts in a “cell-cell contact” fashion. Evidences indicate that the ligand BMP proprotein’s differential proteolytic processing by proprotein convertases (PCs or PCSKs) influences its signaling range. Further, different signaling ranges were found between teloblast lineages (N/Q and O/P), above which make the proteolytic processing of BMP5-8 in leech embryo DV patterning an unsolved question. To characterize the role of PCSK-mediated cleavage in leech DV patterning, I carried out the following experiments. First, by reverse transcription-PCR and whole mount in situ hybridization, expressions of all 12 PCSK-related genes in leech have been confirmed and localized at embryogenesis stage 8. Second, a cell surface-linked indicator of proteolysis assay (CLIP) is prepared to test PC activity in vivo. Third, ectopic expression of cleavage site-mutated BMP5-8 in leech has been used. The above results indicate that the proteolytic processing of BMP5-8 is not required for development of normal neuroectodermal, which is also a key event of body axis formation.