斑馬魚mbnl3之鑑定與功能性分析

Muscleblind-like (MBNL)是一群參與調控特定組織所需差異性剪輯的蛋白家族。在果蠅中，muscleblind蛋白參與了肌肉及感光受器的末端分化調控。在人類及小鼠中都具有3種MBNL的paralogs。先前我們實驗室已在斑馬魚選殖出其3種mbnl基因(zmbnl1-3)以研究其在斑馬魚早期發育所扮演的角色。在本篇論文主要集中於zmbnl3的相關研究。藉由pre-mRNA的差異性剪輯，zmbnl3至少可轉譯出5種不同的蛋白質異構物。zmbnl3除了含有MBNL典型的CCCH zinc finger domains，也含有其他特殊的保留區域(如LEV box, NGR box, Ser/Thr-rich domain)。zmbnl3廣泛表現於成魚組織，但不同的isoforms在各組織的表現量不盡相同。在胚胎發育時期，zmbnl3在24hpf之後才漸漸出現。利用全胚體原位雜交技術觀察(WISH)到zmbnl3在胚胎中呈現廣泛性表現。利用antisense morpholino knockdown zmbnl3，並以不同marker gene(wnt1, tnnt2, anxa5)進行WISH，及分析其可能的下游基因clc-1 and tnnt2，並沒有觀察到與WT明顯的差異。但藉由alcian blue staining發現 zmbnl3 morphants其咽喉鰓弓的軟骨發育有異常的情況，且在48hpf之後，myoD表現量較WT下降。另一方面，顯微注射zmbnl3 cRNA發現其異常表型主要為體軸彎曲及體節縮短。隨著施打劑量增加，亦發現畸形率有dose-dependent的情況。利用頭部及肌肉發育相關marker(wnt1, pax2.1, myoD, myogenin)進行WISH，發現過量表達zmbnl3的胚胎腦部發育受到影響且體節排列嚴重異常。但以RT-PCR分析其可能下游基因(clc-1, myoD, myogenin,)並沒有發現改變。將zmbnl3過量表達於C2C12細胞，可發現zmbnl3會抑制肌肉細胞的分化(如:缺乏融合的多核肌纖維細胞、mef2a 剪輯形式停留在分化前及MHC蛋白表現量下降)。由Dual-luciferase reporter assaye更發現了在斑馬魚胚胎中，zmbnl3可以降低myoD promoter的活性。综合以上結果，我們推測zmbnl3藉由MyoD-dependent途徑來干擾肌肉細胞進行分化。

關鍵字

斑馬魚

並列摘要

Muscleblind-like (MBNL) is a family of proteins that participate in regulation of tissue-specific alternative splicing. In Drosophila, the muscleblind protein was shown to regulate terminal differentiation of photoreceptors and muscles. Three MBNL paralogs have been identified in humans and mice. Previously, we cloned three mbnl genes (zmbnl1 – 3) in zebrafish in order to study their function during fish development. In this study, we focus on zmbnl3. Alternative splicing of the zmbnl3 primary transcripts gives rise to at least 5 protein isoforms. In addition to the characteristic CCCH zinc finger domains, several structural motifs (like LEV box, NGR box, Ser/Thr-rich domain) are also found conserved in zmbnl3. Zmbnl3 is expressed in most adult tissues although the expression of specific spliceforms varies. During embryogenesis, zmbnl3 transcripts are hardly detected until 24hpf. Whole-mount in situ hybridization (WISH) reveals that zmbnl3 expression in the embryo is more ubiquitous rather than specific. zmbnl3 morphants of antisense morpholono knockdown were examined by WISH analysis of several marker genes (wnt1, tnnt2, anxa5), as well as by RT-PCR analysis of the splicing patterns of its suspected targets, clc-1 and tnnt2. The morphants did not show clear difference from WT embryos. However, alcian blue staining revealed overt defects in the pharyngeal arches of zmbnl3 morphants, and myoD expression was also decreased. On the other hand, microinjection of zmbnl3 cRNA into the embryos resulted in defective embryos with crooked body axes and short somites. The defective rate of the injected embryos increased in a dose-dependent manner. WISH analysis of several marker genes (wnt1, pax2.1, myoD, myogenin) revealed that embryos overexpressed with zmbnl3 had disorganized somite formation and abnormal brain development. RT-PCR analysis indicated that the splicing patterns of clc-1 and expression levels of myoD and myogenin were not changed. When introduced into C2C12 cells, zmbnl3 inhibited cell differentiation as judged by lack of cell fusion, change of mef2a splicing pattern and reduction of MHC expression. Dual-luciferase reporter assay further revealed that zmbnl3 down-regulated myoD promoter activity in fish embryos. These data suggest that zmbnl3 may interfere with muscle differentiation through the MyoD-dependent pathway.

並列關鍵字

zmbnl3

參考文獻

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斑馬魚mbnl3之鑑定與功能性分析

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