肝醣生成酵素激酶3ß調控斑馬魚的心臟發育

Abstract

glycogen synthase kinase 3β (GSK3β)是一多功能的serine/threonine蛋白激酶，在生物體內許多器官組織中都會表現。GSK3β參與許多複雜的生理反應，如：細胞存活、發育型態、致癌過程等。基於以上的特性，在活體內研究GSK3β如何參與單一調控機制的實驗進行不易，因此GSK3β在心臟胚胎發育過程及對心臟功能上的影響機制仍未明。在本實驗中，我們使用綠螢光標定心臟的斑馬魚轉殖品系來輔助心臟形態功能實驗觀察的進行。為了有效且專一的抑制GSK3β表現，我們在胚胎一細胞時期注射GSK3β antisense morpholino oligonucleotide（gsk3β-MO）來抑制GSK3β蛋白質生成。在注射gsk3β-MO的胚胎中發現：授精後24小時（24 hours postfertilization；24 hpf），心臟先驅細胞已在中軸位置癒合並形成心管。然而過了36 hpf，管狀心臟卻仍無法完成正確地進行左移（jogging）與彎曲（looping）等左右非對稱發育過程（cardiac left-right asymmetry）。授精後第四天（4 days postfertilization；4 dpf）之後，可觀察到細長的條狀心臟，且心跳速率變慢、心臟收縮力減弱，甚至有圍心腔異常腫大的情形。注射gsk3β-MO導致心臟發育異常，且損害的程度和比例會隨著gsk3β-MO劑量提高而增加；同時注射gsk3β-MO和GSK3β mRNA則可達到救援（rescue）的效果：心臟異常的比例由嚴重異常43％，輕微異常53.5％降為嚴重異常31.8％，輕微異常12.1％。在原位雜交（in situ hybridization）實驗中，得知注射gsk3β-MO胚胎的cardiac myosin light chain（cmlc2）mRNA表現正常； atrial natriuretic factor（ANF）mRNA表現量大增；和bone morphogenetic protein（bmp4）mRNA有異位性表現且表現量大增，顯示心臟左右非對稱發育受到干擾。此外，已知GSK3β在Wnt/β-catenin pathway會與axin、adenomatous polyposis coli （APC）形成複合體，而在注射axin1-MO胚胎中也可觀察到心臟左右非對稱發育異常的情形。因此，經由實驗結果推測GSK3β調控斑馬魚胚胎心臟發育過程中的左右非對稱發育；而其所參與的調控路徑為Wnt/β-catenin pathway。

Glycogen synthase kinase 3β (GSK3β) encodes a multifunctional serine/threonine protein kinase, which is ubiquitously expressed in organisms. Although GSK3β is known to play roles in many biological processes, including cell survival, tumorigenesis and developmental patterning, it remains unclear that the function of GSK3β in cardiogenesis in vivo. We used a GFP-tagged heart transgenic zebrafish to address this question. In order to specifically inhibit GSK3β, gsk3β antisense morpholino oligonucleotide (gsk3β-MO) was injected into one-celled embryos to block the translation of the gsk3β mRNA. In gsk3β-MO -injected embryos, we found that heart precursor cells lined up at the midline at 24 hpf, failed to complete the heart positioning, and then stretched slowly to a thin, ‘string-like’ shape about 96 hpf. In addition, in this morphants, the heart rate was slower, the contraction was weaker, and pericardial edema was commonly observed. Knowdown of GSK3β resulted in a severe disruption of early (jogging) and late (looping) aspects of cardiac left-right asymmetry. The degree of cardiac defects due to GSK3β attenuation was dose-dependent. But these defectives could be rescued by injecting synthetic gsk3β mRNA. Consistent with the morphological change of heart, the expression of bmp4, a heart-asymmetry marker and a target of Wnt/β-catenin signaling pathway, was upregulated in GSK3β morphants: the asymmetry of heart was completely disrupted. Interestingly, we found that cardiac defects happened in the gsk3β-MO-injected embryos were similar to those observed in axin1 morphants. Therefore, our findings strongly suggest that GSK3β plays a role in L/R-biased heart positioning through Wnt/β-catenin pathway during zebrafish cardiogenesis.