肝醣生成酵素激酶 3β 對斑馬魚胚胎血管發育的影響

The Effect of Glycogen Synthase Kinase 3β on the Angiogenesis during Zebrafish Embryogenesis

林依臻(Yi-Zhen Lin)

指導教授 ： 蔡懷楨

國立臺灣大學/生命科學院/分子與細胞生物學研究所/碩士(2010年)

https://doi.org/10.6342/NTU.2010.10325

摘要

Glycogen synthase kinase3β (GSK3β) 是一個具有多種生化特性的蛋白。但是關於 GSK3β 在胚胎血管發育中的角色並不清楚。因此，首先進行 GSK3β 的免疫染色，觀察 GSK3β 在早期胚胎發育中的表現位置，結果顯示 GSK3β 分布於體節，以及在體節間隙具有較強的訊號，並且由細胞移殖實驗中可知 GSK3β 調控胚胎血管發育是非細胞自發性的影響。接著，由於PI3K/AKT 為 GSK3β 的上游訊息調控之一。所以，我們利用血管表現綠螢光之基因轉殖斑馬魚 Tg(fli1:EGFP)，浸泡 PI3K 之抑制劑 (LY294002)，結果顯示血管受到抑制：節間血管只從腹部沿著體節向上生長至脊索以及神經管之間的長度，便停滯生長 ; 腸道靜脈血管以及尾部微血管叢則均未形成。進行西方浸漬法實驗得知磷酸化 GSK3β 減少，得知 PI3K 受到抑制時會使得 GSK3β 活化。當我們進行過度表現 gsk3β mRNA 時發現體內的磷酸化 GSK3β/GSK3β 的比值上升，並且磷酸化的 AKT 也有增加的情形。此時，節間血管沿著正確軌道生長至一半之後便會使血管的領導細胞產生過多的異位性表現以及遷移錯亂。此結果顯示了 PI3K/AKT/GSK3β 的訊息傳遞會誘導胚胎血管發育。另一方面，當使用專一性抑制 GSK3β 轉譯之 gsk3β-MO 注射至胚胎中時，並不會誘導胚胎血管發育，於 30 hpf 時，節間血管只生長至脊索與神經管之間的長度便停滯於體節間隙且並沒有異位性的表現，到了48 hpf 時，則是呈現血管缺失的形態。使用 GSK3β 之抑制劑 LiCl 浸泡胚胎則是呈現與注射 gsk3β-MO 之胚胎相同的節間血管受到抑制，而腸道靜脈血管於 48-72 受到抑制而未生成。進行西方浸漬法結果顯示，注射gsk3β-MO 之胚胎的磷酸化與未磷酸化的 GSK3β 均受到抑制並未表現。故推論無論是 GSK3β 或是 pGSK3β 都需要存在於肌肉細胞中，以維持正常的 angiogenesis 發生。此外，細胞移殖實驗顯示催化位置突變之 GSK3β (GSK3β-KM)，可誘發 angiogenesis;但是 constitutively active 之 GSK3β (GSK3β-S9A) 並不會誘發 angiogenesis，故推論 pGSK3β 可正向調控胚胎發育早期的 angiogenesis 。最後，我們進行 TOPflash 實驗以了解在胚胎發育中 Wnt 及PI3K 訊息傳遞的關連。當給予 GSK3β-KM mRNA 時，會活化 Wnt 訊息傳遞; 當給予 GSK3β-S9A mRNA 以及 抑制 PI3K 時，則是會抑制 Wnt 訊息傳遞。抑制 PI3K 的胚胎中也發現磷酸化的 GSK3β 以及磷酸化的 AKT 均有減少的趨勢。這顯示了 PI3K 訊息傳遞會因為調控 GSK3β 的活性而影響了 Wnt 訊息傳遞。總結以上實驗結果，得知: (1) 無論是 GSK3β 或是 pGSK3β 都必須存在於體節中; (2) pGSK3β 在胚胎發育時的 angiogenesis 中是一個正向的調控; (3) PI3K/AKT 訊息傳遞與 Wnt 訊息傳遞為平行調控 angiogenesis。

關鍵字

肝醣生成酵素激酶斑馬魚 ； 血管新生

並列摘要

Glycogen synthase kinase3β (GSK3β) has multiple biological functions. However, the role of GSK3β in angiogenesis during embryonic development is unclear. To address this issue, we firstly analyzed the expression patterns of GSK3β during early embryogenesis. Immunostaining revealed that GSK3β was distributed in somites, especially at the somite boundary. Cell transplantation studies showed that GSK3β modulated angiogenesis in a non cell-autonomous manner. Next, we treated embryos derived from zebrafish transgenic line Tg(fli1:EGFP), whose vessels are GFP-tagged, with a PI3K inhibitor, Ly294002, and found that angiogenesis was severely inhibited. For example, the intersegmental vessel (Se) grew only halfway through their ventral trajectory and stalled at the boundary between the notochord and neural tube, and the subintestinal venous vessel (SIV) and caudal fin capillary remained undeveloped. Western blot analysis revealed that the level of phosphorylated GSK3β was reduced, suggesting that PI3K inhibitor caused the activation of GSK3β. We also demonstrated that over-expression of gsk3β mRNA in embryos increased the phosphorylated form of both GSK3β and AKT, which caused the Se tip cells across halfway through ventral trajectory and sprouted ectopically and randomly. These results suggested that PI3K/AKT/GSK3β signaling induced angiogenesis during embryogenesis. On the other hand, knockdown of GSK3β by gsk3β-specific morpholino (MO) did not induced angiogenesis processes but caused the Se tip cells to remain at the boundary between the notochord and neural tube without ectopically sprouting during 30 hpf and finally display a defective pattern during 48 hpf. Inhibit GSK3β by LiCl in embryos also displayed phenotypes similar to those of gsk3β morphants, such as severe inhibition of Se development during 12-24 hpf and complete loss of SIV formation during 48-72 hpf. Western blot analysis showed that the levels of both unphosphorylated and phosphorylated GSK3β (pGSK3β) were almost lost in the gsk3β-MO-injected embryos. These results indicated that both GSK3β and pGSK3β were necessarily presented in somites for normally angiogenesis. Furthermore, cell transplant studies showed that the catalytically inactive GSK3β (GSK3β-KM) mRNA in somites could induce the initiation of angiogenesis, but the constitutively active GSK3β (GSK3β-S9A) mRNA could not, suggesting that pGSK3β positively regulates angiogenesis. Finally, we analyzed the interactions between Wnt and PI3K signaling during embryogenesis. TOPflash report assay showed that the Wnt signals were increased if embryos received GSK3β-KM mRNA, but Wnt signals were reduced if embryos received either GSK3β-S9A mRNA or PI3K inhibitor. Western blot analysis revealed that both of pGSK3β and phosphorylated AKT were reduced when treated with PI3K inhibitor. These results suggested that PI3K signaling affects Wnt signaling via regulated GSK3β. Taken together, we concluded that during zebrafish embryogenesis in somites 1) both GSK3β and pGSK3β are necessary; 2) pGSK3β functions as a positive modulator on angiogenesis; and 3) the PI3K/AKT signaling pathway is parallel with Wnt signaling pathway to regulate angiogenesis .

並列關鍵字

gsk3 ； zebrafish ； angiogenesis

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