| 摘要: | DnaJ 家族蛋白 (Heat shock protein 40 family) 是協助蛋白折疊的重要分子,近年來在許多研究中發現其家族蛋白在腫瘤的抑制與細胞凋亡過程中也扮演相當重要的角色。人類 hTid1 (Tumorous imaginal discs 1) 又稱為 DnaJA3,是果蠅抑癌基因 (tumor suppressor gene) Tid56 的同源基因。hTid1 會在全身組織中表達,透過選擇性剪接可轉譯出 hTid1-L 和 hTid1-S 兩種蛋白異構物,此兩種蛋白在細胞內訊息傳遞路徑的功能主要為調節子 (regulator),hTid1-L 具有促進細胞走向細胞凋亡的功能,而 hTid1-S 則抑制細胞凋亡。由於兩種 hTid1 蛋白產物的 C 端胺基酸序列的差異,hTid1-S 較 hTid1-L 更具有停留於粒線體中之特質。在斑馬魚中亦具有兩個 hTid1 旁系同源基因,命名為 DnaJA3a 和 DnaJA3b。在實驗室先前的研究發現 DnaJA3b 主要在受精後 72 和 96 小時 (hpf, hours post fertilization) 及成熟大腦與肌肉組織中有較高的表現。為瞭解 DnaJA3b 在斑馬魚胚胎發育時期可能的功能,本研究先確認 DnaJA3b mRNA 在 24 到 120 hpf 皆有表達,且主要表達在體節肌肉區域。當利用 morpholino oligonucleotides (MO) 抑制 DnaJA3b 基因功能時,結果顯示斑馬魚胚胎體節發育異常,且透過原位雜交 (whole mount in situ hybridization) 發現 myod、her1、fgf1a 表現受到抑制。過去已知 FGF 訊息傳遞路徑在胚胎體節發育過程中扮演重要角色,因此利用 FGF 路徑抑制劑 SU5402 處理胚胎後發現其體節發育的表型與 DnaJA3b MO 處理胚胎非常相似。利用 RT-qPCR 分析 DnaJA3b MO 處理後以及 SU5402 藥物處理後胚胎中,參與體節發育以及 FGF 路徑相關分子表達之變化,結果顯示參與體節發育以及 FGF 路徑的相關分子 mRNA 量皆減少。本研究結果顯示 DnaJA3b 可能透過影響 FGF 訊息傳遞路徑,而在斑馬魚胚胎體節發育過程中扮演重要角色。
DnaJ family (Heat shock protein 40 family) is an important cochaperone of DnaK (Hsp70), involved in protein folding, unfolding, translocation, assembly, disassembly. Human gene Tumorous imaginal discs 1 (hTid1), also named DnaJA3, is a homologue of the Drosophila tumor suppressor gene Tid56. Human Tid1 (hTid1) expresses in all tissues in the human body and encodes two alternative splicing variants, hTid1-L and hTid1-S. Previous studies revealed that deletion of Tid1 in mice leads to early embryonic lethality. This suggests that Tid1 may be associated with the embryonic development process. Zebrafish (Danio rerio) also possess two DnaJA3 paralogues, named DnaJA3a and DnaJA3b that are similar to hTid1-S and hTid1-L, respectively.
Our previous study showed the expression of DnaJA3b mRNA was mainly at 72 and 96 hpf in the developmental stages and adult brain and muscle. Therefore, to identify the possible functions of zebrafish DnaJA3b, we found DnaJA3b mRNA expression gradually increased throughout 24 to 120 hpf. In vivo loss of function of DnaJA3b caused by the injection of morpholino oligonucleotides showed knockdown of DnaJA3b led to somite abnormalities. Meanwhile, DnaJA3b morphants exhibited inhibited expression of myod, her1 and fgf1a, as validated by whole mount in situ hybridization (WISH). FGF signaling pathways are known to involve in the embryonic somite development. We found that treatment with an FGFR inhibitor, SU5402, led to similar phenotypes as those shown in the DnaJA3b morphants. Thus, we sought to investigate the relationship between DnaJA3b and FGF signaling during the embryonic somite development. Data showed that zebrafish embryos treated with FGFR inhibitor SU5402 at 6 hpf for 1 hr also demonstrated somite defects and shorter body length. Changes in gene expression levels of FGF signaling pathway-related genes, such as fgf1a, fgf17, fgf8a, fgfr1, in both the DnaJA3b morphants and SU5402-treated embryos were observed by using RT-qPCR experiments. Therefore, our results suggest that DnaJA3b play an important role during the early zebrafish embryo somite development processes likely by affecting the FGF signaling pathway. |
