arl6ip1基因在斑馬魚胚胎的時空表現及其在視網膜發育過程之功能

Spatiotemporal Expressions during Embryogenesis and Functional Roles during Retinogenesis of Zebrafish arl6ip1 Gene

黃倖妍(Hsing-Yen Huang)

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/博士(2010年)

https://doi.org/10.6342/NTU.2010.10529

摘要

人類遺傳疾病巴德-畢德氏症候群第三型(Bardet-Biedl syndrome (BBS), type 3)被證實與ADP-ribosylation factor-like 6 (Arl6)突變有關，為求更加了解Arl6的功能，一些與Arl6交互作用的蛋白例如ADP-ribosylation factor-like 6 interacting protein 1 (Arl6ip1)、Sec61β、JWA等因此被發現。本文主要利用斑馬魚作為模式動物探討Arl6ip1在胚胎發育過程中所扮演的角色及功能。研究證實arl6ip1 mRNA於一細胞時期就能表現在blastodisc中，其均勻廣泛的表現可以持續至受精後24小時，之後漸漸地只會侷限在某些器官例如腦、眼、腎、心臟和鰭芽；顯微切片更詳細瞭解arl6ip1 mRNA在一些組織例如視網膜、hypochord、spinal cord、myotome也會表現，這些證據顯示Arl6ip1在胚胎發育過程中為一母體效應基因，並且有著動態性的表現分布；而當我們抑制Arl6ip1蛋白則發現上述這些器官或組織出現嚴重缺失例如扁平頭、小眼症、圍心腔腫大、色素異常表現、軀幹彎曲、tectum缺失、腎管發育不全、心管線性化、視網膜細胞不分化、不表現rod opsin和red cone opsin、失去視覺震顫能力且發育至第5天會死亡，這些喪失Arl6ip1的胚胎(arl6ip1-morphants)似乎與BBS病人具有部分相似病徵。為更進一步了解Arl6ip1的功能，我們先著重在上述最明顯易見的小眼症缺陷來探討Arl6ip1在retinogenesis之分子機制。首先我們觀察眼睛特化因子rx1和pax6的表現，證實兩因子並無受到太大影響；但在視網膜分化因子的表現則是嚴重缺失的，同時細胞移植實驗證明Arl6ip1以一non-cell autonomous的行為影響視網膜細胞的分化。為瞭解arl6ip1-morphants視網膜細胞分化是如何受到影響，我們先以視網膜切片計數arl6ip1-morphants的細胞數，結果顯示arl6ip1-morphants視網膜細胞數只有野生型的25%，利用TUNEL或FACS則證實視網膜細胞數少的原因是受到異常細胞週期而非過多細胞凋亡所致。Bromodeoxyuridine (BrdU)和phospho-histone H3 (PH3)更證實arl6ip1-morphants視網膜細胞有細胞週期延遲、大量活躍於S-phase和異常停留在M-phase的現象，由於這些異常的特徵與斑馬魚shh突變型之視網膜缺陷非常相似，因此我們想要利用Tg(shh:GFP)之轉殖品系追蹤胚胎缺失Arl6ip1時是否會影響Shh表現，結果顯示Tg(shh:GFP)缺失Arl6ip1後，在視網膜中觀察不到GFP表現；且Shh下游調控分子p57kip2也不表現，而p57kip2相對立因子cyclinD1則持續的表現於視網膜中，但若加以誘導表現p57kip2則能有效降低異常BrdU表現量，達到拯救因缺乏p57kip2所引起不正常細胞週期的缺陷，這些結果顯示Arl6ip1可能對Shh signaling pathway產生影響進而干擾視網膜細胞週期進行。另一方面，我們也觀察到arl6ip1-morphants視網膜細胞出現膨脹的內質網和高基氏體、形狀變異之粒線體、較大的細胞核以及chop mRNA高度表現，同時也觀察到細胞凋亡的現象出現在發育晚期的胚胎，這些證據顯示胚胎缺失Arl6ip1後可能會引起與內質網壓力有關的細胞凋亡。我們更進一步發現Arl6ip1與Ras-related nuclear protein (Ran)具有直接的交互作用，兩者共同座落於中心體，且在胚胎發育過程中的表現或缺失型態是相似的，尤其在ran-morphants視網膜也出現細胞不分化的現象，因此，我們推測Arl6ip1也可能藉由與Ran之交互作用共同去調節視網膜細胞週期的進行。總結，在胚胎發育過程中arl6ip1基因對斑馬魚胚胎的存活是必需的，且Arl6ip1更是扮演著多功能的角色影響視網膜的細胞增生、分化和存活。

關鍵字

斑馬魚 ； 視網膜 ； 細胞分化 ； 細胞增生 ； 細胞凋亡 ； 細胞週期

並列摘要

ADP-ribosylation factor-like 6 (Arl6) mutation is linked to human disease and that Arl6 interacts with Arl6 interacting protein 1 (Arl6ip1). However, the expression pattern and function of Arl6ip1 during embryogenesis are unknown. To confirm whether abnormal Arl6ip1 function might result in embryonic defects in zebrafish, we examined the expression patterns of arl6ip1 during embryogenesis, and we found that they were maternally expressed and exhibited in the brain, optic primordia, hypochord, spinal cord, myotome, heart, fin-bud, kidney, trunk and retina. Knockdown of Arl6ip1 revealed the following phenotypic defects: microphthalmia, disorganized pigment pattern, flat head, defective tectum, deficient pectoral fins, abnormal pneumatic duct, pericardial edema and deformed trunk. Particularly, histological dissection of the retinae of arl6ip1-morphants revealed defective retinal neurons and disorganized retinal layers. We further confirmed that opsins of arl6ip1-morphants were not transcribed. Based on these evidences, we propose that Arl6ip1 may play important roles in zebrafish ocular, heart and fin-bud development. Next, we focused on the retinal development to discuss the functional role(s) of Arl6ip1 that because small eyes were easily observed in all phenotypic defects. We demonstrated that Arl6ip1 is required for retinal differentiation in a non-cell autonomous manner. Arl6ip1-knockdown inhibits shh expression to block retinal cell differentiation and that loss of Arl6ip1 in embryos leads to defects in retinal cell cycle progression. Unlike the expression patterns of cyclinD1 and p57kip2 in wild-type embryos, arl6ip1-morphants keep cyclinD1 expression throughout the retina, and an undetectable p57kip2, suggesting that the retinal cells of arl6ip1-morphants proliferate abnormally, do not differentiate, and maintain in M-phase. Arl6ip1 maintains the integrity of organelles and the survival of retinal cells in that loss of Arl6ip1 function leads to ER-stress-mediated apoptosis at late developmental stage. We identified that Arl6ip1 binds to Ras-related nuclear protein (Ran) through cell cycle at centrosomes. The phenotypes of ran-morphants were similar to those of arl6ip1-morphants, suggesting that Arl6ip1 interacts with Ran to modulate cell cycle progression during retinogenesis.

並列關鍵字

Arl6ip1 ； zebrafish ； retina ； cell proliferation ； cell differentiation ； apoptosis ； cell cycle

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