神經元中間絲蛋白α-Internexin在雞動物模式中的分子選殖與表現分析

Molecular Cloning and Characterization of Chicken Neuronal Intermediate Filament Protein α-Internexin

劉紀秀(Chi-Hsiu Liu)

指導教授 ： 錢宗良

國立臺灣大學/醫學院/解剖學暨生物細胞學研究所/博士(2013年)

https://doi.org/10.6342/NTU.2013.10414

摘要

α-internexin是一種神經元中間絲蛋白。根據之前的研究，在哺乳動物發育的過程中，α-internexin表現於發育早期的神經系統，且表現時間早於神經元中間絲蛋白三合體(neurofilament triplet proteins：NFL, NFM and NFH)。有別於在細胞中形成異質聚合物(heteropolymers)的神經元中間絲蛋白三合體，α-internexin具有形成均質聚合物(homopolymers)的能力。這些研究結果顯示α-internexin與神經元中間絲蛋白三合體為不同的細胞骨架蛋白，並在早期神經發育扮演重要的角色。
雞是一種古老且重要的模式動物，也是第一個被全基因定序的非哺乳類動物，牠填補了哺乳類與其他脊椎動物在演化學研究上的空缺。目前已知有部分的雞神經中間絲蛋白已被選殖與定性，但沒有相關研究探討α-internexin在鳥類神經發育過程中的的表現與生理機制。因此，於本研究中我們以雞為動物模式來研究α-internexin基因及其特性與功能。首先，我們自雞的基因體中辨別並選殖雞的α-internexin同源基因，確認基因序列、結構與其在雞胚發育過程中之表現情形。同時，我們利用該基因序列製作雞α-internexin的多株抗體，確認其效力與專一性，並應用於後續的實驗。研究結果顯示，雞α-internexin (chkINA)與哺乳類、兩棲類以及魚類的α-internexin具高度的基因序列相似性。在體外基因轉染實驗(transient transfection)中可觀察到chkINA能夠在轉染細胞裡形成絲狀結構，並且能與細胞內生性的中間絲聚合在同一細胞骨架網絡中。利用聚合酶鏈鎖反應及西方點墨轉漬法，我們發現chkINA主要表現在雞的中樞神經系統中；在發育中的雞胚裡，chkINA在腦部的表現量隨生長過程遞增。免疫組織化學染色的結果顯示，chkINA廣泛地分布在雞腦中，並與神經中間絲蛋白三合體形成絲狀結構。我們亦發現chkINA在發育中的小腦表現量相當豐富，且為小腦顆粒神經元(granule neurons)所衍生之平行纖維(parallel fibers)中主要的細胞中間絲蛋白。此外，chkINA也被觀察到會短暫地表現在發育中的視網膜各類神經細胞內，然而其分布情形隨發育過程的推衍而漸趨受限於視網膜之神經節細胞(ganglion cells)、無軸突細胞(amacrine cells)以及水平細胞(horizontal cells)中。
綜合本研究所獲得的結果，我們認為chkINA為神經元特定表現之中間絲蛋白，可作為發育中之神經細胞的標記物，應用於以雞為模式生物之神經科學研究，並將可提供演化發育生物學以及生物資訊學研究更多的線索。

關鍵字

α-internexin ； 神經元中間絲蛋白 ； 雞胚 ； 發育

並列摘要

α-Internexin is one of the neuronal intermediate filament (IF) proteins, which also include peripherin and neurofilament (NF) triplet proteins designated as NFL, NFM and NFH for low, middle and high molecular weight subunits, respectively. It is expressed by most neurons as the differentiation begins and precedes the expression of the NF triplet proteins in mammals. Unlike the NF triplets which are obligate heteropolymers, α-internexin has the ability to form homopolymers. These studies have supported the belief that α-internexin and NF triplet form separate filament systems. The chicken is an important model organism that bridges the evolutionary gap between mammals and other vertebrates. It also represents the first non-mammalian amnitoe to have its genome sequenced. The chicken embryo is a useful model system in developmental biology due to experimental advantages of in ovo embryogenesis. Some neuronal IF genes of chicken were cloned in previous studies. Still, little is known about the gene sequence and physiological function of the neuronal IF α-internexin in avian.
In this study, we aim to identify the mRNA sequence encoding chicken α-internexin and to characterize the gene structures and expression levels during developmental process. Human, mouse, rat, frog and zebrafish α-internexin sequences were used to search for orthologues. The putative chicken α-internexin (chkINA) cDNA was generated via RT-PCR of total chicken brain RNA. The gene structures and predicted amino acid sequence of chkINA revealed a high similarity with the homologues from human, mouse, rat, bovine, frog, and zebrafish. Transient transfection assay showed that the filamentous pattern of chkINA could be found in transfected cells, and colocalized with other endogenous IFs by demonstration of immunocytochemistry with chicken-specific antibody. The expression of chkINA was detected in early stage of development and during developmental process. Moreover, chkINA was widely expressed in chicken brains and retinae, and was colocalized in fibrous patterns with NF triplet proteins. chkINA was also expressed abundantly in the developing cerebellum and was the major IF protein in the parallel fibers of granule neurons. In addition, chkINA could be transiently detected in all neuronal lineages in the developing chicken retina; however, as development progressed, chkINA expression became restricted to the nerve fibers of ganglion, amacrine and horizontal cells. Thus, we conjecture that chkINA might be a neuron-specific IF protein and could be a useful marker to be applied to neurobiological studies in chicken. The annotation of chkINA may be applied for neuroscience research. In addition, this may fill the gaps of the molecular evolution of neural intermediate filaments, and provide hints in genomics, evolutionary developmental biology or bioinformatics.

並列關鍵字

α-internexin ； neurofilament ； chicken embryo ； development

參考文獻

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