虎皮蛙視網膜及松果體中的感光細胞

Abstract

本研究我們選擇虎皮蛙 (學名: Hoplobatrachus rugulosus) 作為實驗動物；依據實驗室先前的研究，我們發現雞的 α-internexin 及斑馬魚的 inaa 不僅會出現在視網膜的感光細胞中，也可以在松果體中的類感光細胞中有表現分布。然而，α-internexin 在兩生類松果體中的表現尚未被探討，作為在演化史上可以彌補斑馬魚及脊椎動物中空缺的角色，虎皮蛙可為一個重要的兩生類神經發育模式動物。 關於一種常用於兩生類發育研究的實驗動物:爪蟾Xenopus laevis的研究指出，Xenopus α-internexin mRNA序列已被命名為“xefiltin”。雖然虎皮蛙和Xenopus 並不是同一物種，但是屬於同一目。因此，本論文研究目的是在探討 α-internexin 的同源基因xefiltin: ina.S 及 nif.S 在虎皮蛙發育中，於視網膜及松果體內之表現與分布狀況。因此，我們特別製作多株抗體可以標定虎皮蛙的 α-internexin 的同源基因 ina.S 及 nif.S 蛋白，並使用西方墨點法與免疫組織染色確認 ina.S 及 nif.S 在發育中視網膜及松果體之表現與分布情況。為了確認 ina.S 及 nif.S 可以在松果體中的類感光細胞中之可能表現分布，另外選擇了兩隻可用於標定感光細胞的抗體: recoverin 及 XAP1 於實驗中來比較其分布情形。 根據免疫染色結果， nif.S 可以於感光細胞的外段 (outer segment) 被觀察到，但是另一隻抗體: ina.S 只有在視網膜神經元 (retinal neurons) 中可檢測到。相對的，並未在感光細胞中檢測到。在松果體中，recoverin 及 XAP1 皆可以標示出感光細胞;另一方面，ina.S 及 nif.S 只能在早期虎皮蛙的松果體少許細胞中被偵測到。綜合本研究結果，我們針對感光細胞在發育中虎皮蛙視網膜及松果體的表達和分布做詳細的觀察及比較。我們認為XAP1，而非ina.S 或 nif.S，可以應用成為研究青蛙松果體中的感光細胞的特定標示物，有助於提供在青蛙的松果體中感光細胞演化發育生物學研究更多線索。

In this study, Hoplobatrachus rugulosus, commonly referred to as tiger-skinned frog, is to be chosen the animal model. In previous studies, it has been found that both chicken α-internexin and zebrafish inaa (an ortholog of mammalian α-internexin) were developmentally distributed not only in photoreceptors of retina but also in the photoreceptor-like cells of the pineal gland. However, from an evolutionary point of view, there is still lack of evidence to illustrate the temporal and spatial distribution of frog α-internexin protein in the developing pineal gland. According to the previous studies about Xenopus laevis, the animal model that is usually used for amphibian developmental research, the mRNA sequence which encodes Xenopus α-internexin had been identified as “xefiltin”. Although H. rugulosus is not the same species with X. laevis, both of them are classified in the same order. The aims of this study is to characterize the expression pattern of α-internexin-like proteins, internexin neuronal intermediate filament protein alpha S homeolog (ina.S) and low molecular weight neuronal intermediate filament S homeolog (nif.S) which might be expressed in the developing retina and the pineal gland of the tiger-skinned frog. Thus, for this study these 2 antibodies were generated to detect the frog α-internexin-like proteins and to confirm the distribution of these proteins in the developing frog retina as well as the pineal gland by western blot and immunohistochemistry. Furthermore, the markers applied to detect the photoreceptor cell in the retina and the pineal gland are recoverin and XAP1 (Clone 3D2), both of them are reported to be specific to photoreceptors. Based on the distribution patterns in frog retina, immunopositive staining of anti-nif.S could be observed in the outer segment of photoreceptors, but anti-ina.S could only be found in retinal neurons. In the pineal gland, both photoreceptor markers, recoverin and XAP1, could be detected. The α-internexin-like protein, ina.S and nif.S, could be detected only in a few photoreceptor-like cells at the early stage of the frog pineal gland. In summary, this thesis has studied the distribution patterns of photoreceptor cells in retina and in pineal gland of developing tiger-skinned frogs. It can be concluded that XAP1 could be a good marker to identify the photoreceptor cells in the developing pineal gland of frogs.