| 摘要: |
| 多板纲软体动物(石鳖)生有8片重复排列的贝壳,与其他类群如腹足类、双壳类等有显著的区别,这些差异蕴含着重要的发育和演化意义。前期研究发现,红条毛肤石鳖(Acanthochitona rubrolineata)幼虫贝壳发育组织的细胞排列欠规则,在普通显微镜下不易识别单个细胞结构。本研究首先利用整装原位杂交手段鉴定出一些参与贝壳发育的细胞群体,发现表达engrailed的细胞分布于贝壳发育区中央区域的突起部位(脊)和边缘区域,其中脊区域的表达呈条纹状,边缘区域的表达则与贝壳发育区的外周吻合,但是对这些细胞的形态和排列模式等细节无法开展深入观察。接下来对石鳖幼虫进行了半薄切片,发现可以识别出壳板发育区细胞及脊细胞的大致轮廓和分布模式,但分辨率仍然受限。为了进一步提升分辨能力,结合了整装原位杂交实验及半薄切片技术,对表达engrailed基因的贝壳发育相关细胞开展了观察研究。结果显示,对整装原位杂交后的幼虫进行半薄切片,组织细胞结构保持完整,细胞轮廓清晰,能够清楚区分engrailed阳性细胞。观察表明,这些细胞具有较高的核质比,总体呈V形排列,且贝壳发育区中央部位与边缘部位的engrailed阳性细胞在形态和排列上存在区别。这些结果证明,将原位杂交与半薄切片结合,可提升对细胞结构观察的分辨率,实现同时从分子和细胞学层面研究石鳖贝壳发育相关细胞。本研究对进一步从细胞水平研究石鳖的贝壳发育过程、解析相关贝壳发育相关基因的功能有重要的支撑作用。研究结果对揭示更多贝壳起源的信息,理解软体动物贝壳的起源和演化有重要意义。 |
| 关键词: 半薄切片 原位杂交 红条毛肤石鳖 贝壳发育 |
| DOI：10.11759/hykx20230227001 |
| 分类号:Q344 |
| 基金项目:国家自然科学基金项目(42076123) |
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| Investigation of chiton cells related to shell development through in situ hybridization combined with semithin section-ing |
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XIA Yuxiu1,2, HUAN Pin1, LIU Baozhong1
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1.Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China;2.University of Chinese Academy of Sciences, Beijing 100049, China
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| Abstract: |
| Polyplacophoran mollusks (chiton) have eight serially arranged shell plates, which have important developmental and evolutionary implications and are considerably different from those in other molluscan lineages, such as gastropods and bivalves. Previous studies have revealed that shell-formation cells of Acanthochitona rubrolineata are irregularly arranged, making it difficult to discern single cells under ordinary microscopy. In this study, we initially investigated shell-formation tissues through in situ hybridization. engrailed-positive cells were observed in the ridges of the central and marginal regions of the shell field. Expressions in the ridges exhibited a striped pattern, and the expression in the marginal region restricted the periphery of the shell field. However, the morphology and arrangement patterns of these cells could not be clearly discerned. Moreover, larval cells could be better recognized in semithin sections, which was still limited. Therefore, we combined semithin sectioning and in situ hybridization to further improve cellular resolution. Semithin sectioning of the larvae after in situ hybridization revealed intact tissue morphology with discernable cell outlines, and the engrailed-positive cells could be clearly distinguished. These cells had high nucleus-to-cytoplasm ratios and exhibited an overall V-shaped arrangement pattern. Additionally, the engrailed-positive cells in the central and marginal regions of the shell field exhibited different arrangement patterns and cell shapes. These results demonstrated that combining in situ hybridization and semithin sectioning could improve the resolution at the cellular level. Thus, the proposed strategy will help investigate the cellular mechanisms underlying chiton shell formation and the roles of shell-formation genes. Moreover, the proposed strategy can help further elucidate the origin and evolution of molluscan shells. |
| Key words: semithin sectioning in situ hybridization Acanthochitona rubrolineata shell development |
