中国科学院海洋研究所机构知识库

  贝壳是软体动物的重要保护器官，研究软体动物贝壳发生机制对于了解软体动物的发育和演化具有重要意义。贝壳发生起始于胚胎发育早期，由背部外胚层细胞形成贝壳形成区，并继而分泌形成贝壳。贝壳发育过程中，贝壳形成区的形态发生是至关重要的一步，具有高度保守性及一定程度的种间多样性。本研究选取了分属软体动物两个亚门的腹足纲动物笠贝(Lottia goshimai)和多板纲动物红条毛肤石鳖(Acanthochitona rubrolineata)为研究对象，研究了其贝壳形成区的发育机制。

  腹足纲动物的贝壳发生机制有一定的研究基础，但缺乏高精度的观察及细胞和分子两个层面的关联性研究。基于上述现状，本研究利用激光共聚焦显微镜等高分辨率技术对笠贝贝壳形成区的形态发生进行了研究。通过扫描电镜观察，发现笠贝贝壳形成区由中央区域及外围区域两部分组成，在发育过程中有两个特点，一是中央区域产生伪足，此区域在随后的发育过程中内陷并扩张；二是外围区域逐渐呈现一种规律化的“玫瑰花样”的同心圆结构。在此基础上，对4个贝壳形成相关基因（engrailed、bmp2/4、gata2/3、hox1）的表达模式进行了分析，发现这些基因表达于贝壳形成区的不同位置（8 hpf），表明贝壳形成区由不同的细胞群体组成。同时，发现这4个基因的表达随发育进程（6-8 hpf）呈现从腹面和侧面向背面聚集的趋势，表明在贝壳形成区发育过程中可能存在细胞迁移现象。通过标记F-actin（纤维状态的肌动蛋白，即微丝），发现贝壳形成区的细胞形态在发育过程中经历了显著的变化。此外，在8 hpf时，贝壳形成区出现两处较强的F-actin聚集信号，其一与中央内陷区域分布的大量伪足具有显著相关性，另一处则可能指示了贝壳形成区与其它外胚层的分界线。通过BrdU（5-溴脱氧尿嘧啶）掺入实验，发现在贝壳形成区扩张过程中没有明显的细胞增殖现象。综上所述，这些结果表明细胞运动及细胞形态变化在贝壳形成区形态发生过程中占主导作用，而细胞增殖在此过程中作用不大。此外，本研究发现笠贝贝壳发生过程中中央区域细胞与中/内胚层细胞始终紧密接触，这一特点不支持现有的“内胚层诱导”假说，提示贝壳发生过程在不同腹足纲动物中可能存在重要差别。

  多板纲动物的贝壳形成区沿身体前后轴重复性地排列于背部，具有“假体节”的形态特点和发育过程。然而，目前尚没有针对这种发育过程的详细研究。首先通过扫描电子显微镜观察，发现石鳖担轮幼虫头部有一部分未覆盖纤毛的区域，此区域为第一个贝壳形成区。背部担轮环下方具有六个重复排列的贝壳形成区，其贝壳形成区间的界限在发育过程中随躯干部的延长而变的更加清晰。在此基础上，考虑到“假体节”和体节的相似性，本文研究了体节形成同源基因在多板纲动物中的表达模式。共研究了5种体节形成相关基因（engrailed、notch、delta、hedgehog、wnt1）在重要时间节点（24，48 hpf）的表达模式，发现这些基因全都以“条纹状”表达于贝壳形成区。进一步的，研究了这些基因在笠贝中的表达模式，以探讨贝壳发育机制在这两种亲缘关系较远的贝类间的差别。结果发现，除notch及一个delta同源基因（delta2）外，笠贝中这些基因（delta1、hedgehog、wnt1）均未表现出与贝壳发育的明显相关性，表明笠贝与石鳖的贝壳发生机制有显著差别。

  本研究揭示了腹足纲及多板纲软体动物贝壳发生过程的独特特点，初步鉴定了参与两种动物贝壳发生的多个基因，展示了贝壳发生区细胞的分化及运动过程，从细胞和分子水平为理解软体动物的贝壳发生机制提供了基础支撑。

  Shell is an essential structure of mollusks. The molluscan shell protects the internal organs and emerges since the Cambrian period. The knowledge of the shell formation is important to understand the development and evolution of mollusks. The larval shell formation begins at the early stage of embryonic development, when some dorsal cells differentiate into the shell field and then secrets the larval shell eventually. Morphogenesis of the shell field is an essential node of this process, showing both high conservation and interlineage variations. Our knowledge of the morphogenesis of molluscan shell filed remain largely elusive. In this study, we investigated the morphogenesis of the shell field in the gastropod Lottia goshimai and the polyplacophoran Acanthochitona rubrolineata.

  Although the morphological changes of larval shell formation are extensively investigated in gastropod, the resolution is limited. Using confocal microscopy and other techniques, we revealed details regarding the morphogenesis of the shell field in Lo. goshimai . The shell field of Lo. goshimai consisted of central and peripheral parts with distinct morphological characters. There were two major characteristics during its development, as revealed by scanning electron microscopy. Firstly, there were evident protrusions on the surface of central area, which we determined to be lamellipodia based on morphological characters. This area invaginated and expanded in subsequent development. Secondly, the arrangement of the peripheral cells became regular and exhibited a characteristic rosette pattern. We then analyzed the expression of several potential shell formation (pSF) genes during shell field morphogenesis, including engrailed, bmp2/4, gata2/3 and hox1. At 8 hpf (hour post fertilization), the four pSF genes were expressed in different regions of the shell field, indicating there were different cell groups. Besides, the expression of these genes changed continuously from 6 to 8 hpf, indicating likely active cell migrations during the period. We also investigated the dynamics of fiber actin (F-actin) from 6 to 8 hpf using phalloidin staining. The results revealed evident changes in the cell shape and showed two aggregations of F-actin at 8 hpf, which may correlate with the distribution of the lamellipodia in central area and the boundary between the shell field and other ectoderm tissues, respectively. Finally, we detected no obvious cell proliferation in the shell field from 6 to 8 hpf using BrdU assay. Together, these results suggest the dominant role of cell movement and cell shape changes in the shell filed morphogenesis, while cell proliferation plays little role in this process. Besides, constant contacts between ectodermal and meso/endodermal tissues did not support the induction of shell field morphogenesis by endodermal tissues in general, suggesting that the morphogenesis of shell field may be significantly different in mollusks.

  The shell fields of polyplacophoran A. rubrolineata are arranged repeatedly on the dorsal side of embryos, showing the characteristics of “pseudosegments” . However, little is known about the mechanism underlying its development. Scanning electron microscopy revealed seven shell fields on the dorsal side, with one of them in the pretrochal region and the other six repeatedly arranged in the posttrochal region. Based on the morphological similarity between polyplacophoran pseudosegments and the segments of other animals (such as insects and vertebrates), we investigated the expression of segment-related genes in A. rubrolineata development, including engrailed, notch, delta, hedgehog and wnt1. All of the genes showed a striped pattern in the shell fields (pseudosegments), indicating that they were involved in the shell (pseudosegment) formation of chiton. However, we found that these genes were not likely involved in the shell formation of Lo. goshimai, indicating that the shell formation mechanism in Lo. goshimai s and A. rubrolineata differs greatly.

  This study revealed the unique characteristics of the shell field in Lo. goshimai and A. rubrolineata. These results would provide fundamental supports for understanding the shell formation mechanism of mollusks.

目  录

第1章  引言       I

1.1  软体动物及贝壳发生简介   1

1.1.1  软体动物的分类       1

1.1.2  软体动物幼虫贝壳发生过程简介    2

1.1.3  软体动物成体贝壳形成过程简介    6

1.2  腹足纲动物贝壳形成的研究      7

1.2.1  贝壳形成区早期发育过程       7

1.2.2  贝壳发生相关基因    12

1.3  多板纲动物贝壳形成的研究      15

1.3.1  多板纲动物背部的细胞群组成及功能    16

1.3.2  多板纲动物壳板的形成过程    18

1.3.3  体节发育机制简介    20

1.4  本研究的目的和意义   29

第2章  材料与方法    31

2.1  实验材料采集及固定   31

2.2  RNA提取、cDNA合成       31

2.3  整装原位杂交      35

2.4  组织化学实验      35

2.5  扫描电镜观察      35

第3章  结果       37

3.1 笠贝贝壳形成区的早期发育研究 37

3.1.1  笠贝贝壳形成区发育过程中形态变化    37

3.1.2  贝壳形成相关基因在笠贝贝壳发育区形成过程中的表达分析    39

3.1.3  笠贝贝壳形成区发育过程中微丝的动态变化       46

3.1.4  贝壳形成区形态发生过程细胞增殖情况       48

3.2  多板纲动物贝壳形成区的早期发育研究   50

3.2.1  石鳖贝壳形成区发育过程       50

3.2.2  体节形成相关基因在石鳖贝壳形成区表达模式研究    52

3.3  体节形成相关基因在腹足纲动物中表达模式研究   58

第4 章  讨论      65

4.1  笠贝贝壳形成区形态发生的基本特点      65

4.2  对笠贝贝壳形成区形态发生机制的启示   66

4.3  多板纲动物贝壳形成区形态发生      68

4.4  多板纲动物贝壳形成区形态发生机制启示      69

第5 章  结论与展望   70

参考文献       73

致  谢    83

作者简历及攻读学位期间发表的学术论文与研究成果    85