实验海洋生物学重点实验室知识库

软体动物身体模式多样，演化地位独特。多板纲（石鳖）作为软体动物中较为独特的一支，同时具有贝壳和棘刺两种钙化结构，是其他软体动物不具备的特征。此外石鳖具有8片贝壳，这一特征与其他有壳软体动物（如腹足纲和双壳纲）也存在很大差异，解析多板纲发育过程对理解软体动物的起源和演化具有重要意义。除贝壳发育外，多板纲发育还包含多个重要的发育事件。第一，软体动物早期发育受组织者细胞的调控，但目前对多板纲组织者的研究较少，对其认知非常有限。第二，变态作为一个重要的发育事件，标志着幼虫从浮游到底栖生活的转变，期间贝壳相关组织也经历了重大的变化。因此，深入研究石鳖的早期卵裂、贝壳发育以及变态过程具有重要的意义。

本文以红条毛肤石鳖（Acanthochitona rubrolineata）为研究对象，采用多种实验方法研究其早期发育过程中的卵裂模式、贝壳发育区的形态发生以及变态过程，重点在细胞和分子层面上关注了贝壳发育区的发生过程。具体结果如下：

1.卵裂模式：采用鬼笔环肽染色的方法追踪了石鳖胚胎的卵裂过程。结果显示该物种呈典型的螺旋卵裂，具有左旋和右旋交替细胞分裂的模式。在32细胞阶段，观察到植物极的一个大分裂球的表面露出部分与其他三个大分裂球相比明显较小，推测该细胞为组织者（organizer，即3D细胞）。

2.贝壳发育区的形态发生：采用扫描电子显微镜观察、鬼笔环肽染色以及整装原位杂交多种实验手段，探究了红条毛肤石鳖贝壳发育区的形态发生，从不同层面揭示了该过程的发育细节，结果如下：

（1）形态学特征：在12-16 hpf期间，幼虫背部的形态特征不明显。从18 hpf开始，担轮环前部区域分化成纤毛区域和无纤毛区域。在24 hpf，中央区域逐渐发育出交替排列的突起与凹陷结构，标志着脊（ridge）和壳板发育区（plate field）的形成。位于中央区域的壳板发育区、脊以及外周组织共同构成了贝壳发育区（shell field）。

（2）F-actin动态变化：在担轮环前后区域均发现F-actin的聚集。在担轮环前部区域，F-actin的聚集勾勒出环带的前体区域，推测其标记了棘刺形成细胞，本文将该区域命名为环带发育区（girdle field，以下简称GF区）。随着发育的进行，F-actin信号由最初的“点”状形式转变为深入组织内部的“管”状结构。在壳板发育区也观察到存在重复排列的F-actin聚集，并且随着发育的进行，信号逐渐增强。

（3）engrailed基因的动态表达：在幼虫早期，engrailed基因就开始表达，随后经历了复杂的变化过程。在此期间，表达区域持续增加，最终在中央区域形成8条条带的表达模式，大部分条带与突起位置相吻合。该区域engrailed的表达存在单条增加以及由一片区域转变为多条条带两种发育模式。同时，在GF区也发现engrailed的表达，该区域的表达构成GF区的内侧边缘。

（4）变态过程的贝壳发育：随着发育的进行，壳板发育区不断扩张，同时脊不断被挤压，直至7片贝壳完全形成。

3.变态：观察了红条毛肤石鳖幼虫附着变态过程，探索了诱导因子，结果如下：

（1）石鳖幼虫的附着和变态属于两个独立发育事件，需要不同的诱导信号。

（2）添加同种或不同种物种的成体组织和未受精卵细胞均可诱导石鳖幼虫附着，表明诱导线索与特定的物种无关，但可能来自组织或细胞腐败释放出的小分子。本文将该未知小分子命名为X因子。同时还发现X因子仅能诱导附着，而钾离子可以诱导附着和变态两个阶段。

综上所述，本研究对石鳖幼虫的早期发育过程进行了全面观察，包括识别了潜在的组织者（3D细胞），在分子和细胞层面揭示了贝壳发育区形态发生过程，探索出两种诱导变态的因子，初步实现了石鳖幼体的培育，这些结果可为理解软体动物的发育和演化机制提供新认知。此外，石鳖与经济软体动物同属一个类群，其发育机制的许多方面可能存在共通之处，因此本研究结果也可为经济软体动物的育种育苗工作提供理论依据和技术支持。

Mollusks show diverse body plans and have unique phylogenetic positions. Polyplacophorans (chitons) represent a distinctive molluscan lineage by showing both shell plates and spicules, which are not observed in other lineages. In addition, their feature of possessing eight shell plates differs significantly from other shelled mollusks such as gastropods and bivalves. As such, the knowledge of polyplacophoran development is essential to understand the origin and evolution of mollusks. In addition to shell development, several key aspects of polyplacophoran development should be emphasized. First, the early development of mollusks is modulated by the organizer. However, polyplacophorans organizers are rarely investigated and the knowledge of organizer is greatly limited. Second, metamorphosis is a key developmental event marking the transition from pelagic to benthic lifestyle, during which the shell-related tissues experience significant changes. Thus, it is of great importance to investigate early cleavage, shell development and metamorphosis of polyplacophorans.

Here, we investigated the early development of the polyplacophoran Acanthochitona rubrolineata using multiple approaches. Essential developmental events including cleavage pattern, shell field morphogenesis, and metamorphosis were explored. In particular, we focused on the development of shell field at the molecular and cellular levels. The results are as follows.

1. Cleavage pattern. Early cleavage was traced through phalloidin staining. The results revealed typical spiral cleavage in this species, characteristic of alternative sinistral and dextral cell division. At the 32-cell stage, we observed a vegetal macromere showing evidently smaller surface compared to the other three, indicating it may be the organizer (the 3D blastomere).

2. Shell field morphogenesis. SEM, phalloidin staining and whole mount in situ hybridization were used to investigate the shell field morphogenesis of A. rubrolineata, which revealed different aspects of the process, as follows.

(1) Morphological characteristics. There was no evident morphological characteristics in dorsal larval tissues between 12 and16 hour post fertilization (hpf). Beginning at 18 hpf, the pretrochal region differentiated into ciliated and non-ciliated regions. At 24 hpf, alternative bulges and grooves were developed in the central region, marking the formation of ridges and shell plate fields. The centrally located ridges and shell plate fields, as well as the marginal tissues, constituted the shell field.

(2) F-actin dynamics. F-actin was aggregated in pretrochal and posttrochal regions. Those in the pretrochal region outlined the precursor of girdle, which presumably marked spicule-forming cells. We designated this area as the girdle field (GF). It initially showed “spot” pattern and subsequently transitioned into tiny “tubes” inserting deeply in the tissue. F-action aggregations in the central region were observed in the plate fields, which got enhanced with the ongoing of development.

(3) Expression dynamics of engrailed. In the early larval stages, the expression of engrailed was started and subsequently undergone a complex process of changes. Generally, the expression area continued to increase, finally transitioned into a pattern of eight stripes in the central region, most of which coincided with the bulges. The expression changes of engrailed in this region showed two patterns: increase of single stripes, and a transformation from a broad area into multiple stripes. At the same time, engrailed expression was also found in the GF, and the expression in this field outlined the inner edge of the GF.

(4) Shell plate production during metamorphosis. The shell plates secreted by the plate field subsequently showed continuous expansion and the ridges shrank, until the seven shell plates were fully developed.

3. Metamorphosis. We explored the factors that could induce settlement and metamorphosis of A. rubrolineata. The results are as follows.

(1) Our results indicate settlement and metamorphosis were two independent developmental events in A. rubrolineata, which required different inductive signals.

(2) Adult tissues and unfertilized oocytes, either from the same or different species, could both induce settlement of A. rubrolineata, indicating the inductive clues were not related to particular species, but might be small molecules derived from the decay of tissues/cells. We called this unidentified molecule(s) to be factor X. We also found that although factor X only induced settlement, potassium (KCl) could induce both settlement and metamorphosis.

In conclusion, in the present study, we comprehensively investigated the early development of the polyplacophoran mollusk A. rubrolineata, including recognizing the presumptive organizer (the 3D blastomere), characterizing the morphogenesis of shell field at both molecular and cellular levels, and identifying two types of factors that induce metamorphosis. Juveniles were produced accordingly. The results enrich the knowledge of molluscan development and evolution. Moreover, the chitons belong to the same taxa as many economic molluscan species, and many aspects of its development mechanism may have commonalities. Therefore, these results would also provide supports for seed production and selective breeding in economic molluscan species.

第1章 绪论........................................................................................ 1

1.1 软体动物简介.................................................................................................... 1

1.1.1 软体动物分类............................................................................................. 1

1.1.2 软体动物贝壳............................................................................................. 2

1.2 软体动物的早期发育........................................................................................ 3

1.2.1 螺旋卵裂简介............................................................................................. 3

1.2.2 组织者细胞................................................................................................. 5

1.2.3 多板纲动物细胞卵裂................................................................................. 6

1.3 多板纲动物贝壳发育区的研究........................................................................ 7

1.3.1 多板纲动物贝壳发育区的组成................................................................. 8

1.3.2 贝壳形成相关基因................................................................................... 10

1.4 软体动物的变态发育...................................................................................... 13

1.4.1 软体动物附着变态简介........................................................................... 14

1.4.2 多板纲动物变态研究............................................................................... 16

1.4.3 影响附着变态的因子............................................................................... 17

1.5 本研究的目的和意义...................................................................................... 18

第2章 材料和方法.......................................................................... 20

2.1 幼虫培养及固定.............................................................................................. 20

2.2 鬼笔环肽染色.................................................................................................. 20

2.3 扫描电子显微镜观察...................................................................................... 20

2.4 半薄切片及染色.............................................................................................. 21

2.4.1 半薄切片................................................................................................... 21

2.4.2 苏木素染色............................................................................................... 21

2.4.3 天青-亚甲蓝染色...................................................................................... 22

2.5 探针合成.......................................................................................................... 22

2.5.1 RNA提取................................................................................................... 22

2.5.2 cDNA合成................................................................................................. 22

2.5.3 引物合成................................................................................................... 22

2.5.4 PCR扩增和胶回收.................................................................................... 23

2.5.5 探针合成和纯化....................................................................................... 24

2.6 整装原位杂交.................................................................................................. 24

第3章 结果...................................................................................... 26

3.1 红条毛肤石鳖发育简介.................................................................................. 26

3.2 红条毛肤石鳖早期细胞分裂.......................................................................... 27

3.3 红条毛肤石鳖贝壳发育区的早期发育.......................................................... 29

3.3.1 贝壳发育区发育过程的形态发生........................................................... 29

3.3.2 贝壳发育区形态发生过程中F-actin变化.............................................. 32

3.3.3 贝壳发育区engrailed基因的表达分析.................................................. 35

3.4 红条毛肤石鳖幼虫的变态.............................................................................. 39

3.4.1 简介........................................................................................................... 39

3.4.2 红条毛肤石鳖附着条件研究................................................................... 40

3.4.3 红条毛肤石鳖变态条件研究................................................................... 43

3.4.4 红条毛肤石鳖变态发育过程的贝壳发育............................................... 44

第4章 讨论...................................................................................... 47

4.1 红条毛肤石鳖的早期卵裂.............................................................................. 47

4.2 红条毛肤石鳖的贝壳发育.............................................................................. 48

4.2.1 早期贝壳发育区的建成........................................................................... 49

4.2.2 变态过程贝壳发育的动态变化............................................................... 52

4.3 影响石鳖变态的因素...................................................................................... 53

第5章 结论和展望.......................................................................... 54

5.1 结论.................................................................................................................. 54

5.2 创新性.............................................................................................................. 54

5.3 问题和展望...................................................................................................... 54

参考文献............................................................................................... 56

致谢................................................................................................... 63

作者简历及攻读学位期间发表的学术论文与其他相关学术成果..... 64