背腹轴形成是两侧对称动物早期发育过程中的重要事件之一。一般认为，BMP信号通路在背腹轴形成过程中具有重要调控作用，这种机制在多种动物中均已得到了验证。然而，不同动物中的背腹轴决定机制存在多样性，呈现出较强的可塑性。作为两侧对称动物的三个主要分支之一，冠轮动物中存在多种背腹轴调控机制，是研究动物背腹轴形成机制保守性和可塑性的理想对象。同时，对冠轮动物背腹轴形成机制的研究也是理解这一大类动物发育和演化的关键环节。本课题组前期研究发现，在腹足纲软体动物笠贝（Lottia goshimai）的背腹轴形成过程中，bmp2/4和chordin起关键的调控作用。但同时也发现，抑制chordin后的胚胎虽然早期阶段完全失去背腹轴，但会随着发育部分恢复背腹极性，提示其他基因协同调控了背腹轴形成过程。基于此研究现状，有必要对笠贝的背腹轴调控网络进行进一步探索，以更加深刻的理解笠贝背腹轴形成的分子机制。

本研究以笠贝为研究对象，利用扫描电镜和原位杂交手段，从形态学水平和分子水平系统展示了背腹轴的形成过程；鉴定了部分BMP信号调节基因并分析了它们的时空表达模式，解析了与背腹轴形成的关联关系；最后对部分基因进行了RNAi抑制实验，探索其在背腹轴形成中的功能。主要结果如下：

1. 通过扫描电镜观察和原位杂交实验，系统分析了笠贝早期胚胎的变化过程，从形态学水平和分子水平上详细展示了背腹轴的形成过程。笠贝胚胎在3.5hpf左右进入原肠作用阶段，细胞剧烈运动，背腹轴随之形成。8hpf后原肠作用基本结束，担轮幼虫特征性结构（壳区和胚孔）开始形成，进入担轮幼虫阶段。在这一时期内（3.5hpf~8hpf），标记基因gata2/3和soxb表达范围的变化指示了背腹侧不同类型细胞群体随着发育不断运动的过程。

2. 通过序列比对和系统发育分析，鉴定了6个可能参与背腹轴形成的基因：admp、bmp5-8、gremlin、tolloid、bambi和nogginb。分析这些基因的时空表达规律，发现以下特点：首先，不同基因出现不对称表达模式的时间不同：bambi、gremlin持续不对称表达；tolloid、bmp5-8早期广泛表达，晚期不对称表达；admp在早期没有表达，在晚期不对称表达；nogginb一直没有表达。其次，不同基因表达于不同的类型组织：bambi，gremlin，tolloid在背侧类型组织中表达，bmp5-8在腹侧类型组织中表达，admp倾向于在背腹交际处表达。有意思的是，即使是表达于相同类型的组织，这些基因的表达区域也不同（如tolloid和gremlin），这种差异提示存在多个细胞群体与背腹轴形成相关。

3. 初步的功能实验证实gremlin和tolloid在笠贝背腹轴形成中具有调控作用。通过RNAi抑制两个基因的功能后，都会使笠贝胚胎的形态与标记基因的表达出现异常。具体而言，抑制gremlin后，幼虫身体拉长、腹侧特征性凹陷变平，标记基因表达位置迁移至身体后部。抑制tolloid则似乎使幼虫停滞于较早期的形态，标记基因表达在底部，且近乎辐射对称。这些结果均提示胚胎的背腹轴形成受到了严重干扰，两个基因都对背腹轴形成过程具有调控作用。两种突变表型的差异提示gremlin和tolloid的作用方式存在差异。结合其分子性质和表达部位，推测tolloid可能直接调控BMP信号，而gremlin则有可能通过间接方式发挥作用。

综上所述，本研究展示了笠贝背腹轴的形成过程，揭示了背腹轴形成中信号网络的更多细节。综合现有研究基础及本研究的结果，基于原位表达和功能实验（MO、RNAi），初步描绘了笠贝背腹轴形成的分子作用网络。该网络包含bmp2/4、chordin、admp、bmp5-8、gremlin、tolloid、bambi共七个基因，存在多个调控节点。在拓扑结构上与其他动物存在相似性，但在admp、bmp5-8、gremlin等节点上存在自身的特点。该网络为进一步解析基因功能提供了基础支持，为探究冠轮动物中背腹轴决定机制提供了重要信息。

The formation of a dorsal-ventral (DV) axis is one of the most important events in the early development of bilaterian animals. It is generally believed that BMP signaling plays an important role in DV patterning, which has been widely verified in a variety of animal lineages. However, there are considerable differences in the DV patterning mechanism of different species, among which is Spiralia. Recent researches have revealed diverse mechanisms in the DV patterning of several spiralian representatives. As one of the three major bilaterian clades, Spiralia is an ideal object to study the plasticity of the DV patterning mechanisms in animals. Moreover, unraveling the DV patterning mechanisms is an essential aspect toward a comprehensive understanding of the development and evolution of this large bilaterian clade. Previous studies in the gastropod mollusk Lottia goshimai have confirmed the conserved role of bmp2/4 and chordin in its DV patterning. However, it was found that the inhibitory effect of chordin on DV patterning was greatly weakened with ontogenesis, indicating additional molecules participate in this process. Based on this result, it is necessary to further explore other candidate regulatory molecules comprising the complete network underlying L. goshimai DV patterning, which would provide a deeper understanding of the DV patterning mechanism in this species.

In this study, we used scanning electron microscopy (SEM), RNAi and in situ hybridization (ISH) to investigate the mechanism underlying L. goshimai DV patterning. We described the formation of the DV axis at the morphological and molecular levels, identified six genes of the BMP signaling pathway that may regulate DV patterning, and analyzed their spatiotemporal expression patterns. Finally, we investigate the roles of two genes using RNAi. The main results are described as follows.

1. Based on scanning electron microscopy and in situ hybridization, we analyzed the changes of the early embryo of L. goshimai, and demonstrated the process of the DV patterning in detail at both morphological and molecular levels. The embryo entered the gastrulation stage at about 3.5 hpf, which generally ended at 8 hpf before the embryo developing into the trochophore. The results indicate that the formation of DV axis is strongly related to the epibolic gastrulation. During this period (3.5~8hpf), the expression of marker genes gata2/3 and soxb reflected the continuous movement of the dorsal-/ventral-type cell populations with the development went. These results provide a basis for further investigations on DV patterning.

2. We identified six genes that may be involved in the formation of the DV axis: admp, bmp5-8, gremlin, tolloid, bambi and nogginb. The results of in situ hybridization revealed that the developmental stages showing asymmetrical (posttrochal) expression varied among different genes. They include: 1) two genes showing constantly asymmetrical expression (bambi, gremlin); 2) two genes showing universal expression at early stages but asymmetrical expression at late stages (bmp5-8, tolloid); 3) one gene showing no expression at early stages and asymmetrical expression at late stages (admp);4) one gene showing no expression during the testing phase (nogginb). These genes were expressed in different types of tissues: bambi, gremlin and tolloid expressed in dorsal-type tissue, and bmp5-8 expressed mainly in ventral-type tissues. The cells expressing admp could not be confidently determined; it seemed that these posterior cells were located between dorsal-and ventral-type tissues. Moreover, most of these genes showed restricted expression in small numbers of cells, indicating distinct cell populations regulating DV patterning.

3. Preliminary functional experiments confirmed the regulatory role of gremlin and tolloid in DV patterning. Inhibition of either of the two genes resulted in abnormal embryonic development and altered expression of the marker gene soxb. After gremlin knockdown, the larval body became elongated, and soxb expression migrated to the posterior part of the body. Inhibition of tolloid caused the larva showing similarities to earlier embryos, indicating developmental arrest, and soxb exhibited a trend of radial expression. These results suggest that both genes regulate the formation of the DV axis. The different knockdown phenotypes indicate varied regulatory modes of the two genes. tolloid may directly regulate BMP signal, while gremlin may play an indirect role.

Based on the results abovementioned as well as those of previous reports, we constructed a gene regulatory network underlying the DV patterning of L. goshimai. The network comprised seven genes, including bmp2/4, chordin, admp, bmp5-8, gremlin, tolloid, bambi. It is generally similar to that of other animals, while showing novel characters of several nodes such as admp, bmp5-8 and gremlin. These results provide a fundamental support for further functional studies and would be essential to explore the intriguing DV patterning mechanisms in spiralians.

第1章  引言... 1

1.1  两侧对称动物及其体轴... 1

1.2  BMP信号通路... 3

1.2.1  BMP信号通路简介... 3

1.2.2  参与背腹轴调控的BMP信号通路分子... 7

1.3  背腹轴调控模式... 13

1.3.1  典型动物的背腹轴调控模式... 13

1.3.2  冠轮动物背腹轴调控模式... 19

1.4  本研究的目的与意义... 25

第2章  材料方法... 29

2.1  样品收集与固定... 29

2.2  扫描电镜观察... 29

2.3  基因鉴定与聚类分析... 29

2.4  探针合成... 29

2.4.1  RNA提取... 29

2.4.2  cDNA合成... 30

2.4.3  引物设计... 30

2.4.4  PCR扩增与胶回收... 31

2.4.5  探针合成与纯化... 31

2.5  整装原位杂交... 32

2.6  RNA干扰... 32

第3章  结果... 35

3.1  笠贝背腹轴形成过程的观察与描述... 35

3.1.1  笠贝早期胚胎发育过程的形态变化... 35

3.1.2  分子水平的背腹轴发育过程... 39

3.2  笠贝背腹轴调控分子的表达分析... 41

3.2.1 背腹轴调控分子的系统发育分析... 41

3.2.2  背腹轴调控分子时空表达分析... 45

3.3  部分背腹轴调控分子的功能初探... 55

第4章  讨论... 59

4.1  笠贝早期发育过程及背腹轴形成过程... 59

4.2  笠贝背腹轴调控基因的表达规律与功能解析... 60

4.3  笠贝的背腹轴调控网络... 65

第5章  结论与展望... 69

参考文献... 71

致  谢... 81

作者简历及攻读学位期间发表的学术论文与研究成果    83