中国科学院海洋研究所机构知识库

冠轮动物（Lophotrochozoa/Spiralia）具有独特的分类地位和演化历史，研究其早期发育机制对于理解动物的发育和演化具有重要的意义。原肠作用（gastrulation）是动物发育过程中极其重要的一个环节，奠定了动物个体发育的蓝图，一直以来是发育和演化领域高度关注的科学问题。本研究以冠轮动物的代表物种笠贝（Lottia goshimai）为对象，解析了笠贝原肠作用过程中重要细胞群体的起源和分化过程，进一步研究了依赖MAPK信号的组织者细胞（organizer）对原肠作用的调控机制。

由于笠贝原肠作用的前期研究主要针对外胚层开展，对中胚层的研究较少，因此首先关注了原肠作用过程中胚层的起源和发育。此外，笠贝原肠作用过程起始于组织者细胞的形成，而MAPK信号在组织者细胞的激活和原肠作用的调控中发挥关键作用。因此，本文继续研究了笠贝组织者细胞中MAPK信号发挥作用的关键阶段及其调控原肠作用和个体发育不同生理过程的差异效应。

本研究的主要结果如下：

1. 对笠贝原肠作用过程中胚层的发育模式以及外中胚层的精确起源进行了详细研究，发现笠贝中胚层组织可以分为endomesoderm（暂译为“内中胚层”）及ectomesoderm（暂译为“外中胚层”）两部分。内中胚层起源于4d细胞，分布于靠近背侧外胚层的胚胎内部，形成了两条左右对称呈“V”字形排布的中胚层带（mesodermal bandlets）。外中胚层分布于腹侧，由胚胎的外胚层内侧延伸到胚胎身体内部，呈左右对称分布。鉴定了五个中胚层发育相关基因（twist1，twist2，snail1，snail2和mox）并研究了其表达模式。结果表明，只有twist1和snail1在部分内中胚层细胞群体表达，而五个基因均表达在外中胚层细胞群体中，这些基因的表达动态反映了早期中胚层细胞的分化过程。snail2在原肠作用早期的表达模式展示了外中胚层前体细胞从胚胎表面进入内部的过程。基于基因表达信息和细胞谱系追踪，确定笠贝外中胚层的起源细胞为3a²¹¹和3b²¹¹。

2. 初步探究了笠贝MAPK信号发挥作用的发育阶段。通过MAPK信号通路U0126抑制剂的处理实验，观察统计了48 hpf（hours postfertilization）面盘幼虫的眼点数量以及贝壳和足等重要器官的发育情况。五个不同的时间窗口处理显示，当处理窗口为2-40细胞期，8-60细胞期和16-64细胞期时，面盘幼虫出现典型的组织者细胞功能被抑制后的发育缺陷；而处理窗口为40细胞期-4 hpf和60细胞期-4.6 hpf时，对面盘幼虫的影响不大。基于此，认为40细胞期之后的抑制剂处理对胚胎的发育没有明显影响。考虑到基本确定笠贝组织者细胞是3D卵裂球，而其形成于32细胞期，因此32-40细胞期应是组织者细胞发挥功能的关键阶段。然而，本研究发现处理窗口为2-40细胞期时产生的抑制效应远大于8-60细胞期的处理组，表明在2-8细胞期抑制MAPK信号通路也可对组织者细胞产生较大影响。这一结果提示笠贝中组织者细胞的形成与MAPK信号通路的激活并不是完全同步的，MAPK远在组织者细胞还未形成时即开始发挥与组织者细胞功能相关的发育调控功能，推测这些功能可能与组织者细胞的激活等发育事件相关。

3. 发现MAPK信号通路对不同发育事件的调控作用存在差异效应。通过细胞、分子水平以及形态发生的研究揭示了MAPK信号通路对不同个体发育事件的调控机制，包括组织者细胞形态、BMP信号梯度、背腹相关的细胞群体及标志性器官（如眼点、贝壳、足）的形成等多个方面。结果表明，高浓度（75 mM）抑制剂（U0126）处理胚胎时，发育受抑制的程度最高，具体表现在组织者细胞从形态上不能区分，pSmad1/5/8信号的强度明显减弱、信号梯度基本消失，背腹相关的细胞群体由两侧对称分布变为辐射对称分布，极高比例的幼虫器官形态异常。中浓度（50 mM）抑制剂处理时，发育受抑制程度略低于高浓度的抑制剂处理组。组织者细胞从形态上不能区分，pSmad1/5/8信号的强度明显减弱、信号梯度减弱但仍存在，背腹相关的细胞群体由两侧对称分布变为辐射对称分布，存在较高比例的幼虫器官发育异常。低浓度（25 mM）抑制剂处理时，对发育的影响最弱，但组织者细胞从形态上仍不能区分，pSmad1/5/8信号的强度稍微减弱、信号梯度略有减小，背腹相关细胞群体的正确分布受到轻微影响，同时仅有较低比例的幼虫器官发育异常。以上结果表明MAPK信号通路对不同发育事件的调控存在明显的差异效应，不同发育过程对MAPK信号的响应不耦合。组织者细胞形态的形成依赖高强度的MAPK信号，背腹相关细胞群体的正确分布及器官形成对MAPK信号强度的要求略低，而低强度的MAPK信号即可形成并维持BMP信号梯度。此外，进一步分析了抑制剂处理后早期胚胎的细胞分化情况，发现抑制剂影响了早期细胞群体的分化。这种细胞分化命运的改变可能是组织者细胞活性被抑制后原肠作用及个体发育异常的根本原因。

本研究解析了笠贝原肠作用过程中胚层组织的早期发育模式和分化规律，进一步研究了组织者细胞这一原肠作用关键环节中MAPK信号通路发挥作用的阶段，解析了MAPK信号对于植物极细胞形态、BMP信号通路的激活模式、背腹侧组织标记基因的表达规律以及关键器官的差异调控效应，研究结果丰富了对笠贝早期发育机制的认知，对于理解软体动物乃至冠轮动物的发育和演化机制有重要意义。

Spiralia comprises a unique clade of bilaterian animals, whose development is essential to understand the developmental and evolutionary mechanisms of animals. Gastrulation is a crucial developmental event in animal development. By generating three germ layers, gastrulation establishes the basis to form the body plan of the species, and thus has always been an important issue in developmental and evolutionary research. In this study, we investigated the different aspects of gastrulation in the patellogastropod mollusk Lottia goshimai, a representative of equal-cleaving spiralian, including the early specification of mesoderm and the mechanisms of organizer function/MAPK signaling.

Previous research primarily focused on the ectoderm of L. goshimai. Therefore, here we turn to the origin and early development of mesoderm. Moreover, the gastrulation of L. goshimai begins with the formation of the D-quadrant organizer, and MAPK signaling plays a key role in organizer function. We thus investigated the essential aspects of organizer function/MAPK signaling, including the stage in which the MAPK signaling executes its roles and the differentiated effects of MAPK signaling on gastrulation and ontogenesis.

The main results are as follows.

1. We investigated the mesodermal development during gastrulation of L. goshimai. The mesoderm could be divided into two categories, the endomesoderm and the ectomesoderm. The endomesoderm derived from 4d was situated beneath the dorsal ectoderm, forming two mesodermal bandlets (MBs) containing one left and one right bandlets, together showing a “V” shape. The ectomesoderm was distributed on the ventral side, showing a bilateral pattern. Five genes (twist1, twist2, snail1, snail2 and mox) related to mesodermal development were identified and their expression patterns were investigated. The results revealed that only twist1 and snail1 were expressed in endomesoderm, while all the five genes were expressed in ectomesoderm. The expression dynamics of these genes reflect the differentiation of various mesodermal subpopulations. The expression of snail2 in early gastrulae demonstrated the process of ectomesodermal internalization, referring to the relocation of the precursors from the surface into the interior of the embryo. By tracing the expression of snail2 and cell lineage analysis, we revealed that 3a²¹¹ and 3b²¹¹ were the precursors of the ectomesoderm in L. goshimai.

2. We explored the developmental stage that is crucial for the regulatory roles of MAPK signaling/organizer in L. goshimai. After the treatment of the MAPK signaling inhibitor U0126, the development of eyes, shells and feet of 48 hpf-larvae were investigated to explore whether organizer function was influenced. Totally five treatments in different time windows were conducted. We found that the time windows of 2-40 cell, 8-60 cell and 16-64 cell all produced typical veliger phenotypes with influenced organizer function, including abnormal eyes development and the lack of shell or foot. However, when the treatments were performed in the terms of 40 cell-4 hpf or 60 cell-4.6 hpf, there was little effect on larval development. Based on these results, we concluded that inhibitor treatment after the 40-cell stage has little effect on embryonic development. Given that the organizer of L. goshimai was generally determined to be the 3D blastomere, which was formed at the 32-cell stage, the 32-40-cell stage should be the key stage for organizer function. However, we found that the inhibition during the 2-40-cell stage caused much greater effects than the 8-60-cell stage, indicating that the inhibition of MAPK signaling during the 2-8-cell stage had a great impact on organizer function. This suggested that the time window for organizer function was not equivalent to that of MAPK signaling. It is attempting to speculate that MAPK signaling begins to play its regulatory roles (related to organizer cell function) long before the formation of organizer. These roles may be related to the activation of organizer.

3. The MAPK signaling has differentiated effects on varied developmental events. Through cellular, molecular and morphogical analysis, we investigated various regulatory effects of MAPK signaling on ontogesis, including the morphological characteristics of organizer and the formation of BMP signaling gradient in early gastrulae, the formation of dorsal and ventral tissues in middle gastrulae and organogenesis in veliger larvae (such as the development of eyes, shells and feet). We found that embryos treated with a high concentration (75 mM) of the specific inhibitor U0126 caused the most serious effects. These were manifested by indistinguishable organizer (the 3D blastomere), the reduction of pSmad1/5/8 intensities as well as the loss of signaling gradient when compared to normal embryos, and the transition from bilateral to radial-symmetrical distribution of the dorsal and ventral tissues. A very high proportion of larvae showed abnormal organogenesis under this treatment. At the medium concentration (50 mM), the effects were weaker. The organizer was still indistinguishable based on morphological characteristics. The intensity of pSmad1/5/8 signal and signal gradient were weakened (compared to control embryos) but still detectable. The dorsal and ventral tissues were still radial-symmetrically distributed, and a high proportion of larvae had abnormal organogenesis. At the low concentration (25 mM), the effect on development was the weakest, but the organizer remained morphologically indistinguishable. The intensity and gradient of pSmad1/5/8 decreased slightly compared to normal embryos. The specification and distribution of dorsal and ventral tissues were only slightly affected, and only a small proportion of larvae had abnormal organogenesis. These results indicated that the MAPK signaling had differentiated effects on the various developmental events. In another word, the developmental processes related to MAPK signaling are uncoupled and require varied levels of the signaling. The formation of the characteristic organizer morphology depends on a high level of MAPK signaling, the formation of dorsal and ventral tissues and normal organogenesis require a medium level of MAPK signaling, while a low level of MAPK signaling is sufficient to establish and maintain the BMP signaling gradient. In addition, the cell differentiation of early embryos treated with the inhibitor was analyzed. We found that inhibitor treatment affected the differentiation of a wide range of blastomeres. These changes in cell fate may have contributed to the altered gastrulation and ontogenesis after inhibiting organizer function.

Conclusively, we analyzed the early mesodermal specification during the gastrulation of the gastropod mollusk L. goshimai and further explored mechanisms of MAPK signaling/organizer function, a key aspect of gastrulation. The results revealed the molecular and cellular mechanisms underlying these developmental events. The findings would provide fundamental supports for understanding the unique developmental characters and evolution of mollusks and spiraliand.

第一章  引言... 1

1.1  两侧对称动物（Bilateria）的早期发育... 1

1.1.1  冠轮动物（Lophotrochozoa/Spiralia）的早期发育... 2

1.1.2  软体动物（Mollusca）的早期发育... 7

1.2  两侧对称动物中胚层的早期发育及标记基因... 14

1.2.1  内中胚层和外中胚层的起源和发育过程... 15

1.2.2  中胚层发育过程相关的标记基因... 20

1.3  冠轮动物组织者细胞... 23

1.4  MAPK信号通路... 25

1.4.1  MAPK信号通路与组织者细胞的关系... 26

1.4.2  MAPK信号通路与背腹轴的调控... 29

1.5  研究目的和意义... 31

第二章  材料与方法... 34

2.1  幼虫的采集与培养... 34

2.2  基因的鉴定及系统发育树的构建... 34

2.3  RNA提取... 35

2.4  反转录 cDNA第一链的合成... 36

2.5  整装原位杂交... 36

2.5.1  引物设计与合成... 36

2.5.2  探针合成... 37

2.5.3  原位杂交... 38

2.6  Phalloidin染色及激光共聚焦观察... 38

2.7  免疫组化... 39

2.8  抑制剂处理... 39

2.9  扫描电子显微镜观察... 40

2.10  技术路线... 40

第三章  结果... 42

3.1  笠贝中胚层标记基因的鉴定及在早期胚胎中的表达模式... 42

3.1.1  中胚层的发育过程的显微观察... 42

3.1.2  中胚层细胞标记基因的鉴定和聚类分析... 44

3.1.3  中胚层标记基因的表达模式... 47

3.1.4  外中胚层细胞的起源... 50

3.2  MAPK通路的作用时间窗口... 55

3.2.1  抑制剂处理时间窗口的选择... 55

3.2.2  不同时间窗口处理时48 hpf幼虫的发育情况... 56

3.3  MAPK通路抑制剂的剂量效应... 59

3.3.1  不同剂量抑制剂对组织者细胞形态的影响... 59

3.3.2  不同剂量抑制剂对BMP信号通路的影响... 61

3.3.3  不同剂量抑制剂对背腹侧组织发育的影响... 63

3.3.4  不同剂量抑制剂对48 hpf幼虫器官发育的影响... 67

3.4  抑制剂处理对早期胚胎细胞分化的影响... 70

3.4.1  早期标记基因的筛选及表达模式... 71

3.4.2  抑制剂处理对早期胚胎细胞分化的影响... 76

3.5  小结... 79

第四章  讨论... 81

4.1  笠贝的原肠作用... 81

4.2  中胚层的早期发育模式... 82

4.2.1  4d卵裂球和中胚层带... 82

4.2.2  中胚层标记基因的表达规律... 83

4.2.3  外中胚层的前体细胞... 85

4.3  组织者细胞/MAPK信号调控笠贝发育的机制... 86

4.3.1  MAPK信号发挥作用的时间窗口... 88

4.3.2  U0126抑制剂的剂量效应... 89

4.3.3  抑制剂处理后早期胚胎的细胞命运改变... 90

第5章  结论... 92

参考文献... 94

附  录... 101

致  谢... 102

作者简历及攻读学位期间发表的学术论文与研究成果... 104