Fstl1在丘脑发育中的功能研究

 2021-12-28 08:12

论文总字数:32939字

摘 要

丘脑作为各种感觉通路的最终中继站及感觉运动的整合中枢,参与脑部的高级功能,诸如调节意识、警觉和注意力以及情绪等等。丘脑最显著的特性是由大量的核团所组成,核团是由功能和结构相同的细胞聚集形成,具有特异的基因表达模式以及独特的功能,核团之间也存在复杂的相互作用。Fstl1(follistatin-like 1)作为一种分泌性的胞外糖蛋白,最早从小鼠的成骨细胞系中克隆获得,因为转录生长因子TGF(Transforming Growth Factor)β 1可以诱导其表达上调,所以又被称为TSC-36(TGF-β-stimulated clone 36)。鸡胚实验的研究发现Flik(FSTL1在鸡中的同源物)在神经诱导中发挥作用并参与维持Shh信号的表达,故此推断Fstl1可能参与小鼠中枢神经系统的发育。实验前期关于Fstl1的表达谱研究中发现, Fstl1在中枢神经系统的多个区域均有较强的表达。在间脑中主要表达在脑室区,尤其在ZLI和松果体区域有较强的表达。由此推测,Fstl1可能对丘脑的早期发育以及松果体的形成起重要的调控作用。因此,我们进一步利用Fstl1基因敲除小鼠来研究丘脑早期的发育。

本课题主要通过原位杂交技术和免疫荧光技术手段,在丘脑发育早期E12.5时检测其模式形成,在丘脑核团初步形成的围产期E18.5检测相关核团的定位。我们发现Fstl1基因被敲除之后,丘脑早期发育过程中的生发中心ZLI区域和p1区域均变小,在发育晚期E18.5天发现ZLI的衍生的结构外髓板(EML,external medullary lamina)向背侧上移,pax6阳性的顶盖结构也相应变小。此外,我们还发现上丘脑结构中的松果体减小、后连合(posterior commissure)的细胞形态异常。这些结果表明Fstl1参与调控早期丘脑的模式形成,其具体的机制调控还待进一步研究。以上结果有助于深入研究丘脑的发育和发生机制。

关键词: follistatin-like 1,ZLI,松果体,模式形成,丘脑

Function of Fstl1 in the Developing Thalamus

Abstract

The thalamus is a major part of the diencephalon that acts as a relay station between the cortex and other sensory systems. It is well characterized by many functionally and morphologically distinct nuclei that have respective genes expression patterning. The thalamus also plays critical roles in sensory, motor, and cognitive functions. Follistatin like 1 (Fstl1) as a secreted extracellular glycoprotein, which was first cloned from the mouse osteoblastic MC3T3-E1 cell line, and can be up-regulated by TGF-β. We also call it TSC-36 (TGF-β-stimulated clone 36). Previous studies reported that Flik, the Fstl1 homologue in chick was play a role in neural induction and subsequent maintenance of Shh signaling. Based on Fstl1 expression pattern Fstl1 was strongly expressed in multiple regions of the CNS. It was expressed in the ventricle zonismse(VZ) of the diencephalon, especially strongly located in the ZLI(zone limitans intrathalamica) and the pineal gland. These data showed that Fstl1 may participate in regulating the development of thalamus and the pineal gland. To better understanding the underlying molecular mechanisms, we ablated Fstl1 by intercrossing Fstl1 hetezygous(Fstl1 /-) mouse.

In this present study, we examined the pattern formation at embyonic 12.5(E12.5) by tissue in situ hybridization and immunofluorescence at E12.5. Furthermore, we detected the position of the thalamic nuclei at E18.5. Our results reveal the ZLI change small and its derivative EML (external medullary lamina) shift dorsally. The progenitor region of p1 is reduce and the pretectal nuclei is shrunken than the wildtype mice. Moreover, the presumptive pineal gland shrink slightly and the cell of the posterior commission are abnormal. These results suggested that Fstl1 plays a role in the developing thalamus and will furthermore improve the comprehension of the mechanism of thalamus development.

Keywords:  Fst1, Zli, pineal gland, pattern formation, thalamus

中文摘要 2

Abstract 3

第一章 绪论 5

1、 丘脑结构概述 5

1.1、丘脑的组织发生 5

1.2、丘脑的早期发育 6

1.3、参与调控丘脑发育的信号分子和转录因子 8

1.4、上丘脑的组成及功能 10

2、 Fstl1基因的研究现状 11

2.1、Fstl1的概况 11

2.2、Fstl1在发育过程中的功能 12

2.3、Fstl1在神经系统发育过程中的作用 12

第二章 实验材料与方法 12

1、 实验材料及试剂 12

1.1、主要仪器 12

1.2、主要材料及试剂 13

2、 实验方法 15

2.1、小鼠饲养、繁殖及组织收取 15

2.2、PCR 16

2.3、免疫荧光 16

2.4、组织原位杂交 17

第三章 结果与讨论 18

1、 实验结果 18

1.1、Fstl1小鼠基因型鉴定(PCR) 18

1.2、Fstl1基因的敲除效率 19

1.3、Fstl1敲除后ZLI区域变小 19

1.4、pTH-R区域的研究结果 24

1.5、p1区域的研究结果 26

1.6、上丘脑的研究结果 28

2、 结果讨论 31

参考文献: 33

致谢 36

  1. 绪论
  2. 丘脑结构概述

1.1、丘脑的组织发生

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