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Title: Lmx1b基因在中脑发育过程中的功能研究
Author: 郭超
Degree Level: 博士
Issued Date: 2008-11-27
Degree Grantor: 中国科学院上海生命科学研究院
Place of Degree Grantor: 上海生命科学研究院
Supervisor: 丁玉强
Keyword: Lmx1b ; 菱脑峡组织中心 ; Fgf8 ; 顶盖 ; 小脑 ; 发育 ; 中脑多巴胺神经元 ; 黒质
Alternative Title: THE FUNCTION OF HOMODOMAIN PROTEIN LMX1B
Major: 神经生物学
Abstract: 第一部分 胚胎发育期转录因子Lmx1b在中后脑结构形成中的作用 摘 要 分泌蛋白FGF8和WNT1在菱脑峡组织中心诱导中后脑的区域化(Regionalization)形成过程中起着关键作用。然而,在发育过程中这些分泌蛋白表达的转录调控机制有待于进一步阐明。我们利用基因敲除的方法研究Lmx1b在小鼠中脑和后脑发育中的作用。发现胚胎第七天Lmx1b开始表达在胚胎的前侧(神经外胚层,将来发育成脑),至第九天表达逐渐局限到菱脑峡组织中心部位,提示Lmx1b基因可能在中后脑的发育过程中起作用。生后Lmx1b基因敲除小鼠的中脑和小脑都变小,组织矢状切片染色观察发现中脑下丘消失,缩小的中脑上丘与小脑直接连在一起;小脑变得很小,中部没有融合。分析基因敲除小鼠胚胎中脑和后脑部位的基因表达,应该在4个体节时期开始表达的Fgf8缺失,同时Wnt1的表达下调。其他基因如转录因子En1和Pax2的表达也发生下调,而Gbx2的表达在4个体节的阶段才下调,而Otx2和Pax6的表达并没有受到影响,说明Lmx1b控制着菱脑峡组织中心的诱导功能,但不影响菱脑峡组织中心的定位。Wnt1驱动的Cre重组酶小鼠区域特异性敲除Lmx1b的研究,进一步证实了在菱脑峡组织中心内表达的Lmx1b对中脑和后脑发育的重要性。以上结果表明,在中脑顶盖(上丘和下丘)和小脑的发育过程中,Lmx1b通过调节菱脑峡组织中心内Fgf8、Wnt1以及其它菱脑峡组织中心发育相关的转录因子的表达,从而参与调控中脑和后脑的发育。 第二部分 Lmx1b调控的菱脑峡组织中心对于中脑多巴胺神经元的发育是必需的 摘 要 过去的研究一直认为LIM同源盒转录因子Lmx1b对于中脑多巴胺(DA)神经元的分化是必需的。然而,Lmx1b-/-小鼠中多巴胺神经元的丢失,是由于在DA神经元自身表达Lmx1b的作用还是早期中后脑模式缺陷引起的后果仍需进一步澄清。我们的研究发现,DA中表达的Lmx1b对于DA的分化和存活不是必需的,Lmx1b-/-胚胎中DA神经元的丢失是由于在胚胎早期发育中菱脑峡组织中心功能受损所引起的二级效应。我们利用在TH-Cre;Lmx1bfloxl- 和DatCre/+;Lmx1bflox/- 小鼠选择性的敲除分化中DA神经元中的Lmx1b,无论在胚胎发育期还是在成年期,DA神经元中TH,Pitx3,Nurr1和多巴胺转运体DAT的表达与对照组相比没有明显差别。当用Wnt1-Lmx1b转基因小鼠恢复Lmx1b-/-小鼠的菱脑峡组织中心的活性时,DA神经元也被恢复过来。而Wnt1Lmx1b;Lmx1b-/-小鼠MHB中Fgf8和Wnt1的重新出现,亦即基本正常的顶盖和小脑进一步显示菱脑峡组织中心功能的恢复。此外,用Nestin-Cre在菱脑峡组织中心形成后敲除全脑的Lmx1b的情况下,DA的发育过程没有受到明显影响。上述结果表明菱脑峡组织中心的诱导活性对于DA的发育是必需的,而DA中表达的Lmx1b对于DA的分化和存活不是必需的。
English Abstract: Lmx1b is essential for Fgf8 and Wnt1 expression in the isthmic organizer during tectum and cerebellum development in mice Abstract Secreted factors FGF8 and WNT1 are essential either for the inductive activity of the isthmus organizer or for the regionalization of the midbrain-hindbrain boundary (MHB). However, transcriptional regulation of these secreted factors during development remains to be elucidated. Here we show that the LIM homeobox gene Lmx1b is expressed in the anterior embryo as early as E7.5 and its expression becomes progressively restricted to the isthmus at E9.0. Analysis of gene expression in the MHB of the mutant embryos showed that many genes were lost by E9.5. In the MHB of Lmx1b-/- embryos, the expression of Fgf8, which normally occurs at the 4-somite stage, was completely absent, whereas Wnt1 was downregulated before the 4-somite stage. Moreover, transcription factors En1 and Pax2 were also downregulated prior to the 4-somite stage, whereas Gbx2 downregulation occurred at the 4-somite stage. By contrast, Otx2 and Pax6 expression was not affected in Lmx1b-/- embryos. The requirement of specific Lmx1b expression in the MHB was further confirmed by Wnt1-Cre-mediated region-specific conditional knockout of Lmx1b. As a result of these molecular defects, the development of the tectum and cerebellum was severely impaired in Lmx1b-/- mice. Taken together, our results indicate that Lmx1b plays an essential role in the development of the tectum and cerebellum by regulating expression of Fgf8, Wnt1 and several isthmus-related transcription factors in the MHB, and is a crucial component of a cross-regulatory network required for the induction activity of the isthmic organizer in the MHB. Lmx1b-controlled isthmic organizer is essential for development of midbrain dopaminergic neurons Abstract The LIM homeodomain transcription factor Lmx1b has been suggested to be required for the differentiation of midbrain dopaminergic (mDA) neurons. However, whether the loss of mDA neurons in Lmx1b-/- mice is due to its intrinsic role in the mDA lineage or to a consequence of the malformations caused by the earlier mid/hindbrain patterning defects remains to be clarified. We report here that Lmx1b expression in mDA neurons is dispensable for their differentiation and maintenance, and the loss of mDA neurons in Lmx1b-/- mice is due to the disruption of inductive activity of the isthmic organizer (IsO) in the absence of Lmx1b at the mid/hindbrain boundary (MHB). We found that mDA neurons revealed by tyrosine hydroxylase (TH), Pitx3, Nurr1 and dopamine transporter were indistinguishable from wild-type controls during embryonic development as well as in adulthood in TH-Cre;Lmx1bflox/- and DatCre/+;Lmx1bflox/- mice, in which Lmx1b was selectively deleted in differentiating mDA neurons. In addition, mDA neurons were recovered in Lmx1b-/- mice, when IsO activity was restored by Wnt1-Lmx1b transgene at MHB. The restored IsO activity was evidenced by apparently normal tectum and cerebellum and recurrence of expression of Fgf8 and Wnt1 at MHB in Wnt1Lmx1b;Lmx1b-/-. Furthermore, when Lmx1b was deleted in the whole brain after the formation of IsO by Nestin-Cre, mDA neurons were normal, whereas serotonergic neurons displayed defective development phenocopying what observed in Lmx1b-/- mice. Thus, our results indicate that the inductive activity of IsO is essential, but Lmx1b expression in mDA neurons is dispensable for their differentiation and maintenance.
Language: 中文
Content Type: 学位论文
URI: http://ir.sibs.ac.cn/handle/331001/2377
Appears in Collections:神经所(总)_学位论文

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Recommended Citation:
Lmx1b基因在中脑发育过程中的功能研究.郭超[d].中国科学院上海生命科学研究院,2008.20-25
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