Functional study of LIM-homeodomain proteins Lhx1 and Lhx5 in the maintenance of cerebellar Purkinje neurons in the postnatal and adult mouse.

• Other Authors: Tam, Wing Yip
• Format: Others
• Language: English; Chinese
• Published: 2012
• Subjects: Cerebral cortex > Growth; DNA-binding proteins; Mice; Cerebral Cortex > physiology; LIM-Homeodomain Proteins
• Online Access: http://library.cuhk.edu.hk/record=b5549493; http://repository.lib.cuhk.edu.hk/en/item/cuhk-328038

蒲金氏細胞(Purkinje cell)是小腦中的一種主要神經元，其主要作用在於協調身體活動及平衡。蒲金氏細胞之早期分化需要兩個密切相關的LIM同源盒結構域基因Lhx1及Lhx5。在胚胎小腦發育期間，這兩個基因的失活化會導致蒲金氏細胞數量大量減少。但有趣的是，就算在蒲金氏細胞完成分化之後，Lhx1/5之表達依然維持在高水平。這顯示Lhx1/5在產後小腦發育過程中可能有更多作用。為了研究這些可能作用，我把條件性Lhx1/5雙基因剔除小鼠和Pcp2-IRES-Cre轉基因小鼠交配，從而令Lhx1/5在產後第二天的蒲金氏細胞失活化。結果顯示Lhx1/5雙突變體老鼠在出生後兩星期即有顯著但程度不太大的運動失調。但在八星期，牠們出現嚴重的運動協調及身體平衡能力缺失。可是，擁有一個正常的Lhx1或Lhx5等位基因的控制小鼠並沒有這些不正常行為出現。在出生後的三個星期內，缺乏Lhx1/5會導致蒲金氏細胞樹突不正常發展，但小腦的整體細胞結構和分層卻維持正常。另外，這兩個基因對維持蒲金氏細胞已發展的樹突並不起作用，而且在六個月大的成年突變小鼠並沒有蒲金氏細胞退化。利用微陣列及逆轉錄聚合酶鏈式反應，我們在成年突變小鼠的小腦中確定了數個參與在麩胺酸及鈣訊息的突觸基因表達量下降。而這些突觸基因也在其他運動失調小鼠有下降的表達量。研究結果說明了Lhx1及Lhx5對蒲金氏細胞樹突發展有著重要、但功能重疊的作用。 === 在探究Lhx1/5如何控制蒲金氏細胞樹突發展時，我們發現Lhx1/5與Foxp4有蛋白質交互作用。Foxp4屬forkhead家族成員轉錄因子，它表達在小腦原基、遷移中及成熟的蒲金氏細胞。為了初步瞭解Foxp4在蒲金氏細胞發展中的作用，我在產後第十天小腦薄片組織培養中，利用siRNA降低Foxp4基因的表達量。結果發現蒲金氏細胞樹突及關聯的伯格曼膠質細胞支架出現結構性受損。這顯示Foxp4對維持蒲金氏細胞樹突有重要作用。 === 為了進一步研究Foxp4在活體蒲金氏細胞及小腦發育的作用，我把條件性Foxp4基因剔除小鼠和不同的Cre轉基因小鼠交配，從而令Foxp4在不同的發育過程階段中失活化。但是有趣地，我只能在同質結合突變小鼠 (Foxp4Δ/Δ)，即Foxp4在生殖細胞時期已經被剔除的情況下，觀察到小腦發育遲緩。當Foxp4在其他發育過程階段中失活化，我並沒有觀察到任何缺陷表型。這個結果顯示了在活體中發生了功能性的彌補，但在小腦薄片組織培養中卻沒有發生。另外，條件性Lhx1/5雙基因剔除小鼠和條件性Foxp4基因剔除小鼠的不同表型意味著在控制蒲金氏細胞及小腦發育過程中，有其他蛋白質可能參與在Lhx1/5及Foxp4的轉錄複合子中。我們需要更多的研究去明白Foxp和 LIM同源盒結構域蛋白質在功能上的聯系及它們在中樞神經系統發育中的作用。 === Purkinje cells (PCs) are one of the principal neurons in the cerebellum that is essential for the coordination of fine-tuning body movement and balancing. Early differentiation of PCs requires two closely related LIM-homeodomain genes Lhx1 and Lhx5, as inactivation of both genes results in significant reduction of PC number in embryonic cerebellum. Interestingly, high levels of Lhx1/5 expressions persist even after PC differentiation in the postnatal cerebellum. Hence, there may be additional roles for these two genes during postnatal PC development. To address this question, conditional inactivation approach was used to inactivate both Lhx1/5 in postnatal PCs specifically beginning at postnatal day 2 (P2). Lhx1/5 double conditional knockout (DKO) mutants were generated by crossing Lhx1/5 conditional null mutant mice with Pcp2-IRES-Cre mice. The mutants initially showed modest but noticeable ataxic locomotion at around two weeks after birth. However at 8 weeks old, the mutants displayed severe deficits in motor coordination and body balance. The control animals with one functional copy of either Lhx1 or Lhx5 did not show any abnormality. Deficiencies of both genes could lead to abnormal PC dendritogenesis during the first three weeks of life although the general cytoarchitectural lamination of cerebellar cortex was maintained. However, the two genes were dispensable for the maintenance of developed dendrites in adult mouse and no PC degeneration was observed in the 6 month-old double mutant mouse. Further microarray and semi-quantitative RT-PCR analysis identified down-regulation of several synaptic genes that involved in glutamate and/or calcium signaling in our Lhx1/5 DKO mutant and such disturbance had also been found in other ataxic mouse models. Overall, our findings suggest that Lhx1/5 are required but functionally redundant in dendritogenesis of PCs. === During investigation on how Lhx1/5 control the dendritogenesis of PCs, Lhx1/5 proteins were found to physically interact with Foxp4. Foxp4 belongs to the forkhead transcription factor family that is expressed in developing cerebellum primordium, migrating and mature PCs. To initially examine the function of Foxp4 in PC development, Foxp4 was knocked down by siRNA in organotypic cerebellar slice culture at P10. Impaired organization of PC dendritic arbors and associated Bergmann glial scaffold were resulted, suggesting that Foxp4 is essential for the maintenance of PC dendritic arborization. === To further investigate the function of Foxp4 during the cerebellum and PCs development in vivo, a Foxp4 CKO mouse line was generated and crossed with different lines of Cre-deleter mice, including Zp3-Cre, Pax2-Cre, En1-Cre and Pcp2-IRES-Cre, to inactivate Foxp4 at different developmental stages. Intriguingly, although developmental delay of cerebellum was found in germline deletion of Foxp4 homozygous recombined null mutant, no defective phenotype was observed when Foxp4 was inactivated at other stages. Hence, functional compensation might take place in vivo but not in the cerebellar slice culture. The phenotypic difference between Lhx1/5 DKO and Foxp4 CKO mice imply potential involvement of other proteins in the transcription complex between Lhx1/5 and Foxp4 in regulating the cerebellum and/or PCs development. Thus further investigation is required to understand the functional association between Foxp and LIM-homeodomain protein families during the development of central nervous system. === Detailed summary in vernacular field only. === Detailed summary in vernacular field only. === Detailed summary in vernacular field only. === Tam, Wing Yip. === Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. === Includes bibliographical references (leaves 187-203). === Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. === Abstract also in Chinese. === Abstract --- p.1 === 摘要 --- p.4 === Acknowledgements --- p.6 === Abbreviations --- p.8 === Figure list --- p.12 === Table list --- p.15 === Chapter Chapter 1 --- General Introduction --- p.16 === Chapter 1.1 --- An overview of cerebellum functions and anatomy --- p.16 === Chapter 1.2 --- Purkinje cell development in the mouse --- p.19 === Chapter 1.2.1 --- Embryonic development of mouse cerebellum --- p.19 === Chapter 1.2.2 --- Postnatal development of mouse cerebellum --- p.21 === Chapter 1.3 --- Degeneration of Purkinje cell leads to spinocerebellar ataxia --- p.22 === Chapter 1.4 --- LIM-homeodomain genes Lhx1 and Lhx5 --- p.24 === Chapter 1.4.1 --- LIM-homeodomain --- p.24 === Chapter 1.4.2 --- Lhx1 and Lhx5 are crucial to Purkinje cell differentiation --- p.25 === Chapter 1.5 --- Hypothesis, aim and strategy of the study --- p.26 === Chapter Chapter 2 --- Generation of Lhx5 conditional knockout allele in the mouse --- p.31 === Chapter 2.1 --- Chapter summary --- p.31 === Chapter 2.2 --- Introduction --- p.32 === Chapter 2.3 --- Materials and methods --- p.35 === Chapter 2.3.1 --- Materials --- p.35 === Chapter 2.3.2 --- Construction of Lhx5-conditional targeting vector by recombineering --- p.39 === Chapter 2.3.3 --- Gene targeting in mouse embryonic stem cells --- p.53 === Chapter 2.3.4 --- Generation of Lhx5 CKO mouse --- p.62 === Chapter 2.3.5 --- Histological examination of Lhx5 CKO mouse brain --- p.63 === Chapter 2.4 --- Results --- p.64 === Chapter 2.4.1 --- Generation of Lhx5 conditional targeting construct --- p.64 === Chapter 2.4.2 --- Screening of targeted ES cell clones --- p.64 === Chapter 2.4.3 --- Karyotyping --- p.65 === Chapter 2.4.4 --- Generation of chimeric mice and maintenance of Lhx5 CKO mice --- p.66 === Chapter 2.4.5 --- Histological examination of Lhx5 recombined null mutant mouse --- p.67 === Chapter 2.4.6 --- Gross anatomical examination of Lhx5 recombined null mutant mouse --- p.69 === Chapter 2.5 --- Discussion --- p.71 === Chapter Chapter 3 --- Generation and characterization of Pcp2-CreER[superscript T]² transgenic mouse --- p.75 === Chapter 3.1 --- Chapter summary --- p.75 === Chapter 3.2 --- Introduction --- p.76 === Chapter 3.3 --- Materials and methods --- p.79 === Chapter 3.3.1 --- Materials --- p.79 === Chapter 3.3.2 --- Construction of pPcp2-IRES-CreER[superscript T]²-FRT-Kan-FRT --- p.81 === Chapter 3.3.3 --- Generation of BAC-Pcp2-IRES-CreER[superscript T]² transgene --- p.85 === Chapter 3.3.4 --- Generation of Pcp2-CreER[superscript T]² transgenic mice --- p.90 === Chapter 3.3.5 --- Characterization of Pcp2-CreER[superscript T]² transgenic mice --- p.91 === Chapter 3.4 --- Results --- p.93 === Chapter 3.4.1 --- Construction of BAC-Pcp2-IRES-CreER[superscript T]² --- p.93 === Chapter 3.4.2 --- Production of Pcp2-CreER[superscript T]² transgenic mice --- p.93 === Chapter 3.4.3 --- Expression of Cre recombinase in Pcp2-CreER[superscript T]² transgenic mice --- p.93 === Chapter 3.4.4 --- Histological examination of Pcp2-CreER[superscript T]² transgenic mice --- p.97 === Chapter 3.4.5 --- Behavioral test of Pcp2-CreER[superscript T]² transgenic mice by rotarod --- p.98 === Chapter 3.5 --- Discussion --- p.100 === Chapter Chapter 4 --- Characterization of Lhx1/5 double conditional knockout mouse --- p.103 === Chapter 4.1 --- Chapter summary --- p.103 === Chapter 4.2 --- Introduction --- p.104 === Chapter 4.3 --- Materials and methods --- p.106 === Chapter 4.3.1 --- Mouse strain --- p.106 === Chapter 4.3.2 --- Behavioral tests --- p.106 === Chapter 4.3.3 --- Histological examination of cerebellum --- p.107 === Chapter 4.3.4 --- CreER[superscript T]² induction by tamoxifen --- p.108 === Chapter 4.3.5 --- Gene expression profiling using microarray --- p.109 === Chapter 4.3.6 --- Transmission electron microscopy --- p.110 === Chapter 4.3.7 --- Statistical analysis --- p.111 === Chapter 4.4 --- Results --- p.112 === Chapter 4.4.1 --- Early postnatal developmental delay in female DKO mutant --- p.112 === Chapter 4.4.2 --- Lhx1/5 DKO mutants displayed significant motor deficit --- p.114 === Chapter 4.4.3 --- Abnormal Purkinje cell dendritic arborization in the adult Lhx1/5 DKO mutant mouse --- p.117 === Chapter 4.4.4 --- Reduction in the number of synaptic vesicles in the adult Lhx1/5 DKO mutant --- p.119 === Chapter 4.4.5 --- Abnormal Purkinje cell dendrite development in the Lhx1/5 DKO mutant mouse --- p.120 === Chapter 4.4.6 --- Lhx1/5 were not required for the maintenance of developed Purkinje cell dendrite --- p.122 === Chapter 4.4.7 --- Comparison of gene expression profiles in the Lhx1/5 DKO mutant and control --- p.128 === Chapter 4.5 --- Discussion --- p.130 === Chapter Chapter 5 --- Foxp4 - a potential interacting partner of Lhx1/5 --- p.137 === Chapter 5.1 --- Chapter summary --- p.137 === Chapter 5.2 --- Introduction --- p.138 === Chapter 5.3 --- Materials and methods --- p.139 === Chapter 5.3.1 --- Co-immunoprecipitation --- p.139 === Chapter 5.3.2 --- Foxp4 expression pattern and knockdown in cerebellar slice culture --- p.140 === Chapter 5.3.3 --- Generation of Foxp4 CKO mouse --- p.146 === Chapter 5.4 --- Results --- p.148 === Chapter 5.4.1 --- Lhx1 and Lhx5 physically interacted with Foxp4 --- p.148 === Chapter 5.4.2 --- Foxp4 expression during mouse cerebellum development --- p.150 === Chapter 5.4.3 --- Effective gene silencing by siRNA in cerebellar slice culture --- p.151 === Chapter 5.4.4 --- Silencing gene expression of Foxp4 at P5 exerted no observable effect on Purkinje cell survival or differentiation --- p.154 === Chapter 5.4.5 --- Developed Purkinje cell dendritic arbors and associated Bergmann glial fibers were impaired when Foxp4 was knockdown at P10 --- p.157 === Chapter 5.4.6 --- Generation of Foxp4 targeting construct and conditional knockout mouse --- p.159 === Chapter 5.4.7 --- Developmental delay of cerebellum in Foxp4 recombined homozygous mutants --- p.163 === Chapter 5.4.8 --- Normal cerebellum development in adult En1-Cre; Foxp4[superscript fx/fx] and Pax2-Cre; Foxp4[superscript fx/fx] mutants --- p.165 === Chapter 5.4.9 --- Purkinje cell-specific knockout of Foxp4 did not impair Purkinje cell maintenance, motor activity and learning --- p.167 === Chapter 5.5 --- Discussion --- p.171 === Chapter 5.6 --- Acknowledgements --- p.177 === Chapter Chapter 6 --- General discussion, future works and conclusion --- p.179 === Chapter 6.1 --- Evolutionary conserved function of Lhx1 and Lhx5 in neurons --- p.180 === Chapter 6.2 --- LHX1 and LHX5 in human diseases --- p.181 === Chapter 6.3 --- Transcription complex between LIM-homeodomain and forkhead domain proteins may be important in the cerebellum development --- p.182 === Chapter 6.4 --- Future works --- p.183 === Chapter 6.5 --- Conclusion --- p.186 === References --- p.187