微小核醣核酸群及微小核醣核酸-34所調控的基因對影響果蠅蘑菇體神經發育的研究

Abstract

探討神經發育(Neural development)的分子調控機制對了解如何建構健全的神經系統極其重要。本論文利用黃果蠅(Drosophila melanogaster)掌管學習與記憶功能的核心結構-蘑菇體(Mushroom body)-研究以下三個主題：神經元命運特化(Neuronal fate specification)，樹突/軸突生長導向(Dendrite/Axon guidance)和神經重塑(Neuronal remodeling)。它們在神經發育扮演重要角色，因為神經幹細胞(Neural stem cell)決定它的子神經元(Daughter neuron)的細胞命運特化後，神經元的樹突/軸突會依特定方向性延展至正確位置，最後當個體逐漸發育成熟時，神經重塑使得特定的神經元具有完整功能或獲取新的功能。 果蠅估計約有13,600個基因，逐一篩選每一個基因是否影響神經發育，需要花費龐大的時間、人力及資源，由於微小核醣核酸(microRNA/miR)能同時對多個基因進行敲落(Knockdown)，使得多重基因表現量下降，因此本論文研究透過過量表現miR對蘑菇體的發育影響作為快速篩選。果蠅目前已知有147個miRs，本論文針對137個miRs過量表現在果蠅蘑菇體中，發現有74個miRs會造成蘑菇體的異常表現型(Phenotype)，其中的25個miRs為本論文感興趣與神經元命運特化，樹突/軸突生長導向及神經重塑相關。 先前本人所參與的一篇文獻指出，過量表達miR-34會導致神經重塑的缺失。本論文透過生物資訊學方式預測出可能被miR-34影響的目標基因(198個基因)，再針對這其中的147基因個別地進行敲落。其中TGF-β受體baboon (babo)基因為miR-34的預測目標基因之一，而過往的研究已經指出babo可調控脫皮激素受體B1 (Ecdysone receptor B1)的表現進而掌控蘑菇體神經重塑。本人所參與的另一篇文獻，由過量表達miR-34以及mir-34基因突變的實驗結果，也證實miR-34的確會調控Babo的表現，所以此調控機制極可能是過量表達miR-34導致蘑菇體的神經重塑缺失的主因之一。在此文獻中，本人也是第一個將Babo在果蠅大腦的各個區域的表現，做了完整的描述。除了babo基因之外，maternal gene required for meiosis (mamo)基因可能是另一個miR-34目標基因，當敲落mamo之後同樣顯示出蘑菇體神經重塑缺失的結果，有趣的是，除了神經重塑外， mamo亦主導著蘑菇體神經元的命運特化，然而mamo基因如何同時參與在神經重塑和細胞命運特化的兩個過程，還需進一步地去釐清。 總之，本論文研究利用過量表現miR來快速篩選對於果蠅蘑菇體神經元發育的影響，進一步選擇欲研究的表現型，然後再去找尋出受到特定的miR調控的可能目標基因，藉此有效率的方式，希望能夠在短時間內找出感興趣的基因以便進行研究分析，最終來瞭解影響神經發育的基因及分子調控的機制。

Investigation of molecular mechanisms underlying neural development is important for the understanding of the construction of the functional nervous system. Mushroom bodies (MBs) of Drosophila melanogaster, which execute the essential function in learning and memory, were used in this thesis to address three major issues of neural development, including neuronal fate specification, dendrite/axon guidance and neuronal remodeling. The importance of these processes is as follows: once daughter neurons, derived from neural stem cells, specify their cell fate, they extend dendrites/axons to proper locations to make neuronal connections. Sometimes, neurons undergo the neuronal remodeling process to remake new neuronal connections at the late developmental stage in order to form the functional nervous system. Since Drosophila contains roughly 13,600 genes, it is time consuming and labor intensive to examine each gene one by one for the causal effect on neural development. In this thesis, by taking advantage of the potential ability of microRNAs (miRs, a class of non-coding RNAs) in knocking down the expression of multiple genes, miRs were overexpressed for their causal effects on MB development. 137 miRs out of 147 known miRs in Drosophila were overexpressed and 74 miRs were found to cause abnormal MB phenotypes. Among those 74 miRs, 25 of them potentially affected neuronal fate specification, dendrite/axon guidance and neuronal remodeling. A previous report (with my contribution) has shown that miR-34 overexpression resulted in neuronal remodeling defect in MB neurons. In this thesis, through the bioinfomatics approach, 147 out of 198 predicted miR-34 target genes were selected for examining their possible roles in MB neuronal remodeling. A putative miR-34 target gene, baboon (babo) encoding a TFG-β receptor, is known for its role in MB neuronal remodeling by regulating the expression of ecdysone receptor B1. A recent report (also with my contribution) via loss- and gain-of-function studies of miR-34 has demonstrated that miR-34 indeed regulated the expression of Babo and the MB neuronal remodeling defect induced by miR-34 overexpression was likely due to downregulation of Babo. In this report, I, for the first time, comprehensively depicted the Babo expression pattern in the Drosophila brain. In addition to babo, maternal gene required for meiosis (mamo), the other putative miR-34 target gene, was found to participate not only in neuronal remodeling but also in neuronal fate specification of MB neurons. Future investigation is needed for deciphering on how mamo regulates both neuronal remodeling and neuronal fate specification in MB neurons. In conclusion, in this thesis, overexpression of miRs was used to quickly identify the ones that affect MB development. Followed by searching for target genes of those identified miRs of interest that affect MB development, this thesis provides an alternative approach to quickly identify genes and molecules of interest to investigate the mechanisms underlying neural development.