斑馬魚cdk10, diras1a和diras1b基因選殖、表現模式與功能性分析

Expression pattern and biological function of zebrafish cyclin-dependent kinase 10, diras1a and diras1b gene

葉期瑋(Chi-Wei Yeh)

指導教授 ： 許立松

中山醫學大學/醫學院/生化暨生物科技研究所/博士(2014年)

https://doi.org/10.6834/CSMU.2014.00166

摘要

在本篇研究中，我們利用斑馬魚作為脊椎動物模式來探討CDK10基因在發育上角色。透過原位雜交實驗指出，CDK10基因大量表現於腦部區域。相似於其哺乳動物的同源基因，CDK10蛋白能與轉錄因子ETS2相互結合，並藉由磷酸化組蛋白H1證實CDK10具有激酶活性。利用反譯核酸抑制內生性cdk10基因的表現進一步顯示會引發SOX2陽性細胞的凋亡，並降低下游神經元指標基因表達。透過乙醯化微管蛋白染色實驗揭示在cdk10 morphants組別中，Rohon-Beard感覺神經元的數量顯著地降低。而上述的實驗結果與islet2基因在背側區域表達下降是相輔相成的。此外，藉由觀察端腦區與整個脊髓皆可發現在cdk10 morphants的組別中，huC陽性神經元具有顯著性地缺失。同時，在cdk10 morphants組別中的視網膜神經節細胞的分布亦有減少的現象。然而，這些表徵可由共同注射cdk10 mRNA而回復如同控制組一般。有趣的是，降低內生性cdk10基因的表現顯著地升高raf1a mRNA的表達。同時，藉由預先處理MEK抑製劑（U0126）可明顯回復cdk10 morphants中SOX2和ngn1基因的轉錄水平。本篇研究揭露cdk10基因在脊椎動物發育過程中的功能性特性且證實cdk10基因藉由調控raf1a基因的表達並顯示其對於神經新生中的重要性。
Small GTPase Ras superfamily在調控細胞增生，細胞移動，細胞骨架重組與細胞凋亡等過程中扮演其重要角色。在本篇研究中，我們鑑定並選殖出斑馬魚的distinct ras 1a和distinct ras 1b（diras1a和diras1b）基因。透過序列比對發現，diras1a和diras1b的氨基酸序列與其哺乳動物的同源基因具有高度的同源性。藉由反轉錄聚合酶連鎖反應分析指出diras1a和diras1b基因的表現起始於受精後第12小時，且表現量在受精後第24小時持續表達。原位雜交實驗揭露diras1a與diras1b基因皆大量表現在腦部區域和視網膜神經節細胞層中，其中，兩者的分佈位置具有獨特性與重疊區。在細胞實驗中利用過度表達diras1a或diras1b基因在小鼠的神經母細胞瘤(Neuroblastoma)中會促進神經元細胞的極化。利用西方墨點法分析顯示diras1a或diras1b基因皆可下調RhoA蛋白的表達，相對地，上調Rac1蛋白的表現量。其中，當細胞大量表達野生型diras1a或diras1b基因亦促使Rac1與PAK1的結合；然而，此現象在diras1a或diras1b基因突變的組別中是缺乏的。共同表達失活的CDK5, Pak1或預先處理ARP2/3抑製劑（CK-548）皆可抑制由diras1a和diras1b基因所誘導的神經元分化現象。在斑馬魚模式中，利用反譯核酸抑制內生性diras1基因的表現不僅減少軸突的導向，亦引起三叉神經節數量的缺失；其中，並不會影響神經前驅細胞的指標基因，如ngn1和neuroD。進一步藉由共同注射小鼠的Diras1基因或人類的持續性活化態Rac1基因可顯著性回復三叉神經節數量的缺失。總結，本篇研究證實diras1基因透過Rac1依賴性路徑參與其神經元的分化及維持三叉神經節數目。

關鍵字

斑馬魚 ； 神經發育

並列摘要

In this study, we used zebrafish as an animal model to elucidate the developmental function of cdk10 in vertebrates. In situ hybridization analyses demonstrated that cdk10 is expressed throughout development with a relative enrichment in the brain in the late stages. Similar to its mammalian ortholog, cdk10 can interact with the transcription factor ETS2 and exhibit kinase activity by phosphorylating histone H1. Morpholino-based loss of cdk10 expression caused apoptosis in sox2-positive cells and decreased the expression of subsequent neuronal markers. Acetylated tubulin staining revealed a significant reduction in the number of Rohon-Beard sensory neurons in cdk10 morphants. This result is similar to that demonstrated by decreased islet2 expression in the dorsal regions. Moreover, cdk10 morphants exhibited a marked loss of huC-positive neurons in the telencephalon and throughout the spinal cord axis. The population of retinal ganglion cells was also diminished in cdk10 morphants. These phenotypes were rescued by co-injection of cdk10 mRNA. Interestingly, the knockdown of cdk10 significantly elevated raf1a mRNA expression. Meanwhile, an MEK inhibitor (U0126) recovered sox2 and ngn1 transcript levels in cdk10 morphants. Our findings provide the first functional characterization of cdk10 in vertebrate development and reveal its critical function in neurogenesis by modulation of raf1a expression.
The small GTPase Ras superfamily of proteins plays an important role in cell proliferation, cell migration, cytoskeleton reorganization, and apoptosis. In the present study, we identified zebrafish distinct ras 1a and 1b (diras1a and diras1b) genes. The protein sequences of diras1a and diras1b displayed high homology with their mammalian orthologues. RT-PCR analysis indicated that both genes were first expressed 12 hours post-fertilization (HPF), enriched at 24 HPF and persistently expressed thereafter. Whole mount in situ hybridization revealed that each gene is abundantly expressed in the brain regions and the ganglion cell layer in the retina with unique and overlapping patterns. Forced expression of diras1a and diras1b in mouse Neuro-2a cells triggered neuronal polarization. Western blot analysis revealed that both diras1a and diras1b downregulated RhoA expression, and in opposite, upregulated of Rac1 expression. Increased interaction of Rac1 with Pak1 (p21-activated kinase 1) was found in cells overexpressing wild type but not truncated diras1a and diras1b. Co-expression of dominant negative CDK5, Pak1 or pre-treated with an Arp 2/3 inhibitor (CK-548) attenuated diras1a- and diras1b-induced neuronal polarization. In zebrafish model, knockdown of diras1a and/or diras1b by morpholino antisense oligonucleotides not only reduced axon guidance but also caused the loss of trigeminal ganglion without affecting the precursor markers such as ngn1 and neuroD. Co-injection with mRNA derived from mouse Diras1 or constitutively active human Rac1 restored the population of trigeminal ganglion. In conclusion, we have provided the first evidences that diras1 is involved in neuronal differentiation and maintains the number of trigeminal ganglions through the Rac1-dependent pathway.

並列關鍵字

zebrafish ； neurodevelopment

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