進(jìn)化上高度保守的Wnt/Wingless信號(hào)通路在動(dòng)物的器官發(fā)育,、能量代謝和干細(xì)胞維持等過程中發(fā)揮重要作用，并與多種疾病的發(fā)生有密切聯(lián)系,。然而,，迄今為止，人們對(duì)于該通路的信號(hào)轉(zhuǎn)導(dǎo)機(jī)制還缺少充分的認(rèn)識(shí)。為了探明其中未知的調(diào)控機(jī)制,，中科院上海生命科學(xué)研究院營養(yǎng)科學(xué)研究所宋海云研究組與瑞士蘇黎世大學(xué)Basler研究組通過聯(lián)合研究,，利用轉(zhuǎn)基因果蠅庫進(jìn)行遺傳學(xué)篩選，從中發(fā)現(xiàn)了Wnt/Wingless信號(hào)通路的新調(diào)節(jié)因子并揭示了其中的調(diào)控機(jī)理,。該研究成果Systematic screening of a Drosophila ORF library in vivo uncovers Wnt/Wg pathway components近日在線發(fā)表于國際學(xué)術(shù)期刊Developmental Cell,。
果蠅由于其豐富的遺傳操縱手段和短暫的生命周期，經(jīng)常被用作篩選新基因的體內(nèi)模型,。然而,，目前流行的RNAi篩選常會(huì)遇上以下困難：不能發(fā)現(xiàn)功能冗余基因；敲低基因功能的效率不一,；脫靶（off-target）影響其它基因功能,。針對(duì)這些問題，Basler研究組挑選了與細(xì)胞生長相關(guān)的基因,，逐一克隆和制備轉(zhuǎn)基因過表達(dá)果蠅,，并用于篩選Wnt/Wingless信號(hào)通路中新的調(diào)控基因。
在初步篩選的基礎(chǔ)上,，宋海云研究組對(duì)發(fā)現(xiàn)的候選基因進(jìn)行了驗(yàn)證,，確定了三個(gè)新調(diào)節(jié)因子。博士生黃大舜和尹定子對(duì)其中一個(gè)調(diào)節(jié)因子Nek2的調(diào)控機(jī)理進(jìn)行了深入研究,，發(fā)現(xiàn)Nek2通過磷酸化修飾關(guān)鍵蛋白Dishevelled影響信號(hào)的傳遞,。有趣的是，Nek2對(duì)Dishevelled的磷酸化有雙重作用：對(duì)其N端的修飾能增強(qiáng)Dishevelled活性和促進(jìn)信號(hào)傳遞,；在信號(hào)強(qiáng)度超過一定閾值后通過對(duì)其C端的多重磷酸化介導(dǎo)Dishevelled的降解和抑制信號(hào)通路的過度激活,。通過這種雙重調(diào)控，既能在信號(hào)通路開啟時(shí)快速傳遞信號(hào),，又能防止信號(hào)通路不適當(dāng)?shù)爻掷m(xù)激活,，從而保證Wingless信號(hào)通路對(duì)下游基因表達(dá)的精細(xì)調(diào)控。最后,，黃大舜還找到了與Nek2有冗余功能的基因dco,，在同時(shí)降低Nek2和dco的功能時(shí)，會(huì)嚴(yán)重阻礙Wingless信號(hào)通路下游基因的表達(dá)和果蠅的器官發(fā)育,。
這些研究得到了中國科學(xué)院,、國家自然科學(xué)基金和科技部重大研究計(jì)劃的資助。（生物谷Bioon.com）
doi:10.1016/j.devcel.2013.02.019
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Systematic Screening of a Drosophila ORF Library In Vivo Uncovers Wnt/Wg Pathway Components
Claus Schertel1, 7, Dashun Huang2, 5, 7, Mikael Björklund3, 4, Johannes Bischof1, Dingzi Yin5, Rongxia Li5, Yi Wu5, Rong Zeng5, Jiarui Wu2, 5, Jussi Taipale4, 6, Haiyun Song5, , , Konrad Basler1, ,
We created a site-directed UAS-ORF library of 655 growth-regulating genes in Drosophila. This library represents a large collection of genes regulating cell cycle, cell size, and proliferation and will be a valuable resource for studying growth regulation in vivo. By using misexpression of genes, we prevent problems arising from genetic redundancy and can uncover novel gene functions. To validate the usefulness of this library, we screened for Wingless (Wg) pathway components. We used a combination of experimental and bioinformatic approaches to predict candidates and identified three serine/threonine kinases as regulators of Wg signaling. We show that one of these, Nek2, optimizes pathway response by direct phosphorylation of Dishevelled. In addition, we describe functional relations for roughly 5% of all Drosophila genes and identify a large number of genes that regulate cell size, proliferation, and final organ size upon misexpression.
