Characterization of a morphogenetic furrow specific Gal4 driver in the developing Drosophila eye.
Characterization of a morphogenetic furrow specific Gal4 driver in the developing Drosophila eye.
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DOI：
10.1371/journal.pone.0196365
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发表时间：
2018
期刊：
影响因子：
3.7
通讯作者：
Singh A
中科院分区：
文献类型：
--
作者：
Sarkar A;Gogia N;Farley K;Payton L;Singh A
利用Gal4-UAS系统以时空方式表达感兴趣的基因的能力允许使用果蝇模型来研究各种生物现象。在果蝇眼睛发育过程中，一种称为形态发生沟（MF）的同步分化波在后缘启动，导致视网膜神经元分化。在脊椎动物的分化视网膜中也观察到这种同步分化。由于MF是高度动态的，它可以作为研究模式和分化的良好模型。然而，没有任何Gal4驱动因子可用于观察基因的功能增益或功能丧失，特别是沿着动态MF。十肢截瘫（dpp）基因编码转化生长因子- β (tgf - β)超家族的分泌蛋白，该蛋白在后缘表达，然后随MF移动。然而，与MF相关的dpp基因表达模式不同，dpp- gal4驱动基因的靶向表达仅限于发育中的眼盘的后缘。我们筛选了含有与Gal4编码区融合的dpp调控区域的GMR系，利用GFP报告基因鉴定了dpp的MF特异性增强子。我们采用免疫组织化学方法检测基因表达。其基本原理是GFP报告基因的表达将与发育中的眼睛中的dpp表达域相对应。我们发现了两个新的dpp- gal4系，即GMR17E04-Gal4和GMR18D08-Gal4，它们携带dpp基因第一个内含子区域的序列。GMR17E04-Gal4在发育期间和后期在整个蛹视网膜中沿MF驱动表达，而GMR18D08-Gal4在整个发育中的眼盘中驱动GFP转基因的表达，随后仅在蛹视网膜的腹侧部分驱动表达。因此，GMR18D08-Gal4将作为一种新的靶向蛹视网膜腹侧基因表达的试剂。我们使用GMR17E04-Gal4、GMR18D08-Gal4和现有的dpp-Gal4驱动因子比较了Wg（眼发育负调节因子）的错误表达表型。使用我们新鉴定的MF特异性驱动产生的眼睛表型与现有dpp-Gal4驱动产生的表型不相似。这表明，沿着MF的错误表达研究需要使用我们研究中产生的新的Gal4驱动因子来重新审视。
The ability to express a gene of interest in a spatio-temporal manner using Gal4-UAS system has allowed the use of Drosophila model to study various biological phenomenon. During Drosophila eye development, a synchronous wave of differentiation called Morphogenetic furrow (MF) initiates at the posterior margin resulting in differentiation of retinal neurons. This synchronous differentiation is also observed in the differentiating retina of vertebrates. Since MF is highly dynamic, it can serve as an excellent model to study patterning and differentiation. However, there are not any Gal4 drivers available to observe the gain- of- function or loss- of- function of a gene specifically along the dynamic MF. The decapentaplegic (dpp) gene encodes a secreted protein of the transforming growth factor-beta (TGF-beta) superfamily that expresses at the posterior margin and then moves with the MF. However, unlike the MF associated pattern of dpp gene expression, the targeted dpp-Gal4 driver expression is restricted to the posterior margin of the developing eye disc. We screened GMR lines harboring regulatory regions of dpp fused with Gal4 coding region to identify MF specific enhancer of dpp using a GFP reporter gene. We employed immuno-histochemical approaches to detect gene expression. The rationale was that GFP reporter expression will correspond to the dpp expression domain in the developing eye. We identified two new dpp-Gal4 lines, viz., GMR17E04-Gal4 and GMR18D08-Gal4 that carry sequences from first intron region of dpp gene. GMR17E04-Gal4 drives expression along the MF during development and later in the entire pupal retina whereas GMR18D08-Gal4 drives expression of GFP transgene in the entire developing eye disc, which later drives expression only in the ventral half of the pupal retina. Thus, GMR18D08-Gal4 will serve as a new reagent for targeting gene expression in the ventral half of the pupal retina. We compared misexpression phenotypes of Wg, a negative regulator of eye development, using GMR17E04-Gal4, GMR18D08-Gal4 with existing dpp-Gal4 driver. The eye phenotypes generated by using our newly identified MF specific driver are not similar to the ones generated by existing dpp-Gal4 driver. It suggests that misexpression studies along MF needs revisiting using the new Gal4 drivers generated in our studies.
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