Generation of GAL4-responsive muscleblind constructs
Generation of GAL4-responsive muscleblind constructs
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DOI：
10.1002/gene.10147
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发表时间：
2002-09-01
期刊：
影响因子：
1.5
通讯作者：
Pérez-Alonso, M
中科院分区：
文献类型：
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作者：
García-Casado, Z;Artero, RD;Pérez-Alonso, M
肌肉盲(MBL)基因编码蛋白质亚型MBLA到MBL D，这两种亚型通过从共同的初级转录本进行选择性剪接而产生。MBL A、B和C包含两个Cys3His类型的锌指结构域，而MBL D只包含一个完整的锌指。该基因的功能缺失突变表明，MBL参与了果蝇的终末光感受器和肌肉分化。在视网膜发育过程中，MBL是光感受器神经元横纹状体分化所必需的。MBL纯合零的克隆完全缺乏这些轻归属结构(Begemann等人，1997年)。同样，在MBL突变胚胎中，幼虫体壁肌肉的末端分化受到损害。在这些突变的胚胎中，肌肉显示肌节具有杂乱的Z线，间接肌肉附着的细胞外基质减少(Artero等人，1998)。此外，旨在了解强直性肌营养不良(DM)这种显性常染色体疾病的分子基础的生化研究发现，人类MBL同源基因MBNL和MBLL是DM发病途径中的关键元素(Miller等，2000)。然而，尽管进行了大量的研究，但MBL蛋白的分子功能尚未被阐明。目前的工作模型表明，MBNL/MBLL蛋白在RNA代谢中发挥作用，可能是在前mRNA剪接或mRNA核输出过程中。这些模型主要由观察到的MBNL/MBLL蛋白与CUG和CCUG扩展结合支持(Miller等人，2000；Mankodi等人，2001；Fardaei等人，2002)。为了产生苍蝇功能缺陷的获得以及用于人类疾病果蝇模型的遗传工具，我们制作了三个UAS结构，包含果蝇蛋白亚型MBLA和MBLC，以及由cDNAKIAA0428编码的人MBNL蛋白异构体，其中包括MBNL基因座上大多数可供选择使用的外显子(Fardaei等人，2002年)。使用标准的种系转化方法(Spradling和Rubin，1982)将UAS构建物(图1)注射到白色1118基因型的果蝇胚胎中。我们建立了5个携带UAS-MBLC的转基因株系、6个携带UAS-MBLC的转基因株系和3个携带UAS-KIAA0428的转基因株系。通过遗传杂交将这些品系定位到指定的染色体上(图1)。通过将UAS-MBL A和UAS-MBL C与Engraded-Gal4驱动器杂交，获得了Gal4驱动这些结构物表达的直接实验证据，这给出了明显不同于内源性MBL转录的片段极性表达模式。在这些胚胎中，原位杂交清楚地检测到由增强启动子驱动的异位MBL表达(图2A-C)。此外，转基因的表达针对成体结构的前体，以揭示其生物学活性的形态缺陷。在内源性Seven less启动子(sev-Gal4)的驱动下，将MBLA和MBLC靶向表达到果蝇的眼睛想象盘上，导致了粗糙的眼睛表型，从UAS-MBLA的非常轻微的缺陷到UAS-MBLC的明显粗糙的眼睛(图2D-F)。UAS-KIAA0428在类似条件下的表达会导致严重粗糙的眼睛表型和致死性，这取决于所使用的转基因。我们使用了我们最弱的转基因(UAS-KIAA0428)。KD。1)和25℃的孵化温度，以获得可存活的后代和更温和的眼睛表型(图2G)。通过这些粗糙的眼睛的切面显示了细胞水平的形态缺陷。在所有病例中，我们都检测到小眼显示典型的平面细胞-…
The muscleblind (mbl) gene encodes protein isoforms Mbl A to Mbl D, which arise by alternative splicing from a common primary transcript. Mbl A, B, and C contain two Zn-finger domains of the type Cys3His, while Mbl D contains only one complete Zn finger. Loss of function mutations in the gene reveal that mbl is involved in both terminal photoreceptor and muscle differentiation in Drosophila. During retina development mbl is essential for rhabdomere differentiation in photoreceptor neuron. Clones homozygous null for mbl completely lack these lightharvesting structures (Begemann et al., 1997). Similarly, the terminal differentiation of the larval body wall muscles is compromised in mbl mutant embryos. In these mutant embryos muscles show sarcomeres with disorganized Z-lines and reduction in the extracellular matrix of indirect muscle attachments (Artero et al., 1998). In addition, biochemical studies aimed at understanding the molecular basis of myotonic dystrophy (DM), a dominant-autosomic disease, identified the human mbl paralogs MBNL and MBLL as critical elements in the DM pathogenesis pathway (Miller et al., 2000). Despite intense research, however, the molecular function of Mbl proteins has not yet been elucidated. The current working models point to a role for MBNL/MBLL proteins in RNA metabolism, possibly during pre-mRNA splicing or mRNA nuclear export. These models are supported mainly by the observation that MBNL/MBLL proteins bind to CUG and CCUG expansions (Miller et al., 2000; Mankodi et al., 2001; Fardaei et al., 2002). To generate both gain of function defects in the fly as well as genetic tools to use in a Drosophila model for the human disease, we made three UAS constructs containing the Drosophila protein isoforms Mbl A and Mbl C and the human MBNL protein isoform encoded by the cDNA KIAA0428, which includes most of the alternatively used exons in the MBNL locus (Fardaei et al., 2002). UAS constructs (Fig. 1) were injected into fly embryos of the genotype white1118 using standard germline transformation methods (Spradling and Rubin, 1982). We established five independent transgenic lines carryingUAS-mbl A, six transgenic lines carrying UAS-mbl C, and three transgenic lines carrying UAS-KIAA0428. These lines were mapped by genetic crosses to the indicated chromosomes (Fig. 1). Direct experimental evidence of Gal4-driven expression for these constructs was obtained by crossing UAS-mbl A and UAS-mbl C to the engrailed-Gal4 driver, which gives a segment polarity pattern of expression clearly different from the endogenous mbl transcription. In situ hybridization in these embryos clearly detected ectopic mbl expression driven by the engrailed promoter (Fig. 2A–C). In addition, expression of the transgenes was targeted to the precursors of adult structures to reveal morphological defects indicative of their biological activity. Targeted expression of Mbl A and Mbl C to the Drosophila eye imaginal disc driven by an endogenous sevenless promoter (sev-Gal4) led to a rough eye phenotype which ranged from a very mild defect in the case of UAS-mbl A to a clearly rough eye in the case of UAS-mbl C (Fig. 2D–F). Expression of UAS-KIAA0428 under similar conditions led to a severely rough eye phenotype and lethality, depending on the transgene used. We used our weakest transgene (UAS-KIAA0428. KD. 1) and an incubation temperature of 25 C for these crosses in order to obtain viable offspring and a milder eye phenotype (Fig. 2G). Tangential sections through these rough eyes revealed morphological defects at the cellular level. In all cases we detected ommatidia showing typical planar cell …