Generation of GAL4-responsive muscleblind constructs.

Abstract

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 white using standard germline transformation methods (Spradling and Rubin, 1982). We established five independent transgenic lines carrying UAS-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 polarity (PCP) defects but also, especially when the UAS-mbl C was overexpressed, photoreceptor loss. In a similar set of experiments, we targeted expression of all three transgenes to the wing imaginal disc using 69B-Gal4 or apterous-Gal4 as drivers (Fig. 2H–K). Offspring from these crosses revealed defects in the mor-