Control of dendritic development by the Drosophila fragile X-related gene involves the small GTPase Rac1

Alan Lee 1 Wenjun Li 0 1 Kanyan Xu 0 1 Brigitte A. Bogert 0 1 Kimmy Su 1 Fen-Biao Gao ) 0 1 0 Neuroscience Graduate Program, University of California , San Francisco, San Francisco, CA 94141-9100, USA 1 Gladstone Institute of Neurological Disease, University of California , San Francisco, San Francisco, CA 94141-9100, USA - Fragile X syndrome is caused by loss-of-function mutations in the fragile X mental retardation 1 gene. How these mutations affect neuronal development and function remains largely elusive. We generated specific point mutations or small deletions in the Drosophila fragile Xrelated (Fmr1) gene and examined the roles of Fmr1 in dendritic development of dendritic arborization (DA) neurons in Drosophila larvae. We found that Fmr1 could be detected in the cell bodies and proximal dendrites of DA neurons and that Fmr1 loss-of-function mutations increased the number of higher-order dendritic branches. Conversely, overexpression of Fmr1 in DA neurons dramatically decreased dendritic branching. In dissecting the mechanisms underlying Fmr1 function in dendrite The normal development of dendritic structures of various neuronal subtypes is essential for the proper function of a nervous system. During the past few years, a number of studies in several model systems have demonstrated the essential roles of neuronal activity, extracellular cues and intrinsic factors in dendritic morphogenesis (for reviews, see McAllister, 2000; Scott and Luo, 2001; Cline, 2001; Jan and Jan, 2001; Whitford et al., 2002; Wong and Ghosh, 2002; Gao and Bogert, 2003). However, it remains largely unknown what limits the growth of a particular neuron, and in particular, the number and length of dendritic branches during development. One family of proteins that may be important in controlling neuronal growth is that of mRNA-binding proteins. These proteins control gene expression at multiple steps of mRNA metabolism, such as splicing, transport, localization, translation and degradation (reviewed by Darnell, 2002; Dreyfuss et al., 2002). Some of these proteins are highly or solely expressed in neurons, and their functions are beginning to be revealed (reviewed by Musunuru and Darnell, 2001; Steward and Schuman, 2001). In this study, we focus on the role of the fly homolog of the fragile X mental retardation 1 (FMR1) gene in dendritic development. The absence of the FMR1 gene activity causes fragile X syndrome, the most common form of inherited mental retardation in humans, with an estimated incidence of 1 in 4000 males and 1 in 8000 females (reviewed by ODonnell and Warren, 2002). FMR1 encodes a putative RNA-binding protein development, we found that the mRNA encoding small GTPase Rac1 was present in the Fmr1-messenger ribonucleoprotein complexes in vivo. Mosaic analysis with a repressor cell marker (MARCM) and overexpression studies revealed that Rac1 has a cell-autonomous function in promoting dendritic branching of DA neurons. Furthermore, Fmr1 and Rac1 genetically interact with each other in controlling the formation of fine dendritic branches. These findings demonstrate that Fmr1 affects dendritic development and that Rac1 is partially responsible for mediating this effect. with two ribonucleoprotein K homology (KH) domains and an arginine- and glycine-rich domain (RGG box). The FMR1 protein preferentially binds to poly(G), poly(U) and a subset of brain mRNAs in vitro (Ashley et al., 1993; Siomi et al., 1993; Brown et al., 1998). In addition, FMR1 is associated with polyribosomes and a large number of mRNAs in vivo, some of which contain G quartet structures as FMR1-binding motifs (Feng et al., 1997a; Corbin et al., 1997; Sung et al., 2000; Darnell et al., 2001; Brown et al., 2001; Zalfa et al., 2003; Miyashiro et al., 2003). It remains to be determined which proteins encoded by mRNAs in FMR1-mRNP complexes are primarily responsible for the morphological and functional deficits caused by the absence of FMR1. The exact molecular function of FMR1 in vivo remains largely unknown. Some studies suggest that FMR1 affects mRNA localization and translation (Darnell et al., 2001; Brown et al., 2001; Zalfa et al., 2003; Miyashiro et al., 2003), although the underlying mechanism is unclear (Antar and Bassell, 2003). The recent demonstration that Drosophila fragile X-related protein (Fmr1; previously dFXR) interacts with components of the RNA interference (RNAi) machinery raises the possibility that Fmr1/FMR1 may also function as part of a gene-silencing mechanism (Ishizuka et al., 2002; Caudy et al., 2002). FMR1 is highly expressed in neuronal perikaryon and dendrites and shuttles between the nucleus and the cytosol (Devys et al., 1993; Fridell et al., 1996; Feng et al., 1997b). Studies of individuals with fragile X syndrome, Fmr1 knockout mice and cultured neurons, although not entirely consistent with each other, raise the possibility that FMR1 is involved in the proper development of spines of central nervous system (CNS) neurons (Hinton et al., 1991; Comery et al., 1997; Braun and Segal, 2000; Nimchinsky et al., 2001). We study the role of Fmr1 in the peripheral nervous system (PNS) of the Drosophila larva, which is relatively simple and consists of 44 sensory neurons in each abdominal hemisegment (Ghysen et al., 1986; Bodmer et al., 1989; Orgogozo et al., 2001). Dendritic arborization (DA) neurons, one subtype of PNS sensory neurons, elaborate extensive dendritic arbors just underneath the epidermis to receive sensory inputs (Bodmer and Jan, 1987; Gao et al., 1999). The ability to visualize the dendritic arbors in living Drosophila larvae allows us to quantitatively examine the effects of Fmr1 on dendritic development of identifiable neurons in vivo. The Fmr1 gene is the only fly homolog of the human FMR1 gene that also has RNA-binding activity in vitro (Adams et al., 2000; Wan et al., 2000). It has been reported that Fmr1 mutation impairs the synaptic function at the neuromuscular junction (Zhang et al., 2001). In addition, Fmr1 is required for normal circadian rhythm of adult flies (Dockendorff et al., 2002; Morales et al., 2002; Inoue et al., 2002). To study the role of Fmr1 in dendritic growth, we isolated Fmr1 protein-deficient mutant fly lines in which specific point mutations or small deletions were introduced into the Fmr1 gene. We report that Fmr1 is expressed in DA sensory neurons and limits dendritic branching during development. In addition, we show that the mRNA encoding the small GTPase Rac1 is present in Fmr1-mRNP complexes and that the function of Fmr1 in dendrite development is partially mediated by Rac1. Materials and methods Fly lines and genetic crosses All the flies were raised at 25C and fed standard food. The P-element insertion line EP(3)3517 was from the Szeged Stock Center (Hungary). Genomic rescue analysis confirmed that the P-element was inserted into the intron between the first and second exon of the Fmr1 gene. The ATG start codon is located in the (...truncated)