Brain patterning defects caused by mutations of the twin of eyeless gene in Drosophila melanogaster.

The Drosophila Pax6 genes, eyeless (ey) and twin of eyeless (toy), are expressed in both eyes and the brain. Previous studies have demonstrated that ey plays important roles in axonal outgrowth and differentiation of mushroom bodies (MBs), which are centers for associative learning and memory in flies. However, the functional significance of toy in brain development is poorly understood. Here, we describe the expression patterns of TOY, and show that TOY expression partially overlaps with EY expression in the embryonic, larval and adult brains. Mutations of toy perturb brain neuromere formation in the embryonic stages, and result in severe deformation of the MB lobes in pharate adult brains. Moreover, we also analyzed toy functions by gain-of-function experiments, and show that overexpression of toy results in degeneration of MB lobes. Thus, our results demonstrate the importance of toy in embryonic brain patterning as well as in post-embryonic development of the major brain structures such as MBs.

The Drosophila fragile X mental retardation gene regulates sleep need.

Sleep need is affected by developmental stage and neuronal plasticity, but the underlying mechanisms remain unclear. The fragile X mental retardation gene Fmr1, whose loss-of-function mutation causes the most common form of inherited mental retardation in humans, is involved in synaptogenesis and synaptic plasticity, and its expression depends on both developmental stage and waking experience. Fmr1 is highly conserved across species and Drosophila mutants carrying dFmr1 loss-of-function or gain-of-function mutations are well characterized: amorphs have overgrown dendritic trees with larger synaptic boutons, developmental defects in pruning, and enhanced neurotransmission, while hypermorphs show opposite defects, including dendritic and axonal underbranching and loss of synapse differentiation. We find here that dFmr1 amorphs are long sleepers and hypermorphs are short sleepers, while both show increased locomotor activity and shortened lifespan. Both amorphs and hypermorphs also show abnormal sleep homeostasis, with impaired waking performance and no sleep rebound after sleep deprivation. An impairment in the circadian regulation of sleep cannot account for the altered sleep phenotype of dFmr1 mutants, nor can an abnormal activation of glutamatergic metabotropic receptors. Moreover, overexpression of dFmr1 throughout the mushroom bodies is sufficient to reduce sleep. Finally, dFmr1 protein levels are modulated by both developmental stage and behavioral state, with increased expression immediately after eclosure and after prolonged wakefulness. Thus, dFmr1 expression dose-dependently affects both sleep and synapses, suggesting that changes in sleep time in dFmr1 mutants may derive from changes in synaptic physiology.

Differential microarray analysis of Drosophila mushroom body transcripts using chemical ablation.

Mushroom bodies (MBs) are the centers for olfactory associative learning and elementary cognitive functions in the Drosophila brain. As a way to systematically elucidate genes preferentially expressed in MBs, we have analyzed genome-wide alterations in transcript profiles associated with MB ablation by hydroxyurea. We selected 100 genes based on microarray data and examined their expression patterns in the brain by in situ hybridization. Seventy genes were found to be expressed in the posterodorsal cortex, which harbors the MB cell bodies. These genes encode proteins of diverse functions, including transcription, signaling, cell adhesion, channels, and transporters. Moreover, we have examined developmental functions of 40 of the microarray-identified genes by transgenic RNA interference; 8 genes were found to cause mild-to-strong MB defects when suppressed with a MB-Gal4 driver. These results provide important information not only on the repertoire of genes that control MB development but also on the repertoire of neural factors that may have important physiological functions in MB plasticity.

Defective neuronal development in the mushroom bodies of Drosophila fragile X mental retardation 1 mutants.

Fragile X mental retardation 1 (Fmr1) is a highly conserved gene with major roles in CNS structure and function. Its product, the RNA-binding protein FMRP, is believed to regulate translation of specific transcripts at postsynaptic sites in an activity-dependent manner. Hence, Fmr1 is central to the molecular mechanisms of synaptic plasticity required for normal neuronal maturation and cognitive ability. Mutations in its Drosophila ortholog, dfmr1, produce phenotypes of brain interneurons and axon terminals at the neuromuscular junction, as well as behavioral defects of circadian rhythms and courtship. We hypothesized that dfmr1 mutations would disrupt morphology of the mushroom bodies (MBs), highly plastic brain regions essential for many forms of learning and memory. We found developmental defects of MB lobe morphogenesis, of which the most common is a failure of beta lobes to stop at the brain midline. A similar recessive beta-lobe midline-crossing phenotype has been previously reported in the memory mutant linotte. The dfmr1 MB defects are highly sensitive to genetic background, which is reminiscent of mammalian fragile-X phenotypes. Mutations of dfmr1 also interact with one or more third-chromosome loci to promote alpha/beta-lobe maturation. These data further support the use of the Drosophila model system for study of hereditary cognitive disorders of humans.