Transient active zone remodeling in the Drosophila mushroom body supports memory

1. Download : Download high-res image (166KB)
2. Download : Download full-size image

     

A mosaic genetic screen for genes necessary for Drosophila mushroom body neuronal morphogenesis

Neurons undergo extensive morphogenesis during development. To systematically identify genes important for different aspects of neuronal morphogenesis, we performed a genetic screen using the MARCM system in the mushroom body (MB) neurons of the Drosophila brain. Mutations on the right arm of chromosome 2 (which contains approximately 20% of the Drosophila genome) were made homozygous in a small subset of uniquely labeled MB neurons. Independently mutagenized chromosomes (4600) were screened, yielding defects in neuroblast proliferation, cell size, membrane trafficking, and axon and dendrite morphogenesis. We report mutations that affect these different aspects of morphogenesis and phenotypically characterize a subset. We found that roadblock, which encodes a dynein light chain, exhibits reduced cell number in neuroblast clones, reduced dendritic complexity and defective axonal transport. These phenotypes are nearly identical to mutations in dynein heavy chain Dhc64 and in Lis1, the Drosophila homolog of human lissencephaly 1, reinforcing the role of the dynein complex in cell proliferation, dendritic morphogenesis and axonal transport. Phenotypic analysis of short stop/kakapo, which encodes a large cytoskeletal linker protein, reveals a novel function in regulating microtubule polarity in neurons. MB neurons mutant for flamingo, which encodes a seven transmembrane cadherin, extend processes beyond their wild-type dendritic territories. Overexpression of Flamingo results in axon retraction. Our results suggest that most genes involved in neuronal morphogenesis play multiple roles in different aspects of neural development, rather than performing a dedicated function limited to a specific process.     

Drosophila Eph receptor guides specific axon branches of mushroom body neurons

The conserved Eph receptors and their Ephrin ligands regulate a number of developmental processes, including axon guidance. In contrast to the large vertebrate Eph/Ephrin family, Drosophila has a single Eph receptor and a single Ephrin ligand, both of which are expressed within the developing nervous system. Here, we show that Eph and Ephrin can act as a functional receptor-ligand pair in vivo. Surprisingly, and in contrast to previous results using RNA-interference techniques, embryos completely lacking Eph function show no obvious axon guidance defects. However, Eph/Ephrin signaling is required for proper development of the mushroom body. In wild type, mushroom body neurons bifurcate and extend distinct branches to different target areas. In Eph mutants, these neurons bifurcate normally, but in many cases the dorsal branch fails to project to its appropriate target area. Thus, Eph/Ephrin signaling acts to guide a subset of mushroom body branches to their correct synaptic targets.     

Testing odor response stereotypy in the Drosophila mushroom body

The mushroom body is an insect brain structure required for olfactory learning. Its principal neurons, the Kenyon cells (KCs), form a large cell population. The neuronal populations from which their olfactory input derives (olfactory sensory and projection neurons) can be identified individually by genetic, anatomical, and physiological criteria. We ask whether KCs are similarly identifiable individually, using genetic markers and whole-cell patch-clamp in vivo. We find that across-animal responses are as diverse within the genetically labeled subset as across all KCs in a larger sample. These results combined with those from a simple model, using projection neuron odor responses as inputs, suggest that the precise circuit specification seen at earlier stages of odor processing is likely absent among the mushroom body KCs.     

Respective roles of the DRL receptor and its ligand WNT5 in Drosophila mushroom body development

In recent decades, Drosophila mushroom bodies (MBs) have become a powerful model for elucidating the molecular mechanisms underlying brain development and function. We have previously characterized the derailed (drl; also known as linotte) receptor tyrosine kinase as an essential component of adult MB development. Here we show, using MARCM clones, a non-cell-autonomous requirement for the DRL receptor in MB development. This result is in accordance with the pattern of DRL expression, which occurs throughout development close to, but not inside, MB cells. While DRL expression can be detected within both interhemispheric glial and commissural neuronal cells, rescue of the drl MB defects appears to involve the latter cellular type. The WNT5 protein has been shown to act as a repulsive ligand for the DRL receptor in the embryonic central nervous system. We show here that WNT5 is required intrinsically within MB neurons for proper MB axonal growth and probably interacts with the extrinsic DRL receptor in order to stop axonal growth. We therefore propose that the neuronal requirement for both proteins defines an interacting network acting during MB development.     

A map of olfactory representation in the Drosophila mushroom body

Neural coding for olfactory sensory stimuli has been mapped near completion in the Drosophila first-order center, but little is known in the higher brain centers. Here, we report that the antenna lobe (AL) spatial map is transformed further in the calyx of the mushroom body (MB), an essential olfactory associated learning center, by stereotypic connections with projection neurons (PNs). We found that Kenyon cell (KC) dendrites are segregated into 17 complementary domains according to their neuroblast clonal origins and birth orders. Aligning the PN axonal map with the KC dendritic map and ultrastructural observation suggest a positional ordering such that inputs from the different AL glomeruli have distinct representations in the MB calyx, and these representations might synapse on functionally distinct KCs. Our data suggest that olfactory coding at the AL is decoded in the MB and then transferred via distinct lobes to separate higher brain centers.     

Foraging behaviour in Drosophila larvae: mushroom body ablation

Drosophila larvae and adults exhibit a naturally occurring genetically based behavioural polymorphism in locomotor activity while foraging. Larvae of the rover morph exhibit longer foraging trails than sitters and forage between food patches, while sitters have shorter foraging trails and forage within patches. This behaviour is influenced by levels of cGMP-dependent protein kinase (PGK) encoded by the foraging (for) gene. Rover larvae have higher expression levels and higher PGK activities than do sitters. Here we discuss the importance of the for gene for studies of the mechanistic and evolutionary significance of individual differences in behaviour. We also show how structure-function analysis can be used to investigate a role for mushroom bodies in larval behaviour both in the presence and in the absence of food. Hydroxyurea fed to newly hatched larvae prevents the development of all post-embryonically derived mushroom body (MB) neuropil. This method was used to ablate MBs in rover and sitter genetic variants of foraging to test whether these structures mediate expression of the foraging behavioural polymorphism. We found that locomotor activity levels during foraging of both the rover and sitter larval morphs were not significantly influenced by MB ablation. Alternative hypotheses that may explain how variation in foraging behaviour is generated are discussed.     

Development of neuronal connectivity in Drosophila antennal lobes and mushroom bodies

Recent advances in the study of the connectivity of Drosophila olfactory system include the demonstration that olfactory receptor neurons project to specific glomeruli according to the receptor type they express, and that their projection neuron partners are prespecified to innervate particular glomeruli by birth order or time. This same theme of sequential generation has been observed in the generation of the three major types of mushroom body neurons.     

Circuit reorganization in the Drosophila mushroom body calyx accompanies memory consolidation

1. Download : Download high-res image (199KB)
2. Download : Download full-size image

     

Molecular chaperones activate the Drosophila ecdysone receptor, an RXR heterodimer

The steroid hormone 20-hydroxyecdysone coordinates the stages of Drosophila development by activating a nuclear receptor heterodimer consisting of the ecdysone receptor, EcR, and the Drosophila RXR receptor, USP. We show that EcR/USP DNA binding activity requires activation by a chaperone heterocomplex like that required for activation of the vertebrate steroid receptors, but not previously shown to be required for activation of RXR heterodimers. Six proteins normally present in the chaperone complex were individually purified and shown to be sufficient for this activation. We also show that two of the six (Hsp90 and Hsc70) are required in vivo for ecdysone receptor activity, and that EcR is the primary target of the chaperone complex.     

Isoform specific control of gene activity in vivo by the Drosophila ecdysone receptor

The steroid hormone 20-hydroxyecdysone induces metamorphosis in insects. The receptor for the hormone is the ecdysone receptor, a heterodimer of two nuclear receptors, EcR and USP. In Drosophila the EcR gene encodes 3 isoforms (EcR-A, EcR-B1 and EcR-B2) that vary in their N-terminal region but not in their DNA binding and ligand binding domains. The stage and tissue specific distribution of the isoforms during metamorphosis suggests distinct functions for the different isoforms. By over-expressing the three isoforms in animals we present results supporting this hypothesis. We tested for the ability of the different isoforms to rescue the lack of dendritic pruning that is characteristic of mutants lacking both EcR-B1 and EcR-B2. By expressing the different isoforms specifically in the affected neurons, we found that both EcR-B isoforms were able to rescue the neuronal defect cell autonomously, but that EcR-A was less effective. We also analyzed the effect of over-expressing the isoforms in a wild-type background. We determined a sensitive period when high levels of either EcR-B isoform were lethal, indicating that the low levels of EcR-B at this time are crucial to ensure normal development. Over-expressing EcR-A in contrast had no detrimental effect. However, high levels of EcR-A expressed in the posterior compartment suppressed puparial tanning, and resulted in down-regulation of some of the tested target genes in the posterior compartment of the wing disc. EcR-B1 or EcR-B2 over-expression had little or no effect.     

Transmembrane/juxtamembrane domain-dependent Dscam distribution and function during mushroom body neuronal morphogenesis

Besides 19,008 possible ectodomains, Drosophila Dscam contains two alternative transmembrane/juxtamembrane segments, respectively, derived from exon 17.1 and exon 17.2. We wondered whether specific Dscam isoforms mediate formation and segregation of axonal branches in the Drosophila mushroom bodies (MBs). Removal of various subsets of the 12 exon 4s does not affect MB neuronal morphogenesis, while expression of a Dscam transgene only partially rescues Dscam mutant phenotypes. Interestingly, differential rescuing effects are observed between two Dscam transgenes that each possesses one of the two possible exon 17s. Axon bifurcation/segregation abnormalities are better rescued by the exon 17.2-containing transgene, but coexpression of both transgenes is required for rescuing mutant viability. Meanwhile, exon 17.1 targets ectopically expressed Dscam-GFP to dendrites while Dscam[exon 17.2]-GFP is enriched in axons; only Dscam[exon 17.2] affects MB axons. These results suggest that exon 17.1 is minimally involved in axonal morphogenesis and that morphogenesis of MB axons probably involves multiple distinct exon 17.2-containing Dscam isoforms.