Mcm3 replicative helicase mutation impairs neuroblast proliferation and memory in Drosophila.

In the developing Drosophila brain, a small number of neural progenitor cells (neuroblasts) generate in a co-ordinated manner a high variety of neuronal cells by integration of temporal, spatial and cell-intrinsic information. In this study, we performed the molecular and phenotypic characterization of a structural brain mutant called small mushroom bodies (smu), which was isolated in a screen for mutants with altered brain structure. Focusing on the mushroom body neuroblast lineages we show that failure of neuroblasts to generate the normal number of mushroom body neurons (Kenyon cells) is the major cause of the smu phenotype. In particular, the premature loss of mushroom body neuroblasts caused a pronounced effect on the number of late-born Kenyon cells. Neuroblasts showed no obvious defects in processes controlling asymmetric cell division, but generated less ganglion mother cells. Cloning of smu uncovered a single amino acid substitution in an evolutionarily conserved protein interaction domain of the Minichromosome maintenance 3 (Mcm3) protein. Mcm3 is part of the multimeric Cdc45/Mcm/GINS (CMG) complex, which functions as a helicase during DNA replication. We propose that at least in the case of mushroom body neuroblasts, timely replication is not only required for continuous proliferation but also for their survival. The absence of Kenyon cells in smu reduced learning and early phases of conditioned olfactory memory. Corresponding to the absence of late-born Kenyon cells projecting to α'/ß' and α/ß lobes, smu is profoundly defective in later phases of persistent memory.

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.

Metamorphosis of the mushroom bodies; large-scale rearrangements of the neural substrates for associative learning and memory in Drosophila.

Paired brain centers known as mushroom bodies are key features of the circuitry for insect associative learning, especially when evoked by olfactory cues. Mushroom bodies have an embryonic origin, and unlike most other brain structures they exhibit developmental continuity, being prominent components of both the larval and the adult CNS. Here, we use cell-type-specific markers, provided by the P[GAL4] enhancer trap system, to follow specific subsets of mushroom body intrinsic and extrinsic neurons from the larval to the adult stage. We find marked structural differences between the larval and adult mushroom bodies, arising as the consequence of large-scale reorganization during metamorphosis. Extensive, though incomplete, degradation of the larval structure is followed by establishment of adult specific alpha and beta lobes. Kenyon cells of embryonic origin, by contrast, were found to project selectively to the adult gamma lobe. We propose that the gamma lobe stores information of relevance to both developmental stages, whereas the alpha and beta lobes have uniquely adult roles.

Neuroblast ablation in Drosophila P[GAL4] lines reveals origins of olfactory interneurons.

Hydroxyurea (HU) treatment of early first instar larvae in Drosophila was previously shown to ablate a single dividing lateral neuroblast (LNb) in the brain. Early larval HU application to P[GAL4] strains that label specific neuron types enabled us to identify the origins of the two major classes of interneurons in the olfactory system. HU treatment resulted in the loss of antennal lobe local interneurons and of a subset of relay interneurons (RI), elements usually projecting to the calyx and the lateral protocerebrum (LPR). Other RI were resistant to HU and still projected to the LPR. However, they formed no collaterals in the calyx region (which was also ablated), suggesting that their survival does not depend on targets in the calyx. Hence, the ablated interneurons were derived from the LNb, whereas the HU-resistant elements originated from neuroblasts which begin to divide later in larval life. Developmental GAL4 expression patterns suggested that differentiated RI are present at the larval stage already and may be retained through metamorphosis.

Expression of Drosophila mushroom body mutations in alternative genetic backgrounds: a case study of the mushroom body miniature gene (mbm).

Mutations in 12 genes regulating Drosophila melanogaster mushroom body (MB) development were each studied in two genetic backgrounds. In all cases, brain structure was qualitatively or quantitatively different after replacement of the "original" genetic background with that of the Canton Special wild-type strain. The mushroom body miniature gene (mbm) was investigated in detail. mbm supports the maintenance of MB Kenyon cell fibers in third instar larvae and their regrowth during metamorphosis. Adult mbm1 mutant females are lacking many or most Kenyon cell fibers and are impaired in MB-mediated associative odor learning. We show here that structural defects in mbm1 are apparent only in combination with an X-linked, dosage-dependent modifier (or modifiers). In the Canton Special genetic background, the mbm1 anatomical phenotype is suppressed, and MBs develop to a normal size. However, the olfactory learning phenotype is not fully restored, suggesting that submicroscopic defects persist in the MBs. Mutant mbm1 flies with full-sized MBs have normal retention but show a specific acquisition deficit that cannot be attributed to reductions in odor avoidance, shock reactivity, or locomotor behavior. We propose that polymorphic gene interactions (in addition to ontogenetic factors) determine MB size and, concomitantly, the ability to recognize and learn odors.

Associative odor learning in Drosophila abolished by chemical ablation of mushroom bodies.

The corpora pedunculata, or mushroom bodies (MBs), in the brain of Drosophila melanogaster adults consist of approximately 2500 parallel Kenyon cell fibers derived from four MB neuroblasts. Hydroxyurea fed to newly hatched larvae selectively deletes these cells, resulting in complete, precise MB albation. Adult flies developing without MBs behave normally in most respects, but are unable to perform in a classical conditioning paradigm that tests associative learning of odor cues and electric shock. This deficit cannot be attributed to reductions in olfactory sensitivity, shock reactivity, or locomotor behavior. The results demonstrate that MBs mediate associative odor learning in flies.

Genetic analysis of the foraging microregion of Drosophila melanogaster.

The rover/sitter polymorphism in Drosophila melanogaster larval behaviour is a unique example of a genetically determined, naturally occurring behavioural polymorphism. Allelic variation at the foraging locus (for) accounts for the rover (long foraging paths) and sitter (short foraging paths) phenotypes. We previously developed lethal tagging and used deficiency mapping to place for in the 24A3-C5 interval on the polytene chromosome map, thereby defining the for microregion. Here, we subjected this microregion to mutational analysis to (i) isolate putative lethal foraging mutations and characterize their behavioural phenotypes to assess whether or not for is a vital locus, (ii) generate cytologically detectable chromosome rearrangements with breakpoints in or near for for more precise localization and for future molecular analysis of the for gene, and (iii) identify other gene loci in the immediate vicinity of the for locus. We recovered 10 gamma-induced and 33 ethyl methanesulfonate (EMS) induced new mutations that define seven complementation groups in 24A3-D4. Two new EMS-induced lethal for alleles and four gamma-induced rearrangements with breakpoints in for were identified, which allowed us to further localize for to 24A3-5. All lethal mutations in for resulted in an altered behavioural phenotype providing evidence that both vital and behavioural functions are encoded by for.

Genetic localization of foraging (for): a major gene for larval behavior in Drosophila melanogaster.

Localizing genes for quantitative traits by conventional recombination mapping is a formidable challenge because environmental variation, minor genes, and genetic markers have modifying effects on continuously varying phenotypes. We describe "lethal tagging," a method used in conjunction with deficiency mapping for localizing major genes associated with quantitative traits. Rover/sitter is a naturally occurring larval foraging polymorphism in Drosophila melanogaster which has a polygenic pattern of inheritance comprised of a single major gene (foraging) and minor modifier genes. We have successfully localized the lethal tagged foraging (for, 2-10) gene by deficiency mapping to 24A3-C5 on the polytene chromosome map.