Tripartite mushroom body architecture revealed by antigenic markers.

We have explored the organization of the axonal lobes in Drosophila mushroom bodies by using a panel of immunohistochemical markers. These markers consist of antibodies to eight proteins expressed preferentially in the mushroom bodies: DAMB, DCO, DRK, FASII, LEO, OAMB, PKA RII, and RUT. Previous to this work, four axonal lobes, two projecting dorsally (alpha and alpha') and two medially (beta and gamma), had been described in Drosophila mushroom bodies. However, our analysis of immunohistochemically stained frontal and sagittal sections of the brain revealed three medially projecting lobes. The newly distinguished lobe, which we term beta', lies along the dorsal surface of beta, just posterior to gamma. In addition to resolving a fifth lobe, our studies revealed that there are specific lobe sets defined by equivalent marker expression levels. These sets are (1) the alpha and beta lobes, (2) the alpha' and beta' lobes, and (3) the gamma lobe and heel (a lateral projection formed by a hairpin turn of some of the peduncle fibers). All of the markers we have examined are consistent with these three sets. Previous Golgi studies demonstrate that each mushroom body cell projects one axon that branches into a dorsal lobe and a medial lobe, or one unbranched axon that projects medially. Taken together with the lobe sets listed above, we propose that there are three major projection configurations of mushroom body cell axons: (1) one branch in the alpha and one in the beta lobe, (2) one branch in the alpha' and one in the beta' lobe, and (3) one unbranched axon projecting to the heel and the gamma lobe. The fact that these neuron types exhibit differential expression levels of a number of mushroom body genes suggests that they may have corresponding functional differences. These functions may be conserved in the larvae, as several of these genes were expressed in larval and embryonic mushroom bodies as well. The basic mushroom body structure, including the denritic calyx, peduncle, and lobes, was already visible by the late stages of embryogenesis. With new insights into mushroom body organization, and the characterization of markers for developing mushroom bodies, we are beginning to understand how these structures form and function.

Efficient isolation and mapping of rad genes of the fungus Coprinus cinereus using chromosome-specific libraries.

We have constructed cosmid libraries from electrophoretically separated chromosomes of the basidiomycete Coprinus cinereus. These libraries greatly facilitate the isolation of genes by complementation of mutant phenotypes and are particularly useful for map-based cloning strategies. From a library constructed from two co-migrating C.cinereus chromosomes, we isolated a clone that complements the C.cinereus rad9-1 mutation. Examination of this clone showed that it complements both the repair and meiotic defects of this mutant. Restriction fragment length polymorphism mapping using a portion of this clone showed that it maps to the rad9 locus. In addition, a single copy of transforming DNA is sufficient to complement the rad9-1 defects. Thus, we believe we have cloned the rad9 gene itself. We also used a chromosome-specific library and backcrossed isolates to rapidly identify a cosmid clone which is tightly linked to the rad11 locus and is therefore a suitable starting point for a chromosome walk. These rapid methods of gene mapping and isolation should be applicable to any organism with separable chromosomes.