INTRODUCTION The embryonic CNS of Drosophila has become an important model system for investigations of neuronal development. Insight into the mechanisms that control neurogenesis, axonal pathfinding and target recognition has been gained by exploit- ing the powerful genetic and molecular genetic techniques available in Drosophila (for reviews see Campos-Ortega and Knust, 1990; Grenningloh et al., 1990; Goodman and Doe, 1993). However, most of the investigations on development in the embryonic CNS of Drosophila have been carried out on the ventral ganglia. Thus, little is known about the develop- ment of the Drosophila brain (see Campos-Ortega and Harten- stein, 1985). Insight into neurogenesis and axogenesis in an insect brain has been attained recently in the grasshopper. Embryonic neu- roblasts in the grasshopper brain have been identified and some of their molecular expression patterns characterized (Zacharias et al., 1993). Moreover, the role of glial cell-bound proliferative clusters in prefiguring brain pathways and the processes that generate the axon tracts in the brain have been studied (Boyan et al., 1995a,b,c). However, it is unclear if the results of these investigations can be applied to Drosophila. In the hemimetabolous grasshopper, embryogenesis gives rise to adult- like structures (Bate, 1976; Chapman, 1982). In contrast, in the holometabolous Drosophila, embryonic development produces a highly derived larval stage, which is refigured postembryoni- cally (Campos-Ortega and Hartenstein, 1985; Truman and Bate, 1988). The degree to which the embryonic brain of Drosophila is restructured postembryonically has not yet been resolved (see Truman et al., 1993). (For a review of older literature on insect brain development see Malzacher, 1968.) In this report, we analyse the cellular and molecular processes that give rise to an initial set of axonal projections in the embryonic brain of Drosophila. We first use histology, immunocytochemistry and enhancer detection in combination with light microscopy, laser confocal microscopy and electron microscopy to determine how the commissural and descend- ing pathways are established. We find that commissural inter- connections are formed by axons that project along an inter- hemispheric cell bridge and that descending interconnections are prefigured by a chain of glial cells along which pioneering axons extend. We then use specific mutants to show that CNS midline cells are involved in the formation of both commis- sural and descending brain pathways. We find that mutations in the commissureless gene, which is involved in growth cone 3849 Development 121, 3849-3860 (1995) Printed in Great Britain © The Company of Biologists Limited 1995 DEV2017 The establishment of initial axonal pathways in the embryonic brain of Drosophila melanogaster was investi- gated at the cellular and molecular level using antibody probes, enhancer detector strains and axonal pathfinding mutants. During embryogenesis, two bilaterally symmetri- cal cephalic neurogenic regions form, which are initially separated from each other and from the ventral nerve cord. The brain commissure that interconnects the two brain hemispheres is pioneered by axons that project towards the midline in close association with an interhemispheric cellular bridge. The descending longitudinal pathways that interconnect the brain to the ventral nerve cord are pre- figured by a chain of longitudinal glial cells and a cellular bridge between brain and subesophageal ganglion; pio- neering descending and ascending neurons grow in close association with these structures. The formation of the embryonic commissural and longitudinal pathways is dependent on cells of the CNS midline. Mutations in the commissureless gene, which affects growth cone guidance towards the midline, result in a marked reduction of the brain commissure. Mutations in the single-minded gene and in other spitz group genes, which affect the differentiation of CNS midline cells, result in the absence or aberrant pro- jection of longitudinal pathways. The analysis of axon pathway formation presented here reveals remarkable sim- ilarities as well as distinct differences in the embryonic development of the brain and the segmental ganglia, and forms the basis for a comprehensive genetic and molecular genetic dissection of axonal pathfinding processes in the developing brain. Key words: axonal pathfinding, CNS, glia, embryo, Drosophila, commissureless, single-minded SUMMARY Embryonic development of the Drosophila brain: formation of commissural and descending pathways Stavros Therianos 1 , Sandra Leuzinger 1 , Frank Hirth 1 , Corey S. Goodman 2 and Heinrich Reichert 1 1 Laboratory of Neurobiology, Institute of Zoology, University of Basel, CH-4051 Basel, Switzerland 2 Howard Hughes Medical Institute, Department of Molecular and Cell Biology, University of California, Berkeley, California 94720, USA