The Drosophila N-CAM homolog Fasciclin II (FasII) is expressed during the embryonic period in a subset of central neurons that pioneer the neuropile of the larval brain. Toward the end of embryogenesis, FasII expression in axon tracts diminishes but resumes from the late first larval instar in an increasingly complex pattern of axon tracts that join the tracts laid down in the embryo. We present evidence that FasII is expressed in a major fraction of the long axon tracts that interconnect different domains of the larval brain. For many tracts, FasII expression remains stable throughout larval development and pupal development. Therefore, the FasII pattern of axon tracts, along with the mushroom body and optic lobe, both of which are also FasII-positive, represents a useful set of landmarks that define different regions in the Drosophila brain throughout development. In this study, serial confocal brain sections were used to generate digital three-dimensional models of larval axon tracts at different stages. These models form part of our effort to generate an anatomic framework of Drosophila larval brain structure required for accurate localization of gene expression and gene function in experimental studies of neural development.

We have studied the formation of the neuroblasts of the Drosophila brain which segregate from the procephalic neurectoderm. The expression domains of the segment polarity gene engrailed (en) allow one to subdivide the procephalic neuroectoderm into tritocerebral, deuterocerebral, and protocerebral neuromeres. Based upon the expression pattern of the proneural gene lethal of scute (l'sc), as well as the pattern of brain neuroblast segregation, the protocerebral and deuterocerebral neuromeres can be further subdivided into a central, anterior, and posterior domain. A total of 75-80 neuroblasts segregate in a stereotyped pattern from the procephalic neurectoderm of each side during stages 9-11. With respect to their position and the expression of the markers asense (ase) and seven-up (svp), 23 small groups of one to five neuroblasts each were identified. The first eight groups (Pc1-4, Dc1-3, Dp1), collectively called SI/II neuroblasts in analogy to the subpopulation of ventral neuroblasts which appear at the same stage), arise from the central domain of the protocerebral and deuterocerebral neurectoderm, respectively. Later groups form anteriorly and posteriorly from the earlier ones, leading to a centrifugal growth of the procephalic neuroblast population. SIII neuroblasts (Pa1-4, Pp1-2, Dp2) arise during stage 10, SIV neuroblasts (Pa5-6, Pp3-4, Da1, T1-2) during early stage 11, and SV neuroblasts (Pp5, Pdm) during late stage 11 and early stage 12. The dorsomedial domain of the procephalic neurectoderm represents a special case. Unlike other procephalic neuroblasts which delaminate from the surface ectoderm as individual cells, cells of the dorsomedial protocerebral domain are internalized during stage 12 as large, coherent clusters by a movement which can be best characterized as a combination of mass-delamination and invagination.

The Drosophila E-cadherin homolog, DE-cadherin, is expressed postembryonically by brain neuroblasts and their lineages of neurons ("secondary lineages"). DE-cadherin appears in neuroblasts as soon as they can be identified by their increase in size and then remains expressed uninterruptedly throughout larval life. DE-cadherin remains transiently expressed in the cell bodies and axons of neurons produced by neuroblast proliferation. In general, axons of neurons belonging to one lineage form tight bundles. The trajectories of these bundles are correlated with the location of the neuronal lineages to which they belong. Thus, axon bundles of lineages that are neighbors in the cortex travel parallel to each other and reach the neuropile at similar positions. It is, therefore, possible to assign coherent groups of neuroblasts and their lineages to the individual neuropile compartments and long axon tracts introduced in the accompanying articles (Nassif et al. [2003] J Comp Neurol 455:417-434; Younossi-Hartenstein et al. [2003] J Comp Neurol 455:435-450). In this study, we have reconstructed the pattern of secondary lineages and their projection in relationship to the compartments and Fasciclin II-positive long axon tracts. Based on topology and axonal trajectory, the lineages of the central brain can be subdivided into 11 groups that can be followed throughout successive larval stages. The map of larval lineages and their axonal projection will be important for future studies on postembryonic neurogenesis in Drosophila. It also lays a groundwork for investigating the role of DE-cadherin in larval brain development.

The neuropile of the late embryonic Drosophila brain can be subdivided into a vertical component (cervical connective), a transverse component (supraesophageal commissure), and a horizontal component for which we propose the term protocerebral connective. The core of each neuropile component is formed by numerous axon fascicles, the trajectory of which follows an invariant pattern. In the present study we have used an antibody against the adhesion molecule Fasciclin II (FasII) that is expressed in a large number of early differentiating neurons of the Drosophila embryo to follow the development of the axon tracts of the brain. The FasII antigen appears on the surface of clusters of neuronal somata prior to axon outgrowth. These clusters, for which we propose the term fibre tract founder clusters, are laid out in a linear pattern that forms an almost uninterrupted longitudinal track reaching from the ventral nerve cord to the "tip" of the brain. After expressing FasII on their soma, neurons of the fibre tract founder clusters extend axons that grow along the surface of the founder clusters and form a simple system of pioneer tracts for each of the components of the brain neuropile. We have reconstructed the FasII-positive fibre tract founder clusters and their axons from optical sections and generated digital 3-D models that illustrate the spatial relationships of the pioneer tracts. Three fibre tract founder clusters, D/T, P1, and P3m, pioneer the cervical connective. P21 and P2m form a transverse track that pioneers the supraesophageal commissure. P4m and P41/P51/VP5m form two tracts that pioneer a medial and a lateral component of the protocerebral connective, respectively. Because FasII expression continues uninterruptedly into the larval period when the "rudiments" of many parts of the adult neuropile are readily identifiable, it was possible to assign several of the embryonic pioneer tracts to definitive neuropile components, including the median bundle, antennocerebral tract, mushroom body, and posterior optic tract.

Glial cells in Drosophila and other insects are organized in an outer layer that envelops the surface of the central and peripheral nervous system (subperineurial glia, peripheral glia), a middle layer associated with neuronal somata in the cortex (cell body glia), and an inner layer surrounding the neuropile (longitudinal glia, midline glia, nerve root glia). In the ventral nerve cord, most glial cells are formed by a relatively small number of neuro-glioblasts; subsequently, glial cell precursors migrate and spread out widely to reach their final destination. By using a glia-specific marker (antibody against the Repo protein) we have reconstructed the pattern of glial cell precursors at successive developmental stages, focusing on the glia of the supraesophageal ganglion and subesophageal ganglion which are not described in previous studies. Digitized images of consecutive optical sections were used to generate 3-D models that show the spatial pattern of glial cell precursors in relationship to the neuropile, brain surface, and peripheral nerves. Similar to their spatial organization in the ventral nerve cord, glial cells of the brain populate the brain nerves and outer surface, cortical cell body layer, and cortex-neuropile interface. Neuropile-associated glial cells arise from a cluster located at the base of the supraesophageal ganglion; from this position, they migrate dorsally along the developing axon tracts and by late embryonic stages form a sheath around all neuropile compartments, including the supraesophageal commissure. Surface and cell body glial cells derive from several discrete foci, notably two large clusters at the deuterocerebrum/protocerebrum boundary and the posterior protocerebrum. From these foci, glial cells then fan out to envelop the surface of the supraesophageal ganglion.