Developmental axon pruning is widely used in constructing the nervous system. Accordingly, diverse mechanisms are likely employed for various forms of axon pruning. In the Drosophila mushroom bodies (MB), gamma neurons initially extend axon branches into both the dorsal and medial MB axon lobes in larvae. Through a well-orchestrated set of developmental events during metamorphosis, axon branches to both lobes degenerate prior to the formation of adult connections. Here, we analyze ultrastructural changes underlying axon pruning by using a genetically encoded electron microscopic (EM) marker to selectively label gamma neurons. By inhibiting axon pruning in combination with the use of this EM marker, we demonstrate a causal link between observed cellular events and axon pruning. These events include changes in axon ultrastructure, synaptic degeneration, and engulfment of degenerating axon fragments by glia for their subsequent breakdown via the endosomal-lysosomal pathway. Interestingly, glia selectively invade MB axon lobes at the onset of metamorphosis; this increase in cell number is independent of axon fragmentation. Our study reveals a key role for glia in the removal of axon fragments during developmental axon pruning.

The diencephalon comprises three functionally distinct regions: synencephalon, dorsal thalamus, and ventral thalamus. Patterning of the diencephalon has been proposed to involve subdivision of its anteroposterior axis into segments, neuromeres or prosomeres (Bergquist and Kallen, 1954;Vaage, 1969;Figdor and Stern, 1993;Rubenstein et al., 1994;Redies et al., 2000;Yoon et al., 2000). However, the number and sequence of diencephalic neuromeres, or even their existence, are uncertain. We have examined the proposed subdivisions by morphology, gene expression, acquisition of boundary-specific phenotypes, and cell lineage restriction. We find that at stage 16 in chick the diencephalon is divided into synencephalon and parencephalon. The synencephalon exhibits neuromeric morphology, expresses Prox, and acquires neuromere boundary properties at its interface with both the midbrain and the parencephalon. Although the mesencephalic/synencephalic boundary restricts cell mixing, the synencephalic/parencephalic boundary does not. Similarly, there is no lineage restriction between the parencephalon and the more rostral forebrain (secondary prosencephalon). Subdivision of the parencephalon into ventral and dorsal thalamus involves the formation of a narrow intraparencephalic territory, the zona limitans intrathalamica (zli). This is correlated with the acquisition of cell lineage restriction at both anterior and posterior borders of the zli, the appearance of boundary-specific properties, and Gbx2 and Dlx2 expression in dorsal thalamic and ventral thalamic territories, respectively. At stage 22, the synencephalon is divided into two domains, distinguished by differential gene expression and tissue morphology, but associated with neither a boundary phenotype nor cell lineage restriction. Our results suggest that the diencephalon does not have an overt segmental pattern.

The Drosophila central brain is composed of approximately 100 paired lineages, with most lineages comprising 100–150 neurons. Most lineages have a number of important characteristics in common. Typically, neurons of a lineage stay together as a coherent cluster and project their axons into a coherent bundle visible from late embryo to adult. Neurons born during the embryonic period form the primary axon tracts (PATs) that follow stereotyped pathways in the neuropile. Apoptotic cell death removes an average of 30–40% of primary neurons around the time of hatching. Secondary neurons generated during the larval period form secondary axon tracts (SATs) that typically fasciculate with their corresponding primary axon tract. SATs develop into the long fascicles that interconnect the different compartments of the adult brain. Structurally, we distinguish between three types of lineages: PD lineages, characterized by distinct, spatially separate proximal and distal arborizations; C lineages with arborizations distributed continuously along the entire length of their tract; D lineages that lack proximal arborizations. Arborizations of many lineages, in particular those of the PD type, are restricted to distinct neuropile compartments. We propose that compartments are ‘scaffolded” by individual lineages, or small groups thereof. Thereby, the relatively small number of primary neurons of each primary lineage set up the compartment map in the late embryo. Compartments grow during the larval period simply by an increase in arbor volume of primary neurons. Arbors of secondary neurons form within or adjacent to the larval compartments, resulting in smaller compartment subdivisions and additional, adult specific compartments.

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In Drosophila embryos, segment boundaries form at the posterior edge of each stripe of engrailed expression. We have used an HRP-CD2 transgene to follow by transmission electron microscopy the cell shape changes that accompany boundary formation. The first change is a loosening of cell contact at the apical side of cells on either side of the incipient boundary. Then, the engrailed-expressing cells flanking the boundary undergo apical constriction, move inwards and adopt a bottle morphology. Eventually,grooves regress, first on the ventral side, then laterally. We noted that groove formation and regression are contemporaneous with germ band retraction and shortening, respectively, suggesting that these rearrangements could also contribute to groove morphology. The cellular changes accompanying groove formation require that Hedgehog signalling be activated, and, as a result, a target of Ci expressed, at the posterior of each boundary (obvious targets like stripe and rhomboid appear not to be involved). In addition, Engrailed must be expressed at the anterior side of each boundary,even if Hedgehog signalling is artificially maintained. Thus, there are distinct genetic requirements on either side of the boundary. In addition,Wingless signalling at the anterior of the domains of engrailed (and hedgehog) expression represses groove formation and thus ensures that segment boundaries form only at the posterior.

SummaryIt is well established that perception is largely multisensory [1]; often served by modalities such as touch, vision, and hearing that detect stimuli emanating from a common point in space [2, 3]; and processed by brain tissue maps that are spatially aligned [4]. However, the neural interactions among modalities that share no spatial stimulus domain yet are essential for robust perception within noisy environments remain uncharacterized. Drosophila melanogaster makes its living navigating food odor plumes. Odor acts to increase the strength of gaze-stabilizing optomotor reflexes [5] to keep the animal aligned within an invisible plume, facilitating odor localization in free flight [6–8]. Here, we investigate the cellular mechanism for cross-modal behavioral interactions. We characterize a wide-field motion-selective interneuron of the lobula plate that shares anatomical and physiological similarities with the “Hx” neuron identified in larger flies [9, 10]. Drosophila Hx exhibits cross-modal enhancement of visual responses by paired odor, and presynaptic inputs to the lobula plate are required for behavioral odor tracking but are not themselves the target of odor modulation, nor is the neighboring wide-field “HSE” neuron [11]. Octopaminergic neurons mediating increased visual responses upon flight initiation [12] also show odor-evoked calcium modulations and form connections with Hx dendrites. Finally, restoring synaptic vesicle trafficking within the octopaminergic neurons of animals carrying a null mutation for all aminergic signaling [13] is sufficient to restore odor-tracking behavior. These results are the first to demonstrate cellular mechanisms underlying visual-olfactory integration required for odor localization in fruit flies, which may be representative of adaptive multisensory interactions across taxa.

Chemotaxis is important for the survival of most animals. How the brain translates sensory input into motor output beyond higher olfactory processing centers is largely unknown. We describe a group of excitatory neurons, termed Odd neurons, which are important for Drosophila larval chemotaxis. Odd neurons receive synaptic input from projection neurons in the calyx of the mushroom body and project axons to the central brain. Functional imaging shows that some of the Odd neurons respond to odor. Larvae in which Odd neurons are silenced are less efficient at odor tracking than controls and sample the odor space more frequently. Larvae in which the excitability of Odd neurons is increased are better at odor intensity discrimination and odor tracking. Thus, the Odd neurons represent a distinct pathway that regulates the sensitivity of the olfactory system to odor concentrations, demonstrating that efficient chemotaxis depends on processing of odor strength downstream of higher olfactory centers.

Using a combination of dye injections, clonal labeling, and molecular markers we have reconstructed the axonal connections between brain and ventral nerve cord of the first instar Drosophila larva. Out of the approximately 1400 neurons that form the early larval brain hemisphere, less than 50 cells have axons descending into the ventral nerve cord. Descending neurons fall into four topologically defined clusters located in the antero-medial, antero-lateral, dorsal, and baso-posterior brain, respectively. The antero-lateral cluster represents a lineage derived from a single neuroblast. Terminations of descending neurons are almost exclusively found in the anterior part of the ventral nerve cord, represented by the gnathal and thoracic neuromeres. This region also contains small numbers of neurons with axons ascending into the brain. Terminals of the ascending axons are found in the same basal brain regions that also contain descending neurons. We have mapped ascending and descending axons to the previously described scaffold of longitudinal fiber tracts that interconnect different neuromeres of the ventral nerve cord and the brain. This work provides a structural framework for functional and genetic studies addressing the control of Drosophila larval behavior by brain circuits.