Full reconstruction of neuron morphology is of fundamental interest for the analysis and understanding of their functioning. We have developed a novel method capable of automatically tracing neurons in three-dimensional microscopy data. In contrast to template-based methods, the proposed approach makes no assumptions about the shape or appearance of neurite structure. Instead, an efficient seeding approach is applied to capture complex neuronal structures and the tracing problem is solved by computing the optimal reconstruction with a weighted graph. The optimality is determined by the cost function designed for the path between each pair of seeds and by topological constraints defining the component interrelations and completeness. In addition, an automated neuron comparison method is introduced for performance evaluation and structure analysis. The proposed algorithm is computationally efficient and has been validated using different types of microscopy data sets including Drosophila’s projection neurons and fly neurons with presynaptic sites. In all cases, the approach yielded promising results.

Because of its genetic, molecular, and behavioral tractability, Drosophila has emerged as a powerful model system for studying molecular and cellular mechanisms underlying the development and function of nervous systems. The Drosophila nervous system has fewer neurons and exhibits a lower glia:neuron ratio than is seen in vertebrate nervous systems. Despite the simplicity of the Drosophila nervous system, glial organization in flies is as sophisticated as it is in vertebrates. Furthermore, fly glial cells play vital roles in neural development and behavior. In addition, powerful genetic tools are continuously being created to explore cell function in vivo. In taking advantage of these features, the fly nervous system serves as an excellent model system to study general aspects of glial cell development and function in vivo. In this article, we review and discuss advanced genetic tools that are potentially useful for understanding glial cell biology in Drosophila.

Pavlovian olfactory learning in Drosophila produces two genetically distinct forms of intermediate-term memories: anesthesia-sensitive memory, which requires the amnesiac gene, and anesthesia-resistant memory (ARM), which requires the radish gene. Here, we report that ARM is specifically enhanced or inhibited in flies with elevated or reduced serotonin (5HT) levels, respectively. The requirement for 5HT was additive with the memory defect of the amnesiac mutation but was occluded by the radish mutation. This result suggests that 5HT and Radish protein act on the same pathway for ARM formation. Three supporting lines of evidence indicate that ARM formation requires 5HT released from only two dorsal paired medial (DPM) neurons onto the mushroom bodies (MBs), the olfactory learning and memory center in Drosophila: (i) DPM neurons were 5HT-antibody immunopositive; (ii) temporal inhibition of 5HT synthesis or release from DPM neurons, but not from other serotonergic neurons, impaired ARM formation; (iii) knocking down the expression of d5HT1A serotonin receptors in α/β MB neurons, which are innervated by DPM neurons, inhibited ARM formation. Thus, in addition to the Amnesiac peptide required for anesthesia-sensitive memory formation, the two DPM neurons also release 5HT acting on MB neurons for ARM formation.

We found that glia secrete myoglianin, a TGF-β ligand, to instruct developmental neural remodeling in Drosophila. Glial myoglianin upregulated neuronal expression of an ecdysone nuclear receptor that triggered neurite remodeling following the late-larval ecdysone peak. Thus glia orchestrate developmental neural remodeling not only by engulfment of unwanted neurites but also by enabling neuron remodeling.

Drosophila brains contain numerous neurons that form complex circuits. These neurons are derived in stereotyped patterns from a fixed number of progenitors, called neuroblasts, and identifying individual neurons made by a neuroblast facilitates the reconstruction of neural circuits. An improved MARCM (mosaic analysis with a repressible cell marker) technique, called twin-spot MARCM, allows one to label the sister clones derived from a common progenitor simultaneously in different colors. It enables identification of every single neuron in an extended neuronal lineage based on the order of neuron birth. Here we report the first example, to our knowledge, of complete lineage analysis among neurons derived from a common neuroblast that relay olfactory information from the antennal lobe (AL) to higher brain centers. By identifying the sequentially derived neurons, we found that the neuroblast serially makes 40 types of AL projection neurons (PNs). During embryogenesis, one PN with multi-glomerular innervation and 18 uniglomerular PNs targeting 17 glomeruli of the adult AL are born. Many more PNs of 22 additional types, including four types of polyglomerular PNs, derive after the neuroblast resumes dividing in early larvae. Although different offspring are generated in a rather arbitrary sequence, the birth order strictly dictates the fate of each post-mitotic neuron, including the fate of programmed cell death. Notably, the embryonic progenitor has an altered temporal identity following each self-renewing asymmetric cell division. After larval hatching, the same progenitor produces multiple neurons for each cell type, but the number of neurons for each type is tightly regulated. These observations substantiate the origin-dependent specification of neuron types. Sequencing neuronal lineages will not only unravel how a complex brain develops but also permit systematic identification of neuron types for detailed structure and function analysis of the brain.

Neurons derived from the same progenitor may acquire different fates according to their birth timing/order. To reveal temporally guided cell fates, we must determine neuron types as well as their lineage relationships and times of birth. Recent advances in genetic lineage analysis and fate mapping are facilitating such studies. For example, high-resolution lineage analysis can identify each sequentially derived neuron of a lineage and has revealed abrupt temporal identity changes in diverse Drosophila neuronal lineages. In addition, fate mapping of mouse neurons made from the same pool of precursors shows production of specific neuron types in specific temporal patterns. The tools used in these analyses are helping to further our understanding of the genetics of neuronal temporal identity.

Full reconstruction of neuron morphology is of fundamental interest for the analysis and understanding of neuron function. We have developed a novel method capable of tracing neurons in three-dimensional microscopy data automatically. In contrast to template-based methods, the proposed approach makes no assumptions on the shape or appearance of neuron’s body. Instead, an efficient seeding approach is applied to find significant pixels almost certainly within complex neuronal structures and the tracing problem is solved by computing an graph tree structure connecting these seeds. In addition, an automated neuron comparison method is introduced for performance evaluation and structure analysis. The proposed algorithm is computationally efficient. Experiments on different types of data show promising results.

Numb can antagonize Notch signaling to diversify the fates of sister cells. We report here that paired sister cells acquire different fates in all three Drosophila neuronal lineages that make diverse types of antennal lobe projection neurons (PNs). Only one in each pair of postmitotic neurons survives into the adult stage in both anterodorsal (ad) and ventral (v) PN lineages. Notably, Notch signaling specifies the PN fate in the vPN lineage but promotes programmed cell death in the missing siblings in the adPN lineage. In addition, Notch/Numb-mediated binary sibling fates underlie the production of PNs and local interneurons from common precursors in the lAL lineage. Furthermore, Numb is needed in the lateral but not adPN or vPN lineages to prevent the appearance of ectopic neuroblasts and to ensure proper self-renewal of neural progenitors. These lineage-specific outputs of Notch/Numb signaling show that a universal mechanism of binary fate decision can be utilized to govern diverse neural sibling differentiations.

Nuclear receptors (NRs) comprise a family of ligand-regulated transcription factors that control diverse critical biological processes including various aspects of brain development. Eighteen NR genes exist in the Drosophila genome. To explore their roles in brain development, we knocked down individual NRs through the development of the mushroom bodies (MBs) by targeted RNAi. Besides recapitulating the known MB phenotypes for three NRs, we found that unfulfilled (unf), an ortholog of human photoreceptor specific nuclear receptor (PNR), regulates axonal morphogenesis and neuronal subtype identity. The adult MBs develop through remodeling of gamma neurons plus de-novo elaboration of both alpha’/beta’ and alpha/beta neurons. Notably, unf is largely dispensable for the initial elaboration of gamma neurons, but plays an essential role in their re-extension of axons after pruning during early metamorphosis. The subsequently derived MB neuron types also require unf for extension of axons beyond the terminus of the pruned bundle. Tracing single axons revealed misrouting rather than simple truncation. Further, silencing unf in single-cell clones elicited misguidance of axons in otherwise unperturbed MBs. Such axon guidance defects may occur as MB neurons partially lose their subtype identity, as evidenced by suppression of various MB subtype markers in unf knockdown MBs. In sum, unf governs axonal morphogenesis of multiple MB neuron types, possibly through regulating neuronal subtype identity.