Predetermined embryonic glial cells form the distinct glial sheaths of the Drosophila peripheral nervous system.

One of the numerous functions of glial cells in Drosophila is the ensheathment of neurons to isolate them from the potassium-rich haemolymph, thereby establishing the blood-brain barrier. Peripheral nerves of flies are surrounded by three distinct glial cell types. Although all embryonic peripheral glia (ePG) have been identified on a single-cell level, their contribution to the three glial sheaths is not known. We used the Flybow system to label and identify each individual ePG in the living embryo and followed them into third instar larva. We demonstrate that all ePG persist until the end of larval development and some even to adulthood. We uncover the origin of all three glial sheaths and describe the larval differentiation of each peripheral glial cell in detail. Interestingly, just one ePG (ePG2) exhibits mitotic activity during larval stages, giving rise to up to 30 glial cells along a single peripheral nerve tract forming the outermost perineurial layer. The unique mitotic ability of ePG2 and the layer affiliation of additional cells were confirmed by in vivo ablation experiments and layer-specific block of cell cycle progression. The number of cells generated by this glial progenitor and hence the control of perineurial hyperplasia correlate with the length of the abdominal nerves. By contrast, the wrapping and subperineurial glia layers show enormous hypertrophy in response to larval growth. This characterisation of the embryonic origin and development of each glial sheath will facilitate functional studies, as they can now be addressed distinctively and genetically manipulated in the embryo.

The Neuronal Circuit of the Dorsal Circadian Clock Neurons in Drosophila melanogaster.

Drosophila's dorsal clock neurons (DNs) consist of four clusters (DN1as, DN1ps, DN2s, and DN3s) that largely differ in size. While the DN1as and the DN2s encompass only two neurons, the DN1ps consist of ∼15 neurons, and the DN3s comprise ∼40 neurons per brain hemisphere. In comparison to the well-characterized lateral clock neurons (LNs), the neuroanatomy and function of the DNs are still not clear. Over the past decade, numerous studies have addressed their role in the fly's circadian system, leading to several sometimes divergent results. Nonetheless, these studies agreed that the DNs are important to fine-tune activity under light and temperature cycles and play essential roles in linking the output from the LNs to downstream neurons that control sleep and metabolism. Here, we used the Flybow system, specific split-GAL4 lines, trans-Tango, and the recently published fly connectome (called hemibrain) to describe the morphology of the DNs in greater detail, including their synaptic connections to other clock and non-clock neurons. We show that some DN groups are largely heterogenous. While certain DNs are strongly connected with the LNs, others are mainly output neurons that signal to circuits downstream of the clock. Among the latter are mushroom body neurons, central complex neurons, tubercle bulb neurons, neurosecretory cells in the pars intercerebralis, and other still unidentified partners. This heterogeneity of the DNs may explain some of the conflicting results previously found about their functionality. Most importantly, we identify two putative novel communication centers of the clock network: one fiber bundle in the superior lateral protocerebrum running toward the anterior optic tubercle and one fiber hub in the posterior lateral protocerebrum. Both are invaded by several DNs and LNs and might play an instrumental role in the clock network.

Neuroanatomical details of the lateral neurons of Drosophila melanogaster support their functional role in the circadian system.

Drosophila melanogaster is a long-standing model organism in the circadian clock research. A major advantage is the relative small number of about 150 neurons, which built the circadian clock in Drosophila. In our recent work, we focused on the neuroanatomical properties of the lateral neurons of the clock network. By applying the multicolor-labeling technique Flybow we were able to identify the anatomical similarity of the previously described E2 subunit of the evening oscillator of the clock, which is built by the 5th small ventrolateral neuron (5th s-LNv ) and one ITP positive dorsolateral neuron (LNd ). These two clock neurons share the same spatial and functional properties. We found both neurons innervating the same brain areas with similar pre- and postsynaptic sites in the brain. Here the anatomical findings support their shared function as a main evening oscillator in the clock network like also found in previous studies. A second quite surprising finding addresses the large lateral ventral PDF-neurons (l-LNv s). We could show that the four hardly distinguishable l-LNv s consist of two subgroups with different innervation patterns. While three of the neurons reflect the well-known branching pattern reproduced by PDF immunohistochemistry, one neuron per brain hemisphere has a distinguished innervation profile and is restricted only to the proximal part of the medulla-surface. We named this neuron "extra" l-LNv (l-LNv x). We suggest the anatomical findings reflect different functional properties of the two l-LNv subgroups.

Bridging the gap between postembryonic cell lineages and identified embryonic neuroblasts in the ventral nerve cord of Drosophila melanogaster.

The clarification of complete cell lineages, which are produced by specific stem cells, is fundamental for understanding mechanisms, controlling the generation of cell diversity and patterning in an emerging tissue. In the developing Central Nervous System (CNS) of Drosophila, neural stem cells (neuroblasts) exhibit two periods of proliferation: During embryogenesis they produce primary lineages, which form the larval CNS. After a phase of mitotic quiescence, a subpopulation of them resumes proliferation in the larva to give rise to secondary lineages that build up the CNS of the adult fly. Within the ventral nerve cord (VNC) detailed descriptions exist for both primary and secondary lineages. However, while primary lineages have been linked to identified neuroblasts, the assignment of secondary lineages has so far been hampered by technical limitations. Therefore, primary and secondary neural lineages co-existed as isolated model systems. Here we provide the missing link between the two systems for all lineages in the thoracic and abdominal neuromeres. Using the Flybow technique, embryonic neuroblasts were identified by their characteristic and unique lineages in the living embryo and their further development was traced into the late larval stage. This comprehensive analysis provides the first complete view of which embryonic neuroblasts are postembryonically reactivated along the anterior/posterior-axis of the VNC, and reveals the relationship between projection patterns of primary and secondary sublineages.

Tracing cells throughout development: insights into single glial cell differentiation.

In the article "Predetermined embryonic glial cells form the distinct glial sheaths of the Drosophila peripheral nervous system" we combined our expertise to identify glial cells of the embryonic peripheral nervous system on a single cell resolution with the possibility to genetically label cells using Flybow. We show that all 12 embryonic peripheral glial cells (ePG) per abdominal hemisegment persist into larval (and even adult) stages and differentially contribute to the three distinct glial layers surrounding peripheral nerves. Repetitive labelings of the same cell further revealed that layer affiliation, morphological expansion, and control of proliferation are predetermined and subject to an intrinsic differentiation program. Interestingly, wrapping and subperineurial glia undergo enormous hypertrophy in response to larval growth and elongation of peripheral nerves, while perineurial glia respond to the same environmental changes with hyperplasia. Increase in cell number from embryo (12 cells per hemisegment) to third instar (up to 50 cells per hemisegment) is the result of proliferation of a single ePG that serves as transient progenitor and only contributes to the outermost perineurial glial layer.