Stem cell-specific ecdysone signaling regulates the development and function of a Drosophila sleep homeostat.

Complex behaviors arise from neural circuits that are assembled from diverse cell types. Sleep is a conserved and essential behavior, yet little is known regarding how the nervous system generates neuron types of the sleep-wake circuit. Here, we focus on the specification of Drosophila sleep-promoting neurons-long-field tangential input neurons that project to the dorsal layers of the fan-shaped body neuropil in the central complex (CX). We use lineage analysis and genetic birth dating to identify two bilateral Type II neural stem cells that generate these dorsal fan-shaped body (dFB) neurons. We show that adult dFB neurons express Ecdysone-induced protein E93, and loss of Ecdysone signaling or E93 in Type II NSCs results in the misspecification of the adult dFB neurons. Finally, we show that E93 knockdown in Type II NSCs affects adult sleep behavior. Our results provide insight into how extrinsic hormonal signaling acts on NSCs to generate neuronal diversity required for adult sleep behavior. These findings suggest that some adult sleep disorders might derive from defects in stem cell-specific temporal neurodevelopmental programs.

A discrete neuronal population coordinates brain-wide developmental activity.

In vertebrates, stimulus-independent activity accompanies neural circuit maturation throughout the developing brain1,2. The recent discovery of similar activity in the developing Drosophila central nervous system suggests that developmental activity is fundamental to the assembly of complex brains3. How such activity is coordinated across disparate brain regions to influence synaptic development at the level of defined cell types is not well understood. Here we show that neurons expressing the cation channel transient receptor potential gamma (Trpγ) relay and pattern developmental activity throughout the Drosophila brain. In trpγ mutants, activity is attenuated globally, and both patterns of activity and synapse structure are altered in a cell-type-specific manner. Less than 2% of the neurons in the brain express Trpγ. These neurons arborize throughout the brain, and silencing or activating them leads to loss or gain of brain-wide activity. Together, these results indicate that this small population of neurons coordinates brain-wide developmental activity. We propose that stereotyped patterns of developmental activity are driven by a discrete, genetically specified network to instruct neural circuit assembly at the level of individual cells and synapses. This work establishes the fly brain as an experimentally tractable system for studying how activity contributes to synapse and circuit formation.

Robo2 Drives Target-Selective Peripheral Nerve Regeneration in Response to Glia-Derived Signals.

Peripheral nerves are divided into multiple branches leading to divergent synaptic targets. This poses a remarkable challenge for regenerating axons as they select their original trajectory at nerve branch-points. Despite implications for functional regeneration, the molecular mechanisms underlying target selectivity are not well characterized. Danio Rerio (zebrafish) motor nerves are composed of a ventral and a dorsal branch that diverge at a choice-point, and we have previously shown that regenerating axons faithfully select their original branch and targets. Here we identify robo2 as a key regulator of target-selective regeneration (sex of experimental subjects unknown). We demonstrate that robo2 function in regenerating axons is required and sufficient to drive target-selective regeneration, and that robo2 acts in response to glia located precisely where regenerating axons select the branch-specific trajectory to prevent and correct axonal errors. Combined, our results reveal a glia-derived mechanism that acts locally via axonal robo2 to promote target-selective regeneration.SIGNIFICANCE STATEMENT Despite its relevance for functional recovery, the molecular mechanisms that direct regenerating peripheral nerve axons toward their original targets are not well defined. Zebrafish spinal motor nerves are composed of a dorsal and a ventral branch that diverge at a stereotyped nerve branch-point, providing a unique opportunity to decipher the molecular mechanisms critical for target-selective regeneration. Using a combination of live cell imaging and molecular-genetic manipulations, we demonstrate that the robo2 guidance receptor is necessary and sufficient to promote target-selective regeneration. Moreover, we demonstrate that robo2 is part of a genetic pathway that generates transient, spatially restricted, and tightly coordinated signaling events that direct axons of the dorsal nerve branch toward their original, pre-injury targets.

SPARC enables genetic manipulation of precise proportions of cells.

Many experimental approaches rely on controlling gene expression in select subsets of cells within an individual animal. However, reproducibly targeting transgene expression to specific fractions of a genetically defined cell type is challenging. We developed Sparse Predictive Activity through Recombinase Competition (SPARC), a generalizable toolkit that can express any effector in precise proportions of post-mitotic cells in Drosophila. Using this approach, we demonstrate targeted expression of many effectors in several cell types and apply these tools to calcium imaging of individual neurons and optogenetic manipulation of sparse cell populations in vivo.

FlpStop, a tool for conditional gene control in Drosophila.

Manipulating gene function cell type-specifically is a common experimental goal in Drosophila research and has been central to studies of neural development, circuit computation, and behavior. However, current cell type-specific gene disruption techniques in flies often reduce gene activity incompletely or rely on cell division. Here we describe FlpStop, a generalizable tool for conditional gene disruption and rescue in post-mitotic cells. In proof-of-principle experiments, we manipulated apterous, a regulator of wing development. Next, we produced conditional null alleles of Glutamic acid decarboxylase 1 (Gad1) and Resistant to dieldrin (Rdl), genes vital for GABAergic neurotransmission, as well as cacophony (cac) and paralytic (para), voltage-gated ion channels central to neuronal excitability. To demonstrate the utility of this approach, we manipulated cac in a specific visual interneuron type and discovered differential regulation of calcium signals across subcellular compartments. Thus, FlpStop will facilitate investigations into the interactions between genes, circuits, and computation.

The lh3 Glycosyltransferase Directs Target-Selective Peripheral Nerve Regeneration.

Functional PNS regeneration requires injured axons to return to their original synaptic targets, yet the mechanisms underlying target-selective regeneration have remained elusive. Using live-cell imaging in zebrafish we find that regenerating motor axons exhibit a strong preference for their original muscle territory and that axons probe both correct and incorrect trajectories extensively before selecting their original path. We show that this process requires the glycosyltransferase lh3 and that post-injury expression of lh3 in Schwann cells is sufficient to restore target-selective regeneration. Moreover, we demonstrate that Schwann cells neighboring the transection site express the lh3 substrate collagen4a5 and that during regeneration collagen4a5 destabilizes axons probing inappropriate trajectories to ensure target-selective regeneration, possibly through the axonal repellant slit1a. Our results demonstrate that selective ECM components match subpopulations of regenerating axons with their original targets and reveal a previously unappreciated mechanism that conveys synaptic target selection to regenerating axons in vivo. VIDEO ABSTRACT.

Schwann cells and deleted in colorectal carcinoma direct regenerating motor axons towards their original path.

After complete nerve transection, a major challenge for regenerating peripheral axons is to traverse the injury site and navigate toward their original trajectory. Denervated Schwann cells distal to the lesion site secrete factors promoting axonal growth and serve as an axonal substrate, yet whether Schwann cells also actively direct axons toward their original trajectory is unclear. Using live-cell imaging in zebrafish, we visualize for the first time how in response to nerve transection distal Schwann cells change morphology as axons fragment, and how Schwann cell morphology reverses once regenerating growth cones have crossed the injury site and have grown along distal Schwann cells outlining the original nerve path. In mutants lacking Schwann cells, regenerating growth cones extend at rates comparable with wild type yet frequently fail to cross the injury site and instead stray along aberrant trajectories. Providing growth-permissive yet Schwann cell-less scaffolds across the injury site was insufficient to direct regenerating growth cones toward the original path, providing compelling evidence that denervated Schwann cells actively direct regenerating axons across the injury site toward their original trajectory. To identify signals that guide regenerating axons in vivo, we examined mutants lacking the deleted in colorectal carcinoma (DCC) guidance receptor. In these dcc mutants, a significant fraction of regenerating motor axons extended along aberrant trajectories, similar to what we observe in mutants lacking Schwann cells. Thus, Schwann cell and dcc-mediated guidance are critical early during regeneration to direct growth cones across the transection gap and onto their original axonal trajectory.

A novel role for Lh3 dependent ECM modifications during neural crest cell migration in zebrafish.

During vertebrate development, trunk neural crest cells delaminate along the entire length of the dorsal neural tube and initially migrate as a non-segmented sheet. As they enter the somites, neural crest cells rearrange into spatially restricted segmental streams. Extracellular matrix components are likely to play critical roles in this transition from a sheet-like to a stream-like mode of migration, yet the extracellular matrix components and their modifying enzymes critical for this transition are largely unknown. Here, we identified the glycosyltransferase Lh3, known to modify extracellular matrix components, and its presumptive substrate Collagen18A1, to provide extrinsic signals critical for neural crest cells to transition from a sheet-like migration behavior to migrating as a segmental stream. Using live cell imaging we show that in lh3 null mutants, neural crest cells fail to transition from a sheet to a stream, and that they consequently enter the somites as multiple streams, or stall shortly after entering the somites. Moreover, we demonstrate that transgenic expression of lh3 in a small subset of somitic cells adjacent to where neural crest cells switch from sheet to stream migration restores segmental neural crest cell migration. Finally, we show that knockdown of the presumptive Lh3 substrate Collagen18A1 recapitulates the neural crest cell migration defects observed in lh3 mutants, consistent with the notion that Lh3 exerts its effect on neural crest cell migration by regulating post-translational modifications of Collagen18A1. Together these data suggest that Lh3-Collagen18A1 dependent ECM modifications regulate the transition of trunk neural crest cells from a non-segmental sheet like migration mode to a segmental stream migration mode.

Heterosexual transmission of human immunodeficiency virus type 1 subtype C: Macrophage tropism, alternative coreceptor use, and the molecular anatomy of CCR5 utilization.

Human immunodeficiency virus type 1 transmission selects for virus variants with genetic characteristics distinct from those of donor quasispecies, but the biological factors favoring their transmission or establishment in new hosts are poorly understood. We compared primary target cell tropisms and entry coreceptor utilizations of donor and recipient subtype C Envs obtained near the time of acute infection from Zambian heterosexual transmission pairs. Both donor and recipient Envs demonstrated only modest macrophage tropism, and there was no overall difference between groups in macrophage or CD4 T-cell infection efficiency. Several individual pairs showed donor/recipient differences in primary cell infection, but these were not consistent between pairs. Envs had surprisingly broad uses of GPR15, CXCR6, and APJ, but little or no use of CCR2b, CCR3, CCR8, GPR1, and CXCR4. Donors overall used GPR15 better than did recipients. However, while several individual pairs showed donor/recipient differences for GPR15 and/or other coreceptors, the direction of the differences was inconsistent, and several pairs had unique alternative coreceptor patterns that were conserved across the transmission barrier. CCR5/CCR2b chimeras revealed that recipients as a group were more sensitive than were donors to replacement of the CCR5 extracellular loops with corresponding regions of CCR2b, but significant differences in this direction were not consistent within pairs. These data show that sexual transmission does not select for enhanced macrophage tropism, nor for preferential use of any alternative coreceptor. Recipient Envs are somewhat more constrained than are donors in flexibility of CCR5 use, but this pattern is not universal for all pairs, indicating that it is not an absolute requirement.

Entry coreceptor use and fusion inhibitor T20 sensitivity of dual-tropic R5X4 HIV-1 in primary macrophage infection.

Macrophages are important targets for HIV-1, and R5X4 strains play a central role in pathogenesis, especially in late-stage patients who may receive the fusion inhibitor T20 (enfuvirtide). Sensitivity to T20 varies markedly among HIV-1 strains and is influenced by viral and cellular factors that affect Env/CD4/coreceptor interactions. We addressed the relation between T20 inhibition and the pathway by which R5X4 HIV-1 infects primary macrophages, which express both coreceptors. In U87/CD4/coreceptor cells, T20 sensitivity for entry through CCR5 and CXCR4 was correlated. In macrophages, the proportion of total entry mediated by each coreceptor differed among isolates. Neither pathway was uniformly more or less sensitive to T20, however, nor did the proportion of entry mediated by each coreceptor predict T20 sensitivity. T20 sensitivity for macrophage infection overall correlated modestly with that for entry through CCR5 but not through CXCR4; however, unlike U87 cells, sensitivity of entry through CCR5 and CXCR4 was not correlated. These results suggest that strain-specific factors influence R5X4 T20 sensitivity regardless of the coreceptor used, an absence of systematic differences in efficiency by which R5X4 strains use the 2 coreceptors, and that efficiency and kinetics of interactions with CCR5 are central determinants of macrophage entry even when both pathways are utilized.