Central projections from Johnston's organ in the locust: Axogenesis and brain neuroarchitecture.

Johnston's organ (Jo) acts as an antennal wind-sensitive and/or auditory organ across a spectrum of insect species and its axons universally project to the brain. In the locust, this pathway is already present at mid-embryogenesis but the process of fasciculation involved in its construction has not been investigated. Terminal projections into the fine neuropilar organization of the brain also remain unresolved, information essential not only for understanding the neural circuitry mediating Jo-mediated behavior but also for providing comparative data offering insights into its evolution. In our study here, we employ neuron-specific, axon-specific, and epithelial domain labels to show that the pathway to the brain of the locust is built in a stepwise manner during early embryogenesis as processes from Jo cell clusters in the pedicel fasciculate first with one another, and then with the two tracts constituting the pioneer axon scaffold of the antenna. A comparison of fasciculation patterns confirms that projections from cell clusters of Jo stereotypically associate with only one axon tract according to their location in the pedicellar epithelium, consistent with a topographic plan. At the molecular level, all neuronal elements of the Jo pathway to the brain express the lipocalin Lazarillo, a cell surface epitope that regulates axogenesis in the primary axon scaffold itself, and putatively during fasciculation of the Jo projections to the brain. Central projections from Jo first contact the primary axon scaffold of the deutocerebral brain at mid-embryogenesis, and in the adult traverse mechanosensory/motor neuropils similar to those in Drosophila. These axons then terminate among protocerebral commissures containing premotor interneurons known to regulate flight behavior.

Single-cell type analysis of wing premotor circuits in the ventral nerve cord of Drosophila melanogaster.

To perform most behaviors, animals must send commands from higher-order processing centers in the brain to premotor circuits that reside in ganglia distinct from the brain, such as the mammalian spinal cord or insect ventral nerve cord. How these circuits are functionally organized to generate the great diversity of animal behavior remains unclear. An important first step in unraveling the organization of premotor circuits is to identify their constituent cell types and create tools to monitor and manipulate these with high specificity to assess their function. This is possible in the tractable ventral nerve cord of the fly. To generate such a toolkit, we used a combinatorial genetic technique (split-GAL4) to create 195 sparse driver lines targeting 198 individual cell types in the ventral nerve cord. These included wing and haltere motoneurons, modulatory neurons, and interneurons. Using a combination of behavioral, developmental, and anatomical analyses, we systematically characterized the cell types targeted in our collection. Taken together, the resources and results presented here form a powerful toolkit for future investigations of neural circuits and connectivity of premotor circuits while linking them to behavioral outputs.

A searchable image resource of Drosophila GAL4 driver expression patterns with single neuron resolution.

Precise, repeatable genetic access to specific neurons via GAL4/UAS and related methods is a key advantage of Drosophila neuroscience. Neuronal targeting is typically documented using light microscopy of full GAL4 expression patterns, which generally lack the single-cell resolution required for reliable cell type identification. Here, we use stochastic GAL4 labeling with the MultiColor FlpOut approach to generate cellular resolution confocal images at large scale. We are releasing aligned images of 74,000 such adult central nervous systems. An anticipated use of this resource is to bridge the gap between neurons identified by electron or light microscopy. Identifying individual neurons that make up each GAL4 expression pattern improves the prediction of split-GAL4 combinations targeting particular neurons. To this end, we have made the images searchable on the NeuronBridge website. We demonstrate the potential of NeuronBridge to rapidly and effectively identify neuron matches based on morphology across imaging modalities and datasets.

Early embryonic development of Johnston's organ in the antenna of the desert locust Schistocerca gregaria.

Johnston's organ has been shown to act as an antennal auditory organ across a spectrum of insect species. In the hemimetabolous desert locust Schistocerca gregaria, Johnston's organ must be functional on hatching and so develops in the pedicellar segment of the antenna during embryogenesis. Here, we employ the epithelial cell marker Lachesin to identify the pedicellar domain of the early embryonic antenna and then triple-label against Lachesin, the mitosis marker phosphohistone-3, and neuron-specific horseradish peroxidase to reveal the sense-organ precursors for Johnston's organ and their lineages. Beginning with a single progenitor at approximately a third of embryogenesis, additional precursors subsequently appear in both the ventral and dorsal pedicellar domains, each generating a lineage or clone. Lineage locations are remarkably conserved across preparations and ages, consistent with the epithelium possessing an underlying topographic coordinate system that determines the cellular organization of Johnston's organ. By mid-embryogenesis, twelve lineages are arranged circumferentially in the pedicel as in the adult structure. Each sense-organ precursor is associated with a smaller mitotically active cell from which the neuronal complement of each clone may derive. Neuron numbers within a clone increase in discrete steps with age and are invariant between clones and across preparations of a given age. At mid-embryogenesis, each clone comprises five cells consolidated into a tightly bound cartridge. A long scolopale extends apically from each cartridge to an insertion point in the epithelium, and bundled axons project basally toward the brain. Comparative data suggest mechanisms that might also regulate the developmental program of Johnston's organ in the locust.

Evidence for the cholinergic markers ChAT and vAChT in sensory cells of the developing antennal nervous system of the desert locust Schistocerca gregaria.

Sensory and motor systems in insects with hemimetabolous development must be ready to mediate adaptive behavior directly on hatching from the egg. For the desert locust S. gregaria, cholinergic transmission from antennal sensillae to olfactory or mechanosensory centers in the brain requires that choline acetyltransferase (ChAT) and the vesicular acetylcholine transporter (vAChT) already be present in sensory cells in the first instar. In this study, we used immunolabeling to demonstrate that ChAT and vAChT are both expressed in sensory cells from identifiable sensilla types in the immature antennal nervous system. We observed ChAT expression in dendrites, neurites and somata of putative basiconic-type sensillae at the first instar stage. We also detected vAChT in the sensory axons of these sensillae in a major antennal nerve tract. We then examined whether evidence for cholinergic transmission is present during embryogenesis. Immunolabeling confirms that vAChT is expressed in somata typical of campaniform sensillae, as well as in small sensory cell clusters typically associated with either a large basiconic or coeloconic sensilla, at 99% of embryogenesis. The vAChT is also expressed in the somata of these sensilla types in multiple antennal regions at 90% of embryogenesis, but not at earlier (70%) embryonic stages. Neuromodulators are known to appear late in embryogenesis in neurons of the locust central complex, and the cholinergic system of the antenna may also only reach maturity shortly before hatching.

Epithelial domains and the primordial antennal nervous system of the embryonic grasshopper Schistocerca gregaria.

The antenna is a key sensory organ in insects. Factors which pattern its epithelium and the spacing of sensillae will play an important role in shaping its contribution to adaptive behavior. The antenna of the grasshopper S. gregaria has three major articulations: scape, pedicel, and flagellum. During postembryonic development, the flagellum lengthens as segments (so-called meristal annuli) are added at each molt. However, the five most apical annuli do not subdivide; thus, their epithelial domains must already be defined during embryogenesis. We investigated epithelial compartmentalization and its relationship to the developing primordial nervous system of the antenna by simultaneous immunolabeling against the epithelial cell surface molecule Lachesin, against neuron-specific horseradish peroxidase, and against the mitosis marker phospho-histone 3. We found that Lachesin is initially expressed in a highly ordered pattern of "rings" and a "sock" in the apical antennal epithelium of the early embryo. These expression domains appear in a stereotypic order and prefigure later articulations. Proliferative cells segregate into these developing domains and pioneer- and sensory-cell precursors were molecularly identified. Our study allows pioneer neurons, guidepost cells, and the earliest sensory cell clusters of the primordial nervous system to be allocated to their respective epithelial domain. As the apical-most five domains remain stable through subsequent development, lengthening of the flagellum must originate from more basal regions and is likely to be under the control of factors homologous to those which regulate boundary and joint formation in the antenna of Drosophila.

Dysregulation of axogenesis in the antennal nervous system of the embryonic grasshopper Schistocerca gregaria.

The antennal nervous system of the grasshopper Schistocerca gregaria features two parallel axon tracts each established early in embryogenesis by discrete pairs of pioneer neurons located at the antennal tip and whose growth cones contact so-called base pioneers en route to the brain. Here we present two antennal phenotypes in which a stereotypic dysregulation of axogenesis in a given tract is observed when only the base pioneer associated with that pathway is missing, consistent with a role for this cell type in guided axogenesis. Dysregulation involves defasciculation and aberrant navigation by pioneer axons resulting in a missing or depleted primordial antennal nerve to the brain. The dysregulated phenotypes reveal that axogenesis in each pathway is regulated independently. Previously unseen discrepancies in the navigational decisions made by pioneer neurons which derive sequentially from the same mother cell demonstrate that these progeny have separate identities. Possible mechanisms for the dysregulated phenotypes are considered.

Patterns of cell death in the embryonic antenna of the grasshopper Schistocerca gregaria.

We have investigated the pattern of apoptosis in the antennal epithelium during embryonic development of the grasshopper Schistocerca gregaria. The molecular labels lachesin and annulin reveal that the antennal epithelium becomes subdivided into segment-like meristal annuli within which sensory cell clusters later differentiate. To determine whether apoptosis is involved in the development of such sensory cell clusters, we examined the expression pattern of the cell death labels acridine orange and TUNEL in the epithelium. We found stereotypic, age-dependent, wave-like patterns of cell death in the antenna. Early in embryogenesis, apoptosis is restricted to the most basal meristal annuli but subsequently spreads to encompass almost the entire antenna. Cell death then declines in more basal annuli and is only found in the tip region later in embryogenesis. Apoptosis is restricted throughout to the midregion of a given annulus and away from its border with neighboring annuli, arguing against a causal role in annular formation. Double-labeling for cell death and sensory cell differentiation reveals apoptosis occurring within bands of differentiating sensory cell clusters, matching the meristal organization of the apical antenna. Examination of the individual epithelial lineages which generate sensory cells reveals that apoptosis begins peripherally within a lineage and with age expands to encompass the differentiated sensory cell at the base. We conclude that complete lineages can undergo apoptosis and that the youngest cells in these lineages appear to die first, with the sensory neuron dying last. Lineage-based death in combination with cell death patterns in different regions of the antenna may contribute to odor-mediated behaviors in the grasshopper.

Ontogeny of pioneer neurons in the antennal nervous system of the grasshopper Schistocerca gregaria.

The nervous system of the antenna of the grasshopper Schistocerca gregaria consists of two nerve tracts in which sensory cells project their axons to the brain. Each tract is pioneered early in embryogenesis by a pair of identified cells located apically in the antennal lumen. The pioneers are thought to originate in the epithelium of the antenna and then delaminate into the lumen where they commence axogenesis. However, unambiguous molecular identification of these cells in the epithelium, of an identifiable precursor, and of their mode of generation has been lacking. In this study, we have used immunolabeling against neuron-specific horseradish peroxidase and against Lachesin, a marker for differentiating epithelial cells, in combination with the nuclear stain DAPI, to identify the pioneers within the epithelium of the early embryonic antenna. We then track their delamination into the lumen as differentiated neurons. The pioneers are not labeled by the mesodermal/mesectodermal marker Mes3, consistent with an epithelial (ectodermal) origin. Intracellular dye injection, as well as labeling against the mitosis marker phospho-histone 3, identifies precursor cells in the epithelium, each associated with a column of cells. Culturing with the S-phase label 5-ethynyl-2'-deoxyuridine (EdU) shows that both a precursor and its column have incorporated the label, confirming a lineage relationship. Each set of pioneers can be shown to belong to a separate lineage of such epithelial cells, and the precursors remain and are proliferative after generating the pioneers. Analyses of mitotic spindle orientation then enable us to propose a model in which a precursor generates its pioneers asymmetrically via self-renewal.

Fates of identified pioneer cells in the developing antennal nervous system of the grasshopper Schistocerca gregaria.

In the early embryonic grasshopper, two pairs of sibling cells near the apex of the antenna pioneer its dorsal and ventral nerve tracts to the brain. En route, the growth cones of these pioneers contact a so-called base pioneer associated with each tract and which acts as a guidepost cell. Both apical and basal pioneers express stereotypic molecular labels allowing them to be uniquely identified. Although their developmental origins are largely understood, the fates of the respective pioneers remain unclear. We therefore employed the established cell death markers acridine orange and TUNEL to determine whether the apical and basal pioneers undergo apoptosis during embryogenesis. Our data reveal that the apical pioneers maintain a consistent molecular profile from their birth up to mid-embryogenesis, at which point the initial antennal nerve tracts to the brain have been established. Shortly after this the apical pioneers undergo apoptosis. Death occurs at a developmental stage similar to that reported elsewhere for pioneers in a leg - an homologous appendage. Base pioneers, by contrast, progressively change their molecular profile and can no longer be unequivocally identified after mid-embryogenesis. At no stage up to then do they exhibit death labels. If they persist, the base pioneers must be assumed to adopt a new role in the developing antennal nervous system.

Pioneer neurons of the antennal nervous system project to protocerebral pioneers in the grasshopper Schistocerca gregaria.

The twin nerve tracts of the antenna of the grasshopper Schistocerca gregaria are established early in embryogenesis by sibling pairs of pioneers which delaminate from the epithelium into the lumen at the antennal tip. These cells can be uniquely identified via their co-expression of the neuronal labels horseradish peroxidase and the lipocalin Lazarillo. The apical pioneers direct axons toward the antennal base where they encounter guidepost-like cells called base pioneers which transiently express the same molecular labels as the apical pioneers. To what extent the pioneer growth cones then progress into the brain neuropil proper, and what their targets there might be, has remained unclear. In this study, we show that the apical antennal pioneers project centrally beyond the antennal base first into the deutocerebral, and then into the protocerebral brain neuropils. In the protocerebrum, we identify their target circuitry as being identified Lazarillo-positive cells which themselves pioneer the primary axon scaffold of the brain. The apical and base antennal pioneers therefore form part of a molecularly contiguous pathway from the periphery to an identified central circuit of the embryonic grasshopper brain.

A method for immunolabeling neurons in intact cuticularized insect appendages.

The antennae of the grasshopper Schistocerca gregaria possess a pair of nerve pathways which are established by so-called pioneer neurons early in embryonic development. Subsequently, sensory cell clusters mediating olfaction, flight, optomotor responses, and phase changes differentiate from the antennal epithelium at stereotypic locations and direct their axons onto those of the pioneers to then project to the brain. Early in embryonic development, before the antennae become cuticularized, immunolabeling can be used to follow axogenesis in these pioneers and sensory cells. At later stages, immunolabeling becomes problematical as the cuticle is laid down and masks internal antigen sites. In order to immunolabel the nervous system of cuticularized late embryonic and first instar grasshopper antennae, we modified a procedure known as sonication in which the appendage is exposed to ultrasound thereby rendering it porous to antibodies. Comparisons of the immunolabeled nervous system of sectioned and sonicated antennae show that the cellular organization of sensory clusters and their axon projections is intact. The expression patterns of neuron-specific, microtubule-specific, and proliferative cell-specific labels in treated antennae are consistent with those reported for earlier developmental stages where sonication is not necessary, suggesting that these molecular epitopes are also conserved. The method ensures reliable immunolabeling in intact, cuticularized appendages so that the peripheral nervous system can be reconstructed directly via confocal microscopy throughout development.

Axogenesis in the antennal nervous system of the grasshopper Schistocerca gregaria revisited: the base pioneers.

The antennal nervous system of the grasshopper Schistocerca gregaria comprises two parallel pathways projecting to the brain, each pioneered early in embryogenesis by a pair of sibling cells located at the antennal tip. En route, the growth cones of pioneers from one pathway have been shown to contact a guidepost-like cell called the base pioneer. Its role in axon guidance remains unclear as do the cellular guidance cues regulating axogenesis in the other pathway supposedly without a base pioneer. Further, while the tip pioneers are known to delaminate from the antennal epithelium into the lumen, the origin of this base pioneer is unknown. Here, we use immunolabeling and immunoblocking methods to clarify these issues. Co-labeling against the neuron-specific marker horseradish peroxidase and the pioneer-specific cell surface glycoprotein Lazarillo identifies not only the tip pioneers but also a base pioneer associated with each of the developing antennal pathways. Both base pioneers co-express the mesodermal label Mes3, consistent with a lumenal origin, whereas the tip pioneers proved Mes3-negative confirming their affiliation with the ectodermal epithelium. Lazarillo antigen expression in the antennal pioneers followed a different temporal dynamic: continuous in the tip pioneers, but in the base pioneers, only at the time their filopodia and those of the tip pioneers first recognize one another. Immunoblocking of Lazarillo expression in cultured embryos disrupts this recognition resulting in misguided axogenesis in both antennal pathways.