Summary Bilaterally symmetric motor patterns—those in which left-right pairs of muscles contract synchronously and with equal amplitude (such as breathing, smiling, whisking, locomotion)—are widespread throughout the animal kingdom. Yet surprisingly little is known about the underlying neural circuits. We performed a thermogenetic screen to identify neurons required for bilaterally symmetric locomotion in Drosophila larvae, and identified the evolutionarily-conserved Even-skipped+ interneurons (Eve/Evx). Activation or ablation of Eve+ interneurons disrupted bilaterally symmetric muscle contraction amplitude, without affecting the timing of motor output. Eve+ interneurons are not rhythmically active, and thus function independently of the locomotor CPG. GCaMP6 calcium imaging of Eve+ interneurons in freely-moving larvae showed left-right asymmetric activation that correlated with larval behavior. TEM reconstruction of Eve+ interneuron inputs and outputs showed that the Eve+ interneurons are at the core of a sensorimotor circuit capable of detecting and modifying body wall muscle contraction.

Animals generate diverse motor behaviors, yet how the same motor neurons (MNs) generate two distinct or antagonistic behaviors remains an open question. Here, we characterize Drosophila larval muscle activity patterns and premotor/motor circuits to understand how they generate forward and backward locomotion. We show that all body wall MNs are activated during both behaviors, but a subset of MNs change recruitment timing for each behavior. We used TEM to reconstruct a full segment of all 60 MNs and 236 premotor neurons (PMNs), including differentially-recruited MNs. Analysis of this comprehensive connectome identified PMN-MN ‘labeled line’ connectivity; PMN-MN combinatorial connectivity; asymmetric neuronal morphology; and PMN-MN circuit motifs that could all contribute to generating distinct behaviors. We generated a recurrent network model that reproduced the observed behaviors, and used functional optogenetics to validate selected model predictions. This PMN-MN connectome will provide a foundation for analyzing the full suite of larval behaviors.

Command-like descending neurons can induce many behaviors, such as backward locomotion, escape, feeding, courtship, egg-laying, or grooming (we define ‘command-like neuron’ as a neuron whose activation elicits or ‘commands’ a specific behavior). In most animals, it remains unknown how neural circuits switch between antagonistic behaviors: via top-down activation/inhibition of antagonistic circuits or via reciprocal inhibition between antagonistic circuits. Here, we use genetic screens, intersectional genetics, circuit reconstruction by electron microscopy, and functional optogenetics to identify a bilateral pair of Drosophila larval ‘mooncrawler descending neurons’ (MDNs) with command-like ability to coordinately induce backward locomotion and block forward locomotion; the former by stimulating a backward-active premotor neuron, and the latter by disynaptic inhibition of a forward-specific premotor neuron. In contrast, direct monosynaptic reciprocal inhibition between forward and backward circuits was not observed. Thus, MDNs coordinate a transition between antagonistic larval locomotor behaviors. Interestingly, larval MDNs persist into adulthood, where they can trigger backward walking. Thus, MDNs induce backward locomotion in both limbless and limbed animals.

More than 30 years of studies into Drosophila melanogaster neurogenesis have revealed fundamental insights into our understanding of axon guidance mechanisms, neural differentiation, and early cell fate decisions. What is less understood is how a group of neurons from disparate anterior-posterior axial positions, lineages and developmental periods of neurogenesis coalesce to form a functional circuit. Using neurogenetic techniques developed in Drosophila it is now possible to study the neural substrates of behavior at single cell resolution. New mapping tools described in this review, allow researchers to chart neural connectivity to better understand how an anatomically simple organism performs complex behaviors.

The mechanisms specifying neuronal diversity are well-characterized, yet it remains unclear how or if these mechanisms regulate neural circuit assembly. To address this, we mapped the developmental origin of 160 interneurons from seven bilateral neural progenitors (neuroblasts), and identify them in a synapse-scale TEM reconstruction of the Drosophila larval CNS. We find that lineages concurrently build the sensory and motor neuropils by generating sensory and motor hemilineages in a Notch-dependent manner. Neurons in a hemilineage share common synaptic targeting within the neuropil, which is further refined based on neuronal temporal identity. Connectome analysis shows that hemilineage-temporal cohorts share common connectivity. Finally, we show that proximity alone cannot explain the observed connectivity structure, suggesting hemilineage/temporal identity confers an added layer of specificity. Thus, we demonstrate that the mechanisms specifying neuronal diversity also govern circuit formation and function, and that these principles are broadly applicable throughout the nervous system.

21The mechanisms specifying neuronal diversity are well-characterized, yet it remains unclear how or if these 22 mechanisms regulate neuronal morphology and connectivity. Here we map the developmental origin of 78 23 bilateral pairs of interneurons from seven identified neural progenitors (neuroblasts) within a complete TEM 24 reconstruction of the Drosophila newly-hatched larval CNS. This allows us to correlate developmental 25 mechanism with neuronal projections, synapse targeting, and connectivity. We find that clonally-related 26 neurons from project widely in the neuropil, without preferential circuit formation. In contrast, the two 27 Notch ON /Notch OFF hemilineages from each neuroblast project to either dorsal motor neuropil (Notch ON ) or 28 ventral sensory neuropil (Notch OFF ). Thus, each neuroblast contributes both motor and sensory processing 29 neurons. Lineage-specific constitutive Notch transforms sensory to motor hemilineages, showing hemilineage 30 identity determines neuronal targeting. Within a hemilineage, temporal cohorts target processes and synapses 31 to different sub-domains of the neuropil, effectively "tiling" the hemilineage neuropil, and 32 hemilineage/temporal cohorts are enriched for shared connectivity. Thus, neuroblast lineage, hemilineage, 33 and temporal identity progressively restrict neuropil targeting, synapse localization, and connectivity. We 34 propose that mechanisms generating neural diversity are also determinants of neural circuit formation. 35 36 37 38Tremendous progress has been made in understanding the molecular mechanisms generating neuronal 39 diversity in both vertebrate and invertebrate model systems. In mammals, spatial cues generate distinct pools 40 of progenitors which generate a diversity of neurons and glia appropriate for each spatial domain (1). The 41 same process occurs in invertebrates like Drosophila, but with a smaller number of cells, and this process is 42 particularly well-understood. Spatial patterning genes act combinatorially to establish single, unique 43 progenitor (neuroblast) identity; these patterning genes include the dorsoventral columnar genes vnd, ind, msh 44 (2-4) and the orthogonally expressed wingless, hedgehog, gooseberry, and engrailed genes (5-8). These factors endow 45 each neuroblast with a unique spatial identity, the first step in generating neuronal diversity ( Figure 1A, left). 46Here we focus on the left and right sides of abdominal segment 1 (A1L, A1R) and so segment-specific 47 patterning due to Hox gene expression is not relevant. The second step occurs as each neuroblast "buds off" 48 a series of ganglion mother cells (GMCs) which acquire a unique identity based on their birth-order, due to 49 inheritance from the neuroblast of a "temporal transcription factor"-Hunchback (Hb), Krüppel (Kr), Pdm, 50 and Castor (Cas) -which are sequentially expressed by nearly all embryonic neuroblasts (9). The combination 51 of spatial and temporal factors leads to the production of a unique GMC with each neuroblast division 52 (F...

SUMMARY Combinations of transcription factors (TFs) instruct precise wiring patterns in the developing nervous system; however, how these factors impinge on surface molecules that control guidance decisions is poorly understood. Using mRNA profiling, we identified the complement of membrane molecules regulated by the homeobox TF Even-skipped (Eve), the major determinant of dorsal motor neuron (dMN) identity in Drosophila. Combinatorial loss- and gain-of-function genetic analyses of Eve target genes indicate that the integrated actions of attractive, repulsive, and adhesive molecules direct eve-dependent dMN axon guidance. Furthermore, combined misexpression of Eve target genes is sufficient to partially restore CNS exit and can convert the guidance behavior of interneurons to that of dMNs. Finally, we show that a network of TFs, comprised of eve, zfh1, and grain, induces the expression of the Unc5 and Beaten-path guidance receptors and the Fasciclin 2 and Neuroglian adhesion molecules to guide individual dMN axons.

SUMMARYTranscription factor codes play an essential role in neuronal specification and axonal guidance in both vertebrate and invertebrate organisms. However, how transcription codes regulate axon pathfinding remains poorly understood. One such code defined by the homeodomain transcription factor Even-skipped (Eve) and by the GATA 2/3 homologue Grain (Grn) is specifically required for motor axon projection towards dorsal muscles in Drosophila. Using different mutant combinations, we present genetic evidence that both Grn and Eve are in the same pathway as Unc-5 in dorsal motoneurons (dMNs). In grn mutants, in which dMNs fail to reach their muscle targets, dMNs show significantly reduced levels of unc-5 mRNA expression and this phenotype can be partially rescued by the reintroduction of unc-5. We also show that both eve and grn are required independently to induce expression of unc-5 in dMNs. Reconstitution of the eve-grn transcriptional code of a dMN in dMP2 neurons, which do not project to lateral muscles in Drosophila, is able to reprogramme those cells accordingly; they robustly express unc-5 and project towards the muscle field as dMNs. Each transcription factor can independently induce unc-5 expression but unc-5 expression is more robust when both factors are expressed together. Furthermore, dMP2 exit is dependent on the level of unc-5 induced by eve and grn. Taken together, our data strongly suggests that the eve-grn transcriptional code controls axon guidance, in part, by regulating the level of unc-5 expression.