Neuropil is a fundamental form of tissue organization within brains 1 . In neuropils, densely packed neurons synaptically interconnect into precise circuit architecture 2 , 3 , yet the structural and developmental principles governing this nanoscale precision remain largely unknown 4 , 5 . Here, we use diffusion condensation, an iterative data coarse-graining algorithm 6 , to identify nested circuit structures within the C. elegans neuropil (called the nerve ring). We show that the nerve ring neuropil is largely organized into four strata composed of related behavioral circuits. The stratified architecture of the neuropil is a geometrical representation of the functional segregation of sensory information and motor outputs, with specific sensory organs and muscle quadrants mapping onto particular neuropil strata. We identify groups of neurons with unique morphologies that integrate information across strata and that create neural structures that cage the strata within the nerve ring. We use high resolution light-sheet microscopy 7 , 8 , coupled with lineage-tracing and cell-tracking algorithms 9 , 10 , to resolve the developmental sequence and reveal principles of cell position, migration and outgrowth that guide stratified neuropil organization. Our results uncover conserved structural design principles underlying nerve ring neuropil architecture and function, and a pioneer-neuron-based, temporal progression of outgrowth that guides the hierarchical development of the layered neuropil. Our findings provide a systematic blueprint for using structural and developmental approaches to understand neuropil organization within brains.
To visualize DC cluster relationships we developed C-PHATE, an extension of PHATE 30 capable of generating a 3D representation of the multigranular structures in the DC hierarchical tree ( Fig. 1a,b; see Methods). Quantitative comparisons of DC/C-PHATE results revealed similar clustering behaviors between the two separately analyzed nervering reconstructions (Adjusted Rand Index (ARI) of 0.7; Extended Data Fig. 1a,b), consistent with the long-accepted qualitative descriptions of the stereotyped structure for the C. elegans nerve ring 3,31 , and recent analyses of neurite adjacencies 22 . Examination of the cluster patterns through the iterations of DC revealed known cell-cell interactions and behavioral circuits ( Fig. 1b,c; Extended Data Fig. 2a,b; [32][33][34][35][36][37][38][39] ), consistent with behavioral and connectomic studies 10,19,20 . The multigranular outputs of the DC/C-PHATE results enabled understanding of the cell-cell interactions within the context of functional circuits, and the functional circuits within the context of the larger bundles of the neuropil.Comparisons of the modularity score, a measure of cluster robustness 40,41 , was highest when the majority of neurites were grouped into four super-clusters ( Fig. 1b; Extended Data Fig. 1a,b; Supplemental Video 1,2; see Methods). Overlaying our analysis of these four super-clusters (see Methods) onto the anatomy of the nerve ring revealed that they correspond to four distinct and tightly grouped bundles within the greater neuropil, circling the pharynx isthmus and stacked along the anterior-posterior axis of the animal. These clusters are herein called S1, S2, S3 and S4 for Stratum 1 etc. (Fig. 1c; Extended Data Fig. 1c-h; Supplemental Video 3 show details of these structures and single-cell identities). The stratified organization of the nerve ring neuropil, resolved here at single neurite level, is reminiscent of laminar organizations in the Drosophila nervous system 42,43 , and in the vertebrate retina and the brain cortex 44,45 .We observed that not all computationally clustered neurites followed simple bundled paths through the neuropil. In Strata 1 (S1), 32 anterior sensory neurons project perpendicular to the nerve ring bundle before making a 180潞 curl and returning to the anterior limits of the neuropil, where they terminate as synaptic endplates (Fig. 1d; Extended Data Fig. 3a-d; 3,46 ). These uniquely shaped S1 neurons looped at the borders of the S2/S3/S4 bundles. The anterior loops encase ~90% of S2, and the posterior loops encase ~84% of S3 and 100% of S4 ( Fig. 1d-g; Extended Data Fig. 3e-k ; SupplementalVideo 5; Supplemental Table 1). We found that the 32 axons form a six-fold symmetrical honeycomb structure along the neuropil's arc, creating a scaffold that encapsulates the different strata ( Fig. 1g; Extended Data Fig. 3e-h; Supplemental Video 4).The architecture of the neuropil reflects functional segregation of distinct sensory information streams and motor outputs.C. elegans neurons can be divided into three categories ...
The sensory nervous system of C. elegans comprises cells with varied molecular and functional characteristics, and is, therefore, a powerful model for understanding mechanisms that generate neuronal diversity. We report here that VAB-3, a C. elegans homolog of the homeodomain-containing protein Pax6, has opposing functions in regulating expression of a specific chemosensory fate. A homeodomain-only short isoform of VAB-3 is expressed in BAG chemosensory neurons, where it promotes gene expression and cell function. In other cells, a long isoform of VAB-3, comprising a Paired homology domain and a homeodomain, represses expression of ETS-5, a transcription factor required for expression of BAG fate. Repression of ets-5 requires the Eyes Absent homolog EYA-1 and the Six-class homeodomain protein CEH-32. We determined sequences that mediate high-affinity binding of ETS-5, VAB-3 and CEH-32. The ets-5 locus is enriched for ETS-5-binding sites but lacks sequences that bind VAB-3 and CEH-32, suggesting that these factors do not directly repress ets-5 expression. We propose that a promoterselection system together with lineage-specific expression of accessory factors allows VAB-3/Pax6 to either promote or repress expression of specific cell fates in a context-dependent manner.This article has an associated 'The people behind the papers' interview.
The internalization of the central nervous system, termed neurulation in vertebrates, is a critical step in embryogenesis. Open questions remain regarding how force propels coordinated tissue movement during the process, and little is known as to how internalization happens in invertebrates. We show that in C. elegans morphogenesis, apical constriction in the retracting pharynx drives involution of the adjacent neuroectoderm. HMR-1/cadherin mediates this process via inter-tissue attachment, as well as cohesion within the neuroectoderm. Our results demonstrate that HMR-1 is capable of mediating embryo-wide reorganization driven by a centrally located force generator, and indicate a non-canonical use of cadherin on the basal side of an epithelium that may apply to vertebrate neurulation. Additionally, we highlight shared morphology and gene expression in tissues driving involution, which suggests that neuroectoderm involution in C. elegans is potentially homologous with vertebrate neurulation and thus may help elucidate the evolutionary origin of the brain.
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