Structural and developmental principles of neuropil assembly in C. elegans

Abstract

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.

The Prop1-like homeobox gene unc-42 specifies the identity of synaptically connected neurons

“…The main worm neuropil can be abstracted into a laminar structure with specific clusters (‘strata’) or neighborhoods of neurons defined by ultrastructural adjacencies of neuron processes ( Brittin et al, 2021 ; Moyle et al, 2021 ). Despite all UNC-42(+) neurons projecting into this neuropil ( Figure 1E ), UNC-42(+) axons are not exclusively part of one stratum ( Brittin et al, 2021 ; Moyle et al, 2021 ; Supplementary file 2 ). However, we noted a striking theme of the 40 neurons (15 neuron classes) that express UNC-42(+): they form a network of densely connected neurons ( Figure 1D, E ).…”

“…This suggests that unc-42 may also have a role in assembling neurons into functional circuitry. Interestingly, the processes of unc-42(+) neurons traverse distinct neighborhoods (‘strata’) of the nerve ring ( Moyle et al, 2021 ; Brittin et al, 2021 ), indicating that unc-42 may facilitate intra-strata connectivity, thereby ensuring the coordination of information flow through distinct neighborhoods. We provide evidence for a role of unc-42 in indeed defining synaptic connectivity by demonstrating synaptic connectivity defects in unc-42 mutants, as inferred by an ultrastructural analysis, but also by the loss of expression of genes involved in electrical synapse formation (innexins) as well as loss of genes possibly involved in synaptic targeting/synapse formation.…”

“… Clustering outputs of Brittin et al, 2021 , Moyle et al, 2021 are shown for each neuron. Discordant clustering results of neuronal subclasses are shown for RMD and SIB.…”

A developmental framework linking neurogenesis and circuit formation in the Drosophila CNS

“…Neurons initially target their axons to broad regions (e.g. thalamus/cortex), followed by targeting to a neuropil domain (glomeruli/layer), and finally forming highly specific synapses within the targeted domain (Kolodkin and Tessier-Lavigne, 2011;Moyle et al, 2021).…”

“…A great deal of progress has also been made in understanding neural circuit formation in both vertebrates and invertebrate model systems, revealing a multi-step mechanism. Neurons initially target their axons to broad regions (e.g., thalamus/cortex), followed by targeting to a neuropil domain (glomeruli/layer), and finally forming highly specific synapses within the targeted domain ( Kolodkin and Tessier-Lavigne, 2011 ; Moyle et al, 2021 ).…”