Comparative connectomics of two distantly related nematode species reveals patterns of nervous system evolution

Abstract

Understanding the evolution of the bilaterian brain requires a detailed exploration of the precise nature of cellular and subcellular differences between related species. We undertook an electron micrographic reconstruction of the brain of the predatory nematode Pristionchus pacificus and compared the results with the brain of Caenorhabditis elegans, which diverged at least 100 million years ago. We revealed changes in neuronal cell death, neuronal cell position, axodendritic projection patterns, and synaptic connectivity of homologous neurons that display no obvious changes in overall neurite morphology and projection patterns. These multiscale patterns of evolutionary changes show no bias to specific brain regions or neuron types.

Fig. 1:. The reconstructed Pristionchus pacificus brain.

A: A left lateral view 3D rendering of all P. pacificus neurons reconstructed from serial section electron micrographs of the head. Neurons are shown in different colors for contrast; the pharynx is shown in teal. B: A left lateral 3D rendering of all neuronal nuclei in the P. pacificus head. Nuclei are colored by neuronal function experimentally determined in nematodes: Sensory – red, interneuron – yellow, motor – blue; the pharynx is shown in teal. C: Left lateral views of easily-homologized P. pacificus neurons, including: IL2D, CEPD, BAG, RIA, RID, FLP, RMH, and RIG. Left and right homologous neurons are shown in magenta and blue, respectively, and the pharynx is shown in teal.

Fig. 2:. Heterochronic cell death in P. pacificus.

A: Differential interference contrast image of the P. pacificus embryo, with an arrow pointing to the dying progenitor cell.. B: Lineaging diagram (generated by Simi BioCell) illustrating the death of AVH’s sister cell in C. elegans (left) and AVH’s progenitor cell death in P. pacificus (right) during embryogenesis. Cell fates are defined by nuclear morphology (N= neuron-like nuclear morphology; D = cell death; U = muscle; M = mitosis; P = pharynx; H = hypodermal; duplicated letter means: first letter = predicted fate; second letter = observed fate). C: Phylogenetic tree of the PAS domain of bHLH-PAS proteins from various nematode species.

Fig. 3:. Morphological evolution of neurite trajectories.

In each panel (A-E), the top section shows a schematic comparison of the neuron’s morphology in C. elegans (red) and P. pacificus (blue). Schematics are left lateral views (anterior to the left and dorsal up) for all panels except C (AQR), which is a ventral view. The pharynx is shown in light green and the nerve ring (neuropil region) in grey, outlined by a dashed line. Below the schematics are a connectivity diagram and 3D rendering of the P. pacificus neuron (blue), in some cases with a partner neuron (magenta). Connectivity diagrams show chemical synapses that are shared in all adult nematode samples (black), are C. elegans-specific (red), or P. pacificus-specific (blue). Line width is proportional to synaptic weight and arrowheads show directionality. A connectivity diagram is not relevant for panel E and is not included. A: URB morphology. The 3D rendering of P. pacificus URB (blue) is shown with a conserved synaptic partner of URB, the inner labial sensory neuron IL1 (magenta), which retains its dendrite in P. pacificus, extending from the nerve ring region to beyond the anterior tip of the pharynx. B: PVD morphology. The double slash indicates the PVD soma is posterior to the reconstruction. The 3D rendering shows PVD (blue) with P. pacificus-specific synaptic partner AVD (magenta), a closeup of the nerve ring region. C: RIP morphology. The 3D rendering shows RIP (blue) r with P. pacificus-specific synaptic partner AIZ (magenta), a closeup of the nerve ring region. D: AQR morphology (ventral view). The 3D rendering shows AQR (blue) entering the nerve ring on the left (top), extending over dorsally to the right side (bottom). The dashed line indicates that the posterior-most AQR connecting neurite is not included in the reconstruction. E. ASJ morphology. 3D renderings of both ASJL (blue) and ASJR (magenta) are shown in the lateral posterior view (left) and dorsal posterior view (right, with nearly transparent pharynx), showing that these branches, indicated by arrows, project near the pseudocoelom. F. ALM and AVM morphologies. The double slash indicates the AVM and ALM somas are posterior to the reconstruction. The 3D rendering shows the simpler P. pacificus AVM neurite (blue) contacted by an extended ALM neurite (magenta).

Fig. 4:. Brain-wide comparison reveals widespread connectivity changes.

Panels A-D: Network (wiring) diagrams showing all neurons in the nerve ring and their synaptic connections: shared (black), variable (gray), P. pacificus-specific (blue), C. elegans-specific (red). Arrow widths are proportional to the average number of synapses across datasets; arrowheads represent directionality. A. Complete wiring diagram showing all P. pacificus and C. elegans connections. Legend for color code is provided. B: Shared wiring diagram highlighting shared, non-variable connections in black. Legend for synaptic edge weights is provided. C. Diagram highlighting P. pacificus-specific connections in blue. D. Diagram highlighting C. elegans-specific connections in red.

Fig. 5:. Analysis of species-specific adult chemical connectivity

All embryonically born sex-shared nerve ring neurons that make a species-specific synaptic connection are shown. Stacked bar graphs of the percentage of species-specific undirected synaptic connections relative to all other synapses made by the respective neuron class (left). Stacked bar graphs of the number of sex-specific directed inputs and outputs by neuron class (right).

Fig. 6:. Neurite neighborhood shifts and quantitative analysis of synaptic connectivity differences.

A: Schematic comparison of ASH neuron morphology in C. elegans (red) and P. pacificus (blue). Left lateral view (see legend for Figure 3). B: Schematic of nerve ring cross section illustrating lateral displacement of ASH neurite in P. pacificus (blue) compared to C. elegans (red). C: Adult wiring diagram of ASH. C. elegans-specific connections are shown in red. D: Histogram of adjacency conservation of neuron pairs (Number of EM series with edge present) among all nematode nerve ring series (C. elegans-specific edges are red, P. pacificus-specific edges are blue, shared edges are black, and variable edges are gray). Legend applies to panels D-G. E: Histogram of directed connectivity conservation of neuron pairs (Number of EM series with edge present) among all nematode nerve ring series. F: Histogram of adjacency conservation of neuron pairs among adult nerve ring series. G: Histogram of directed connectivity conservation of neuron pairs among adult nerve ring series. H. Violin plot comparing mean scaled adjacency and connectivity specificity classification Condition: No Connection (no statistical test performed), Not specific, U statistic: 541864, Corrected p-value: 3.11e-27, Condition: Shared, U statistic: 3694, Corrected p-value: 1.80e-9 Condition: P. pacificus-specific, U statistic: 2871, Corrected p-value: 7.56e-5 Condition: C. elegans-specific, U statistic: 180.0, Corrected p-value: 1.73e-4. Legend applies to panels H and I. I. Violin plot comparing log+1 adjusted number of synapses and connectivity specificity classification. Condition: not specific, U statistic: 1842058, Corrected p-value: 9.87e-67 Condition: shared, U statistic: 1804.0, Corrected p-value: 2.72e-2 Condition: P. pacificus-specific, U statistic: 0.0, Corrected p-value: 2.33e-36 Condition: C. elegans-specific, U statistic: 12100.0, Corrected p-value: 1.02e-42. For underlying data, see Supplementary Data 7 and 8.

Fig. 7:. Glial rewiring across species.

A: Lateral posterior view of 3D rendering of IL sheath (ILshDL, magenta) and socket (ILsoDL, blue) cells with the pharynx shown in green. B: Lateral posterior view of 3D rendering of the CEPshDL (magenta) and AWCL (blue) cells with the pharynx shown in green. C: Posterior view rendering of CEPshDL/R (magenta) and AWCL cells (blue) with the pharynx shown in green. D: Chemical synaptic connectivity of CEPsh in adults. Blue lines are connections found only in P. pacificus, red lines are connections found only in C. elegans, and black lines are connections shared in both species. E: Lateral posterior view rendering of 3D reconstruction of the GLR cells with the pharynx shown in green. F: Posterior lateral view of the GLRL (magenta) and RIPL (blue) cells, showing close fasciculation of processes along the pharynx (green). The asterisk denotes the location of the synapse shown in G. G: Example GLRL synapse onto the pharyngeal epithelium. The micrograph shows presynaptic specializations including a presynaptic density and clear vesicles. H: Chemical synaptic connectivity of GLR in adults. Blue lines are connections found only in P. pacificus, red lines are connections found only in C. elegans, and black lines are connections found in both species.