DC/C-PHATE representations of contactome-based relationships.

DC/C PHATE graphs enable representations of neuronal contact relationships. To build DC/C-PHATE graphs we (A) analyzed serial section EM datasets of the C. elegans nerve ring neuropil (located in the head of the animal). (B) Single cross section of the nerve ring (surrounding the pharynx), with segmented neurites pseudo-colored. Dark box corresponds to the zoomed-in image in (C). The cross section is from the JSH dataset digitally segmented (Brittin et al., 2021). (C) Zoom-in cross section with three arbitrary neurons (called A, B, C) highlighted by overlaying opaque cartoon (2-D, left image) and 3-D shapes (middle image) to represent the segmentation process in the z-axis (arrow) and the neuronal contact sites (highlighted Yellow, Yellow dashed, Red). Contacts are quantified for all neuron pairs across the contactome (See Methods), to generate a Contact Matrix (represented here as a table, schematized for the three arbitrary neurons selected and in which specific contact quantities are represented by a color scale and not numerical values). (D) Schematic of how the Diffusion Condensation algorithm (visualized with C-PHATE) works. DC/C-PHATE makes use of the contact matrix to group neurons based on similar adjacency profiles (Brugnone et al. 2019; 2019; Moyle et al. 2021), schematized here for the three neurons in (C). (E) Screenshot of the 3-D C-PHATE graph from a Larva stage 1 (L1; 0 hours post hatching;) contactome, with individual neurons represented as spheres at the periphery. Neurons were iteratively clustered towards the center, with the final iteration containing the nerve ring represented as a sphere in the center of the graph (Highlighted in maroon). (F) Integration in NeuroSCAN of the DC/C-PHATE and EM-derived 3-D neuron morphology representations allow users to point to each sphere in the graph and determine cellular or cluster identities for each iteration. Shown here and circled in Red, an arbitrarily selected cluster (in E), with the identities of the neurons belonging to that cluster (four letter codes in the column to the left of F) and the corresponding neuronal morphologies (right) of this group of neurons in the EM-reconstructed nerve ring (with individual neurons pseudo-colored according to their names to the left). Compass: Anterior (A), Posterior (P), Dorsal (D), Ventral (V), Left (L), Right (R).

Implementation of DC/C-PHATE to developmental contactomes reveal a conserved layered organization maintained during post-embryonic growth.

(A) Cartoon of the C. elegans head and nerve ring (outlined with black box). Below, nerve ring reconstruction from EM data of an L1 animal (5 hours post hatching), with all neurons in gray. Scale bar 2 µm. (B-F) DC/C-PHATE plots generated for available contactomes across C. elegans larval development, colored by stratum identity as described (Moyle et al., 2021). Individual neurons are located at the edges of the graph and condense centrally. The four super-clusters identified and all iterations before are colored accordingly. The identity of the individual neurons belonging to each stratum, and at each larval stage, were largely preserved, and are provided in (Supplemental Table 1). Some datasets contain 5 or 6 super-clusters (colored dark purple, yellow and orange), which are classified as groups of neurons that are differentially categorized across the developmental connectomes. (G-K) Volumetric reconstruction of the C. elegans neuropil (from EM serial sections for the indicated larval stages (columns)) with the neurons colored based on their strata identity. Scale bar 2 m; Anterior (A) left, Dorsal (D) up.

Examination of the architectural motifs underlying the distinct strata across development.

Visualization of (A-F) Stratum 1 (Red) and (G-L) Strata 3 and 4 (Blue and Green) reveal motifs that are preserved (Strata 1) and change (Strata 3 and 4) across developmental contactomes (L1 to Adult, left to right, as indicated by labels on top). (B-F) Cropped view of Stratum 1 at each developmental stage showing a similar shape of two ‘horn-like’ clusters in the C-PHATE graphs (as seen by orange and blue shaded areas). These two clusters have similar neuronal memberships, which are largely invariant across developmental contactomes (Supplemental Table 3). (H-L) Cropped view of Strata 3 and 4 at each developmental stage highlighting differences in the organization and number of neurons contained in each of the Blue and Green strata, which is particularly distinct when comparing (H) L1 and (K) L4 (Supplemental Tables 5, 6). There is an additional supercluster (Yellow in (I-J)) at stages L2 and L3 that contains neurons of S3 and S4 identity.

Case study: AIML and PVQL neurons change clustering patterns across the developmental contactomes.

(A-E) C-PHATE plots across development, with the trajectories of AIM neurons (in purple) and the rest of the spheres colored by stratum identity (see Figure 2). (F-G) Zoom in of the AIM, PVQ, and AVF trajectories corresponding to Larval Stage 1 (A, dotted box) and in (G), Larval Stage 3 (C, dashed box). Note how the relationship between AIM and PVQ neurons in the C-PHATE graph varies for each of the examined contactomes across development, as seen by the iterations before co-clustering (Supplemental Figure 1, Supplemental Table 7).

Case Study: Visualization of contact profiles in individual neurons.

(A) Cartoon schematic of the head of the animal with the AIM neurons (purple) and pharynx (gray), and (dotted box) a 3-D reconstruction of the AIM neuron morphology from the L1 (0 hours post-hatching) dataset. (B) Zoom-in of the simplified DC/C-PHATE clustering of the AIM (purple), PVQ (orange), and AVF (green) neurons for the contactome of an L3 animal. (C) 3-D representation of all contacts onto the AIM neuron morphology in an L1 animal, colored based on contacting partner identity, as labeled (right) in the detailed inset (black box) region. (D) AIM-PVQ contacts (in orange) and AIM-AVF contacts (in green), projected onto the AIM neurons (light purple) across developmental stages and augmented for clarity in the figure (see non-augmented contacts in (Supplemental Figure 5). Scale bar 2 µm.

Case study: Segmented morphologies of AIM, PVQ and AVF across larval development.

(A) Cartoon schematic of the C. elegans head, pharynx (gray) and examined neurons with dashed black box representing the nerve ring region. (B) Schematic representation of the outgrowth path of the AVF neurons as observed by EM (Witvliet et al., 2021). AVFL and AVFR (green) grow along the AIML neuron (purple) onto the AIMR neurite. The distal end of the AVF neurite is highlighted with a black arrowhead in the schematic. (C) Neuronal morphologies of AIM (purple), PVQ (orange), AVF (green) across post embryonic development, as indicated, with black arrowhead pointing to AVF outgrowth. Scale bar = 2 µm. Regions for insets (L1, dotted box; L2, dashed box) correspond to (D). (D) Morphologies of these neurons (rotated to the posterior view) display the AVF neurons’ positions between the AIM and PVQ neurons at the L1 and L2 stage. Indicated outgrowth between neurons continues to the Adult stage (Supplemental Video 2). Note how AVF outgrowth alters contact between PVQ and AIM (Figure 5D).

Case study: AIM-PVQ and AIM-AVF synaptic positions across development.

(A) AIM-PVQ synaptic sites (dark orange arrowheads) and AIM-AVF synaptic sites (dark green arrowheads) in the segmented AIM neurons and reconstructed across post embryonic development from original connectomics data. Scale bar = 2 µm. (B) Schematic of the AIM, PVQ and AVF circuitry across development based on synaptic connectivity and focusing on the stage before AVF outgrowth (L1), during AVF outgrowth (L2) and Adult; arrow direction indicates pre to post synaptic connection, and arrow thickness indicates relative number of synaptic sites (finest, <5 synapses; medium, 5-10 synapses; thickest, 11-30 synapses). (C) Zoom in of synaptic sites (green) in the Adult connectome and embedded into the AIM neuron morphology (light purple). In NeuroSCAN, presynaptic sites are displayed as blocks and postsynaptic sites as spheres, and a scaling factor is applied to the 3-D models (References Materials and Methods).

NeuroSCAN is a tool that enables integrated comparisons of neuronal relationships across development.

With NeuroSCAN, users have integrated access to: C-PHATE plots, 3-D morphological renderings, neuronal contact sites and synaptic representations. Through stage-specific C-PHATE renderings, users can explore neuronal relationships from high dimensional contactome data. (Top) On C-PHATE plots, schematized here, each sphere represents an individual neuron, like AVF or AIM, or a group of neurons clustered together during algorithm iterations. (Right) 3D renderings of AIM neurons (Purple), PVQ neurons (Orange), AVF neurons (Green) can be visualized in the context of the entire nerve ring or other circuits (gray). (Left) AIM:AVF contact sites (green) onto the AIM neuron (purple) with the AIM-AVF synaptic sites (orange). Inset shows zoomed in of contacts and synapses-presynaptic sites (blocks) postsynaptic sites (spheres). Data depicted here are from the L3 stage (27 hours post hatching).

DC/C-PHATE clustering of AIM, PVQ, and AVF across postembryonic development.

(A-E) A cropped view of the DC/C-PHATE plot colored to identify individual neurons and clustering events in (A) Larva stage 1 (5 hours post hatching); (B) Larva stage 2 (23 hours post hatching); (C) Larva Stage 3 (27 hours post hatching); (D) Larva stage 4 (36 hours post hatching); and (E) Adult (48 hours post hatching). See also Video S1 and Table S7.

Projecting contact profiles onto the segmented neuronal shapes.

(A-C) Graphical representations of the strategy utilized for creating the contact profiles for each of the adjacent neurons (purple, red, cyan) onto a cross section of the neuron of interest (Neuron A, yellow). (D-F) Electron micrograph from the L4 dataset with two adjacent neurons colored yellow and cyan. To build 3-D reconstructions of contact sites from adjacent neurons, we analyzed segmented neurons from the electron microscopy datasets in each slice (A, D). Each adjacent neuron is expanded in all directions to the pixel threshold distance (specified for each dataset; Table S1; Methods; CytoSHOW.org) (B, E). A new ROI (region of interest; purple, red, cyan in C; green in F) is created from the overlapping areas between the neuron of interest (yellow) and the adjacent neurons (C,F). (G-I) 3-D reconstruction of neuron (yellow) (G) with adjacent neuron (cyan), (H) with contact sites captured (green) across all slices, and (I) with contact areas from the adjacent neuron augmented (green) as seen in Figure 5 D.

AIM contact sites.

Contact sites from PVQ (Orange and highlighted with orange arrowheads) and from AVF (Green and highlighted with green arrowheads) across developmental stages (as indicated) and projected onto the segmented AIM neurons (transparent purple). This figure is the unmodified NeuroSCAN outputs of contact profiles that corresponds to Figure 5D. In Figure 5D these contact profiles were augmented. Scale bar = 2 um. See also Figure 5 and Video S4.

AVF synaptic sites.

Synaptic sites displayed onto transparent (green) AVF neurons across developmental stages. Presynaptic sites (spheres) and postsynaptic sites (Blocks) arevisualized between the AVF neurons and the AIM (Purple) neurons, PVQ (Orange) neurons and other AVF (either AVFL or AVFR; opaque green) neuron; Scale bar = 2 um.

Visualization of contact sites in NeuroSCAN.

(A) Search for a specific neuron (here, AIM) to filter (B) the list of contacts corresponding to the developmental slider. Neuron A (AIML, here) is the neuron onto which the contacts will be mapped. The Contacts dropdown menu sorts neurons alphabetically (here, colored according to the contact patch color in C). (C) 3-D reconstruction of all AIM contacts at L3 stage. See also Video S3-S4. In the Figure 5D, contacts are augmented.

C-PHATE tutorial in NeuroSCAN.

(A) Add the C-PHATE plot corresponding to the position of the purple circle on the developmental slider (yellow box) by clicking (B) the + sign. (C) Screenshot of C-PHATE plot at L4 (36 hours post hatching), spheres represent individual neurons at the outer edge of the plot and DC iterations increase towards the center where spheres represent clusters of neurons and eventually the entire nerve ring. (D) Screenshot of C-PHATE plot at L4 (36 hours post hatching) with the spheres/clusters containing the AIM neurons highlighted (Blue) by selecting the AIM neurons within the lightbulb menu (red box). See also Video S1. NeuroSCAN features in this figure are not shown to scale.

Visualization of synaptic sites with NeuroSCAN.

(A) Search for synaptic sites for specific neuron(s) (e.g., AIM, PVQ) and choose a developmental time point with the slider. (B) Synapses dropdown menu contains a list of objects representing pre- and postsynaptic sites corresponding to all neuron names in the search bar and sorted alphabetically. Searched neurons can be used with the synaptic filter (C) to select for synapse type (electrical or chemical; Note: only use this feature for L4_36 hours post hatching and Adult_48 hours post hatching) and to filter objects by synaptic specialization (pre or post; gray dotted box), (D) which will follow the filter logic (example shown for AIM and PVQ). (E) To enable visualization of subsets of synapses and differentiate between pre- and postsynaptic sites, each synapse contains object(s) representing the postsynaptic site(s) as spheres (Blue and Purple) and the presynaptic site as a block (Orange). These are ordered “by synapse”, with all postsynaptic objects, then the presynaptic object. This specific example corresponds to a 3-D representation of the PVQL (Orange, Pre) AIAL (Blue, Post), AIML (Purple, Post) synapse. (F-G) All synaptic sites contain the name of the presynaptic neuron (Orange), neuron type (chemical, electrical, or undefined), list of postsynaptic neuron(s) (Blue), and Unique identifier (Black; Section, letter) for cases with multiple synapses between the same neurons. The ‘section’ is unique to each synapse between specified neurons and at that specific developmental stage. It is listed in order of its antero-posterior position in the neuron. Synapse names are not linked through developmental datasets. If the synapse is polyadic, there will be multiple postsynaptic neuron names and objects associated with a single presynaptic site. See also Video S4.

Opening page view and menu.

(A) View of opening page. (B) Menu for access to the ‘About’ window for referencing source information, the Tutorial, and the developmental Promoter database. See also Video S3.

The NeuroSCAN interface enables interrogation of neuronal relationships across development.

(A) The left facing arrow to minimize the left panel and optimize space for the viewer windows. The interface contains four main parts: (B-E) Filters, (F-J) Results, (K-M) Viewer Navigation, and (N-Q) viewer windows. Filter Results by (C) searching for neuron names, (D) selecting a dataset with the developmental slider (in hours posthatching), (E) and filtering synapses based on the pre- or post-synaptic partner on the neurons that are on the search bar. (F) Results drop down menus (filtered by B) for (G) Neuronal morphologies (shown in the viewer as purple in (O)), (H) Contacts (shown in green (O)); (I) Synapses (shown in Orange in (O)); and (J) C-PHATE (shown in (Q)), which gets filtered by the developmental slider in (D). (K) Viewer Navigation to rotate the 3-D projections in all viewers simultaneously (Play All) and which contains a drop-down menu for each viewer (L,M). The viewers are named as Viewer 1 (L, N) or CPHATE viewer (M, P) and followed by information of the developmental stage and the hours post hatching for the objects in the viewer. (O) Reconstruction of the AIM neurons with AVF contacts and synapses at L3 (27 hours post hatching; scale bar = 2 um. (Q) C-PHATE plot at L1 (0 hours post hatching). See also Video S3.

Select and Add objects to viewers.

(A) Click “select (number) items” to select all items in the dropdown list (green box), or (A’) click the hexagon next to each item (green box). (B) Click “Add Selected” (purple box) to add all selected items or (B’) click “Add to” (purple box) to add each item individually. (C) To add the selected item(s) to an existing viewer of the same developmental stage or to a new viewer, choose a viewer as indicated. (D) Click “Deselect (number) items” (orange box) to deselect items. See also Video S3 and S4.

In-viewer toolbar features

(A) In-viewer toolbar for Neurons, Contacts and Synapses and C-PHATE (shown here, only Neurons). (B, K) Change the background color of viewer from dark (white box, moon) to white (white box, sun). (C, L) Change the color of any objects by selecting a desired color, transparency or color code and selecting the object (or instance) name (here, AIML and AIMR). (D, M) Change developmental stage for items in the viewer by using the in-viewer developmental slider. (N) Add 3-D representations of the Nerve Ring for that developmental stage. (E, O) Record and download movies for the viewer. (F,P) Download .gltf files and viewer screenshot (png). (G) Rotate objects around the y-axis. (H) Zoom in and (I) zoom out, and (J) reset objects to original positions in the viewer. See also Video S3.

Viewer navigation menu.

((A) Navigation bar contains a drop-down menu for each viewer (shown here, six viewers at varied developmental stages) and a “Play all” button for simultaneously rotating all objects in each viewer around the y-axis (Video S3). Each viewer dropdown menu contains a dropdown menu for Neurons (green box), Contacts and Synapses. (B) Viewer 6 with reconstructions of three neurons (AIML and AIMR, purple; PVQL, orange) at Larval Stage 4 (L4), 36 hours post hatching. (C) Browse and Select objects in the viewer by navigating the nested dropdown menus. (D) Manage objects in viewers with options to select, group, hide, and delete objects in each viewer. Objects can be deleted with “select” and keyboard “delete”. See also Video S3.

NeuroSCAN architecture.

(A) Source data is defined in a file tree structure that contains various assets such as .gltf files representing various entities, as well as CSVs storing relationships across entities (Data model in Figure S14). The directory structure outlines a vertical hierarchy starting at the developmental stages, then branching downwards through neuron, C-PHATE, contact and synapse data. A python script can be invoked to traverse the directory tree and parse the files, writing to the database accordingly. This enables verification of the ingested data and quick search times through the datasets to identify the related items. The architecture uses Geppetto backend and frontend (Cantarelli et al. 2018). (B) The backend uses a Postgres Database to store underlying data, a Persistent Storage Volume that houses and serves static assets, and the User Interface is a React application that filters, sorts, and searches through the Neurons to be added to an interactive canvas. (C) A variable number of Virtual Machines run the frontend and backend application code, scaling as needed to accommodate traffic. The frontend React/Javascript bundle that is delivered to the (D) client, rendering the neuron data and assets, and a NodeJS application that exposes a JSON API, serving the neuron data and assets based on user interactions.

NeuroSCAN data model.

(A) Reference scheme for B-F; Instance refers to the category (e.g., B, Neuron; C, Developmental Stage), which contains a name or identifier (id) for each object, lists of files associated with the instance (C, Developmental Stage does not have files), and metadata to further describe each instance, which is usually a string (str) or an integer (int). (B) The neuron name is the foundation for the Contacts, Synapses, and C-PHATE, which enables integration across each of these representations and across developmental stages (timepoints) with metadata from WormAtlas (wormatlas.org/MoW_built0.92/MoW.html). (C) The Developmental Stages are named by the larval stages (L1, L2, L3, L4, Adult), and the metadata captures the list of timepoints within those developmental stages (i.e., L1, 0 hours post hatching, and L1, 5 hours post hatching). (D) C-PHATE objects are named with a list of Neurons. (E) Contacts link to the Neuron names (Neuron A and Neuron B nomenclature in Figure S5), and metadata annotates the weight or the number of pixels of contact quantified in the source Electron Microscopy micrographs. (F) Synapses link to the Neuron names (Pre, Post, type, and section described in Figure S7).