Morphology of the aboral organ in Mnemiopsis leidyi

(A) Whole-body image of a 5-day-old M. leidyi cydippid larva viewed in the sagittal plane (lateral view). The boxed region indicates the aboral organ. Abbreviations: ao, aboral organ; tb, tentacle bulb. A–O indicates the aboral–oral body axis. (B) Schematic diagram of a cydippid larva in the aboral view. The body is illustrated with four colours and divided into four quadrants, designated as the first (Q1) through fourth (Q4). The two primary viewing angles are referred to as the sagittal plane (S) and the tentacular plane (T). Abbreviations: ao, aboral organ; cg, ciliated groove; tb, tentacle bulb. (C) Aboral views of the aboral organ highlighting its spatial organization. The left panel presents a schematic representation of the aboral organ, illustrating the four body quadrants (Q1–Q4), colour-coded consistently with panel (B). The right panel shows a corresponding differential interference contrast (DIC) image from the same perspective. The region enclosed by the dotted line delineates the boundary of the aboral organ. The sagittal (S) and tentacular (T) eye icons indicate the viewing directions corresponding to those planes. Abbreviations: bal, balancer; cg, ciliated groove; li, lithocyte. (D) Lateral views of the aboral organ in two orthogonal planes captured by DIC microscopy. The left panel shows a view in the sagittal plane, the right panel displays a view in the tentacular plane. The statocyst is a cavity-like organ enclosed by the dome cilia (do), which contains the statolith formed by lithocytes (li) and supported by the balancer cilia (bal). A–O indicates the aboral–oral body axis. (E) Schematics of the aboral organ in sagittal (S, left) and tentacular (T, right) planes, corresponding to the views in (D). Colours follow the quadrant scheme in (B) and (C). A–O indicates the aboral–oral body axis. (F) Overview of the volume traced in CATMAID with spheres indicating the position of nuclei in the reconstructed cells. Scale bar: 10 µm. (G) Morphological rendering of all cells in the aboral organ displayed in the aboral (left pannel), sagittal plane (middle panel) and tentacular plane views (right panel). Cells are colour-coded according to quadrants. Lithocytes (li) are represented as three grey spheres, balancers (bal) are depicted as grey lines.

Organisation of the aboral synaptic nerve net

(A) Morphological rendering of the large aboral nerve-net neuron ANN_Q1-4 spanning all four quadrants, in aboral (left), sagittal (middle panel) and tentacular view (right panel). Spheres indicate the positions of nuclei. (B) Morphological rendering of the two smaller ANN neurons ANN_Q1Q2 (magenta) and ANN_Q3Q4 (orange), each spanning two quadrants, in aboral (left), sagittal (middle panel) and tentacular (right panel). Spheres indicate the positions of nuclei. (C) Number of mitochondria per cell for each cell type. The number of synapse-associated mitochondria is shown in green, the number of mitochondria outside synapses is shown in grey. N > 2 cells for each cell type except lithocytes (only 2 cells are fully within the volume). Abbreviations: bal, balancer; brg, bridge; lgc, large granular cell; cg, ciliated groove; dv, dense vesicle cells; imc, intra-multiciliated cells; la, lamellate bodies; li, lithocytes; pl, plumose; ANN, apical nerve net; ef, epithelial floor cells; noc, nonciliated; 1c, monociliated; 2c, biciliated; muc, multiciliated cells. (D) Positions of mitochondria within the ANN neurons. Green marks mitochondria associated with presynaptic triad structures. Black marks mitochondria categorized as “Not forming synapses,” which includes cases where synaptic vesicles were present but without a clear triad, vesicle identification was uncertain, or vesicles were absent. (E) Representative electron micrographs of presynaptic triad structures observed in the dataset and their schematic diagrams. The left diagram illustrates synaptic projections from the central ANN (ANN Q1-4) to the lateral ANN (ANN Q3Q4), while the right diagram shows synaptic projections from the lateral ANN (ANN Q1Q2) to the central ANN (ANN Q1-4). Abbreviations: mi, mitochondrion; er, endoplasmic reticulum; sv, synaptic vesicles. Scale bar: 500 nm. (F) Position of synapses. Synapses from the central ANN to the lateral ANN are shown in magenta, while synapses from the lateral ANN to the central ANN are shown in cyan. Blue dots indicate the locations of autapses within ANN_Q1-4.

Synaptic connectivity of the balancer and bridge cells with the ANN.

(A) Number of synaptic inputs from the ANNs (ANN_Q1-4: blue, ANN_Q1Q2: orange, ANN_Q3Q4: magenta) to each cell type. Abbreviations: bal, balancer; brg, bridge; lgc, large granular cell; cg, ciliated groove; dv, dense vesicle cells; imc, intra-multiciliated cells; la, lamellate bodies; li, lithocytes; pl, plumose; ANN, apical nerve net; ef, epithelial floor cells; noc, nonciliated; 1c, monociliated; 2c, biciliated; muc, multiciliated cells. (B) Morphological rendering of balancer cells, with each cell colour-coded by quadrant: Q1 (yellow), Q2 (blue), Q3 (orange), and Q4 (magenta). Each sphere represents the position of an individual nucleus. The fine projections extending from the cells represent traced skeletons that follow the cell body and continue into a single cilium emerging from the soma. Views are shown from three orthogonal perspectives: aboral view (left), sagittal plane (middle), and tentacular plane (right). The spatial arrangement highlights the quadrant-specific organization of balancer clusters within the aboral organ. (C) Morphological rendering of bridge cells spanning the Q1Q2 and Q3Q4 quadrants. The Q1Q2-side bridge cells are shown in blue, while the Q3Q4-side bridge cells are shown in magenta. The morphology of individual bridge cells extending across tentacular-axis-symmetric quadrant regions is depicted. The spheres represent the positions of individual nuclei. (D) Positions of mitochondria within bridge cells. Green marks mitochondria associated with presynaptic triad structures; black marks mitochondria categorized as “Not forming synapses” (including cases with vesicles but no clear triad, uncertain vesicles, or no vesicles). (E) Synaptic connections from ANN neurons to balancer ciliated cells. The positions of synapses from ANNs to balancers (coloured spheres) are indicated. The three ANN cells and their respective synapses are coloured differently (ANN Q1-4, blue; ANN Q1Q2, orange; ANN Q3Q4, magenta). Balancer ciliated cells are shown in light grey. (F) Synaptic connections between ANNs and bridge cells. The positions of synapses from ANNs to bridge cells (magenta spheres) and from bridge cells to ANNs (light blue spehere) are indicated. The skeletons of the three ANN neurons are shown (ANN Q1-4, blue; ANN Q1Q2, orange; ANN Q3Q4, magenta). Bridge cells are shown in light grey. (G) Connectivity matrix of the gravity-sensing neural circuit. Columns represent presynaptic cell groups, while rows represent postsynaptic cell groups. The numbers and varying shades of blue correspond to the number of synapses. (H) Complete synaptic wiring diagram of the gravity-sensing neural circuit. Cells belonging to the same group are shown as diamonds, with the number of cells shown in square brackets. The number of synapses is shown on the arrows. In panels B-F, the left view shows a dorsal view of the aboral organ, the middle panel a sagittal plane view and the right panel a tentacular plane view.

Analysis of ciliary beating, arrests and re-beat across balancers in the four quadrants

(A) Schematic diagram of the differential interference contrast (DIC) microscopy setup used to image the movement of balancer cilia. The microscope was tilted 90 degrees so that the stage was positioned vertically. We used a 40x objective lens and a monochrome CMOS camera sensitive to near-infrared (NIR) light that was synchronized with an 850 nm strobe light source. (B) Representative DIC images of the aboral organ viewed along the sagittal (left) and tentacular (right) planes. Arrowheads indicate the balancer cilia selected for kymograph-based analysis in each orientation. Scale bar: 25 µm. (C) Representative kymographs showing arrest and re-beat events of balancer cilia. Left and right balancer cilia were simultaneously recorded in either the sagittal (left) or tentacular plane (right). Arrowheads mark the onset of arrest (open) and re-beat (filled); horizontal arrows represent 1-second intervals. (D) Boxplots showing time differences in the onset of arrest (white boxes) and re-beat (grey boxes) between left and right balancer cilia. colours of the box outlines indicate the imaging plane: sagittal (blue) or tentacular (orange). Each dot represents a single larva. (E) Bar plots showing ciliary beat frequency (CBF) dynamics of left and right balancers (top and bottom, respectively) recorded from the sagittal (left) or the tentacular (right) planes. Positive and negative values represent opposite sides within each plane. (F) Mean rolling Pearson correlation of left–right balancer CBF calculated with a 20-frame window. Each dot represents an individual larva. Balancers on the tentacular plane showed significantly higher correlation than those on the sagittal plane (t-test, p = 0.014).

Comparison of neural circuits regulating ciliary movement

(A) Neurons are represented by circles, colour-coded to indicate analogous functions in ciliary control. Ciliated cells are shown in grey. Synapses are indicated by arrows, with magenta representing synapses that induce ciliary arrest and blue representing synapses that induce ciliary re-beat or an increase in ciliary beating frequency. (Left) Neural circuit of the M. leidyi gravi-sensory organ. Bridge cells (yellow squares) are suggested to be electrically coupled (indicated by yellow zigzag lines), implying a potential involvement in feedback mechanisms between neurons and ciliated cells. (Right) Neural circuit regulating the beating of cilia in the prototroch ciliary band of larval P. dumerilii. Serotonergic neurons (Ser-h1 neurons, blue) activate ciliary movement, while a cholinergic neuron (MC neuron, magenta) induces ciliary arrest.

Cell-type composition of the ctenophore aboral organ

Morphological rendering of skeletonised cell types, (A) balancer cells (B) bridge cells, (C) large granular cells, (D) ciliated groove cells, (E) dense vesicle cells, (F) dome cells, (G) intracellular multiciliated cells, (H) lamellate bodies, (I) lithocytes, (J) plumose cells, (K) aboral nerve-net neurons (L) epithelial floor cells.

3D reconstruction of all cells comprising the aboral organ

(A) Morphological rendering of skeletonised cells in the ctenophore aboral organ. Each colour represents a distinct cell type. The balancer cells are shown in orange. Some cell groups lack distinguishing subcellular features required for identification and are shown in white.

Ultrastructural features and synaptic organization of the ANN, in comparison to the subepithelial nerve net (SNN)

(A) Registration and annotation of synapses in the actual cellular traces. Pink dots indicate the approximate center of each individual cell, marked within its plasma membrane boundary. Mitochondria involved in synaptic contacts are labeled with orange dots. Red arrows point from these mitochondria to the presynaptic cell body, marking presynaptic structures. Cyan arrows point from the orange-labeled mitochondria to the corresponding postsynaptic cell. (B) Electron microscopy (EM) images showing representative presynaptic structures. The left panel shows a monadic synapse, while the right panel shows a polyadic (byadic) synapse, in which a single presynaptic site contacts two postsynaptic cells. (C) Nuclei within two different types of ANN cells are shown in purple in a single EM section. On the left, the ANN_Q1-4 cell extends widely across the aboral organ and appears as a continuous structure in 3D, despite having nuclei located distantly from each other. In contrast, ANN_Q1Q2 (right) has two adjacent nuclei. (D) Three-dimensional reconstruction of the subepithelial nerve net (SNN). The SNN neurons extend broadly, enveloping the aboral organ. Some portions of the network remain as unconnected fragments due to incomplete reconstruction. (E) Morphological comparison between the ANN in EM images. The SNN shows a “beads-on-a-string” morphology, with discrete bead-like swellings connected by faint electron-dense fibrous structures. (F) Three-dimensional reconstruction of the SNN and ANNs. (G) Morphological comparison between the SNN in EM images. The ANN exhibits a more diffuse distribution, often appearing to interdigitate between surrounding cells. Relatively large mitochondria are frequently observed in the ANN processes.

Full synaptic connectome of the ctenophore apical organ

Full synaptic graph showing individual cells as nodes connected by synaptic contacts. Arrows point from the presynaptic to the postsynaptic cell. Edge thickness is proportional to the number of synapses.

Ciliary beat frequency (CBF) dynamics of balancer cells in additional samples

Bar plots showing CBF dynamics of left and right balancers (top and bottom panels, respectively) recorded from the sagittal (A–C) and tentacular (D–F) planes. Positive and negative values indicate opposing sides within each imaging plane. These data correspond to additional samples not shown in the main figure and support the results presented in Figure 4E.