D. lineata pigmentation at the body wall and oral surface is an externally visible indicator of internal organs
In D. lineata, orange-striped pigmentations only appear on the body column at the levels of P1s and P2s (Fig. 2a) [27]. Therefore, we examined whether pigmentation (Figs. 2a, b) could serve as an externally visible indicator of the arrangement of internal organs. Histological analysis confirmed that the orange stripes are associated with all P1s and P2s but not P3s and further revealed that they are restricted to the endodermal cell layer (Figs. 2c, d). As all mesenteries were externally visible through the transparent ectoderm, the mesenteries not adjacent to the orange stripes were identified as M3s (Fig. 2a, d). Moreover, at the oral surface around the actinopharynx, we found fan-shaped white pigmentation (with the same color as the tentacles) at the bases of tentacles surrounding each siphonoglyph (Fig. 2b). Although white pigmentation appeared intermittently in several other places (Fig. 3b) and orange pigmentation weakly appeared at P3 in several individuals (Fig. 2a), these pigmentations could be distinguished from fan-shaped patterns and orange stripes, respectively. Altogether, orange stripes and fan-shaped patterns can serve as external indicators of P1s and P2s, and siphonoglyphs, respectively.
For further histological analyses, we stained the horizontal sections with DAPI to discern the organ outlines (Figs. 2g-k). We revealed that the red fluorescence was always colocalized with orange-striped pigmentations (Figs. 2c, g, Supplementary Fig. S1B), indicating the positions of the P1s and P2s. Red fluorescence was also found at the bases of tentacles initiated between the M1s and M2s (Fig. 2f, Supplementary Fig. S1C). We purified the fluorescent protein from D. lineata and found distinctive features of GFP-like proteins; the absorbance and emission peaks were at 561 and 577 nm, respectively, closely resembling the dsRed spectrum (Supplementary Fig. S1A). Thus, the red fluorescence as well as the orange stripes and fan-shaped pattern are externally visible indicators for defining organ arrangement.
Intraspecific variation in the number of mesenteries and siphonoglyphs with positive correlations
In line with a previous report [17], we confirmed that the number of orange stripes that indicate P1s and P2s varied between 10 and 18, with 12 at the highest frequency (Fig. 3a). In a total of 114 individuals examined, the siphonoglyph number also varied among one (n = 27, 24%), two (n = 77, 68%), and three (n = 10, 8.8%, Fig. 3b), a frequency that was relatively similar to that found in a previous study [17]. Consistently, the stripe numbers in the diglyphic individuals varied from 10 to 16, with a peak at 12 (69% of the 77 samples; Fig. 3c), whereas that in the monoglyphic individuals varied from 10 to 13, with a peak at 10 (44% of the 27 samples), and that in the triglyphic individuals varied from 11 to 18 (Fig. 3c). Thus, we confirmed the intraspecific variation with the predominance of 12 stripes in diglyphic individuals and 10 in monoglyphic individuals. Moreover, we revealed that the siphonoglyph number positively correlated with that of the stripes (Pearson correlation coefficient r = 0.62; Fig. 3c).
Symmetry polymorphism in the mesenteries and siphonoglyphs
To examine the variations in body symmetry, we analyzed the positional arrangement of gastric pouches, retractor muscles, and siphonoglyphs (Figs. 2d and 4a). First, the symmetry type in the arrangements of the gastric pouches was analyzed. Since the P1s and P2s were alternately arranged at an equal interval distance in the majority of the individuals (74% of the 98 samples), their arrangement was symmetrical to each plane that passes through the center of the actinopharynx (Fig. 4a, upper). Therefore, the number of symmetry planes was equal to half the sum of the number of P1s and P2s, indicating penta-radial (5-radial), hexa-radial (6-radial), and hepta-radial (7-radial) symmetries for 10-, 12-, and 14-striped individuals, respectively (Fig. 4a, upper). Each of the P3s was positioned at every gap between the P1s and P2s, thereby following symmetry patterns (Figs. 2d, g). The remaining individuals exhibited irregularity in the alternating arrangement (Fig. 4a, upper), e.g., adjacent positioning of two P1s (Supplementary Fig. S2A).
Next, the symmetry in the arrangement of the longitudinal retractor muscles and gastric pouches was analyzed. Since retractor muscles are positioned outside of the PDs and inside of the other gastric pouches [14] (Fig. 2h-k), the arrangement of the PDs is key to distinguishing symmetry (Fig. 4a, center). Indeed, all the 10-striped individuals exhibiting 5-radial symmetry possessed a single PD showing bilateral symmetry, whereas the other pouches were arranged symmetrically to the plane passing through PD and P2 (Fig. 4b). A few 12-striped individuals also possessed a PD showing bilateral symmetry (Supplementary Fig. S4A), in which the PD and P1 were positioned on either side of the symmetry plane. In contrast, most of the 12-striped individuals had two PDs, each placed opposite to its counterpart (Fig. 4c). The other pouches were arranged symmetrically to the plane passing through the PDs and to the other (perpendicular to the former, Fig. 4c). Therefore, the gastric pouch and muscle arrangement indicated biradial symmetry (Fig. 4a, center).
The symmetry was maintained even when the siphonoglyph arrangement was included (Fig. 4a, lower); a PD and a siphonoglyph were colocalized on the same symmetry plane (Fig. 2d). Thus, the diglyphic individuals exhibited biradial symmetry, whereas the monoglyphic individuals exhibited bilateral symmetry (Fig. 4d). Notably, irregular arrangements (i.e., an absence of symmetry) appeared in a fraction of monoglyphic (35%) and diglyphic (29%) individuals, mainly for the gastric pouches (e.g., alternating arrangement of P1 or P2) and occasionally on two siphonoglyphs (e.g., a few individuals with 14 stripes, as shown in Fig. 4a upper panel and Supplementary Figure S2B). We concluded that body symmetry can be determined as either bilateral or biradial depending on the siphonoglyph organ number and arrangement.
Lateral inhibition and activation model for gastric pouch specification based on observed gastric pouch arrangement
The correlated polymorphism between the siphonoglyph number and symmetry (Fig. 4d) prompted us to speculate a causal relationship between these two factors. Symmetric arrangements of the gastric pouches (Figs. 4b, c) emerged after irreversible specification of permanent pouches to PDs, P1s, or P2s during the asexual reproduction process (Figs. 1e and 5a) [29]. After the specification completes, each nonspecified gastric pouch (Fig. 5a) provides space for the formation of P3 (Figs. 2d and 5a). Since the positioning of the specified gastric pouches sufficiently reflects that of the mesenteries (M1, M2, and M3), muscles, and siphonoglyphs (Figs. 2d, g), spatial patterning of the gastric pouch specification should clarify how symmetry emerges in the arrangement of internal organs. To this end, we built a mathematical model for gastric pouch specification based on the observed arrangements.
After the specification, each of the P1s was arranged in every fourth pouch between nonspecified pouches (Fig. 5a, t = 1–4) [28], suggesting lateral inhibition of the specification to P1s in the long range (thick inhibitory arrows in cyan, Fig. 5b). Likewise, the arrangement of the P2s in every fourth pouch (Fig. 5a; t = 1–4) suggests another lateral inhibition on the specification to P2s in the same range (thick inhibitory arrow in magenta, Fig. 5b). In addition, P1s and P2s were alternately arranged, leaving one nonspecified pouch between each specified pouch, suggesting that the specification of P1s and P2s would be weakly inhibited by the presence of P2s and P1s (Fig. 5a), respectively (thin inhibitory arrows in cyan and magenta, Fig. 5b).
Taken together, these results show there are at least two types of inhibitory effects on the specification of P1s and P2s, which are released from both P1s and P2s. Such lateral inhibition of gastric pouch specification has been previously formulated by assuming that morphogens are secreted from gastric pouches during early anthozoan development [15]. Based on the model and the alternative arrangement in D. lineata, we introduced two morphogens (B and C) into our model for the asexual reproduction process (Eqs. 2 and 3). This model assumes that the gastric pouches (P1, P2, and PD) serve as signaling centers. Both B and C are secreted from P1s and P2s, inhibiting the specification of the neighboring nonspecified pouches to P1s and P2s, respectively. The secretion (production) rate of B was higher at P1s than at P2s (sb1 > sb2), and the rate of C was higher at P2s than at P1s (sc1 < sc2).
In addition, the temporal order of specification is different between the early development and the present processes; PDs are specified last in the former but first in the latter, preceding the specification of nonspecified pouches to the other P1s and P2s (Fig. 5a) [28], suggesting an inductive effect of PDs on the specification throughout the body (dark blue arrows in Fig. 5b, left). Accordingly, we additionally introduced lateral activation into the model by assuming that another morphogen (A; Eq. 1) is secreted from PDs and diffuses more broadly than the inhibitors (B and C). When the concentration of activator A exceeds a threshold (a > Ta; activator threshold in Fig. 5c) and that of either inhibitor B or C is below the other threshold (Ti; inhibitor threshold) at a nonspecified pouch, it can be specified (Fig. 5c, left panels). In the case of subthreshold B and suprathreshold C (b < Ti and c > Ti), specification to P2 is selectively sufficiently inhibited, resulting in specification to P1 (left upper panel, Fig. 5c). In contrast, P2 is specified in the opposite case (b > Ti and c < Ti; Fig. 5c, left bottom panel). These specifications occur irreversibly, following observations [29]. Thus, D. lineata mesentery arrangements provide a lateral inhibition and activation model representing two inhibitors and an activator for mesentery specification during the asexual reproduction process.
The initial conditions of the present model follow the early stage of asexual reproduction. In many cases, in earlier observations, a lacerated pedal disk (Fig. 1e) includes one stripe (53%, 215 in 402 pedal disks) corresponding to a single pouch of either P1, P2, or PD [17]. In contrast to the gastric pouch, a PD is subsequently specified during reproduction (Fig. 5a, t = 0) [28]. These two initially specified pouches and the total number of gastric pouches appeared to be limited to three combinations in our observations, except for the irregular arrangements (Supplementary Figure S2): a PD and a P2 initially specified in 20 pouches (Figs. 4b and 5d, initial condition 1), a PD and a P1 in 24 pouches (Supplementary Figure S4, Fig. 5e, initial condition 2), and two PDs in 24 pouches (Figs. 4c and 5f, initial condition 3). Importantly, the arrangement of the specified and nonspecified pouches under initial conditions 1 and 2 is bilaterally symmetric, whereas that under initial condition 3 is biradially symmetric. Hence, we adopted the three observed initial conditions with either bilateral or biradial symmetry; two gastric pouches are already specified, whereas the others are nonspecified.
Siphonoglyph number encodes symmetry through lateral inhibition and activation
To examine whether the lateral inhibition and activation model accounts for the emergence of biradial and bilateral symmetry, we performed model simulations using the three combinations of initial conditions (Figs. 5d–f). We first examined initial condition 1 (Fig. 5d), in which P2 and PD among 20 pouches were already specified on opposite sides (Fig. 5g, t = 0; Supplementary Figure S3A). The pouches adjacent to the PD consistently remained nonspecified due to suprathresholds b and c, which were secreted from the PD despite suprathreshold a being secreted from the PD (Fig. 5g, t = 1, gastric pouch index = 10 and 12). Conversely, the second adjacent pouches were specified irreversibly as P2s due to the subthreshold of c (Fig. 5g, t = 1, gastric pouch index = 9 and 13). These P2s subsequently began to secrete inhibitors B and C. These morphogens suppressed the specification of the adjacent pouches due to the suprathreshold of b and c (Fig. 5g, t = 2, gastric pouch index = 8 and 14) but allowed the irreversible specification of the second neighbors to P1s due to subthreshold b and suprathreshold a (Fig. 5g, t = 2, gastric pouch index = 7 and 15). While repeating such sequential specification processes, P1s and P2s were alternately specified every two pouches (Fig. 5g, t = 3 and 4). Importantly, the series of specification orders (Fig. 5g, t = 3 and 4) agreed with the observations of a previous study [28], further validating the model. Given an equal diffusion of these morphogens in clockwise and counterclockwise directions (Supplementary Figure S3A), the specification occurred symmetrically to a single plane, passing through the initially specified two pouches (i.e., a PD and a P2), and thereby reproduced the observed arrangement of bilateral symmetry (Figs. 4b and 5g, t = 4).
Moreover, under the second initial condition of 24 pouches, in which P1 and PD are specified on opposite sides (Fig. 5e), our model succeeded in reproducing the other arrangement of bilateral symmetry to the plane passing through the two pouches initially specified (Supplementary Figure S4B). Thus, the arrangements of the specified and nonspecified pouches, starting from the first and second initial conditions with a single PD, developed to the observed arrangements, retaining bilateral symmetry under 20 and 24 pouches through lateral activation and inhibitions.
Under the third initial condition of two PDs being specified among 24 pouches (Fig. 5f), activator A, inhibitor B, and C morphogens were released equally from these two PDs (Fig. 6, t = 0; Supplementary Figure S3B). Therefore, the sequential specification of pouches occurred from their proximal sides (Fig. 6, t = 1) and, more importantly, proceeded in a symmetric manner to both the plane passing through the two initially specified PDs and the other perpendicularly oriented plane. Following the same specification rules mentioned above, the arrangement of specified and nonspecified pouches developed to the observed pouch, retaining the biradial symmetry to the abovementioned two planes under 24 pouches.
Altogether, the lateral activation and inhibition model supported that the number of PDs (i.e., siphonoglyphs) is sufficient to determine either bilateral or biradial symmetry in the observed specification arrangements. This is because the lateral activation and inhibition mediated by their equal diffusion from specified pouches allowed the irreversible specification of nonspecified pouches, retaining the symmetry of their specification arrangements. The bilateral symmetry initiated with a single plane passing through a PD (Fig. 5d, e). Biradial symmetry is initiated in the case of two PDs due to a mirror image of lateral activation and inhibition (Fig. 5f). These theoretical results recapitulate that the polymorphism in the symmetric arrangement of specified and unspecified pouches arises from the variation in siphonoglyphs.