After 3 days of octanol exposure, worms were allowed to complete regeneration (10 days total). Remarkably, this process resulted in worms that exhibited head shapes highly similar to the heads appropriate to other species of planarians ( Figure 1 E–H). 8-OH treated fragments regenerated both head and tail correctly, but in many cases, head shape was drastically altered. Wild-type(GD) possess a very pointed head shape, with two elongated auricles at the plane of the eyes ( Figure 1 A). Fragments subjected to the same 8-OH treatment scheme regenerated one of: entirely rounded heads like the planarian(SM) ( Figure 1 H), heads with thick necks and “cat-like” auricles like those of the planarian(PF) ( Figure 1 G), heads that are triangular like the planarian(DJ) ( Figure 1 F), or heads that resemble wild-type Figure 1 E). We will refer to the head shapes of regenerates as “pseudo” of the morphologically most similar species. Variant, other species-specific head morphologies were never observed after amputation or during regeneration in water (the control condition)—the normal process of head regeneration has 100% fidelity to the species-specific shape ( Figure S1 A). The same schedule of exposure to hexanol (6-OH), a closely-related compound to octanol which does not effectively block GJs and thus can be used as a control [ 69 ], had no effect ( Figure S1 B). Likewise, intact worms soaked in 8-OH for >3 days did not exhibit any unusual morphological outcomes. Based on these results, we conclude that discrete, species-specific head shapes can be achieved by manipulating the connectivity of physiological networks in the planarian flatworm during head regeneration.

Figure 1. Characterization of varied head morphologies produced by octanol treatment. ( A – D ) Wild-type morphologies of four species of planaria flatworm. Arrows indicate auricle placement and general head shape; ( E – H ) pre-tail (PT) fragments of G. dorotocephala , treated in 8-OH for three days, and then moved into water for the remainder of regeneration ( n > 243). Arrows indicate auricle placement and general head shape. Scale bar 0.5 mm; ( I ) Experimental scheme of octanol treatment. PT fragments are amputated from G. dorotocephala worms. Fragments are treated in octanol (8-OH) for three days, and allowed to regenerate in water for seven days.

Figure 1. Characterization of varied head morphologies produced by octanol treatment. ( A – D ) Wild-type morphologies of four species of planaria flatworm. Arrows indicate auricle placement and general head shape; ( E – H ) pre-tail (PT) fragments of G. dorotocephala , treated in 8-OH for three days, and then moved into water for the remainder of regeneration ( n > 243). Arrows indicate auricle placement and general head shape. Scale bar 0.5 mm; ( I ) Experimental scheme of octanol treatment. PT fragments are amputated from G. dorotocephala worms. Fragments are treated in octanol (8-OH) for three days, and allowed to regenerate in water for seven days.

Planarian flatworm species display a broad range of head shapes, from the very rounded to the almost triangular, with varied shapes of auricles ( Figure 1 A–D). To interrogate the mechanisms responsible for regeneration and maintenance of head shape,planarians were amputated along a plane positioned posterior to the pharynx but anterior to the tail, to produce a pre-tail (PT) fragment. PT fragments were then treated with the gap junction (GJ) communication blocker octanol (8-OH). 8-OH is a commonly-used pan-GJ blocker [ 20 71 ], altering the physiological connectivity between populations of cells, and thereby perturbing the rate and pattern of transmission of bioelectrical and other small molecule signals. Dosage was titrated to a level low enough to enable interference with regenerative signaling without organismic toxicity. 8-OH exposure has been validated to be transient by GC-MS: drug levels are undetectable after a few hours of worm wash-out in water, and octanol does not alter genetic sequences in the worm [ 20 72 ].

Figure 2. Canonical variate analysis of head shape. ( A ) Graphical output, showing confidence ellipses for means, at a 0.9 probability, of shape data from wild-type and experimentally derived morphologies. Ellipses are colored to correspond with phenotype and treatment. n = 10 WT G. dorotocephala , n = 8 WT G. dorotocephala 10 days after amputation and regenerated in water, n = 9 WT D. japonica , n = 6 WT P. felina , n = 8 WT S. mediterranea , n = 7 pseudo G. dorotocephala , n = 13 pseudo D. japonica , n = 5 pseudo P. felina , and n = 6 S. mediterranea flatworms were measured; ( B ) Legend of landmark placement on a wild-type G. dorotocephala head shape (see Materials and Methods); ( C ) Schematic demonstrating alteration of morphology to better resemble another species after 8-OH treatments. Procrustes distances between wild-type G. dorotocephala , 8-OH treated G. dorotocephala with D. japonica head shape, and wild-type D. japonica show objective alteration of morphology to be more similar to the non-native species.

Figure 2. Canonical variate analysis of head shape. ( A ) Graphical output, showing confidence ellipses for means, at a 0.9 probability, of shape data from wild-type and experimentally derived morphologies. Ellipses are colored to correspond with phenotype and treatment. n = 10 WT G. dorotocephala , n = 8 WT G. dorotocephala 10 days after amputation and regenerated in water, n = 9 WT D. japonica , n = 6 WT P. felina , n = 8 WT S. mediterranea , n = 7 pseudo G. dorotocephala , n = 13 pseudo D. japonica , n = 5 pseudo P. felina , and n = 6 S. mediterranea flatworms were measured; ( B ) Legend of landmark placement on a wild-type G. dorotocephala head shape (see Materials and Methods); ( C ) Schematic demonstrating alteration of morphology to better resemble another species after 8-OH treatments. Procrustes distances between wild-type G. dorotocephala , 8-OH treated G. dorotocephala with D. japonica head shape, and wild-type D. japonica show objective alteration of morphology to be more similar to the non-native species.

The quantification was used to determine whether the shapes that looked like other species objectively resembled those species, and to suggest a continuous morphospace within which octanol-induced shape change can be visualized along a continuum (from normal to that of a different species). Canonical variate analysis supported the statistical significance of the given pre-defined morphological groupings (in this case, groupings were based on experimental treatment and morphology) ( Figure 2 A). Comparisons of the Procrustes distances between shape groups ( Figure 2 A, Table 1 ) showed that the experimentally derived morphologies were closer in shape to the wild-type morphologies they resembled than the wild-typehead morphology. Analysis of variance (ANOVA) of both centroid size and shape between wild-type morphology and pseudo morphology groups also confirmed significant differences between groupings (= 7.94,< 0.0001, and= 7.40,< 0.0001, respectively). We conclude that amputation and treatment in 8-OH can produce regenerated worms whose morphology has changed to become significantly more like that of another species of planarian.

Geometric morphometrics [ 73 ] was used to quantify similarities and differences between head shapes of true species, as well as the experimentally derived pseudo morphologies. In brief, geometric morphometric analysis involves placement of a series of landmarks, which are both biologically significant and reproducible across all samples, removal of non-shape variation (size, rotation,), and performance of a set of statistical analysis [ 74 ]. Landmarks were chosen based on the common biological landmarks that existed across samples, and semi-landmarks were placed with prescribed relations to these landmarks ( Figure 2 B). Landmark data was recorded for> 60 worms, including GD worms whose head shape had been experimentally perturbed by 8-OH, control GD worms who had regenerated in water for 10 days, and adult wild-type worms from each of the three species. Principal components analyses (data not shown) and canonical variate analyses were run on the data set. This enabled visualization of mean shape changes between wild-type species morphologies, and between experimentally derived head shapes. Both analyses resulted in the separation of pseudo morphologies from the wild-typemorphology. Procrustes distances between each of the groups were calculated, in order to produce a quantified metric for comparison of shape differences.

Figure 3. Percentage of head shape outcomes correlates with evolutionary distance. ( A ) Evolutionary tree, constructed from rRNA data, showing relationships between species of interest. Species names in red are those that were analyzed in this work; ( B ) Frequency of head shapes obtained in the octanol exposure experiments ( n > 243). Failure to regenerate is defined as the loss of anterior-posterior polarity, and the failure to regenerate any head at all after octanol treatment. Error bars are standard deviations.

Figure 3. Percentage of head shape outcomes correlates with evolutionary distance. ( A ) Evolutionary tree, constructed from rRNA data, showing relationships between species of interest. Species names in red are those that were analyzed in this work; ( B ) Frequency of head shapes obtained in the octanol exposure experiments ( n > 243). Failure to regenerate is defined as the loss of anterior-posterior polarity, and the failure to regenerate any head at all after octanol treatment. Error bars are standard deviations.

Obtaining distinct regenerated morphologies, at different frequencies, despite the same treatment conditions, led us to explore the evolutionary relationship between the four species represented. We mapped out an evolutionary tree based on rRNA homology [ 75 76 ], and compared this tree ( Figure 3 A) to frequencies of the different species’ heads arising from 8-OH treatment ( Figure 3 B). Interestingly, morphologies corresponding to species that are most closely related to(SM and DJ, which are removed from GDs by at least 100 million years of evolutionary distance) occur with a much higher frequency than morphologies corresponding to less-related species (PF). We conclude that not only are phenotypic outcomes from physiological network perturbation stochastic (as the same treatment leads to one of several discrete shapes among individuals), but the frequencies are not equal and correlate roughly with evolutionary distance between the worm species these heads resemble.

Figure 4. Brain morphology is altered after 8-OH treatment. ( A – C ) Brain morphology visualized by anti-synapsin staining of wild-type G. dorotocephala ( n = 10), D. japonica ( n = 15), and S. mediterranea ( n = 6) planarians. Arrows indicate brain morphologies, and dotted lines indicate measurements used for calculation of length/width ratio; ( D – F ) Brain morphologies by anti-synapsin staining of G. dorotocephala regenerates treated in 8-OH that resembled G. dorotocephala heads ( n = 4), D. japonica heads ( n = 4), and S. mediterranea heads ( n = 6). Arrows indicate brain morphologies, and dotted lines indicated measurements used for calculation of length/width ratio. Scale bar 0.5 mm; ( G ) Average brain length/width ratios of wild-type, and 8-OH treated worms (ANOVA p < 4.9 × 10 −14 ). Error bars are standard deviations.

Figure 4. Brain morphology is altered after 8-OH treatment. ( A – C ) Brain morphology visualized by anti-synapsin staining of wild-type G. dorotocephala ( n = 10), D. japonica ( n = 15), and S. mediterranea ( n = 6) planarians. Arrows indicate brain morphologies, and dotted lines indicate measurements used for calculation of length/width ratio; ( D – F ) Brain morphologies by anti-synapsin staining of G. dorotocephala regenerates treated in 8-OH that resembled G. dorotocephala heads ( n = 4), D. japonica heads ( n = 4), and S. mediterranea heads ( n = 6). Arrows indicate brain morphologies, and dotted lines indicated measurements used for calculation of length/width ratio. Scale bar 0.5 mm; ( G ) Average brain length/width ratios of wild-type, and 8-OH treated worms (ANOVA p < 4.9 × 10 −14 ). Error bars are standard deviations.

We next asked whether internal structures were likewise converted to a different shape, as was external morphology. Few aspects of the planarian internal anatomy differ appreciably between species; however, brain size and shape offer an interesting exception to this rule. The brains of wild-typeare elongated and narrow, whileandhave appreciably shorter and wider brain morphologies ( Figure 4 A–C). No living wild-typecould be obtained for this work, and the low frequency of pseudo PF occurrence limited the number available for analysis. Thus, we focused on, and. We performed immunostaining using an anti-synapsin antibody, in order to visualize both the brain, and ventral nerve cords of pseudo and wild-type worms. As recapitulation of wild-type head shape after ten days of regeneration in water had been confirmed by geometric morphometrics, we chose to compare pseudo morphologies to adult worms of other species, in order to minimize confounding data due to variability of regeneration time between species. Overall shape differences were captured by measurement of the brain length/width ratio. These calculations were used to quantify shape differences between species, and to quantify brain remodeling in “pseudo” worms ( Figure 4 G). Strikingly, we found that pseudo worms possessed brain morphologies that look like the brain morphologies of wild-type worms whose head shapes they resembled ( Figure 4 D–F) (ANOVA,< 0.001). We conclude that the patterning processes that are disrupted after gap junction communication perturbation are also responsible for producing the morphology of the brain, and that the altered shapes are not limited to the overall head geometry but include the patterning of the central nervous system within.

In wild-type, very few neoblasts reach into the most anterior 1/6th of the worm ( Figure 5 A). In wild-type, the number of neoblasts in the anterior portion of the body is increased in comparison to, however it is still relatively low ( Figure 5 B). Wild-typeplanarians have an abundant neoblast population in the anterior-most region ( Figure 5 C). All neoblasts in the anterior 1/6th of the worm’s anatomy were counted by hand ( Figure 5 G). Remarkably, the distribution of neoblasts in pseudo worms mirrored precisely the distribution of neoblasts in the wild-type species that they resembled ( Figure 5 D‒F) (ANOVA< 0.05). We conclude that the transformation ofworms to resemble other species also extends to the species-specific, characteristic internal distribution of their stem cells, and that the patterning processes that are disrupted after GJC perturbation are also responsible for organizing the distribution of mitotically active cells.

Planarians derive much of their remarkable regenerative power from a population of heterogeneous adult stem cells, called neoblasts, which comprise the only mitotically active cell population in the body of the flatworm [ 56 77 ]. We next investigated whether or not the spatial distribution of neoblasts was appreciably different between species of planarians, and whether GJ-inhibited worms acquired the neoblast distribution characteristic of the species whose morphology they had taken on. As neoblasts are the only mitotically active cells within the planarian body, we performed immunostaining of phosphorylated histone H3, a standard neoblast marker in planaria [ 78 ], in order to visualize neoblast populations.

Figure 6. Membrane voltage reporter assay demonstrates long-term change of bioelectrical connectivity in octanol-exposed planaria. ( A – D ) Domains of relative membrane potential visualized using DiBAC 4 (3) in wild-type G. dorotocephala , wild-type S. mediterranea , wild-type D. japonica , and wild-type P. feline ; ( E – H ) Domains of relative membrane potential visualized using DiBAC(3) in pseudo G. dorotocephala (GDs), pseudo S. mediterranea (SMs), pseudo D. japonica (DJs), and pseudo P. felina (PFs), respectively. Scale bar 0.5 mm; ( I ) Number of isopotential regions in wild-type GD worms (control), all pseudo morphologies, and pseudo morphologies that have remodeled back to WT (wild-type) GD morphology after 30 days. After octanol treatment, the number of isopotential regions in pseudo morphologies is increased, but decreases to WT levels after remodeling. Black dots indicate worms with GD morphologies, blue dots indicate DJ morphologies, red dots indicate PF morphologies, green dots indicate SM morphologies, and pink dots indicate pseudo worms that have remodeled their morphologies to resemble wild-type GDs. n = 7 wild-type GD, n = 5 pseudo GD, n = 5 pseudo SM, n = 5 pseudo DJ, n = 2 pseudo PF, and n = 6 remodeled worms. Error bars are standard deviations. Non-parametric statistical analysis was done using a post-hoc comparison of all groups by Kruskal-Wallis test ( p = 0.0021), and then between groups using a Dunn’s Multiple Comparison test, which showed differences in voltage domain number between remodeled GDs and pseudo morphologies are statistically significant at p < 0.05.

Figure 6. Membrane voltage reporter assay demonstrates long-term change of bioelectrical connectivity in octanol-exposed planaria. ( A – D ) Domains of relative membrane potential visualized using DiBAC 4 (3) in wild-type G. dorotocephala , wild-type S. mediterranea , wild-type D. japonica , and wild-type P. feline ; ( E – H ) Domains of relative membrane potential visualized using DiBAC(3) in pseudo G. dorotocephala (GDs), pseudo S. mediterranea (SMs), pseudo D. japonica (DJs), and pseudo P. felina (PFs), respectively. Scale bar 0.5 mm; ( I ) Number of isopotential regions in wild-type GD worms (control), all pseudo morphologies, and pseudo morphologies that have remodeled back to WT (wild-type) GD morphology after 30 days. After octanol treatment, the number of isopotential regions in pseudo morphologies is increased, but decreases to WT levels after remodeling. Black dots indicate worms with GD morphologies, blue dots indicate DJ morphologies, red dots indicate PF morphologies, green dots indicate SM morphologies, and pink dots indicate pseudo worms that have remodeled their morphologies to resemble wild-type GDs. n = 7 wild-type GD, n = 5 pseudo GD, n = 5 pseudo SM, n = 5 pseudo DJ, n = 2 pseudo PF, and n = 6 remodeled worms. Error bars are standard deviations. Non-parametric statistical analysis was done using a post-hoc comparison of all groups by Kruskal-Wallis test ( p = 0.0021), and then between groups using a Dunn’s Multiple Comparison test, which showed differences in voltage domain number between remodeled GDs and pseudo morphologies are statistically significant at p < 0.05.

Figure 5. Neoblast (red dots) distribution is altered after octanol treatment. ( A – C ) Analysis of neoblast distribution by staining of phosphorylated histone H3 in adult, wild-type G. dorotocephala ( n = 9), D. japonica ( n = 9), and S. mediterranea ( n = 9) planarians. Dotted lines indicate edges of worm anatomy, as well as 1/6th of the length of the worm body from the anterior tip of the worm. This distance was used to define the posterior boundary of the head. Arrows indicate the region in which neoblasts were counted; ( D – F ) Analysis of neoblast distribution in G. dorotocephala regenerates treated in 8-OH that resembled G. dorotocephala heads ( n = 7), D. japonica heads ( n = 8), and S. mediterranea heads ( n = 7), by anti-phosphorylated histone H3 staining. Arrows indicate region in which neoblasts were counted. Scale bar 0.5 mm; ( G ) Average number of neoblasts in the anterior 1/6th of wild-type, and 8-OH treated worms (ANOVA p < 0.05). Error bars are standard deviations.

Figure 5. Neoblast (red dots) distribution is altered after octanol treatment. ( A – C ) Analysis of neoblast distribution by staining of phosphorylated histone H3 in adult, wild-type G. dorotocephala ( n = 9), D. japonica ( n = 9), and S. mediterranea ( n = 9) planarians. Dotted lines indicate edges of worm anatomy, as well as 1/6th of the length of the worm body from the anterior tip of the worm. This distance was used to define the posterior boundary of the head. Arrows indicate the region in which neoblasts were counted; ( D – F ) Analysis of neoblast distribution in G. dorotocephala regenerates treated in 8-OH that resembled G. dorotocephala heads ( n = 7), D. japonica heads ( n = 8), and S. mediterranea heads ( n = 7), by anti-phosphorylated histone H3 staining. Arrows indicate region in which neoblasts were counted. Scale bar 0.5 mm; ( G ) Average number of neoblasts in the anterior 1/6th of wild-type, and 8-OH treated worms (ANOVA p < 0.05). Error bars are standard deviations.

One of the physiological signals propagated within GJ-mediated cell networks is electric current: patterns of GJ-dependent connectivity can determine isopotential cell fields [ 19 79 ], and we observed that octanol indeed increased the number of regions with distinctpatterns ( Figure 6 ). Because analytical pipelines to read out encoded pattern states from bioelectrical measurements (as has been done for the human brain [ 80 ]) do not yet exist, we sought to begin to establish physiological metrics that could reveal permanent changes of the somatic bioelectric network induced by GJ blockade and could distinguish pseudo worms from those with the original (wild-type) morphology. Thus, we next examined the distribution of isopotential cell groups among the different worm shape outcomes, as such groups are established by the function of gap junctions and are a readout of the topology (connectivity) of developmental bioelectrical networks [ 20 82 ]. Patterns of endogenous relative membrane depolarization and hyperpolarization were visualized using a DiBAC (-(1,3-dibarbituric acid)-trimethine oxonol) dye [ 79 83 ]. DiBAC is anionic, so dye enters cell membranes based on relative degrees of depolarization [ 84 ]. Therefore, increased fluorescence indicates regions of depolarization, and decreased fluorescence indicates relative hyperpolarization. Wild-type worms of the species, and, and GJ-perturbed pseudo worms were imaged with DiBAC dye in order to assess potential differences in relative membrane potential. Images were analyzed with a custom image analysis program (as described in Methods) to determine the number of distinct isopotential regions present in the entire worm. Analysis of worms 10 days after 8-OH treatment (after regeneration was complete, Figure 6 A–H) revealed that transient perturbation of gap junction communication alters body-wide patterns of voltage distribution for many days after the end of 8-OH treatment. We detected an increased number of isopotential regions in the pseudo worms compared to the states of control worms; interestingly, the numbers of isopotential regions return to those of a wild-type state after 30 days of morphological remodeling ( Figure 6 I). Although we cannot be certain that every single cell had been penetrated by the dye, the pattern of isopotential regions and variability among animals suggests that octanol action is stochastic and not 100% effective, only partially disrupting electrical coupling (revealed as regions that differ in) throughout animals treated in drug. We conclude that octanol exposure alters the normal pattern ofresting potential toward a pattern that persists even after 8-OH is withdrawn and regenerative repair occurs, producing an increased concentration of isopotential regions throughout the worm.

In, physiological perturbations can stably change the basic architecture of the planarian body-plan, producing two-headed worms that continue to regenerate as two-headed in perpetuity across future rounds of regeneration in plain water [ 20 ]. Thus, we next asked whether our observed head shape changes inwere permanent. Photographs of worms after treatment with 8-OH were taken daily, from day 10 (when the morphologies of the worms were scored), through day 30 (the time necessary for complete cellular turnover in the planarian flatworm). We found that the induced morphologies were remodeled, long after regenerative processes had ended, to produce morphologies that bore closer resemblance to wild-type. Interestingly, the time course and result of this non-regenerative remodeling differed depending on the starting head shapes. Regenerates that had rounded heads, which resemble, began to develop a more triangular head shape by day 17, and by day 30, bore more of a resemblance tothan. Over the same time course, regenerates that had triangular heads, resembling, developed pronounced auricles, and by day 30 were indistinguishable from wild-type Figure 7 A,B). These findings are fascinating in two respects. First, the change in morphology in absence of a trauma highlights dynamic and robust mechanisms underlying morphological homeostasis—the anatomical state is remodeled over the long-term from an abnormal configuration existing after regenerative repair was complete. Secondly, the remodeling occurs via “paths” through the shape space illustrated by canonical variate analysis ( Figure 7 C). DJ morphology lies between GD and SM morphologies in this space, and we observe remodeling that moves from SM morphology to DJ morphology to GD morphology. From these data, we conclude that morphology is both plastic and robust, restoration of the “target” morphology can occur without trauma to the organism, and that the CV shape space is informative in illustrating parameters and boundaries to morphological variation.

2.8. A Model of Planarian Head Shapes Arising from Cell Interactions

85,86,87,88,62,59,93,96, One of the key challenges facing developmental biology and regenerative medicine is linking large-scale patterning outcomes to the individual activity of cells guided by genetic networks and signaling pathways [ 2 89 ]. Most of the work in the planarian field deals with anterior-posterior fragment polarity [ 60 90 ] or stem cell differentiation [ 57 91 ], and does not address the actual morphology of the head or the rest of the body. Recent quantitative, genetically-grounded models of regeneration [ 92 94 ] likewise use anterior-posterior identity as a binary readout, which does not address or explain changes to the shape of these anatomical regions. Our study of the bioelectrical control of regeneration reported alterations of head remodeling [ 25 ], but was limited to scaling and did not model the detailed shape of the head. To begin to mechanistically link individual cell behaviors (such as those regulated by GJ-mediated signals) to large-scale anatomical outcomes, we next constructed an agent-based model of cell signaling and morphogenesis. Our model focused on cell migration and cell-cell signaling, as these are clearly important for implementing different morphogenetic outcomes [ 95 97 ], and also known to be regulated by GJ connectivity [ 98 ] and bioelectric properties of neighboring cells [ 99 ].

We considered two cell types, A and B. Cells of type A have fixed positions—they cannot move. They produce a substance whose concentration C spreads in space. Its distribution can be described by a reaction-diffusion equation or by some other models. Cells of the type B can move. They do not produce any substance but they receive the substance C produced by cells A and their motion is determined by its concentration distribution. We modeled the planarian head with the following elements: cells of type A, deactivated cells of type A, cells of type B, and a surface boundary. Deactivated A cells are fixed but they do not produce substance C. If there is more than one cell B, then they repulse each other in the same way as they are repulsed by cells A. We choose deactivated cells A symmetrically with respect to the anterior-posterior axis, in order to preserve symmetry of outcome (which implies that B cells move in a similar way on the left and right sides). The outer boundary is composed of points and elastic “springs” connecting them. When a cell B approaches the boundary, a repulsive force acts on it from the boundary. This force is proportional to the distance from the particles and from the intervals of the boundary. Full details of the modeling are given in Figure S2 . In this model, we hypothesize that instructive (GJ-dependent) signaling occurs from the somatic tissues to the neoblasts or their progeny, to guide the new tissue generation and shaping during regeneration. Thus, the red cells are migrating neoblasts (or their progeny) while the black cells are somatic cells interconnected by GJs ( Figure 8 ). In the model, octanol disruption of cell:cell communication is thus modeled by the deactivation of signaling from a specific cell type.

in silico , this model produced planarian head shapes observed in the experiments. These different shapes were achieved from the same initial cell configuration but different deactivation pattern of cells A and different elastic properties of the boundary. These deactivation patterns correspond to octanol treatments that reduces cell-cell communication among a subset of cells. We hypothesize that this results in deactivation of some of the cells’ signaling. The initial cells location configuration and its resulting equilibrium configuration are shown in When simulated, this model produced planarian head shapes observed in the experiments. These different shapes were achieved from the same initial cell configuration but different deactivation pattern of cells A and different elastic properties of the boundary. These deactivation patterns correspond to octanol treatments that reduces cell-cell communication among a subset of cells. We hypothesize that this results in deactivation of some of the cells’ signaling. The initial cells location configuration and its resulting equilibrium configuration are shown in Figure S2 H,I. Cells of type B remain surrounded by A cells. This configuration is chosen in such a way that after deactivation of some of cells A, cells B either remain inside or they migrate outside and produce one of the required four configurations ( Figure S2 A–D). Specific head shape emerges because cells B are pushed away by cells A and by other cells B. They escape through the lower concentration levels of substance C left by deactivated cells A, arriving at the outer membrane and changing its shape.

Figure 8. Computational model reproduces the four discrete outcomes observed experimentally. (A–D) Four types of planarian heads obtained from the computational model. Different shapes result due to deactivation of different cells. Red lines show the trajectories of the red cells; (E–H) Morphometric measurement of different planarian heads, for comparison with real heads (I–L) measured (see M) Definitions of lengths of measurements L1 and L2, used in comparison of real worms to those produced by the model in Computational model reproduces the four discrete outcomes observed experimentally. () Four types of planarian heads obtained from the computational model. Different shapes result due to deactivation of different cells. Red lines show the trajectories of the red cells; () Morphometric measurement of different planarian heads, for comparison with real heads () measured (see Table 2 ). Scale bar 0.5mm. Cells of type A are shown in black, deactivated cells of type A white, cells of type B are red. The outer boundary has two parts differing by its rigidity (soft part of the boundary is shown in red, more rigid part in green). Thus, the red cells represent migrating neoblasts (or their progeny) while the black cells represent somatic cells interconnected by GJs. () Definitions of lengths of measurements L1 and L2, used in comparison of real worms to those produced by the model in Table 2

Figure 8. Computational model reproduces the four discrete outcomes observed experimentally. (A–D) Four types of planarian heads obtained from the computational model. Different shapes result due to deactivation of different cells. Red lines show the trajectories of the red cells; (E–H) Morphometric measurement of different planarian heads, for comparison with real heads (I–L) measured (see M) Definitions of lengths of measurements L1 and L2, used in comparison of real worms to those produced by the model in Computational model reproduces the four discrete outcomes observed experimentally. () Four types of planarian heads obtained from the computational model. Different shapes result due to deactivation of different cells. Red lines show the trajectories of the red cells; () Morphometric measurement of different planarian heads, for comparison with real heads () measured (see Table 2 ). Scale bar 0.5mm. Cells of type A are shown in black, deactivated cells of type A white, cells of type B are red. The outer boundary has two parts differing by its rigidity (soft part of the boundary is shown in red, more rigid part in green). Thus, the red cells represent migrating neoblasts (or their progeny) while the black cells represent somatic cells interconnected by GJs. () Definitions of lengths of measurements L1 and L2, used in comparison of real worms to those produced by the model in Table 2

Table 2. Morphometric comparison between computational model outcomes and real head shape measurements in 4 species.