EvoDevo

The bilaterian through gut with an anal opening is a key invention in animals, since it facilitates effective food processing, which allows animals to grow to a larger body size. However, because non-bilaterian animals lack a through gut, the evolution of anus is still debated. The formation of bilaterian hindgut is governed by the spatial expression of several transcription factors (e.g. Caudal and Brachyury) under the control of Wnt signaling. This conserved pattern has been used to support the homology of the anus of protostomes (insects, snails) and deuterostomes (sea urchins, humans). Here we show, that these bilaterian "hindgut" marker genes are expressed around the male gonopore of several xenacoelomorphs, which have a blind gut without an anal opening. These findings suggest a deep evolutionary relationship between the xenacoelomorph male gonopore and the bilaterian anus. Since xenacoelomorphs are the potential sister group to all remaining Bilateria, our results suggest that the bilaterian anus evolved from a male gonopore that came in contact with the digestive endoderm to form the posterior opening.

Developmental gene regulatory networks (GRNs) describe the interactions among gene products that drive the differential transcriptional and cell regulatory states that pattern the embryo and specify distinct cell fates. GRNs are often deeply conserved, but whether this is the product of constraint inherent to the network structure or stabilizing selection remains unclear. We have constructed the first formal GRN for early development in Heliocidaris erythrogramma, a species with dramatically accelerated, direct development. This life history switch has important ecological consequences, arose rapidly, and has evolved independently many times in echinoderms, suggesting it is a product of selection. We find that H. erythrogramma exhibits dramatic differences in GRN topology compared with ancestral, indirect-developing sea urchins. In particular, the GRN sub-circuit that directs the early and autonomous commitment of skeletogenic cell precursors in indirect developers appears to be absent in H. erythrogramma, a particularly striking change in relation to both the prior conservation of this sub-circuit and the key role that these cells play ancestrally in early development as the embryonic signaling center. These results show that even highly conserved molecular mechanisms of early development can be substantially reconfigured in a relatively short evolutionary time span, suggesting that selection rather than constraint is responsible for the striking conservation of the GRN among other sea urchins.

Although the adult pentaradial body plan of echinoderms evolved from a bilateral ancestor, identifying axial homologies between the morphologically divergent echinoderms and their bilaterian relatives has been an enduring problem in zoology. The expression of conserved bilaterian patterning genes in echinoderms provides a molecular framework for resolving this puzzle. Recent studies in juvenile asteroids suggest that the bilaterian antero-posterior axis maps onto the medio-lateral axis that is perpendicular to each of the five rays of the pentaradial body plan. Here we test this hypothesis in another echinoderm class, the ophiuroids, using the cosmopolitan brittle star Amphipholis squamata. Our results show that the general principles of axial patterning are similar to those described in asteroids, and comparisons with existing molecular data from other echinoderm taxa support the idea that medio-lateral deployment of the AP patterning program across the rays predates the evolution of the asterozoan and likely the echinoderm crown-groups. Our data also reveal expression differences between A. squamata and asteroids, which we attribute to secondary modifications specific to ophiuroids. Together, this work provides important comparative data to reconstruct the evolution of axial properties in echinoderm body plans.

To cope with extreme environmental conditions diverse marine species have developed mechanisms that allow them to permanently or temporarily attach to substrates. In the intertidal zone of marine habitats, where tidal ranges and currents may drift organisms away from their habitat, temporary adhesive systems such as the one inherent the arrow worm Spadella cephaloptera (Chaetognatha) constitute an essential trait for the survival of this taxon. The underlying molecular mechanism of this system has not been described yet, and the existing morphological information is limited to adults. Furthermore, a relationship between the nervous system and the attachment in S. cephaloptera remains to be demonstrated. In this study, single-nuclei sequencing of S. cephaloptera hatchlings was performed, using as a reference a newly sequenced and assembled genome to identify the transcriptomic profiles of the cells mediating attachment, neuronal populations, and the main cell types of chaetognath hatchlings. Our findings, supported by previous studies, suggest that the chaetognath adhesive system evolved convergently to those of other other metazoans. Moreover, diverse cell types were identified in the ventral nerve center and multiple ciliated cell types previously described from anatomical observations were validated. Ongoing in-depth investigation of these data, together with datasets from other developmental stages, will provide further insights into the evolutionary origins of the unique chaetognath body plan.

Medusozoan cnidarians (e.g., jellyfish) metamorphose from a benthic juvenile polyp into a pelagic adult medusa, providing a well-known example of a clade that uses tissue remodeling to create distinct juvenile and adult body plans. Staurozoans (i.e., stalked jellyfish) are an atypical lineage of medusozoans that have lost their medusa stage; thus, their juvenile and adult body plans look remarkably alike. Their limited metamorphosis is characterized by the regression of primary (juvenile) tentacles and the development of secondary (adult) tentacles. In some staurozoan lineages, metamorphosis also involves development of novel adhesive structures (anchors), which are built on top of the regressing primary tentacles. Understanding how cells are partitioned from making juvenile tissues to making adult tissues is important for understanding how animals can make adult structures in the absence of complete metamorphosis. We compared the abundance and distribution of proliferative cells in tissues undergoing regression (primary tentacles) and development (secondary tentacles and anchors) during the juvenile to adult transition in the San Juan Island stalked jellyfish, Haliclystus sanjuanensis. We show that proliferative cells are lost in regressing primary tentacles but are gained in anchors, consistent with a shift in investment from juvenile to adult tissue. Prior to regression, primary and secondary tentacles show similar patterns in their proliferative cell distribution and in the identity of their cnidocytes (stinging cells), indicating that adult tentacles are made by re-deploying a juvenile tentacle program. Finally, we demonstrate that unlike secondary tentacles, primary tentacles cannot regenerate, illustrating that the temporary investment in this tissue is tied to their loss of proliferative cells. Thus, we propose that continued investment in a population of proliferating cells is an important mechanism for segregating temporary tissues (primary tentacles) from long-term tissues (secondary tentacles). These observations of cell dynamics in H. sanjuanensis suggest that temporary investment into juvenile structures may be used to pattern novel adult tissues, providing an important mechanism for diversifying adult body plans.

Across the animal kingdom, metamorphosis transforms a free-swimming larva into a morphologically distinct juvenile, yet how conserved cellular processes are spatiotemporally coordinated to execute this rapid body plan switch remains poorly understood. Using the marine sponge Amphimedon queenslandica -- a member of one of the earliest-diverging animal phyletic lineages -- we characterise the cellular and morphogenetic events of the first six hours of metamorphosis. We show that metamorphosis proceeds through a tightly ordered sequence of events orchestrated by a stereotypic wave of epithelial infoldings that propagates from the basal to the apical pole within the first hour post-settlement. This wave acts as a morphogenetic pacemaker, spatially and temporally inducing subsequent cellular transitions, which include coordinated mucus secretion, cilia resorption, epithelial-mesenchymal transition (EMT), mesenchymal-epithelial transition (MET), transdifferentiation and targeted programmed cell death. Lineage tracing further reveals the stepwise transformation of larval cells at metamorphosis, with labelled epithelial flask cells transdifferentiating into internal archaeocyte stem cells via a transitory amoeboid cell state associated with EMT. These findings demonstrate that rapid metamorphosis comprises a spatially pre-patterned, stepwise program, and provide a cellular framework for understanding body plan transformations with broad implications for the evolution of metazoan metamorphosis.

Cell proliferation is a key driver of morphogenesis and body plan transformation in multicellular animals, yet its spatial organization remains poorly understood in many non-segmented spiralians. In this study, we examine the dynamics of cell division during larval growth and metamorphosis in the larvae and early juveniles of the phoronid Phoronopsis harmeri, using EdU incorporation, anti-phospho-histone H3 immunostaining, confocal laser scanning microscopy, and electron microscopy. Early larval development is characterized by widespread proliferative activity across ectodermal and mesodermal tissues, which becomes progressively compartmentalized as the larva matures. Two structured ring-shaped posterior proliferative zones, pre- and post-telotrochal, emerge within the telotroch and persist through metamorphosis, supporting both larval elongation and the juvenile development of ascending gut branch. In contrast, the metasomal sac and future adult trunk epidermis expand via broadly distributed epithelial proliferation, without forming a localized growth zone. This suggests that P. harmeri combines conserved features, such as a posterior growth zone, with lineage-specific innovations in regional growth. In addition, we identify atypical mitotic characteristics in this species, including unconventional metaphase organization and signs of interkinetic nuclear migration in larval epithelia. Our findings highlight the coexistence of ancestral and derived proliferative mechanisms in phoronids and provide new insights into the evolution of axial elongation and morphogenetic compartmentalization in Lophotrochozoa.

Tapeworms are parasitic flatworms that lack key features conventionally used to define the head-tail axis in free-living organisms, resulting in long standing questions regarding the true orientation of their main body axis. As adults, most tapeworms also exhibit a segmented body which has been considered an adaptation unique to the group. Anteroposterior (AP) patterning in free-living flatworms is controlled by {beta}-catenin-dependent Wnt signalling and positional control genes are expressed by their musculature in highly regionalised domains. Here we investigate the expression of Wnt and Hedgehog components during the strobilar phase of the tapeworm life cycle during which larval tissues are lost and replaced through the continuous production of new tissues. Results reveal previously unidentified centres of signalling associated with their neuromuscular system and show that segments are marked by secondary, AP axes in agreement with the polarity of the primary body axis. Neuromuscular expression and communication between Wnt and Hedgehog signalling is consistent with embryonic and regenerative growth in planarians and is a common mechanism for establishing AP-polarised boundaries in a diverse range of segmented animals. Taken together our results suggest that segmentation in tapeworms represents a modified form of posterior regeneration, a common feature among flatworms.

The evolution of visual systems has compelled numerous investigations of developmental processes underlying eye patterning across Bilateria. It is well-established that homologs of the transcription factor Pax6 play a highly conserved role in eye fate specification and are at the top of the retinal determination gene network (RDGN) hierarchy. In insects, the two Pax6 homologs eyeless (ey) and twin of eyeless (toy) are required for the development of the two visual systems broadly found within the phylum (i.e., median and lateral eyes). Curiously, Pax6 homologs do not appear to maintain this function in well-studied chelicerate models, with emphasis on spiders, a lineage of arachnids with great diversity of eye form and acuity. It was recently proposed that the gene Pax2 (shaven; sv) may have subsumed the role of eye fate specification in chelicerates, a hypothesis predicated upon the observation that one of two spider Pax2 copies is strongly expressed in the developing lateral eyes during embryogenesis. However, no functional data are available for any Pax homologs across Chelicerata. We examined the incidence of Pax family genes across Chelicerata, as well as interrogated the expression and function of Pax2 and Pax6 homologs in the daddy-longlegs Phalangium opilio, an arachnid recently discovered to bear a highly plesiomorphic arrangement of visual systems. Here, we show that ey and toy are expressed early in the developing head lobes of P. opilio, whereas sv is not expressed until well after stages when downstream RDGN members (eyes absent and sine oculis) are already activated. Gene silencing of ey, toy, and sv individually had no discernible effect on eye development. By contrast, double knockdown of ey and toy resulted in an array of median eye defects, spanning loss of some cells of the eye to total loss of the median eyes. Gene expression assays also showed that depletion of the two Pax6 copies resulted in failure of the vestigial median and vestigial lateral eyes. These data are consistent with a conserved role for Pax6 homologs in patterning both visual systems and all three eye pairs in the daddy-longlegs. Our results comprise the first functional data for Pax6 genes in any chelicerate and suggest that heterochronic shifts in expression, rather than changes in function, underlie the atypical dynamics of Pax genes in derived arachnid groups such as spiders.

Metridium senile is a clonal anemone that engages in fighting interactions to defend its territory from non-clonemates utilizing an inducible fighting organ, the catch tentacle. Upon contact with a non-clonemate, the catch tentacle tip detaches onto the non-clonal individual, resulting in necrosis where the tip attaches to the non-clone. The incapacitating function of the catch tentacle is driven by a unique type of cnidocyte, the holotrich, which is not found elsewhere in M. senile, including the feeding tentacles from which catch tentacles develop. Metridium farcimen, the sister species to M. senile, never develops catch tentacles despite their close phylogenetic relationship, as exemplified by their ability to hybridize. Here, we compare the feeding tentacles of both species to the catch tentacles in M. senile to determine how catch tentacles achieve their unusual function. We found that the feeding tentacles of M. senile and M. farcimen house similar types of cnidocytes that develop from proliferative cells distributed throughout the tentacle. By contrast, catch tentacles house distinct cnidocyte types from feeding tentacles and restrict proliferative cells to the base of the tentacle. This suggests immature cnidocytes migrate from base to tip to replace lost cells after an aggressive interaction in the catch tentacle. Additionally, we observed two morphologically and chemically distinct types of holotrichs in the catch tentacles that appear to use different cues to induce firing. Together, our data suggests that the novelty of catch tentacle aggression is mediated by distinct cell and tissue dynamics.

Alveolar echinococcosis (AE) is a lethal zoonosis caused by infiltrative growth of the metacestode larva of the tapeworm Echinococcus multilocularis in host organs. We previously showed that the Echinococcus metacestode is an evolutionarily unique, broadly posteriorized tissue, leading us to hypothesize that canonical WNT (cWNT) signalling--which patterns the body axis across metazoans--might be critical for metacestode formation. Here, we report effective RNAi-mediated knockdown of the E. multilocularis {beta}-catenin gene (bcat-1), the central effector of cWNT signalling, in a primary parasite cell culture system that produces metacestode vesicles. bcat-1(RNAi) cultures were markedly impaired in vesicle formation, exhibited stem-cell hyperproliferation, and displayed disrupted muscle-fibre organisation. Genome-wide transcriptomics revealed a general anteriorization of gene expression, and in situ hybridization showed an overproduction of cells expressing head-inducing factors such as sfrp upon bcat-1 knockdown. Conversely, metacestode-specific genes--including the tegumental factors muc-1, TNFR, and antigen B--as well as the posterior marker post2b were significantly downregulated, consistent with the observed vesicle-formation defects. In situ analyses further identified anterior markers--frizzled-10, nou-darake, notum, and follistatin--that were overexpressed in bcat-1(RNAi) cultures and localized to the future anterior pole at the earliest stages of protoscolex formation. Finally, pharmacological inhibition of WNT signalling with pyrvinium pamoate caused complete loss of posterior tissue in Echinococcus protoscoleces, killed metacestode vesicles, and reduced stem-cell proliferation at nanomolar concentrations. Together, these findings establish a central role for cWNT signalling in directing Echinococcus body-axis formation and the posteriorization events driving metacestode growth within the host, providing insight into asexual parasite proliferation mediated by this biologically unique larval stage and pointing to potential targets for chemotherapy against AE. Author SummaryAlveolar echinococcosis (AE) is a lethal disease caused by the cancer-like growth of the metacestode larva of the tapeworm Echinococcus multilocularis. From a developmental perspective, the Echinococcus metacestode is an unusual biological structure and even atypical among tapeworms. Previous work indicated that metacestode formation involves re-patterning of the body axis, eliminating head structures and producing broadly posteriorized tissue. How this is controlled at the molecular and cellular levels, however, was unknown. In this study, we perturbed expression of the {beta}-catenin gene (bcat-1), a central regulator of canonical WNT signalling, using RNA interference (RNAi). bcat-1(RNAi) parasite cultures failed to generate metacestode vesicles and instead showed stem-cell hyperproliferation and muscle-cell distortion. Genes required for posteriorized metacestode tissue were downregulated, whereas genes directing head formation in adult worms (follistatin, sfrp, fz10, ndk) were upregulated, indicating a general anteriorization of the culture system. Pharmacological inhibition of WNT signalling with pyrvinium pamoate caused complete loss of posterior structures in protoscoleces, reduced stem-cell proliferation, and killed metacestode tissue. These findings identify {beta}-catenin and the canonical WNT pathway as crucial regulators of the posteriorization that underlies metacestode formation. Given that WNT signalling is deregulated in many human cancers and that small-molecule inhibitors are available, our results suggest new avenues for anti-AE drug development.

In tunicates, larvaceans represent a fascinating case of evolution, where the chordate body plan has been maintained despite a rapidly evolving genome characterized by strong In contrast to other tunicates, larvaceans keep the chordate body plan during their entire life. They have acquired a highly specialized epithelium in charge of producing the "house", a complex extracellular apparatus used for filter feeding in the plankton. To what extent the house and this epithelium represent true molecular innovations withing chordates is a question for which thorough transcriptomics can bring novel insights. We conducted a developmental profiling of gene expression at the single-cell level in the larvacean Oikopleura dioica. We provide detailed descriptions of cellular transcriptomes associated with the house-synthesizing organ, which permits to define the molecular specifics of epithelial cell territories. We followed their emergence during development, and we identified genes that represent key candidate molecules for regulating the morphogenesis of the house-producing organ. Dynamic changes in gene expression and cell identities during major developmental transitions of the lifecycle illustrate that our dataset effectively allows access to the diversity of O. dioicas cell types in embryos and in adults. The resources presented here constitute critical assets to investigate larvacean biology and evolution for mechanistic and comparative goals.

Sexual dimorphism is common in animals. The most complete model of sex determination comes from Drosophila melanogaster, where the relative dosage of autosomes and X chromosomes leads indirectly to sex-specific transcripts of doublesex. Female Dsx interacts with a mediator complex protein encoded by intersex to activate female development. In males the transcription factor encoded by fruitless promotes male-specific behavior. The genetics of sex determination have been examined in a small number of other insects, yet several questions remain about the pleisomorphic state. Is doublesex required for female and male development? Is fruitless conserved in male behavior or morphology? Are other components such as intersex functionally conserved? To address these questions, we report expression and functional tests of doublesex, intersex and fruitless in the hemipteran Oncopeltus fasciatus, characterizing three sexual dimorphisms. doublesex prevents intersex phenotypes in all sexes and dimorphic traits in the milkweed bug. intersex and fruitless are expressed across the body, in females and males. fruitless and intersex also affect the genitalia of both sexes, but have effects limited to different dimorphic structures in different sexes. These results reveal roles for intersex and fruitless distinct from other insects, and demonstrate distinct development mechanisms in different sexually dimorphic structures.

BackgroundEarly embryogenesis is characterized by dramatic cell proliferation and movement. In most insects, early embryogenesis includes a phase called the uniform blastoderm, during which cells evenly cover the entirety of the egg. However, the embryo of the German cockroach, Blattella germanica, like those of many insects within the super order Polyneoptera, does not have a uniform blastoderm; instead, its first cells condense rapidly at the site of a future germband. We investigated early development in this species in order to understand how early gene expression is or is not conserved in these insect embryos with distinct early cell behaviors. ResultsWe present a detailed time series of nuclear division and distribution from fertilization through germband formation and report patterns of expression for the early patterning genes hunchback, caudal, and twist in order to understand early polarization and mesoderm formation. We show a detailed time course of the spatial expression of two genes involved in the segmentation cascade, hedgehog and even-skipped, and demonstrate two distinct dynamics of the segmentation process. ConclusionsDespite dramatic differences in cell distribution between the blastoderms of many Polyneopteran insects and those of more well-studied developmental models, expression patterns of early patterning genes are mostly similar. Genes associated with axis determination in other insects are activated relatively late and are probably not maternally deposited. The two phases of segmentation - simultaneous and sequential - might indicate a broadly conserved mode of morphological differentiation. The developmental time course we present here should be of value for further investigation into the causes of this distinct blastoderm type.

Hox genes are transcriptional regulators that elicit cell positional identity along the anterior-posterior region of the body plan across different lineages of Metazoan. Comparison of Hox gene expression across distinct species reveals their evolutionary conservation, however their gains and losses in different lineages can correlate with body plan modifications and morphological novelty. We compare the expression of eleven Hox genes found within Streblospio benedicti, a marine annelid that produces two types of offspring with distinct developmental and morphological features. For these two distinct larval types, we compare Hox gene expression through ontogeny using HCR (hybridization chain reaction) probes for in-situ hybridization and RNA-seq data. We find that Hox gene expression patterning for both types is typically similar at equivalent developmental stages. However, some Hox genes have spatial or temporal differences between the larval types that are associated with morphological and life-history differences. This is the first comparison of developmental divergence in Hox genes expression within a single species and these changes reveal how body plan differences may arise in larval evolution.

Making living machines using biological materials (cells, tissues, and organs) is one of the challenges in developmental biology and modern biomedicine. Constraints in regeneration potential and immune self-defense mechanisms limit the progress in the field. Here, we present unanticipated features related to self-recognition and ancestral neuro-immune architectures of new emerging reference species - ctenophores or comb jellies. These are descendants of the earliest survival metazoan lineage with unique tissues, organs and independent origins of major animal traits such as neurons, muscles, mesoderm, and through-gut. Thus, ctenophores convergently evolved complex organization, compared to bilaterians. Nevertheless, their neural and immune systems are likely functionally coupled, enabling designs and experimental construction of hybrid neural systems and even entire animals. This report illustrates impressive opportunities to build both chimeric animals and neurobots using ctenophores as models for bioengineering. The obtained neurobots and chimeric animals from three ctenophore species (Bolinopsis, Mnemiopsis, and Pleurobrachia) were able to be autonomous and survive for days. In sum, the unification of biodiversity, cell biology, and neuroscience opens unprecedented opportunities for experimental synthetic biology.

The apical pole of eumetazoan ciliated larvae acts as a neurosensory structure and is principally composed of sensory-secretory cells. Cnidarians like the sea anemone Nematostella vectensis are the only non-bilaterian group to evolve ciliated larvae with a neural integrated sensory organ that is likely homologous to bilaterians. Here, we uncovered the molecular signature of the larval sensory organ in Nematostella by generating a transcriptome of the apical tissue. We characterised the cellular identity of the apical domain by integrating larval single-cell data with the apical transcriptome and further validated this through in-situ hybridisation. We discovered that the apical domain comprises a minimum of 6 distinct cell types, including apical cells, neurons, peripheral flask-shaped gland/secretory cells, and undifferentiated cells. By profiling the spatial expression of neuronal genes, we showed that the apical region has a unique neuronal signature distinct from the rest of the body. By combining the planula cilia proteome with the apical transcriptome data, we revealed the sheer complexity of the non-motile apical tuft. Overall, we present comprehensive spatial/molecular data on the Nematostella larval sensory organ and open new directions for elucidating the functional role of the apical organ and larval nervous system.

The evolution of flippers for wing-powered diving in penguins is a striking example of tetrapod limb specialization. The modern penguin flipper is structurally reinforced by a characteristic dorsoventral flattening of the long bones accompanied by a reduction in distal forelimb musculature, features which emerged convergently in flightless diving birds and aquatic mammals. While an extensive fossil record informs the morphological sequence through which these changes occurred, the evolutionary pressures and developmental mechanisms underlying these modifications are unknown. We find that in avian and mammalian forelimbs, a flattened bone morphology only emerged in aquatic lineages that lost ancestral modes of locomotion, including in flightless diving birds, pinnipeds, and cetaceans. Using penguin embryos as an accessible model for investigating flipper development, we demonstrate that early patterning of forelimb musculoskeletal morphology is similar to that seen in forelimbs of non-aquatic birds. Instead, later modifications of gene expression and cell and tissue behaviors underlie flipper phenotypes. Thus, we find that in the early penguin forelimb, the initial cues that pattern the muscle do not differ from other avian species, however late embryonic changes in proliferation result in dramatic reduction of muscle. Likewise, forelimb bones in penguins initially have similar cross-sectional proportions to those in flighted birds. The shape of these bones is, however, remodeled late in embryonic development through a process that shares molecular hallmarks with bone ridge formation at tendon attachment sites. In these bones, ridge-forming tissue initiates at the ends of the bones (the epiphyses) and extends into tendon-like connective tissue along the lateral edges of the bone, widening the long bones along the anterior-posterior axis and producing a flattened bone. Using spatial transcriptomics and comparative genomic tools we determine that differentially expressed genes between the ridge-forming tissues and long bone cartilage are significantly enriched for signals of selection in the penguin lineage and that these genes may also be convergently evolving in marine mammals. Together, these data show that the evolution of musculoskeletal morphology in the penguin flipper occurred through expansion or novel deployment of molecular programs typically associated with tendon-attachment sites during late embryonic development.

The evolution of multicellularity involved the transformation of a simple cell wall of unicellular ancestors into a complex, multifunctional extracellular matrix (ECM). A suitable model organism to study the formation and expansion of an ECM during ontogenesis is the multicellular green alga Volvox carteri, which, along with the related volvocine algae, produces a complex, self-organized ECM composed of multiple substructures. These self-assembled ECMs primarily consist of hydroxyproline-rich glycoproteins, a major component of which is pherophorins. To investigate the geometry of the growing ECM, we fused the yfp gene with the gene for pherophorin II (PhII) in V. carteri. Confocal microscopy reveals PhII:YFP localization at key structures within the ECM, including the boundaries of compartments surrounding each somatic cell and the outer surface of the organism. Image analysis during the life cycle allows the stochastic geometry of those growing compartments to be quantified. We find that their areas and aspect ratios exhibit robust gamma distributions and exhibit a transition from a tight polygonal to a looser acircular packing geometry with stable eccentricity over time, evoking parallels and distinctions with the behavior of hydrated foams. These results provide a quantitative benchmark for addressing a general, open question in biology: How do cells produce structures external to themselves in a robust and accurate manner?

Adult pluripotent stem cells are found in diverse animals, including cnidarians, acoels, and planarians, and confer remarkable abilities such as whole-body regeneration. The mechanisms by which these pluripotent stem cells orchestrate the replacement of all lost cell types, however, remains poorly understood. Underlying heterogeneity within the stem cell populations of these animals is often obscured when focusing on certain tissue types or life history stages, which tend to have indistinguishable spatial expression patterns of stem cell marker genes. Here, we focus on the adult pluripotent stem cells (i-cells) of Hydractinia symbiolongicarpus, a colonial marine cnidarian with distinct polyp types and stolonal tissue. Recently, a single-cell expression atlas was generated for H. symbiolongicarpus which revealed two distinct clusters with i-cell signatures, potentially representing heterogeneity within this species stem cell population. Considering this finding, we investigated eight new putative stem cell marker genes from the atlas including five expressed in both i-cell clusters (Pcna, Nop58, Mcm4, Ubr7, and Uhrf1) and three expressed in one cluster or the other (Pter, FoxQ2-like, and Zcwpw1). We characterized their expression patterns in various contexts - feeding and sexual polyps, juvenile feeding polyps, stolon, and during feeding polyp head regeneration - revealing context-dependent gene expression patterns and a transcriptionally dynamic i-cell population. We uncover previously unknown differences within the i-cell population of Hydractinia and demonstrate that its colonial nature serves as an excellent system for investigating and visualizing heterogeneity in pluripotent stem cells.