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Comparisons of cell proliferation and cell death from tornaria larva to juvenile worm in the hemichordate Schizocardium californicum

Comparisons of cell proliferation and cell death from tornaria larva to juvenile worm in the... Background: There are a wide range of developmental strategies in animal phyla, but most insights into adult body plan formation come from direct-developing species. For indirect-developing species, there are distinct larval and adult body plans that are linked together by metamorphosis. Some outstanding questions in the development of indirect-developing organisms include the extent to which larval tissue undergoes cell death during the process of metamorphosis and when and where the tissue that will give rise to the adult originates. How do the processes of cell division and cell death redesign the body plans of indirect developers? In this study, we present patterns of cell proliferation and cell death during larval body plan development, metamorphosis, and adult body plan formation, in the hemichordate Schizocardium californium (Cameron and Perez in Zootaxa 3569:79–88, 2012) to answer these questions. Results: We identified distinct patterns of cell proliferation between larval and adult body plan formation of S. cali- fornicum. We found that some adult tissues proliferate during the late larval phase prior to the start of overt metamor- phosis. In addition, using an irradiation and transcriptomic approach, we describe a genetic signature of proliferative cells that is shared across the life history states, as well as markers that are unique to larval or juvenile states. Finally, we observed that cell death is minimal in larval stages but begins with the onset of metamorphosis. Conclusions: Cell proliferation during the development of S. californicum has distinct patterns in the formation of lar- val and adult body plans. However, cell death is very limited in larvae and begins during the onset of metamorphosis and into early juvenile development in specific domains. The populations of cells that proliferated and gave rise to the larvae and juveniles have a genetic signature that suggested a heterogeneous pool of proliferative progenitors, rather than a set-aside population of pluripotent cells. Taken together, we propose that the gradual morphological transfor- mation of S. californicum is mirrored at the cellular level and may be more representative of the development strate- gies that characterize metamorphosis in many metazoan animals. Keywords: Metamorphosis, Hemichordate, Tornaria, Cell proliferation, Cell death Background The development of animal body plans has largely been informed by research in a few key model species that pattern the adult body plan during embryogenesis, a *Correspondence: clowe@stanford.edu strategy termed direct development. However, this type Hopkins Marine Station, Department of Biology, Stanford University, Pacific of development is not representative of many animal Grove, CA, USA Full list of author information is available at the end of the article groups, where embryogenesis gives rise to a larva with a © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Bump et al. EvoDevo (2022) 13:13 Page 2 of 20 body plan distinct from that of the adult, a strategy called of tornaria larvae and their counterpart adult bodies can indirect development [2–4]. During direct development, be traced back to the late 1800s [29, 30] and more recent the adult is formed directly from the embryo, while in morphological descriptions of larval and adult body plans indirect development, embryonic processes give rise to a have been carried out in a range of enteropneust species; larval body plan that later transforms into the adult. This Ptychodera flava [31, 32], Balanoglossus misakiensis [33], transformation between larvae and adults is a develop- Balanoglossus simodensis [34] and S. californicum [35]. mental process known as metamorphosis, which is char- In these species, the tornaria larva is formed following acterized by the loss of larval-specific structures and the embryogenesis, while the benthic adult body plan forms emergence of adult structures [5–11]. The prevalence of by metamorphosis following an extended planktonic this developmental strategy across animal phyla clearly period [31, 33–36]. Studies of hemichordate complex life demonstrates that a better mechanistic understanding cycles have largely been based on morphological charac- of indirect development is critical for a more complete ters, with some descriptive patterning studies [37–53]. understanding of the developmental basis of body plan However, the cellular and developmental mechanisms evolution. through metamorphosis remain largely uncharacterized. Many marine organisms utilize indirect development, For example, we do not know whether the adult is formed developing first as larvae that feed and grow before by transformation of larval tissues via transdifferentation reaching metamorphosis [12, 13]. In species, such as or by proliferation of adult-specific cells following large- gastropods with veliger larvae, the morphological dif- scale larval cell death. ference between larval and adult body plans is not very To begin to address these questions, we characterized pronounced, because in these organisms, metamorpho- proliferation and cell death through the development sis represents a major shift in ecological niche but not a and metamorphosis of S. californicum. For the purpose large morphological change [14, 15]. At the other end of of our study, we define metamorphosis in S. californicum the spectrum, as is found in some echinoids, larval and as an overt morphological event. In a rapid 48-h period a adult body plans can be radically different in organiza - swimming planktonic larva transforms into a burrowing tion with a “catastrophic metamorphosis.” In this case, benthic adult as larval ectoderm compacts and condenses the adult develops as a rudiment within the larva and over the underlying mesoderm and endoderm[35]. We metamorphosis results in a complete reorganization of found distinct patterns of cell proliferation between lar- the body around new developmental axes in addition to val and adult body plans and that the start of a clear overt the loss of larval structures [5, 16, 17]. Similarly, in some metamorphosis corresponded with an increase in cell nemertean worms, with a pilidium larva, the adult devel- death. To then determine if there were distinct genetic ops from several rudiments, and metamorphosis culmi- markers of proliferative cells, and if those markers dif- nates with the juvenile consuming the larval tissues [17, fered between life history stages, we deployed an irra- 18]. However, metamorphosis in species with distinct diation strategy to deplete proliferative cells and found a larval and adult body plans does not always involve a number of differentially expressed transcripts. segregated rudiment or cataclysmic metamorphosis — instead larval tissue seems to be remodeled rapidly into Results the adult without obvious drastic histolysis of the larval Patterns of proliferation in larval and adult body plans. body plan [2, 15, 19]. In this type of metamorphosis, do We wanted to test whether patterns of cellular prolifera- adult structures originate from a small population of pro- tion involved in the development of the planktonic larva liferative cells? What is the fate of larval tissues? Indirect- were similar or different to those during the develop - developing hemichordates represent this particular type ment of the benthic juvenile. To describe the distribution of metamorphosis and provide an opportunity to explore of proliferative cells throughout the development of S. this type of developmental strategy. californicum, we assessed the incorporation of the thy- Hemichordates are composed of two classes, the soli- midine analog 5-ethynyl-29-deoxyuridine (EdU), which tary enteropneust worms and the largely colonial, tube- labels cells in S phase [54], during a range of developmen- dwelling pterobranchs [20–23]. While the position of tal stages: early larval development (Fig.  1B), mid larval hemichordates as sister to the echinoderms and closely development (Fig. 1C), late larval development (Fig. 1D), related to chordates has been well established [24–27], metamorphosis (Fig.  1E–I), and in juvenile development new studies have challenged this position [28]. Within (Fig. 1J). the enteropneusts, one family, the Harrimaniiidae, are The earliest larval developmental stage consists of a direct developers, while the families Spengelidae and tightly packed ciliary band that loops around the larva, Ptychoderidae, are indirect developers with a distinct lar- a thin wide squamous epithelium, an apical tuft, and val body plan called the tornaria. Morphological studies a tripartite gut (Figs.  1B, 2). On the ventral surface we Bump  et al. EvoDevo (2022) 13:13 Page 3 of 20 Fig. 1 Larval development and metamorphosis of S. californicum. A Schematic of the complex life cycle of the indirect developing S. californicum (modified from Ref. [35]. B–J Light microscopy of the complex life cycle of the indirect developing S. californicum (from [35]). B early tornaria larva, C mid tornaria larva, D late tornaria larva, E–I process of metamorphosis, J juvenile Fig. 2 Cell proliferation throughout early larval development of S. californicum. All: anterior up; blue = Hoechst, green = EdU; scale bar is 100um. Schematics of ventral (A), dorsal (D), and medial (E) views with key structures labeled. EdU staining with maximum intensity projections showing ventral (B), dorsal (E), and medial H sections. C–I insets. White arrowheads denote preoral ciliary band and postoral ciliary band detected EdU cells throughout the preoral and postoral had higher cell densities. The ciliary bands were densely loops of the circumoral ciliary band (Fig. 2B, C). The cili - packed with nuclei: there are ~ 59% greater number of ary band is used for both swimming and particle capture cells in the ciliary bands versus all other tissues (paired at this stage [55–57] and makes up a large percentage of t test, p = 0.016) and they were also more proliferative the ectoderm. We tested whether the ciliary bands were with ~ 22% more Edu cells than all other tissues (paired more proliferative than the general ectoderm, or simply t test, p = 0.008) (Additional file  1: S1A, B). This suggests Bump et al. EvoDevo (2022) 13:13 Page 4 of 20 that while the ciliary bands are nuclei-dense regions, proliferative at this stage (Fig.  3E, F). This structure is a they appear to be some of those most proliferative struc- portion of the larval protonephridial system, an excretory tures at this stage. This pattern aligns with what has been system that uses a cilia-driven flow for ultrafiltration of observed in the ciliary bands of other Ambulacrarians, coelomic fluid from the protocoel [63, 64]. Finally, at this such as the bipinnaria larvae of Pisaster ochraceus and stage the last notable structure is the tripartite gut, com- Patiria miniata [58]. On the dorsal side of the larva in the posed of pharynx, stomach and intestine, which contin- most anterior regions, EdU cells were detected around ued to proliferate and grow (Fig. 3H, I). the apical organ (Fig.  2E, F), a prominent structure of Close to metamorphosis, the tornaria larva reach full the larval nervous system [59–62]. Other important pro- size (~ 3  mm) and form two additional coelom pairs, liferative structures of larvae include the digestive tract, the mesocoels and metacoels, and the precursors to the where microalgae that have been captured by the ciliary gill slits [65, 66] (Figs. 1D, 4), and we observed a nota- bands pass from the mouth into the pharynx, and finally ble shift in proliferative patterns from earlier develop- into the stomach, where they are digested (Fig. 2H, I). In mental stages. Proliferative cells were still distributed general, at this early larval stage most regions and tissues throughout the ventral ectoderm, both in the ciliary contain proliferative cells. bands, but now also more broadly in the squamous As the tornaria continues to grow and reaches the mid- epithelium between the ciliary bands (Fig.  4B). There dle of larval development (Figs.  1C, 3), defined by the were also a number of EdU cells distributed broadly differentiation of dorsal and ventral saddles as well as throughout the posterior ectoderm of the larva, which the emergence of the posterior telotroch, proliferation is a territory that will compact and elongate during continued throughout the ciliary bands. This was most metamorphosis (Fig.  4C). Across the dorsal surface apparent ventrally in the preoral and post-oral ciliary of the late larva, there were numerous proliferative bands (Fig.  3B). Proliferative cells were detected in the cells distributed throughout the epithelium (Fig.  4E). developing telotroch, the posterior locomotory ciliary There were EdU cells throughout the telotroch and band (Fig.  3C). The telotroch is one of the most distinc - on either side of the dorsal midline, where the dorsal tive structures of the hemichordate tornaria with long cord was beginning to form (Fig.  4F). Perhaps most compound cilia that beat to propel the larva through the interestingly, at this stage, the gut stopped proliferat- water [57]. On the dorsal surface, the protocoel pore was ing and EdU cells were detected within the forming Fig. 3 Cell proliferation throughout mid larval development of S. californicum. All: anterior up; blue = Hoechst, green = EdU; scale bar is 100um. Schematics of ventral (A), dorsal (D), and medial (E) views with key structures labeled. EdU staining with maximum intensity projection showing ventral (B), dorsal (E), and medial (H) sections. C–I insets. White arrowhead in (B and C) denote postoral ciliary band, white asterisk denotes telotroch. White arrowhead in (F) denotes protocoel pore Bump  et al. EvoDevo (2022) 13:13 Page 5 of 20 Fig. 4 Cell proliferation in late larval development of S. californicum. All: anterior up; blue = Hoechst, green = EdU; scale bar is 100 um. Schematics of ventral (A), dorsal (D), and medial (G) views with key structures labeled. EdU staining with maximum intensity projection ventral (B), dorsal (E), and medial (H) sections. B late larva ventral surface, C inset of (B), highlights ventral posterior epidermis and ciliary band E late larva dorsal surface, F inset of (E) highlights dorsal cord, marked by white asterisk, H late larva medial section, arrowheads highlight regions that will give rise to protocoel, mesocoel, and metacoel. I inset of lateral view of late larva medial section, arrowhead highlights gill bars. J protocoel, mesoderm that will form the proboscis, K mesocoel, mesoderm that will form the collar, L metacoel, mesoderm that will form the trunk. M, N distribution of anti-histone H3 (phospho S10) and EdU positive cells in ciliary bands, magenta = pHH3.3, grey = acetylated tubulin Bump et al. EvoDevo (2022) 13:13 Page 6 of 20 Proliferative patterns shift at metamorphosis adult structures (Fig.  4H). In particular, we observed The first morphological indication of the onset of meta - EdU cells in the anlage of the gill slits, which are a morphosis in S. californicum is the compaction and reor- prominent endomesoderm feature of the juvenile body ganization of the larval epidermis and an expansion of plan that are not yet functional in the late larva [67] all the coeloms, which results in a decrease of the blasto- (Fig.  4I). EdU cells were also enriched in the single coelar space (Fig. 1E, F) [35]. Early in metamorphosis, the anterior protocoel (Fig.  4J), and more posterior paired ectoderm of the primary ventral lobe and primary dorsal mesocoels (Fig.  4K) and metacoels (Fig.  4L), which lobe compact around the lateral food groove, as has been will later form the adult mesodermal derivatives of the observed in P. flava [66], and EdU cells were distributed proboscis, collar, and trunk, respectively. In line with throughout several regions of the ectoderm (Fig.  5B). previous morphological observations, in late larvae, EdU cells were detected in the postoral field and pri - structures of the juvenile body plan began to prolifer- mary dorsal saddle that give rise to both the proboscis ate to build the adult anatomical structures ahead of and around the thickening collar (Fig.  5B). At this stage, metamorphosis [35]. EdU cells were also found in the collar and posterodor- We also looked in more detail at the proliferative sally in the region of the developing dorsal cord (Fig. 5C). patterns in the ciliary band (Fig.  4M, N). To achieve On the ventral surface, EdU cells showed a similar dis- this, we coupled our EdU detection with immunofluo- tribution to the dorsal side with proliferative cells in the rescence staining of acetylated tubulin to visualize cilia preoral field, around the collar, in the anlage of the gill and phosphorylated serine 10 of histone H3 (pHH3.3), slits and in the epidermis, where the ventral cord eventu- which marks cells in G2/M phase. We found that pro- ally forms (Additional file 1: S1C). liferative cells display distinct spatial distribution with Metamorphosis then proceeded with the prospective a row of EdU cells at the base, then a row of differ- proboscis ectoderm continuing to thicken as the blasto- entiating phosophohistone h3.3 cells that are lateral coel was reduced, bringing it in contact with the expand- to the cilia (Fig.  4M, N). This regional localization of ing anterior coelom (Fig. 1G, H). The posterior ectoderm EdU cells in relationship to the differentiating phos- continued to expand as the forming trunk continued to ophohistone h3.3 cells suggested that there could be a elongate. At this stage, ectodermal proliferation contin- specific population of proliferative cells that give rise ued in the general epidermis of the proboscis but was to the ciliary bands. Fig. 5 Cell proliferation throughout the metamorphosis of S. californicum. All: anterior up; dorsal view; blue = Hoechst, green = Edu; scale bar is 100um. Schematics of ventral (A), dorsal (D), and medial (G) views with key structures labeled. EdU staining with maximum intensity projection: B Early in metamorphosis, white asterisk marks lateral food groove C Inset of (B), EdU positive cells are distributed around and in the dorsal cord. E Middle of metamorphosis, white arrowhead marks lateral food groove. F Inset of (E), EdU positive cells are found in the dorsal cord and mesocoel. H End of metamorphosis, asterisk marks dorsal cord. I Inset of (H), white arrowhead highlights EdU positive cells are distributed throughout the lateral grooves Bump  et al. EvoDevo (2022) 13:13 Page 7 of 20 absent from the remnants of the ciliary bands (Fig.  5E). As S. californicum transitioned from a distinct larva The epidermis of the proboscis transformed into a through metamorphosis and into a juvenile, proliferative columnar organization as the larva began to take on a cells shifted in their distribution, restricting to specific more vermiform shape. Other proliferative regions at this regions in the juvenile body. Overall, our data suggests stage included the developing gill slits, the metacoels, that the proliferation of the adult body plan begins at and the dorsal cord (Fig.  5F). At this stage, proliferation late larval stages prior to the start of the metamorphosis on the ventral surface occurred in the anterior ectoderm, itself. similar to the dorsal surface, absent from where the cili- ary bands had been (Additional file  1: S1D). EdU cells RNAseq after irradiation reveals the genetic signature were also detected around the collar and around the field, of proliferative cells in two distinct life history states where the ventral cord forms (Additional file 1: S1D). To further explore the molecular characteristics of pro- Finally, metamorphosis concluded as the blastocoelar liferative cells in S. californicum, we exploited the sen- space of the proboscis was eliminated bringing the meso- sitivity of proliferative cells to irradiation [69–73]. We derm and ectoderm in direct contact, the ectoderm of the hypothesized that the transcripts of irradiation-sensitive proboscis and collar transformed into a columnar epithe- genes would be restricted to our EdU , proliferative cell lium, and the posterior coeloms expanded and differenti - population. When we inspected the morphology of EdU ated as the trunk was elongating and narrowing (Fig. 1I). proliferative cells with fluorescent in  situ hybridization At this stage, we detected proliferative cells specifically in (FISH) to detect histone h2b messenger RNA, a known the proboscis, the collar, dorsal cord, and more broadly cell cycle gene, we found that EdU cells possess a nar- below the telotroch in the most posterior ectoderm and row rim of cytoplasm of h2b mRNA surrounding their mesoderm (Fig.  5H). In the proboscis ectoderm there nucleus and these cells often display a cytoplasmic pro- were EdU cells distributed throughout as well as a clear jection (Additional file  1: S1G, H). This morphology is enrichment of EdU cells in the lateral groove, the region reminiscent of the proliferative cells studied in other that had previously been the larval food groove (Fig.  5I). organisms, such as planarian neoblasts, which have been A lateral view of this stage at metamorphosis, highlighted characterized as rounded mesenchymal cells with a high cell proliferation in the gill slits and gut as well as the dor- nuclear-to-cytoplasmic ratio that often extend a cyto- sal and ventral midlines that give rise to the nerve cords plasmic projection [74, 75]. With these additional charac- (Additional file 1: S1E). terizations we next wanted to know if these proliferative In three main regions of the newly formed juvenile cells might share any core genetic signatures with prolif- (Fig.  1J), cell proliferation was detected in the probos- erative cells in other organisms. One hypothesis was that cis, collar, gill pores, gill bars, and trunk (Fig.  6B). In the S. californicum would have a stem-cell-like population anterior of the juvenile, EdU cells were localized to the that expresses many of the classic multipotency or ger- epidermis and line the lateral groove and anterior collar mline multipotency factors, such as piwi, vasa, and nanos (Fig.  6C). This region of the animal is highly innervated [71, 76]. [35]. Proliferative cells were also found in the dorsal gill To do this, we treated larvae and juveniles with irradia- pores which have perforated to allow water flow through tion. Three days after treatment, animals looked morpho - the gill slits and out the gill pores (Fig. 6D). Finally in the logically the same as controls, but EdU incorporation was posterior of the newly formed juvenile, proliferative cells eliminated in both larvae and juveniles (Fig.  7A–D). We were located along the dorsal cord of the trunk (Fig. 6E). extracted total RNA from this same stage of three days At this stage on the ventral surface, we detected EdU post-irradiation from 5 pooled individuals in three inde- cells in the proboscis, the gill slits, and in the ventral pendent biological replicates and made RNA sequenc- cord (Additional file  1: S1F). To see if these patterns of ing libraries (Nugen-Tecan Genomics). RNAseq analysis juvenile growth continued well after metamorphosis, we of irradiated versus non-irradiated identified 20 genes grew animals in sand for several weeks and repeated the in larvae and 123 genes in juveniles showing signifi - EdU labeling, clearing the tissue to make it possible to cant differential expression (log2 fold change ≥ −  2) and visualize the distribution of proliferation in larger, thicker p-adjusted value ≤ 10–6 juveniles (Fig.  7E), with 5 genes tissue. In continued juvenile growth (Fig.  6F) prolifera- that were downregulated at both stages. tive cells were found at the base of the collar coincident In the larval stage, twenty candidate genes were spe- with serotonergic neurons [35]. Interestingly, at this later cific, including fgfr-B (fibroblast growth factor receptor stage cell proliferation in the gill slits and dorsal cord was B) and a number of genes involved in cell division, such less pronounced, but a large number of EdU cells were as ince (inner centromere protein) [77] aspm-1 (abnor- found mid-intestinal in the trunk, potentially identifying mal spindle microtubule assembly) [78–80], and dlgp5 a new region of posterior growth [68]. (disks large-associated protein 5) [81, 82] (Additional Bump et al. EvoDevo (2022) 13:13 Page 8 of 20 Fig. 6 Cell proliferation in juveniles of S. californicum. All: anterior up; dorsal view; blue = Hoechst, green = Edu; scale bar is 100 um. Schematic of juvenile (A) with key structures labeled. EdU staining with maximum intensity projection: B End of metamorphosis. C Highlights regions of (B), EdU positive cells are distributed throughout the proboscis. D Highlights regions of (B), EdU positive cells are distributed throughout the gill bars. E Highlights regions of (B), EdU positive cells are distributed throughout the dorsal cord. F Continuing development of the juvenile body plan. Arrowheads mark the base of the collar and expanding trunk file  2: S2A). In juveniles, 123 genes showed significant immune response traf2 (TNF receptor-associated factor differential expression including a potential germline 2), tlr2 (Toll-like receptor 2), and tlr6 (Toll-like receptor marker spne-2 (spindle-E), genes involved in prolifera- 6) (Additional file 2: S2B). tion, such as anln (anilin), and genes related to a potential Bump  et al. EvoDevo (2022) 13:13 Page 9 of 20 Finally, five genes with differential expression were it may be a universal marker of proliferative cells in S. cal- shared between larva and juvenile: lbr-1 (Lamin B Recep- ifornicum. There are two types of chromatin attachment tor), nusap (Nucleolar And Spindle Associated Protein to lamina, one type is executed by the lamin B receptor 1), tenr-5 (Tenascin-R), tlr6-1 (Toll-like receptor 6), and in embryonic and non-differentiated cells, and the other unchar_4293 (an uncharacterized gene). nusap plays by specific lamin a/c binding proteins in differentiated a role in spindle microtubule organization and also has cells [91]. Previous work in ascidians and echinoderms been implicated in WNT signaling and metastasis [83], has identified lbr-1 orthologs and suggested that this and tenr-5 (Tenascin-R), which belongs to a group of gene may be unique to deuterostomes [92]. We exam- extracellular matrix proteins, tenascins, which are impor- ined the expression of lbr-1 in larvae and found it local- tant in vertebrates stem cell niches for tissue formation, ized in the ciliary bands (Fig. 7L, M), which we previously cell adhesion modulation, and the regulation of prolifera- demonstrated were regions of active cellular proliferation tion and differentiation [84]. (Fig.  3A). Similarly, at the juvenile stage we found lbr-1 Among the larval irradiation-sensitive transcripts, expression in a similar territory, where we had observed fgfr-B was most notable. FGF receptors in vertebrates the distribution of EdU cells (Figs. 5F, 6B, C), such as the are known to regulate cell proliferation, differentia - lateral grooves in the proboscis (Fig.  7N, O) and in the tion, and play a key role in pluripotent stem cells [85]. gill bars (Fig. 7P). Our findings suggest that expression of The two hemichordate FGF receptors Fgfr-A and Fgfr- lbr-1 might serve as a useful marker of labeling prolifera- B arose from a hemichordate-specific duplication [86] tive cells across both life history states. and in the direct developing hemichordate Saccoglossus Finally, the classic multipotency or germline multipo- kowalevskii, fgfr-B is expressed in the endomesoderm at tency factors, such as piwi, vasa, and nanos, [71, 76] did early gastrula stage and also in the ectoderm beginning not have significant differential expression (Additional at late gastrula into later stages [87]. In S. californicum, file  2: S2C, D). Instead, what we recovered were genes we examined the distribution of fgfr-B mRNA and found related more to specific proliferative populations (fgfr-B, expression throughout regions, where we also had pre- spne-2, and lbr-1) and thereby, revealed a possible hetero- viously observed EdU cells, particularly in the ciliary geneity among proliferative progenitor cells. bands (Fig. 7G, H). In the juvenile transcriptomes, the differential expres - Cell death remodels larval tissue at metamorphosis sion of spne-2 (spindle-E) was most notable. In Drosoph- After an investigation of cell proliferation throughout ila melanogaster spindle-E is involved in the generation the life cycle and metamorphosis of S. californicum, we of germ cell piwi-interacting-RNAs (piRNAs) and the next tested if patterns of proliferation were correlated DExD-box helicase domain of spindle-E is required with patterns of cell death. One larval structure that is for silencing of transposable elements in the germline lost or extensively remodeled at metamorphosis is the [88, 89]. In S. californicum, spindle-E was specifically circumoral ciliary band, also called the longitudinal cili- expressed in mesenchymal cells around the posterior of ary band, a larval specific feeding structure [57, 61] that the gills bars (Fig.  7I, K), which is consistent with Vasa is not retained in the juvenile. We investigated the distri- expression in P. flava [90]. Given that spindle-E was bution of cell death with TUNEL (terminal deoxynucle- expressed in a similar region and has been implicated in otidyl transferase dUTP nick end labeling), which detects germline regulation, we hypothesize that spindle-E could breaks in DNA as a proxy for cells undergoing apopto- potentially be a marker of proliferative germline cells in sis [93]. The TUNEL assay labels all free 3′-hydoxyl ter - hemichordates. mini meaning that TUNEL staining will detect apoptosis, The lamin B receptor gene, lbr-1, which plays an programmed cell death, but also necrosis [94, 95]. We important role in tethering chromatin, was differentially overcame previously limitations of TUNEL detection by expressed in the larval and juvenile stages. This suggests taking advantage of Click-iT technology, which utilizes a (See figure on next page.) Fig. 7 Genetic signature of irradiation sensitive EdU cells in both larvae and juveniles. All; blue = Hoechst, green = EdU; scale bar is 100um. A Control larva representing the normal EdU pattern at this stage, representative of 5/5 animals. B Experimental larva representing the EdU pattern at this stage after receiving 120 Gy of X-ray irradiation, representative of 5/5 animals. C Control juvenile representing the normal EdU pattern at this stage, representative of 4/4 animals. D Experimental juvenile representing the EdU pattern at this stage after receiving 200 Gy of X-ray irradiation, representative of 2/2 animals. E Volcano plot showing expression differences in control versus irradiated larva. n = 3 for each group. F Volcano plot showing expression differences in control versus irradiated juvenile. n = 3 for each group. G A larva with HCR probes for Fgfr-B. H Higher magnification of ciliary band with Fgfr-B transcripts. I A juvenile with HCR probes for Spindle-E. J, K Higher magnification of Spindle-E transcripts expressed between the ectoderm and the gills bars. L A larva with HCR probes for Lbr-1. M Inset of H with Lbr-1 transcripts distributed throughout the ciliary band. N A juvenile with HCR probes for Lbr-1. O Inset of I with Lbr-1 transcripts distributed throughout the lateral grooves. P Lbr-1 transcripts distributed in the gill bars Bump et al. EvoDevo (2022) 13:13 Page 10 of 20 Fig. 7 (See legend on previous page.) Bump  et al. EvoDevo (2022) 13:13 Page 11 of 20 modified dUTP with a small, bio-orthogonal alkyne moi - in and around the circumoral ciliary band (Fig.  8C). The ety (EdUTP) and a copper catalyzed covalent click reac- circumoral ciliary band was labelled with many T UNEL tion between that alkyne and a picolyl azide dye [96, 97]. cells, supporting the morphological observation that this Throughout larval development and in late larva we structure begins to break down at this stage. There were + + detected few TUNEL cells, suggesting very limited cell a small number of T UNEL at the anterior end of the death at larval stages (Additional file  3: S3A–C). How- protocoel (Additional file  3: S3D, E). T UNEL cells were ever, once metamorphosis began, indicated by the thick- also absent from the gut at this stage, which is consistent ening of the larval epithelium, there was a large increase with the morphological observation that the gut is main- + + in TUNEL cells (Fig.  8B). TUNEL cells were distrib- tained throughout the transition from larvae to adult uted broadly throughout the ectoderm, with most of [35]. To confirm adequate penetration of the TUNEL them on either side of the developing dorsal cord, and labeling into the deeper tissue layers, we performed a Fig. 8 Cell death throughout the metamorphosis of S. californicum. All: anterior up; blue = Hoechst, grey = TUNEL; scale bar is 100um. Schematics of early in metamorphosis (A), during metamorphosis (D), and end of metamorphosis (G) with key structures labeled. B Start of metamorphosis with an increase in TUNEL cells. C Highlights regions of (B), specifically around lateral grooves. E Middle of metamorphosis. F Highlights regions of E, specifically around the dorsal cord. H End of metamorphosis. I Highlights regions of (H), specifically around the dorsal chord and collar. J Overlap + + + + + + of serotonin cells and TUNEL cells. K Inset of (G), highlighting TUNEL and serotonin positive cells. L Overlap of Elav cells and TUNEL cells. M + + Inset of (L), highlighting TUNEL and Elav cells Bump et al. EvoDevo (2022) 13:13 Page 12 of 20 positive control by artificially nicking the ends of DNA we cannot rule out additional forms of tissue remodeling with DNAse-1 (Additional file 3: S3G, H). or histolysis, our findings suggest that cell death plays At the mid-metamorphosis stage, TUNEL cells were an important role in remodeling larval structures spe- distributed throughout the epidermis and continue to cifically in the anterior ciliary bands, which fused during label the disintegrating ciliary bands that fuse with each metamorphosis. other (Fig. 8E). At this stage we continued to detect very few TUNEL cells in the mesoderm and endoderm. The other region of the ectoderm, where the greatest num- Discussion ber of T UNEL cells were found is directly lateral to the While the development of an adult by transformation dorsal nerve cord (Fig.  8F), a region where we observed of a larva is very common in bilaterians, we understand many proliferative cells at the same stage. These gen - very little about the details of how this process occurs at a eral patterns that we see on the dorsal side are broadly cellular level, particularly given how the process of meta- similar to what was observed on the ventral surface morphosis differs across groups with different life history with TUNEL cells distributed in the ciliary bands and strategies [11, 100–102]. This study in the hemichordate broadly in the ectoderm but excluded from the ven- S. californicum focuses on characterizing the patterns of tral nerve cord (Additional file  3: S3F). The presence of cellular proliferation and cell death during the two differ - TUNEL cells in and around the ciliary bands was con- ent life history stages and during metamorphosis. Unlike sistent with a previous observation that the circumoral the model species D. melanogaster, where metamorpho- ciliary band degenerates and the serotonergic nervous sis results in a major histolysis of larval tissues and the system in this region undergoes extensive reorganization adult emerging from imaginal discs [103], morphological during metamorphosis, as neurite bundles that are pre- studies in S. californicum [35], and indirect developing sent in the ciliary grooves disappear as the ciliary bands hemichordates broadly [36], suggest that metamorpho- fuse [35, 98, 99]. sis occurs by remodeling of larval tissues and the trans- At the end of metamorphosis, there were fewer formation of larva into the adult. Our work illustrates TUNEL cells detected. In the anterior, there were very the similarities and differences in patterns of prolifera - few TUNEL cells in either the ectoderm or mesoderm tion and cell death across distinct life history states in S. (Fig.  8H). Those TUNEL cells that remain were scat- californicum revealing how and when these unique body tered throughout the epidermis in the proboscis, but no plans form. longer in the lateral grooves, which from our EdU study we have observed becoming proliferative at this stage (Fig.  5F). In the posterior, the remaining T UNEL cells The larval body plan shaped by proliferation were detected on either side of the dorsal cord, most For an organism with indirect development, rapid growth prominently in the posterior, where the larval epidermis of the larva is essential. Eggs are small, yet juvenile size at had compacted (Fig. 8I). metamorphosis is a good indicator of individual fitness, To understand the interaction of cell death with so larval growth before metamorphosis is critical [13, the nervous system, we examined serotonin localiza- 104]. We observed in early larval development (Fig.  1B) tion along with TUNEL early in metamorphosis and that the tornaria larva is formed primarily through cell found there is colocalization of s erotonergic cells with proliferation and limited amounts of cell death (Figs.  2, TUNEL cells (Fig.  8J, K). We also examined expression 3, Additional file  3: S3A–C). The patterns of proliferation of elv, a pan-neuronal marker, with TUNEL (Fig.  8L, M) we observed highlight regional differences of growth; + + and found several colocalized elv and T UNEL cells at EdU cells were distributed throughout the larval epi- the edge of the epidermis. Our findings suggest that por - dermis, the gut, and prominently in the ciliary bands tions of the larval nervous system undergo cell death at (Figs.  2A, 3A). At this stage in larval development, we metamorphosis and that the nervous system of the ante- observed little cell death with the use of the TUNEL assay rior ciliary bands may not be maintained in the juvenile (Additional file  3: S3A). There were some TUNEL cells body plan as previously proposed [66]. distributed throughout the larva, but it does not appear Overall, from our characterization of cell death, we that these cells were concentrated to any structure or tis- found that an increase in TUNEL cells correlated with sue. While cell death in bilaterian larval development has metamorphosis. We observed TUNEL cells broadly dis- not been surveyed broadly across taxa, cell proliferation tributed in the epidermis and that their restriction over in larval development has been assessed in a number of time, from anterior to posterior, correlated with the mor- marine larvae and the patterns we observe in S. californi- phological observation of an anterior to posterior tem- cum confirms and extends what has been found in other poral progression of ectodermal thickening [35]. While species [58]. Bump  et al. EvoDevo (2022) 13:13 Page 13 of 20 Origin of the adult body plan begins in the late larva specific structures were remodeled (Fig.  8B, C) and likely The morphological discontinuity between larvae and is important in shaping the morphogenesis of emerging adults has been a consistent source of great curios- adult structures, such as the dorsal cord of the forming ity for zoologists [30, 36, 105]. While metamorphosis adult nervous system (Fig.  8E, F, H, I). Clearly the onset is often thought of as the time when the adult animal of adult morphogenesis, and the initiation of overt meta- emerges from the vestiges of larval anlage, cell prolifera- morphosis, results in a major shift in the patterns of pro- tion may precede more overt morphological change at liferation and cell death. metamorphosis. In the late larva (Fig.  1D) of S. califor- Cell death within larval-specific structures has long nicum, specific regions of the developing adult body are been implicated in studies of metamorphosis, indeed one characterized by cell proliferation prior to the organism of the first recorded observation of apoptosis was in the undergoing the transition from planktonic to benthic metamorphosis of the toad, Alytes obstetricans, in which during metamorphosis (Fig.  4). At this stage, larvae are it was noted that cells of the notochord disappear and competent [9] to begin metamorphosis, but may remain are replaced by cells of the vertebrae [113]. Since then, as swimming tornaria for weeks or months. This is most there have been important findings about the role of cell obvious in the proliferation of the coeloms: the proto- death during anuran metamorphosis that have extended coel, mesocoel, and metacoel that give rise to the probos- the importance of timing in this process [114–116]. The cis mesoderm, collar mesoderm, and trunk mesoderm, mechanism of metamorphosis in insects such as butter- respectively (Fig. 4H–L). The formation of some of these flies and fruit flies have also provided important com - morphological landmarks prior to metamorphosis has parative perspectives into the role of programmed cell been described previously in S. californicum [35] and death as a key event in this process [117, 118]. Finally, in enteropneusts broadly [36, 65, 99, 105] but our study cell death is implicated in the metamorphosis of marine clarifies that these structures originate via broad prolif - invertebrates as they transition from planktonic larvae to eration. Our work describing the patterns of proliferation benthic juveniles [119], in particular in the remodeling illustrates the importance of proliferation in the origin of the larval nervous system in gastropods [120, 121]. In of these tissues prior to the start of metamorphosis and S. californicum we detected TUNEL cells at the start supports long-standing hypotheses that the initiation of of the morphological metamorphosis, most obviously adult structures before metamorphosis is essential in pre- around the anterior ciliary bands, which were involved in paring an organism for a major life history transition [2, larval feeding, and fuse during metamorphosis (Fig.  8B). 105, 106]. This is similar to what has been observed in sea urchins, In indirect developing species, questions of adult ori- where apoptotic cells were detected in the arms and cili- gins revolve around when and where. The timing of adult ary bands of competent larvae [122–124]. Similar to what formation in the sea urchin Strongylocentrotus purpura- has been observed in the enteropneust P. flava [66], in S. tus had been described as occurring through “set-aside californicum the serotonergic neurons associated with cells” in which the juvenile grows from a small rudiment ciliary bands are lost at metamorphosis and we observed within the larval body from a population of sequestered TUNEL and serotonin double-positive cells suggesting cells [17]. Instead of a cellular identify definition of “set- cell death is one way these systems may change at meta- aside cells”, we prefer a broader concept of heterochrony morphosis (Fig. 8J, K). [107, 108] or “deferred development”, which focuses on While cell death plays an important role in removing timing of specification and terminal differentiation of larval-specific structures at metamorphosis, it may also some cell populations relative to others [109]. The para - be involved in sculpting larval tissue during metamor- digm of heterochrony or “deferred development” frames phosis, reminiscent of digit development in vertebrates the adult development of S. californicum as more similar [125], and the formation of leg joints and head segments to the delayed life history shift of marine annelids than in D. melanogaster [126]. One defining feature of meta - that of sea urchins [110–112]. In S. californicum, we morphosis in S. californicum is an overall decrease in observe this deferral of the adult in the late larva, where size that occurs from anterior to posterior. Our finding proliferation of structures of the adult body plan occurs that cell death proceeds from the anterior to posterior prior to the start of metamorphosis (Fig. 4). in the dorsal epidermis (Fig. 8E, F) suggest this might be an important process in integrating and removing larval Metamorphosis integrates cell proliferation and cell death tissue, similar to the apoptosis observed in the mouse We found that cell death correlated with the onset of paw or fly larva [127]. However, a more complete pic - metamorphosis, and regionalized cell proliferation that ture of the type of cell death, apoptosis or necrosis, or began during late larval development continues into the programmed cell removal [128] would need to be tested adult. Cell death was detected in regions, where larval more rigorously with more sophisticated functional Bump et al. EvoDevo (2022) 13:13 Page 14 of 20 approaches. Overall, while we find that cell death occurs When we looked at the differential expression in irra - both in larval-specific structures but also broadly in lar - diated vs. non-irradiated larvae, we found a relatively val tissue during metamorphosis, there are also regions limited number of significantly differentially expressed with more limited cell death, such as the anterior ecto- genes, such as those involved in cell division (such as derm, collar ectoderm, and tripartite gut, leading to the ince, asmp-1, dlgp5) and most interestingly fgfr-B (fibro - possibility that not all larval cells die and may instead blast growth factor receptor B). A similar approach in the incorporated into the adult body plan. Our characteri- parasite Schistosoma mansoni found FGF receptors were zation of cell death in S. californicum supports classical downregulated in response to irradiation, and further morphological descriptions that enteropneusts do not showed that the inhibition of FGF signaling with RNAi have a “catastrophic metamorphosis” [129], and is now resulted in reduced EdU incorporation and down regu- confirmed at a cellular level. lation of cell-cycle-associated transcripts [138]. In juve- nile animals, while we did recover the expression of what may be a germline specific marker in spindle-E, there Proliferative patterns of growth differ in the adult body were also a number of transcripts from our irradiation plan, as do the markers of these proliferative populations experiment that suggest a potential immune response, Juvenile S. californicum (Fig. 1J) have distinct patterns of such as traf2, tlr2 and tlr6. We conclude that the differ - growth—proliferation continues to be enriched in struc- ential expression of these genes is likely related less to the tures that were not functionally part of the larva. The depletion of irradiation-sensitive transcripts and more dorsal and ventral cords, gill bars, proboscis ectoderm likely an immune reaction in response to the irradiation. and lateral groove are all clear examples of regional pro- While additional work will need to be done to deter- liferation of adult structures (Fig.  6B, Additional file  1: mine if markers, such as fgr-B and spindle-E are lineage S1F). We also looked later in juvenile development and and life history-specific proliferation markers, our results found that proliferation was most striking in the trunk certainly support the hypothesis that formation of larval region of the animal that continues to grow (Fig.  6F). and juvenile structures draws on distinct sets of prolif- This pattern of post-metamorphic growth is reminiscent erative populations. The strong focus on species with of the posterior axis elongation by an extended period direct development may miss some interesting regulatory of posterior growth described in the direct-developing features of distinct proliferative cell populations related hemichordate S. kowalevskii [68]. to the development of complex life cycles. For a fuller Given that patterns of cell proliferation differed understanding of developmental diversity and how it has between larvae and juveniles, we wanted to test whether shaped animal body plans, we need both a broader phylo- the genetic signature of proliferative cells was similar or genetic sampling but also greater representation of com- different between the life history states, and if there were plex developmental strategies. specific populations of pluripotent stem cells or broad populations of proliferative progenitors. In organisms Integration of larval and adult body plans such as colonial ascidians [130], acoels [131], flatworms Our study of the balance and timing between cell prolif- [132, 133], cnidarians [134, 135] and sponges [136], adult eration and cell death illustrates that adult morphological stem cells retain the potential to produce both the ger- elements proliferate prior to the start of metamorphosis, mline and several somatic cell types, and suggest that and that the onset of metamorphosis correlates with the there may be an ancestral animal stem cell [137]. We did onset of cell death. However, this study has also raised not recover a clear pluripotent stem cell population, in more questions, particularly the mysterious fate of most contrast to organisms with clear neoblast populations, larval cells that are seemingly maintained through the such as platyhelminthes and acoels [71]. Genes associ- threshold of metamorphosis. The most provocative and ated with multipotency or germline multipotency also exciting possibility is the potential of larval cells taking did not have significant differential expression (Addi - on new identities in the adult body plan. In sea urchins, tional file  4: S4C, S4D). Instead, we found differential which were the key example of catastrophic metamor- expression of genes, such as lbr-1 in both larvae and phosis with set-aside cells, the organization of the larval juveniles, pointing towards the importance of chromatin epithelium is preserved as regionalized apoptosis occurs state independent of the type of proliferative cell. It was in the larval arms that are resorbed [124]. Morphologi- previously suggested that the patterns of lbr and lamA/C cal studies of other echinoderms such as sea cucumbers expression in a number of different mammalian cell types and cidaroids suggest that much of their larval epider- may be indicative of peripheral heterochromatin teth- mis is maintained into the juvenile stage and is not lost ers regulating differentiation and perhaps this is a larger at metamorphosis [139, 140]. Even in examples, such as uniting trend across deuterostomes [91]. nemerteans, which are described as “maximally-indirect Bump  et al. EvoDevo (2022) 13:13 Page 15 of 20 developers’’ [141], we now know that the cells that cre- californicum, an important species for understanding this ate the imaginal discs also contribute to the larval body transformational process through the lens of cell, devel- [142]. Even in D. melanogaster with its specialized imagi- opmental, and evolutionary biology. nal disc cells, differentiated larval tracheal cells become proliferative and form the adult trachea and also adult- specific air sacs [143, 144]. In the tobacco hornworm, Methods Manduca sexta, differentiated cells of the larval legs Collecting, spawning, and larval rearing contribute to the adult legs [145]. Perhaps this linkage Adult Schizocardium californicum were collected in between larval and adult cells is quite common. While Morro Bay State Park, California, in a mudflat located at genetic tools would need to be developed to study this in 35°20′56.7″N 120°50′35.6″W with appropriate state per- detail, studies of transformational metamorphosis have mitting. Animals were spawned as described in [35] with the potential test whether transdifferentiation of cell individual females transferred in bowls of filtered seawa - types occurs as part of normal ontological development ter and placed in an illuminated incubator at 24–26  °C. in organisms with complex life histories. If this is indeed Once hatched, larvae were transferred to 1 gallon glass true, we are left with a tantalizing question, when many jars with continuous stirring and fed larvae with a 1:1 mix larval cells remain, how do they take on the appropriate of Dunaliella tertiolecta and Rhodomonas lens. Every two function in the adult? to four days, containers are washed, water was replaced Almost all our understanding of adult development with clean filtered seawater, and fresh algae was added. comes from direct developers, where the adult body plan To grow larger numbers of animals, some larvae were emerges from the embryo. Despite the prevalence and placed on a continuous flow-through system by being phylogenetic breadth of species that represent indirect transferred into diffusion tubes [146]. Once animals development, we understand very little about how adult began metamorphosis, they were transferred into glass development occurs by transformation. Clearly a greater bowls with terrarium sand. focus is needed on the range of development strategies that characterize metamorphosis in metazoans. Only through this broader sampling of life history strategies EdU labeling can we hope for a more comprehensive understanding Labeling and detection of proliferating cells were per- of the developmental mechanisms responsible for adult formed using the Click-it Plus EdU 488 Imaging Kit body plan formation. (Life Technologies), with the following modifications. Larva and juvenile worms were cultured in FSW supple- mented with 10 μM EdU diluted from a 10 mM stock in Conclusions DMSO. Unless otherwise noted, animals were pulsed for Our study describes cell proliferation and cell death 30 min with EdU then fixed with 3.7% paraformaldehyde through the development of the indirect developing S. in MOPS fix buffer (0.1  M MOPS, 0.5  M NaCl, 2  mM californicum. This species has distinct larval and adult EGTA, 1  mM MgCl2, 1× PBS) for 1  h at room temper- body plans and like other indirect-developing hemichor- ature (RT). For detection of EdU incorporation, labeled dates [31, 33, 98] a metamorphosis that is more transfor- embryos were transferred to a solution of PBS and the mational than catastrophic. Our data represent a cellular detection was performed following the manufacturer’s investigation into the common, yet understudied, bilate- protocol with an increased permeabilization time in 0.5% rian developmental strategy of formation of adult body Triton X-100 in PBS of 40 min and an increased detec- plan by transformation of a larval body plan. Despite tion time of 45 min. the prevalence of this life history strategy, we have lit- tle developmental insights into how this process occurs. Our study uncovered that the broad proliferation of adult TUNEL detection body plan components starts prior to any overt meta- Detection of apoptotic cells were performed using the morphosis. Although cell death was a prominent feature Click-iT Plus TUNEL Assay for in  situ apoptosis detec- of metamorphosis and adult body plan development, it tion with Alexa FluorTM dyes, with the following modi- is unlikely that the entire cell complement of the adult fications. Animals were fixed with the standard protocol can be explained by larval cell death and proliferation (3.7% paraformaldehyde in MOPS fix buffer), washed of a distinct adult stem cell population. Future studies twice in 1 × PBS and permeabilized with proteinase-K for will be needed to clarify the fate of larval cells through 15  min at room temperature. TdT reaction mixture was metamorphosis. Altogether, our study establishes a cellu- incubated for 60 min at 37 °C and Click-iT Plus reaction lar characterization of the formation of larval and adult was carried out for 30 min at 37 °C. body plans and transitions through metamorphosis in S. Bump et al. EvoDevo (2022) 13:13 Page 16 of 20 Antibody labeling described previously [52, 149]. RNA probes were diluted Fixation and antibody labeling was performed as to 0.1–1  ng/ml and hybridized overnight at 60  °C and described previously [35]. To visualize proliferative cells, visualized using an Anti-DIG AP antibody and TSA-Cy3. we used a rabbit polyclonal anti-histone H3 (phospho S10) (Abcam ab5176) diluted 1:200 in blocking solution. To visualize cilia, we used a mouse monoclonal anti-acet- In situ HCR version 3.0. ylated tubulin antibody (Sigma T7451) diluted 1:400 in Complementary DNA sequences specific to genes of blocking solution. To visualize the serotonergic nervous interest were submitted to the in  situ probe generator system, we used a rabbit anti-serotonin antibody (Sigma from the Ozpolat Lab [150]. Gene orthology was deter- S5545) diluted 1:300 in blocking solution. Secondary mined by collecting sequences of interest from related antibodies (ThermoFisher, Alexa Fluor) were added at species and then building gene trees. Sequences were 1:1000 dilution to the blocking solution. aligned with MUSCLE [151] and trees were calculated with Bayesian inference trees using MrBayes version 3.1.2 [152] in 1,000,000 generations with sampling of Imaging trees every 100 generations and a burn-in period of 25% Nuclei were stained with Hoechst 33,342 (1:1000) in PBS (Additional file  4: S4). The sequences generated by the and mounted in PBS using coverslips elevated with clay software were used to order DNA oligo pools (50  μmol feet. For juvenile worms, samples were transferred into DNA oPools Oligo Pool) from Integrated DNA Technol- 50% glycerol for 30–60 min, then 70% glycerol for imag- ogies, resuspended to 1 μmol/μl in 50 mM Tris buffer, pH ing. Samples were imaged on a Zeiss LSM 700 with 10X, 7.5. HCR amplifiers with fluorophores B1-Alexa Fluor- 20X and 40× objectives. For samples larger than the field 546, B2-Alexa Fluor-488, and B3-Alexa Fluor-647 were of view, maximal intensity projections from several stacks ordered from Molecular Instruments, Inc. The HCR was were stitched together (Fiji). performed based on Choi et  al., 2018 and the Hybridi- zation Chain Reaction (HCR) In  Situ Protocol from the Irradiation and transcriptional profiling Patel Lab [153, 154]. For experiments that involved HCR Larva and juvenile worms were exposed to 120 and and TUNEL labeling, HCR was conducted first, and then 200 Gy of X-ray irradiation on a CellRad Faxitron source. additional labeling was performed after. Animals were cultured in FSW after irradiation for 3 days and purified total RNA was prepared from pools of 5 ani - Supplementary Information mals using Qiagen RNeasy. Three independent biological The online version contains supplementary material available at https:// doi. replicates were performed for both control and irradiated org/ 10. 1186/ s13227- 022- 00198-1. experimental groups. Individually tagged libraries for RNA-seq were prepared (Nugen-Tecan Genomics Uni- Additional file 1: S1. Additional characterization of proliferative cells in S. californicum. A) Bar chart of number of Hoesch + in the ciliary bands versal mRNA-seq Kit), pooled in a single lane, and 75-bp vs. non ciliary bands, error bars are ± 1 SD (66% Confidence interval). B) pair-reads were generated using an Illumina HiSeq2000 Bar chart of EdU + cells in the ciliary bands vs. non ciliary bands, error at the Chan-Zuckerberg Biohub. The resulting reads were bars are ± 1 SD (66% Confidence interval). C–F) All: anterior up; scale bar is 100um. blue = Hoechst, green = EdU. C) Ventral view of EdU distribu- mapped to the annotated S. californicum genome (v2.0) tion early in metamorphosis. D) Ventral view of EdU distribution in the using CLC Genomics Workbench (CLC Bio) and differ - middle of metamorphosis. E) Lateral view of EdU distribution at the end ential expression was conducted with DeSeq2. apeglm of metamorphosis. F) Ventral view of EdU distribution in juveniles. G–H) Expression of h2b mRNA and EdU positive cells in the juvenile proboscis was used for log fold change shrinkage [147] and vst (var- blue = Hoechst, yellow = h2b mRNA, green = EdU. iance stabilizing transformation) was used for visualiza- Additional file 2: S2. Differential expression of larval and juvenile tion [148]. transcriptomes, irradiated versus non-irradiated. A) Differential expression of larval transcriptomes with baseMean, log2FoldChange, lfcSE, pvalue, padj. B) Differential expression of juvenile transcriptomes with baseMean, In‑situ hybridization log2FoldChange, lfcSE, pvalue, padj. C) Differential expression of piwi, Samples were relaxed using 3.5% MgCl2 prior to fixation vasa, and nanos in larvae with baseMean, log2FoldChange, lfcSE, pvalue, and fixed in 3.7% formaldehyde in MOPS fix buffer for padj. D) Differential expression of piwi, vasa, and nanos in juveniles with baseMean, log2FoldChange, lfcSE, pvalue, padj. 1 h at room temperature (RT), washed in fix buffer, dehy - Additional file 3: S3. Additional characterization of TUNEL during larval drated in 100% ethanol and stored at − 20 °C. Genes were development and metamorphosis. All: blue = Hoechst, grey = TUNEL, amplified from stage specific cDNA with random hexam - scale bar is 100um. A) lateral of view of mid tornaria body plan. B) Late ers and cloned into pGEM-T Easy (Promega). Digoxy- larva with very few TUNEL + cells. C, Highlights regions of B) a few TUNEL + cells. C). D) Early in metamorphosis from Fig. 7C with an increase genin labeled antisense probes were synthesized using in TUNEL + cells. E) TUNEL + cells found in the mesodermal protocoel. SP6 or T7 RNA polymerase (Promega). In  situ hybridi- F) Ventral view during the middle of metamorphosis. G) Positive control zation was performed a combination of what has been of TUNEL labeling by artificially nicking the ends of DNA with DNAse-1 Bump  et al. EvoDevo (2022) 13:13 Page 17 of 20 2. Jäegersten G. Evolution of the metazoan life cycle; a comprehensive metamorphosis H) Inset of positive control with TUNEL detected in theory. New York: Academic Press; 1972. deeper tissue layers 3. Nielsen C, Nørrevang A. The trochaea theory: an example of life cycle phylogeny. In: Conway Morris S, George JD, Gibson R, Platt HM, editors. Additional file 4: S4. Gene Trees of HCR candidate Genes. Gene trees for The origins and relationships of lower invertebrates, vol. The systemat- A) Lbr-1 B) Fgfr C) Spne-2 ics association special volume; 28. Oxford: Oxford University Press; 1985. p. 28–41. Acknowledgements 4. Raff RA. Origins of metazoan body plans: the larval revolution. Anim We would like to thank the staff of the Hopkins Marine Station and the staff Evol. 2009;43:1473–9. of Morro Bay State Park in particular Vince Cicero, John Sayers, and Katie 5. Hyman LH. The invertebrates, vol. 4. New York: McGraw-Hill; 1955. Drexhage for facilitating our collections. We would like to thank David Rank, 6. Nielsen C. Animal evolution: interrelationships of the living Phyla. 3rd Paul Peluso, and Greg Conception from Pacific Biosciences, Dan Rokhsar from ed. Oxford: Oxford University Press; 2012. UC Berkeley, and Norma Neff from Biohub for supporting the development 7. Gilbert L, Frieden E, editors. Metamorphosis, a problem in developmen- of genomic resources for Schizocardium. We thank members of the Lowe tal biology. 2nd ed. New York: Plenum Press; 1981. 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Comparisons of cell proliferation and cell death from tornaria larva to juvenile worm in the hemichordate Schizocardium californicum

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Copyright © The Author(s) 2022
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10.1186/s13227-022-00198-1
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Abstract

Background: There are a wide range of developmental strategies in animal phyla, but most insights into adult body plan formation come from direct-developing species. For indirect-developing species, there are distinct larval and adult body plans that are linked together by metamorphosis. Some outstanding questions in the development of indirect-developing organisms include the extent to which larval tissue undergoes cell death during the process of metamorphosis and when and where the tissue that will give rise to the adult originates. How do the processes of cell division and cell death redesign the body plans of indirect developers? In this study, we present patterns of cell proliferation and cell death during larval body plan development, metamorphosis, and adult body plan formation, in the hemichordate Schizocardium californium (Cameron and Perez in Zootaxa 3569:79–88, 2012) to answer these questions. Results: We identified distinct patterns of cell proliferation between larval and adult body plan formation of S. cali- fornicum. We found that some adult tissues proliferate during the late larval phase prior to the start of overt metamor- phosis. In addition, using an irradiation and transcriptomic approach, we describe a genetic signature of proliferative cells that is shared across the life history states, as well as markers that are unique to larval or juvenile states. Finally, we observed that cell death is minimal in larval stages but begins with the onset of metamorphosis. Conclusions: Cell proliferation during the development of S. californicum has distinct patterns in the formation of lar- val and adult body plans. However, cell death is very limited in larvae and begins during the onset of metamorphosis and into early juvenile development in specific domains. The populations of cells that proliferated and gave rise to the larvae and juveniles have a genetic signature that suggested a heterogeneous pool of proliferative progenitors, rather than a set-aside population of pluripotent cells. Taken together, we propose that the gradual morphological transfor- mation of S. californicum is mirrored at the cellular level and may be more representative of the development strate- gies that characterize metamorphosis in many metazoan animals. Keywords: Metamorphosis, Hemichordate, Tornaria, Cell proliferation, Cell death Background The development of animal body plans has largely been informed by research in a few key model species that pattern the adult body plan during embryogenesis, a *Correspondence: clowe@stanford.edu strategy termed direct development. However, this type Hopkins Marine Station, Department of Biology, Stanford University, Pacific of development is not representative of many animal Grove, CA, USA Full list of author information is available at the end of the article groups, where embryogenesis gives rise to a larva with a © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Bump et al. EvoDevo (2022) 13:13 Page 2 of 20 body plan distinct from that of the adult, a strategy called of tornaria larvae and their counterpart adult bodies can indirect development [2–4]. During direct development, be traced back to the late 1800s [29, 30] and more recent the adult is formed directly from the embryo, while in morphological descriptions of larval and adult body plans indirect development, embryonic processes give rise to a have been carried out in a range of enteropneust species; larval body plan that later transforms into the adult. This Ptychodera flava [31, 32], Balanoglossus misakiensis [33], transformation between larvae and adults is a develop- Balanoglossus simodensis [34] and S. californicum [35]. mental process known as metamorphosis, which is char- In these species, the tornaria larva is formed following acterized by the loss of larval-specific structures and the embryogenesis, while the benthic adult body plan forms emergence of adult structures [5–11]. The prevalence of by metamorphosis following an extended planktonic this developmental strategy across animal phyla clearly period [31, 33–36]. Studies of hemichordate complex life demonstrates that a better mechanistic understanding cycles have largely been based on morphological charac- of indirect development is critical for a more complete ters, with some descriptive patterning studies [37–53]. understanding of the developmental basis of body plan However, the cellular and developmental mechanisms evolution. through metamorphosis remain largely uncharacterized. Many marine organisms utilize indirect development, For example, we do not know whether the adult is formed developing first as larvae that feed and grow before by transformation of larval tissues via transdifferentation reaching metamorphosis [12, 13]. In species, such as or by proliferation of adult-specific cells following large- gastropods with veliger larvae, the morphological dif- scale larval cell death. ference between larval and adult body plans is not very To begin to address these questions, we characterized pronounced, because in these organisms, metamorpho- proliferation and cell death through the development sis represents a major shift in ecological niche but not a and metamorphosis of S. californicum. For the purpose large morphological change [14, 15]. At the other end of of our study, we define metamorphosis in S. californicum the spectrum, as is found in some echinoids, larval and as an overt morphological event. In a rapid 48-h period a adult body plans can be radically different in organiza - swimming planktonic larva transforms into a burrowing tion with a “catastrophic metamorphosis.” In this case, benthic adult as larval ectoderm compacts and condenses the adult develops as a rudiment within the larva and over the underlying mesoderm and endoderm[35]. We metamorphosis results in a complete reorganization of found distinct patterns of cell proliferation between lar- the body around new developmental axes in addition to val and adult body plans and that the start of a clear overt the loss of larval structures [5, 16, 17]. Similarly, in some metamorphosis corresponded with an increase in cell nemertean worms, with a pilidium larva, the adult devel- death. To then determine if there were distinct genetic ops from several rudiments, and metamorphosis culmi- markers of proliferative cells, and if those markers dif- nates with the juvenile consuming the larval tissues [17, fered between life history stages, we deployed an irra- 18]. However, metamorphosis in species with distinct diation strategy to deplete proliferative cells and found a larval and adult body plans does not always involve a number of differentially expressed transcripts. segregated rudiment or cataclysmic metamorphosis — instead larval tissue seems to be remodeled rapidly into Results the adult without obvious drastic histolysis of the larval Patterns of proliferation in larval and adult body plans. body plan [2, 15, 19]. In this type of metamorphosis, do We wanted to test whether patterns of cellular prolifera- adult structures originate from a small population of pro- tion involved in the development of the planktonic larva liferative cells? What is the fate of larval tissues? Indirect- were similar or different to those during the develop - developing hemichordates represent this particular type ment of the benthic juvenile. To describe the distribution of metamorphosis and provide an opportunity to explore of proliferative cells throughout the development of S. this type of developmental strategy. californicum, we assessed the incorporation of the thy- Hemichordates are composed of two classes, the soli- midine analog 5-ethynyl-29-deoxyuridine (EdU), which tary enteropneust worms and the largely colonial, tube- labels cells in S phase [54], during a range of developmen- dwelling pterobranchs [20–23]. While the position of tal stages: early larval development (Fig.  1B), mid larval hemichordates as sister to the echinoderms and closely development (Fig. 1C), late larval development (Fig. 1D), related to chordates has been well established [24–27], metamorphosis (Fig.  1E–I), and in juvenile development new studies have challenged this position [28]. Within (Fig. 1J). the enteropneusts, one family, the Harrimaniiidae, are The earliest larval developmental stage consists of a direct developers, while the families Spengelidae and tightly packed ciliary band that loops around the larva, Ptychoderidae, are indirect developers with a distinct lar- a thin wide squamous epithelium, an apical tuft, and val body plan called the tornaria. Morphological studies a tripartite gut (Figs.  1B, 2). On the ventral surface we Bump  et al. EvoDevo (2022) 13:13 Page 3 of 20 Fig. 1 Larval development and metamorphosis of S. californicum. A Schematic of the complex life cycle of the indirect developing S. californicum (modified from Ref. [35]. B–J Light microscopy of the complex life cycle of the indirect developing S. californicum (from [35]). B early tornaria larva, C mid tornaria larva, D late tornaria larva, E–I process of metamorphosis, J juvenile Fig. 2 Cell proliferation throughout early larval development of S. californicum. All: anterior up; blue = Hoechst, green = EdU; scale bar is 100um. Schematics of ventral (A), dorsal (D), and medial (E) views with key structures labeled. EdU staining with maximum intensity projections showing ventral (B), dorsal (E), and medial H sections. C–I insets. White arrowheads denote preoral ciliary band and postoral ciliary band detected EdU cells throughout the preoral and postoral had higher cell densities. The ciliary bands were densely loops of the circumoral ciliary band (Fig. 2B, C). The cili - packed with nuclei: there are ~ 59% greater number of ary band is used for both swimming and particle capture cells in the ciliary bands versus all other tissues (paired at this stage [55–57] and makes up a large percentage of t test, p = 0.016) and they were also more proliferative the ectoderm. We tested whether the ciliary bands were with ~ 22% more Edu cells than all other tissues (paired more proliferative than the general ectoderm, or simply t test, p = 0.008) (Additional file  1: S1A, B). This suggests Bump et al. EvoDevo (2022) 13:13 Page 4 of 20 that while the ciliary bands are nuclei-dense regions, proliferative at this stage (Fig.  3E, F). This structure is a they appear to be some of those most proliferative struc- portion of the larval protonephridial system, an excretory tures at this stage. This pattern aligns with what has been system that uses a cilia-driven flow for ultrafiltration of observed in the ciliary bands of other Ambulacrarians, coelomic fluid from the protocoel [63, 64]. Finally, at this such as the bipinnaria larvae of Pisaster ochraceus and stage the last notable structure is the tripartite gut, com- Patiria miniata [58]. On the dorsal side of the larva in the posed of pharynx, stomach and intestine, which contin- most anterior regions, EdU cells were detected around ued to proliferate and grow (Fig. 3H, I). the apical organ (Fig.  2E, F), a prominent structure of Close to metamorphosis, the tornaria larva reach full the larval nervous system [59–62]. Other important pro- size (~ 3  mm) and form two additional coelom pairs, liferative structures of larvae include the digestive tract, the mesocoels and metacoels, and the precursors to the where microalgae that have been captured by the ciliary gill slits [65, 66] (Figs. 1D, 4), and we observed a nota- bands pass from the mouth into the pharynx, and finally ble shift in proliferative patterns from earlier develop- into the stomach, where they are digested (Fig. 2H, I). In mental stages. Proliferative cells were still distributed general, at this early larval stage most regions and tissues throughout the ventral ectoderm, both in the ciliary contain proliferative cells. bands, but now also more broadly in the squamous As the tornaria continues to grow and reaches the mid- epithelium between the ciliary bands (Fig.  4B). There dle of larval development (Figs.  1C, 3), defined by the were also a number of EdU cells distributed broadly differentiation of dorsal and ventral saddles as well as throughout the posterior ectoderm of the larva, which the emergence of the posterior telotroch, proliferation is a territory that will compact and elongate during continued throughout the ciliary bands. This was most metamorphosis (Fig.  4C). Across the dorsal surface apparent ventrally in the preoral and post-oral ciliary of the late larva, there were numerous proliferative bands (Fig.  3B). Proliferative cells were detected in the cells distributed throughout the epithelium (Fig.  4E). developing telotroch, the posterior locomotory ciliary There were EdU cells throughout the telotroch and band (Fig.  3C). The telotroch is one of the most distinc - on either side of the dorsal midline, where the dorsal tive structures of the hemichordate tornaria with long cord was beginning to form (Fig.  4F). Perhaps most compound cilia that beat to propel the larva through the interestingly, at this stage, the gut stopped proliferat- water [57]. On the dorsal surface, the protocoel pore was ing and EdU cells were detected within the forming Fig. 3 Cell proliferation throughout mid larval development of S. californicum. All: anterior up; blue = Hoechst, green = EdU; scale bar is 100um. Schematics of ventral (A), dorsal (D), and medial (E) views with key structures labeled. EdU staining with maximum intensity projection showing ventral (B), dorsal (E), and medial (H) sections. C–I insets. White arrowhead in (B and C) denote postoral ciliary band, white asterisk denotes telotroch. White arrowhead in (F) denotes protocoel pore Bump  et al. EvoDevo (2022) 13:13 Page 5 of 20 Fig. 4 Cell proliferation in late larval development of S. californicum. All: anterior up; blue = Hoechst, green = EdU; scale bar is 100 um. Schematics of ventral (A), dorsal (D), and medial (G) views with key structures labeled. EdU staining with maximum intensity projection ventral (B), dorsal (E), and medial (H) sections. B late larva ventral surface, C inset of (B), highlights ventral posterior epidermis and ciliary band E late larva dorsal surface, F inset of (E) highlights dorsal cord, marked by white asterisk, H late larva medial section, arrowheads highlight regions that will give rise to protocoel, mesocoel, and metacoel. I inset of lateral view of late larva medial section, arrowhead highlights gill bars. J protocoel, mesoderm that will form the proboscis, K mesocoel, mesoderm that will form the collar, L metacoel, mesoderm that will form the trunk. M, N distribution of anti-histone H3 (phospho S10) and EdU positive cells in ciliary bands, magenta = pHH3.3, grey = acetylated tubulin Bump et al. EvoDevo (2022) 13:13 Page 6 of 20 Proliferative patterns shift at metamorphosis adult structures (Fig.  4H). In particular, we observed The first morphological indication of the onset of meta - EdU cells in the anlage of the gill slits, which are a morphosis in S. californicum is the compaction and reor- prominent endomesoderm feature of the juvenile body ganization of the larval epidermis and an expansion of plan that are not yet functional in the late larva [67] all the coeloms, which results in a decrease of the blasto- (Fig.  4I). EdU cells were also enriched in the single coelar space (Fig. 1E, F) [35]. Early in metamorphosis, the anterior protocoel (Fig.  4J), and more posterior paired ectoderm of the primary ventral lobe and primary dorsal mesocoels (Fig.  4K) and metacoels (Fig.  4L), which lobe compact around the lateral food groove, as has been will later form the adult mesodermal derivatives of the observed in P. flava [66], and EdU cells were distributed proboscis, collar, and trunk, respectively. In line with throughout several regions of the ectoderm (Fig.  5B). previous morphological observations, in late larvae, EdU cells were detected in the postoral field and pri - structures of the juvenile body plan began to prolifer- mary dorsal saddle that give rise to both the proboscis ate to build the adult anatomical structures ahead of and around the thickening collar (Fig.  5B). At this stage, metamorphosis [35]. EdU cells were also found in the collar and posterodor- We also looked in more detail at the proliferative sally in the region of the developing dorsal cord (Fig. 5C). patterns in the ciliary band (Fig.  4M, N). To achieve On the ventral surface, EdU cells showed a similar dis- this, we coupled our EdU detection with immunofluo- tribution to the dorsal side with proliferative cells in the rescence staining of acetylated tubulin to visualize cilia preoral field, around the collar, in the anlage of the gill and phosphorylated serine 10 of histone H3 (pHH3.3), slits and in the epidermis, where the ventral cord eventu- which marks cells in G2/M phase. We found that pro- ally forms (Additional file 1: S1C). liferative cells display distinct spatial distribution with Metamorphosis then proceeded with the prospective a row of EdU cells at the base, then a row of differ- proboscis ectoderm continuing to thicken as the blasto- entiating phosophohistone h3.3 cells that are lateral coel was reduced, bringing it in contact with the expand- to the cilia (Fig.  4M, N). This regional localization of ing anterior coelom (Fig. 1G, H). The posterior ectoderm EdU cells in relationship to the differentiating phos- continued to expand as the forming trunk continued to ophohistone h3.3 cells suggested that there could be a elongate. At this stage, ectodermal proliferation contin- specific population of proliferative cells that give rise ued in the general epidermis of the proboscis but was to the ciliary bands. Fig. 5 Cell proliferation throughout the metamorphosis of S. californicum. All: anterior up; dorsal view; blue = Hoechst, green = Edu; scale bar is 100um. Schematics of ventral (A), dorsal (D), and medial (G) views with key structures labeled. EdU staining with maximum intensity projection: B Early in metamorphosis, white asterisk marks lateral food groove C Inset of (B), EdU positive cells are distributed around and in the dorsal cord. E Middle of metamorphosis, white arrowhead marks lateral food groove. F Inset of (E), EdU positive cells are found in the dorsal cord and mesocoel. H End of metamorphosis, asterisk marks dorsal cord. I Inset of (H), white arrowhead highlights EdU positive cells are distributed throughout the lateral grooves Bump  et al. EvoDevo (2022) 13:13 Page 7 of 20 absent from the remnants of the ciliary bands (Fig.  5E). As S. californicum transitioned from a distinct larva The epidermis of the proboscis transformed into a through metamorphosis and into a juvenile, proliferative columnar organization as the larva began to take on a cells shifted in their distribution, restricting to specific more vermiform shape. Other proliferative regions at this regions in the juvenile body. Overall, our data suggests stage included the developing gill slits, the metacoels, that the proliferation of the adult body plan begins at and the dorsal cord (Fig.  5F). At this stage, proliferation late larval stages prior to the start of the metamorphosis on the ventral surface occurred in the anterior ectoderm, itself. similar to the dorsal surface, absent from where the cili- ary bands had been (Additional file  1: S1D). EdU cells RNAseq after irradiation reveals the genetic signature were also detected around the collar and around the field, of proliferative cells in two distinct life history states where the ventral cord forms (Additional file 1: S1D). To further explore the molecular characteristics of pro- Finally, metamorphosis concluded as the blastocoelar liferative cells in S. californicum, we exploited the sen- space of the proboscis was eliminated bringing the meso- sitivity of proliferative cells to irradiation [69–73]. We derm and ectoderm in direct contact, the ectoderm of the hypothesized that the transcripts of irradiation-sensitive proboscis and collar transformed into a columnar epithe- genes would be restricted to our EdU , proliferative cell lium, and the posterior coeloms expanded and differenti - population. When we inspected the morphology of EdU ated as the trunk was elongating and narrowing (Fig. 1I). proliferative cells with fluorescent in  situ hybridization At this stage, we detected proliferative cells specifically in (FISH) to detect histone h2b messenger RNA, a known the proboscis, the collar, dorsal cord, and more broadly cell cycle gene, we found that EdU cells possess a nar- below the telotroch in the most posterior ectoderm and row rim of cytoplasm of h2b mRNA surrounding their mesoderm (Fig.  5H). In the proboscis ectoderm there nucleus and these cells often display a cytoplasmic pro- were EdU cells distributed throughout as well as a clear jection (Additional file  1: S1G, H). This morphology is enrichment of EdU cells in the lateral groove, the region reminiscent of the proliferative cells studied in other that had previously been the larval food groove (Fig.  5I). organisms, such as planarian neoblasts, which have been A lateral view of this stage at metamorphosis, highlighted characterized as rounded mesenchymal cells with a high cell proliferation in the gill slits and gut as well as the dor- nuclear-to-cytoplasmic ratio that often extend a cyto- sal and ventral midlines that give rise to the nerve cords plasmic projection [74, 75]. With these additional charac- (Additional file 1: S1E). terizations we next wanted to know if these proliferative In three main regions of the newly formed juvenile cells might share any core genetic signatures with prolif- (Fig.  1J), cell proliferation was detected in the probos- erative cells in other organisms. One hypothesis was that cis, collar, gill pores, gill bars, and trunk (Fig.  6B). In the S. californicum would have a stem-cell-like population anterior of the juvenile, EdU cells were localized to the that expresses many of the classic multipotency or ger- epidermis and line the lateral groove and anterior collar mline multipotency factors, such as piwi, vasa, and nanos (Fig.  6C). This region of the animal is highly innervated [71, 76]. [35]. Proliferative cells were also found in the dorsal gill To do this, we treated larvae and juveniles with irradia- pores which have perforated to allow water flow through tion. Three days after treatment, animals looked morpho - the gill slits and out the gill pores (Fig. 6D). Finally in the logically the same as controls, but EdU incorporation was posterior of the newly formed juvenile, proliferative cells eliminated in both larvae and juveniles (Fig.  7A–D). We were located along the dorsal cord of the trunk (Fig. 6E). extracted total RNA from this same stage of three days At this stage on the ventral surface, we detected EdU post-irradiation from 5 pooled individuals in three inde- cells in the proboscis, the gill slits, and in the ventral pendent biological replicates and made RNA sequenc- cord (Additional file  1: S1F). To see if these patterns of ing libraries (Nugen-Tecan Genomics). RNAseq analysis juvenile growth continued well after metamorphosis, we of irradiated versus non-irradiated identified 20 genes grew animals in sand for several weeks and repeated the in larvae and 123 genes in juveniles showing signifi - EdU labeling, clearing the tissue to make it possible to cant differential expression (log2 fold change ≥ −  2) and visualize the distribution of proliferation in larger, thicker p-adjusted value ≤ 10–6 juveniles (Fig.  7E), with 5 genes tissue. In continued juvenile growth (Fig.  6F) prolifera- that were downregulated at both stages. tive cells were found at the base of the collar coincident In the larval stage, twenty candidate genes were spe- with serotonergic neurons [35]. Interestingly, at this later cific, including fgfr-B (fibroblast growth factor receptor stage cell proliferation in the gill slits and dorsal cord was B) and a number of genes involved in cell division, such less pronounced, but a large number of EdU cells were as ince (inner centromere protein) [77] aspm-1 (abnor- found mid-intestinal in the trunk, potentially identifying mal spindle microtubule assembly) [78–80], and dlgp5 a new region of posterior growth [68]. (disks large-associated protein 5) [81, 82] (Additional Bump et al. EvoDevo (2022) 13:13 Page 8 of 20 Fig. 6 Cell proliferation in juveniles of S. californicum. All: anterior up; dorsal view; blue = Hoechst, green = Edu; scale bar is 100 um. Schematic of juvenile (A) with key structures labeled. EdU staining with maximum intensity projection: B End of metamorphosis. C Highlights regions of (B), EdU positive cells are distributed throughout the proboscis. D Highlights regions of (B), EdU positive cells are distributed throughout the gill bars. E Highlights regions of (B), EdU positive cells are distributed throughout the dorsal cord. F Continuing development of the juvenile body plan. Arrowheads mark the base of the collar and expanding trunk file  2: S2A). In juveniles, 123 genes showed significant immune response traf2 (TNF receptor-associated factor differential expression including a potential germline 2), tlr2 (Toll-like receptor 2), and tlr6 (Toll-like receptor marker spne-2 (spindle-E), genes involved in prolifera- 6) (Additional file 2: S2B). tion, such as anln (anilin), and genes related to a potential Bump  et al. EvoDevo (2022) 13:13 Page 9 of 20 Finally, five genes with differential expression were it may be a universal marker of proliferative cells in S. cal- shared between larva and juvenile: lbr-1 (Lamin B Recep- ifornicum. There are two types of chromatin attachment tor), nusap (Nucleolar And Spindle Associated Protein to lamina, one type is executed by the lamin B receptor 1), tenr-5 (Tenascin-R), tlr6-1 (Toll-like receptor 6), and in embryonic and non-differentiated cells, and the other unchar_4293 (an uncharacterized gene). nusap plays by specific lamin a/c binding proteins in differentiated a role in spindle microtubule organization and also has cells [91]. Previous work in ascidians and echinoderms been implicated in WNT signaling and metastasis [83], has identified lbr-1 orthologs and suggested that this and tenr-5 (Tenascin-R), which belongs to a group of gene may be unique to deuterostomes [92]. We exam- extracellular matrix proteins, tenascins, which are impor- ined the expression of lbr-1 in larvae and found it local- tant in vertebrates stem cell niches for tissue formation, ized in the ciliary bands (Fig. 7L, M), which we previously cell adhesion modulation, and the regulation of prolifera- demonstrated were regions of active cellular proliferation tion and differentiation [84]. (Fig.  3A). Similarly, at the juvenile stage we found lbr-1 Among the larval irradiation-sensitive transcripts, expression in a similar territory, where we had observed fgfr-B was most notable. FGF receptors in vertebrates the distribution of EdU cells (Figs. 5F, 6B, C), such as the are known to regulate cell proliferation, differentia - lateral grooves in the proboscis (Fig.  7N, O) and in the tion, and play a key role in pluripotent stem cells [85]. gill bars (Fig. 7P). Our findings suggest that expression of The two hemichordate FGF receptors Fgfr-A and Fgfr- lbr-1 might serve as a useful marker of labeling prolifera- B arose from a hemichordate-specific duplication [86] tive cells across both life history states. and in the direct developing hemichordate Saccoglossus Finally, the classic multipotency or germline multipo- kowalevskii, fgfr-B is expressed in the endomesoderm at tency factors, such as piwi, vasa, and nanos, [71, 76] did early gastrula stage and also in the ectoderm beginning not have significant differential expression (Additional at late gastrula into later stages [87]. In S. californicum, file  2: S2C, D). Instead, what we recovered were genes we examined the distribution of fgfr-B mRNA and found related more to specific proliferative populations (fgfr-B, expression throughout regions, where we also had pre- spne-2, and lbr-1) and thereby, revealed a possible hetero- viously observed EdU cells, particularly in the ciliary geneity among proliferative progenitor cells. bands (Fig. 7G, H). In the juvenile transcriptomes, the differential expres - Cell death remodels larval tissue at metamorphosis sion of spne-2 (spindle-E) was most notable. In Drosoph- After an investigation of cell proliferation throughout ila melanogaster spindle-E is involved in the generation the life cycle and metamorphosis of S. californicum, we of germ cell piwi-interacting-RNAs (piRNAs) and the next tested if patterns of proliferation were correlated DExD-box helicase domain of spindle-E is required with patterns of cell death. One larval structure that is for silencing of transposable elements in the germline lost or extensively remodeled at metamorphosis is the [88, 89]. In S. californicum, spindle-E was specifically circumoral ciliary band, also called the longitudinal cili- expressed in mesenchymal cells around the posterior of ary band, a larval specific feeding structure [57, 61] that the gills bars (Fig.  7I, K), which is consistent with Vasa is not retained in the juvenile. We investigated the distri- expression in P. flava [90]. Given that spindle-E was bution of cell death with TUNEL (terminal deoxynucle- expressed in a similar region and has been implicated in otidyl transferase dUTP nick end labeling), which detects germline regulation, we hypothesize that spindle-E could breaks in DNA as a proxy for cells undergoing apopto- potentially be a marker of proliferative germline cells in sis [93]. The TUNEL assay labels all free 3′-hydoxyl ter - hemichordates. mini meaning that TUNEL staining will detect apoptosis, The lamin B receptor gene, lbr-1, which plays an programmed cell death, but also necrosis [94, 95]. We important role in tethering chromatin, was differentially overcame previously limitations of TUNEL detection by expressed in the larval and juvenile stages. This suggests taking advantage of Click-iT technology, which utilizes a (See figure on next page.) Fig. 7 Genetic signature of irradiation sensitive EdU cells in both larvae and juveniles. All; blue = Hoechst, green = EdU; scale bar is 100um. A Control larva representing the normal EdU pattern at this stage, representative of 5/5 animals. B Experimental larva representing the EdU pattern at this stage after receiving 120 Gy of X-ray irradiation, representative of 5/5 animals. C Control juvenile representing the normal EdU pattern at this stage, representative of 4/4 animals. D Experimental juvenile representing the EdU pattern at this stage after receiving 200 Gy of X-ray irradiation, representative of 2/2 animals. E Volcano plot showing expression differences in control versus irradiated larva. n = 3 for each group. F Volcano plot showing expression differences in control versus irradiated juvenile. n = 3 for each group. G A larva with HCR probes for Fgfr-B. H Higher magnification of ciliary band with Fgfr-B transcripts. I A juvenile with HCR probes for Spindle-E. J, K Higher magnification of Spindle-E transcripts expressed between the ectoderm and the gills bars. L A larva with HCR probes for Lbr-1. M Inset of H with Lbr-1 transcripts distributed throughout the ciliary band. N A juvenile with HCR probes for Lbr-1. O Inset of I with Lbr-1 transcripts distributed throughout the lateral grooves. P Lbr-1 transcripts distributed in the gill bars Bump et al. EvoDevo (2022) 13:13 Page 10 of 20 Fig. 7 (See legend on previous page.) Bump  et al. EvoDevo (2022) 13:13 Page 11 of 20 modified dUTP with a small, bio-orthogonal alkyne moi - in and around the circumoral ciliary band (Fig.  8C). The ety (EdUTP) and a copper catalyzed covalent click reac- circumoral ciliary band was labelled with many T UNEL tion between that alkyne and a picolyl azide dye [96, 97]. cells, supporting the morphological observation that this Throughout larval development and in late larva we structure begins to break down at this stage. There were + + detected few TUNEL cells, suggesting very limited cell a small number of T UNEL at the anterior end of the death at larval stages (Additional file  3: S3A–C). How- protocoel (Additional file  3: S3D, E). T UNEL cells were ever, once metamorphosis began, indicated by the thick- also absent from the gut at this stage, which is consistent ening of the larval epithelium, there was a large increase with the morphological observation that the gut is main- + + in TUNEL cells (Fig.  8B). TUNEL cells were distrib- tained throughout the transition from larvae to adult uted broadly throughout the ectoderm, with most of [35]. To confirm adequate penetration of the TUNEL them on either side of the developing dorsal cord, and labeling into the deeper tissue layers, we performed a Fig. 8 Cell death throughout the metamorphosis of S. californicum. All: anterior up; blue = Hoechst, grey = TUNEL; scale bar is 100um. Schematics of early in metamorphosis (A), during metamorphosis (D), and end of metamorphosis (G) with key structures labeled. B Start of metamorphosis with an increase in TUNEL cells. C Highlights regions of (B), specifically around lateral grooves. E Middle of metamorphosis. F Highlights regions of E, specifically around the dorsal cord. H End of metamorphosis. I Highlights regions of (H), specifically around the dorsal chord and collar. J Overlap + + + + + + of serotonin cells and TUNEL cells. K Inset of (G), highlighting TUNEL and serotonin positive cells. L Overlap of Elav cells and TUNEL cells. M + + Inset of (L), highlighting TUNEL and Elav cells Bump et al. EvoDevo (2022) 13:13 Page 12 of 20 positive control by artificially nicking the ends of DNA we cannot rule out additional forms of tissue remodeling with DNAse-1 (Additional file 3: S3G, H). or histolysis, our findings suggest that cell death plays At the mid-metamorphosis stage, TUNEL cells were an important role in remodeling larval structures spe- distributed throughout the epidermis and continue to cifically in the anterior ciliary bands, which fused during label the disintegrating ciliary bands that fuse with each metamorphosis. other (Fig. 8E). At this stage we continued to detect very few TUNEL cells in the mesoderm and endoderm. The other region of the ectoderm, where the greatest num- Discussion ber of T UNEL cells were found is directly lateral to the While the development of an adult by transformation dorsal nerve cord (Fig.  8F), a region where we observed of a larva is very common in bilaterians, we understand many proliferative cells at the same stage. These gen - very little about the details of how this process occurs at a eral patterns that we see on the dorsal side are broadly cellular level, particularly given how the process of meta- similar to what was observed on the ventral surface morphosis differs across groups with different life history with TUNEL cells distributed in the ciliary bands and strategies [11, 100–102]. This study in the hemichordate broadly in the ectoderm but excluded from the ven- S. californicum focuses on characterizing the patterns of tral nerve cord (Additional file  3: S3F). The presence of cellular proliferation and cell death during the two differ - TUNEL cells in and around the ciliary bands was con- ent life history stages and during metamorphosis. Unlike sistent with a previous observation that the circumoral the model species D. melanogaster, where metamorpho- ciliary band degenerates and the serotonergic nervous sis results in a major histolysis of larval tissues and the system in this region undergoes extensive reorganization adult emerging from imaginal discs [103], morphological during metamorphosis, as neurite bundles that are pre- studies in S. californicum [35], and indirect developing sent in the ciliary grooves disappear as the ciliary bands hemichordates broadly [36], suggest that metamorpho- fuse [35, 98, 99]. sis occurs by remodeling of larval tissues and the trans- At the end of metamorphosis, there were fewer formation of larva into the adult. Our work illustrates TUNEL cells detected. In the anterior, there were very the similarities and differences in patterns of prolifera - few TUNEL cells in either the ectoderm or mesoderm tion and cell death across distinct life history states in S. (Fig.  8H). Those TUNEL cells that remain were scat- californicum revealing how and when these unique body tered throughout the epidermis in the proboscis, but no plans form. longer in the lateral grooves, which from our EdU study we have observed becoming proliferative at this stage (Fig.  5F). In the posterior, the remaining T UNEL cells The larval body plan shaped by proliferation were detected on either side of the dorsal cord, most For an organism with indirect development, rapid growth prominently in the posterior, where the larval epidermis of the larva is essential. Eggs are small, yet juvenile size at had compacted (Fig. 8I). metamorphosis is a good indicator of individual fitness, To understand the interaction of cell death with so larval growth before metamorphosis is critical [13, the nervous system, we examined serotonin localiza- 104]. We observed in early larval development (Fig.  1B) tion along with TUNEL early in metamorphosis and that the tornaria larva is formed primarily through cell found there is colocalization of s erotonergic cells with proliferation and limited amounts of cell death (Figs.  2, TUNEL cells (Fig.  8J, K). We also examined expression 3, Additional file  3: S3A–C). The patterns of proliferation of elv, a pan-neuronal marker, with TUNEL (Fig.  8L, M) we observed highlight regional differences of growth; + + and found several colocalized elv and T UNEL cells at EdU cells were distributed throughout the larval epi- the edge of the epidermis. Our findings suggest that por - dermis, the gut, and prominently in the ciliary bands tions of the larval nervous system undergo cell death at (Figs.  2A, 3A). At this stage in larval development, we metamorphosis and that the nervous system of the ante- observed little cell death with the use of the TUNEL assay rior ciliary bands may not be maintained in the juvenile (Additional file  3: S3A). There were some TUNEL cells body plan as previously proposed [66]. distributed throughout the larva, but it does not appear Overall, from our characterization of cell death, we that these cells were concentrated to any structure or tis- found that an increase in TUNEL cells correlated with sue. While cell death in bilaterian larval development has metamorphosis. We observed TUNEL cells broadly dis- not been surveyed broadly across taxa, cell proliferation tributed in the epidermis and that their restriction over in larval development has been assessed in a number of time, from anterior to posterior, correlated with the mor- marine larvae and the patterns we observe in S. californi- phological observation of an anterior to posterior tem- cum confirms and extends what has been found in other poral progression of ectodermal thickening [35]. While species [58]. Bump  et al. EvoDevo (2022) 13:13 Page 13 of 20 Origin of the adult body plan begins in the late larva specific structures were remodeled (Fig.  8B, C) and likely The morphological discontinuity between larvae and is important in shaping the morphogenesis of emerging adults has been a consistent source of great curios- adult structures, such as the dorsal cord of the forming ity for zoologists [30, 36, 105]. While metamorphosis adult nervous system (Fig.  8E, F, H, I). Clearly the onset is often thought of as the time when the adult animal of adult morphogenesis, and the initiation of overt meta- emerges from the vestiges of larval anlage, cell prolifera- morphosis, results in a major shift in the patterns of pro- tion may precede more overt morphological change at liferation and cell death. metamorphosis. In the late larva (Fig.  1D) of S. califor- Cell death within larval-specific structures has long nicum, specific regions of the developing adult body are been implicated in studies of metamorphosis, indeed one characterized by cell proliferation prior to the organism of the first recorded observation of apoptosis was in the undergoing the transition from planktonic to benthic metamorphosis of the toad, Alytes obstetricans, in which during metamorphosis (Fig.  4). At this stage, larvae are it was noted that cells of the notochord disappear and competent [9] to begin metamorphosis, but may remain are replaced by cells of the vertebrae [113]. Since then, as swimming tornaria for weeks or months. This is most there have been important findings about the role of cell obvious in the proliferation of the coeloms: the proto- death during anuran metamorphosis that have extended coel, mesocoel, and metacoel that give rise to the probos- the importance of timing in this process [114–116]. The cis mesoderm, collar mesoderm, and trunk mesoderm, mechanism of metamorphosis in insects such as butter- respectively (Fig. 4H–L). The formation of some of these flies and fruit flies have also provided important com - morphological landmarks prior to metamorphosis has parative perspectives into the role of programmed cell been described previously in S. californicum [35] and death as a key event in this process [117, 118]. Finally, in enteropneusts broadly [36, 65, 99, 105] but our study cell death is implicated in the metamorphosis of marine clarifies that these structures originate via broad prolif - invertebrates as they transition from planktonic larvae to eration. Our work describing the patterns of proliferation benthic juveniles [119], in particular in the remodeling illustrates the importance of proliferation in the origin of the larval nervous system in gastropods [120, 121]. In of these tissues prior to the start of metamorphosis and S. californicum we detected TUNEL cells at the start supports long-standing hypotheses that the initiation of of the morphological metamorphosis, most obviously adult structures before metamorphosis is essential in pre- around the anterior ciliary bands, which were involved in paring an organism for a major life history transition [2, larval feeding, and fuse during metamorphosis (Fig.  8B). 105, 106]. This is similar to what has been observed in sea urchins, In indirect developing species, questions of adult ori- where apoptotic cells were detected in the arms and cili- gins revolve around when and where. The timing of adult ary bands of competent larvae [122–124]. Similar to what formation in the sea urchin Strongylocentrotus purpura- has been observed in the enteropneust P. flava [66], in S. tus had been described as occurring through “set-aside californicum the serotonergic neurons associated with cells” in which the juvenile grows from a small rudiment ciliary bands are lost at metamorphosis and we observed within the larval body from a population of sequestered TUNEL and serotonin double-positive cells suggesting cells [17]. Instead of a cellular identify definition of “set- cell death is one way these systems may change at meta- aside cells”, we prefer a broader concept of heterochrony morphosis (Fig. 8J, K). [107, 108] or “deferred development”, which focuses on While cell death plays an important role in removing timing of specification and terminal differentiation of larval-specific structures at metamorphosis, it may also some cell populations relative to others [109]. The para - be involved in sculpting larval tissue during metamor- digm of heterochrony or “deferred development” frames phosis, reminiscent of digit development in vertebrates the adult development of S. californicum as more similar [125], and the formation of leg joints and head segments to the delayed life history shift of marine annelids than in D. melanogaster [126]. One defining feature of meta - that of sea urchins [110–112]. In S. californicum, we morphosis in S. californicum is an overall decrease in observe this deferral of the adult in the late larva, where size that occurs from anterior to posterior. Our finding proliferation of structures of the adult body plan occurs that cell death proceeds from the anterior to posterior prior to the start of metamorphosis (Fig. 4). in the dorsal epidermis (Fig. 8E, F) suggest this might be an important process in integrating and removing larval Metamorphosis integrates cell proliferation and cell death tissue, similar to the apoptosis observed in the mouse We found that cell death correlated with the onset of paw or fly larva [127]. However, a more complete pic - metamorphosis, and regionalized cell proliferation that ture of the type of cell death, apoptosis or necrosis, or began during late larval development continues into the programmed cell removal [128] would need to be tested adult. Cell death was detected in regions, where larval more rigorously with more sophisticated functional Bump et al. EvoDevo (2022) 13:13 Page 14 of 20 approaches. Overall, while we find that cell death occurs When we looked at the differential expression in irra - both in larval-specific structures but also broadly in lar - diated vs. non-irradiated larvae, we found a relatively val tissue during metamorphosis, there are also regions limited number of significantly differentially expressed with more limited cell death, such as the anterior ecto- genes, such as those involved in cell division (such as derm, collar ectoderm, and tripartite gut, leading to the ince, asmp-1, dlgp5) and most interestingly fgfr-B (fibro - possibility that not all larval cells die and may instead blast growth factor receptor B). A similar approach in the incorporated into the adult body plan. Our characteri- parasite Schistosoma mansoni found FGF receptors were zation of cell death in S. californicum supports classical downregulated in response to irradiation, and further morphological descriptions that enteropneusts do not showed that the inhibition of FGF signaling with RNAi have a “catastrophic metamorphosis” [129], and is now resulted in reduced EdU incorporation and down regu- confirmed at a cellular level. lation of cell-cycle-associated transcripts [138]. In juve- nile animals, while we did recover the expression of what may be a germline specific marker in spindle-E, there Proliferative patterns of growth differ in the adult body were also a number of transcripts from our irradiation plan, as do the markers of these proliferative populations experiment that suggest a potential immune response, Juvenile S. californicum (Fig. 1J) have distinct patterns of such as traf2, tlr2 and tlr6. We conclude that the differ - growth—proliferation continues to be enriched in struc- ential expression of these genes is likely related less to the tures that were not functionally part of the larva. The depletion of irradiation-sensitive transcripts and more dorsal and ventral cords, gill bars, proboscis ectoderm likely an immune reaction in response to the irradiation. and lateral groove are all clear examples of regional pro- While additional work will need to be done to deter- liferation of adult structures (Fig.  6B, Additional file  1: mine if markers, such as fgr-B and spindle-E are lineage S1F). We also looked later in juvenile development and and life history-specific proliferation markers, our results found that proliferation was most striking in the trunk certainly support the hypothesis that formation of larval region of the animal that continues to grow (Fig.  6F). and juvenile structures draws on distinct sets of prolif- This pattern of post-metamorphic growth is reminiscent erative populations. The strong focus on species with of the posterior axis elongation by an extended period direct development may miss some interesting regulatory of posterior growth described in the direct-developing features of distinct proliferative cell populations related hemichordate S. kowalevskii [68]. to the development of complex life cycles. For a fuller Given that patterns of cell proliferation differed understanding of developmental diversity and how it has between larvae and juveniles, we wanted to test whether shaped animal body plans, we need both a broader phylo- the genetic signature of proliferative cells was similar or genetic sampling but also greater representation of com- different between the life history states, and if there were plex developmental strategies. specific populations of pluripotent stem cells or broad populations of proliferative progenitors. In organisms Integration of larval and adult body plans such as colonial ascidians [130], acoels [131], flatworms Our study of the balance and timing between cell prolif- [132, 133], cnidarians [134, 135] and sponges [136], adult eration and cell death illustrates that adult morphological stem cells retain the potential to produce both the ger- elements proliferate prior to the start of metamorphosis, mline and several somatic cell types, and suggest that and that the onset of metamorphosis correlates with the there may be an ancestral animal stem cell [137]. We did onset of cell death. However, this study has also raised not recover a clear pluripotent stem cell population, in more questions, particularly the mysterious fate of most contrast to organisms with clear neoblast populations, larval cells that are seemingly maintained through the such as platyhelminthes and acoels [71]. Genes associ- threshold of metamorphosis. The most provocative and ated with multipotency or germline multipotency also exciting possibility is the potential of larval cells taking did not have significant differential expression (Addi - on new identities in the adult body plan. In sea urchins, tional file  4: S4C, S4D). Instead, we found differential which were the key example of catastrophic metamor- expression of genes, such as lbr-1 in both larvae and phosis with set-aside cells, the organization of the larval juveniles, pointing towards the importance of chromatin epithelium is preserved as regionalized apoptosis occurs state independent of the type of proliferative cell. It was in the larval arms that are resorbed [124]. Morphologi- previously suggested that the patterns of lbr and lamA/C cal studies of other echinoderms such as sea cucumbers expression in a number of different mammalian cell types and cidaroids suggest that much of their larval epider- may be indicative of peripheral heterochromatin teth- mis is maintained into the juvenile stage and is not lost ers regulating differentiation and perhaps this is a larger at metamorphosis [139, 140]. Even in examples, such as uniting trend across deuterostomes [91]. nemerteans, which are described as “maximally-indirect Bump  et al. EvoDevo (2022) 13:13 Page 15 of 20 developers’’ [141], we now know that the cells that cre- californicum, an important species for understanding this ate the imaginal discs also contribute to the larval body transformational process through the lens of cell, devel- [142]. Even in D. melanogaster with its specialized imagi- opmental, and evolutionary biology. nal disc cells, differentiated larval tracheal cells become proliferative and form the adult trachea and also adult- specific air sacs [143, 144]. In the tobacco hornworm, Methods Manduca sexta, differentiated cells of the larval legs Collecting, spawning, and larval rearing contribute to the adult legs [145]. Perhaps this linkage Adult Schizocardium californicum were collected in between larval and adult cells is quite common. While Morro Bay State Park, California, in a mudflat located at genetic tools would need to be developed to study this in 35°20′56.7″N 120°50′35.6″W with appropriate state per- detail, studies of transformational metamorphosis have mitting. Animals were spawned as described in [35] with the potential test whether transdifferentiation of cell individual females transferred in bowls of filtered seawa - types occurs as part of normal ontological development ter and placed in an illuminated incubator at 24–26  °C. in organisms with complex life histories. If this is indeed Once hatched, larvae were transferred to 1 gallon glass true, we are left with a tantalizing question, when many jars with continuous stirring and fed larvae with a 1:1 mix larval cells remain, how do they take on the appropriate of Dunaliella tertiolecta and Rhodomonas lens. Every two function in the adult? to four days, containers are washed, water was replaced Almost all our understanding of adult development with clean filtered seawater, and fresh algae was added. comes from direct developers, where the adult body plan To grow larger numbers of animals, some larvae were emerges from the embryo. Despite the prevalence and placed on a continuous flow-through system by being phylogenetic breadth of species that represent indirect transferred into diffusion tubes [146]. Once animals development, we understand very little about how adult began metamorphosis, they were transferred into glass development occurs by transformation. Clearly a greater bowls with terrarium sand. focus is needed on the range of development strategies that characterize metamorphosis in metazoans. Only through this broader sampling of life history strategies EdU labeling can we hope for a more comprehensive understanding Labeling and detection of proliferating cells were per- of the developmental mechanisms responsible for adult formed using the Click-it Plus EdU 488 Imaging Kit body plan formation. (Life Technologies), with the following modifications. Larva and juvenile worms were cultured in FSW supple- mented with 10 μM EdU diluted from a 10 mM stock in Conclusions DMSO. Unless otherwise noted, animals were pulsed for Our study describes cell proliferation and cell death 30 min with EdU then fixed with 3.7% paraformaldehyde through the development of the indirect developing S. in MOPS fix buffer (0.1  M MOPS, 0.5  M NaCl, 2  mM californicum. This species has distinct larval and adult EGTA, 1  mM MgCl2, 1× PBS) for 1  h at room temper- body plans and like other indirect-developing hemichor- ature (RT). For detection of EdU incorporation, labeled dates [31, 33, 98] a metamorphosis that is more transfor- embryos were transferred to a solution of PBS and the mational than catastrophic. Our data represent a cellular detection was performed following the manufacturer’s investigation into the common, yet understudied, bilate- protocol with an increased permeabilization time in 0.5% rian developmental strategy of formation of adult body Triton X-100 in PBS of 40 min and an increased detec- plan by transformation of a larval body plan. Despite tion time of 45 min. the prevalence of this life history strategy, we have lit- tle developmental insights into how this process occurs. Our study uncovered that the broad proliferation of adult TUNEL detection body plan components starts prior to any overt meta- Detection of apoptotic cells were performed using the morphosis. Although cell death was a prominent feature Click-iT Plus TUNEL Assay for in  situ apoptosis detec- of metamorphosis and adult body plan development, it tion with Alexa FluorTM dyes, with the following modi- is unlikely that the entire cell complement of the adult fications. Animals were fixed with the standard protocol can be explained by larval cell death and proliferation (3.7% paraformaldehyde in MOPS fix buffer), washed of a distinct adult stem cell population. Future studies twice in 1 × PBS and permeabilized with proteinase-K for will be needed to clarify the fate of larval cells through 15  min at room temperature. TdT reaction mixture was metamorphosis. Altogether, our study establishes a cellu- incubated for 60 min at 37 °C and Click-iT Plus reaction lar characterization of the formation of larval and adult was carried out for 30 min at 37 °C. body plans and transitions through metamorphosis in S. Bump et al. EvoDevo (2022) 13:13 Page 16 of 20 Antibody labeling described previously [52, 149]. RNA probes were diluted Fixation and antibody labeling was performed as to 0.1–1  ng/ml and hybridized overnight at 60  °C and described previously [35]. To visualize proliferative cells, visualized using an Anti-DIG AP antibody and TSA-Cy3. we used a rabbit polyclonal anti-histone H3 (phospho S10) (Abcam ab5176) diluted 1:200 in blocking solution. To visualize cilia, we used a mouse monoclonal anti-acet- In situ HCR version 3.0. ylated tubulin antibody (Sigma T7451) diluted 1:400 in Complementary DNA sequences specific to genes of blocking solution. To visualize the serotonergic nervous interest were submitted to the in  situ probe generator system, we used a rabbit anti-serotonin antibody (Sigma from the Ozpolat Lab [150]. Gene orthology was deter- S5545) diluted 1:300 in blocking solution. Secondary mined by collecting sequences of interest from related antibodies (ThermoFisher, Alexa Fluor) were added at species and then building gene trees. Sequences were 1:1000 dilution to the blocking solution. aligned with MUSCLE [151] and trees were calculated with Bayesian inference trees using MrBayes version 3.1.2 [152] in 1,000,000 generations with sampling of Imaging trees every 100 generations and a burn-in period of 25% Nuclei were stained with Hoechst 33,342 (1:1000) in PBS (Additional file  4: S4). The sequences generated by the and mounted in PBS using coverslips elevated with clay software were used to order DNA oligo pools (50  μmol feet. For juvenile worms, samples were transferred into DNA oPools Oligo Pool) from Integrated DNA Technol- 50% glycerol for 30–60 min, then 70% glycerol for imag- ogies, resuspended to 1 μmol/μl in 50 mM Tris buffer, pH ing. Samples were imaged on a Zeiss LSM 700 with 10X, 7.5. HCR amplifiers with fluorophores B1-Alexa Fluor- 20X and 40× objectives. For samples larger than the field 546, B2-Alexa Fluor-488, and B3-Alexa Fluor-647 were of view, maximal intensity projections from several stacks ordered from Molecular Instruments, Inc. The HCR was were stitched together (Fiji). performed based on Choi et  al., 2018 and the Hybridi- zation Chain Reaction (HCR) In  Situ Protocol from the Irradiation and transcriptional profiling Patel Lab [153, 154]. For experiments that involved HCR Larva and juvenile worms were exposed to 120 and and TUNEL labeling, HCR was conducted first, and then 200 Gy of X-ray irradiation on a CellRad Faxitron source. additional labeling was performed after. Animals were cultured in FSW after irradiation for 3 days and purified total RNA was prepared from pools of 5 ani - Supplementary Information mals using Qiagen RNeasy. Three independent biological The online version contains supplementary material available at https:// doi. replicates were performed for both control and irradiated org/ 10. 1186/ s13227- 022- 00198-1. experimental groups. Individually tagged libraries for RNA-seq were prepared (Nugen-Tecan Genomics Uni- Additional file 1: S1. Additional characterization of proliferative cells in S. californicum. A) Bar chart of number of Hoesch + in the ciliary bands versal mRNA-seq Kit), pooled in a single lane, and 75-bp vs. non ciliary bands, error bars are ± 1 SD (66% Confidence interval). B) pair-reads were generated using an Illumina HiSeq2000 Bar chart of EdU + cells in the ciliary bands vs. non ciliary bands, error at the Chan-Zuckerberg Biohub. The resulting reads were bars are ± 1 SD (66% Confidence interval). C–F) All: anterior up; scale bar is 100um. blue = Hoechst, green = EdU. C) Ventral view of EdU distribu- mapped to the annotated S. californicum genome (v2.0) tion early in metamorphosis. D) Ventral view of EdU distribution in the using CLC Genomics Workbench (CLC Bio) and differ - middle of metamorphosis. E) Lateral view of EdU distribution at the end ential expression was conducted with DeSeq2. apeglm of metamorphosis. F) Ventral view of EdU distribution in juveniles. G–H) Expression of h2b mRNA and EdU positive cells in the juvenile proboscis was used for log fold change shrinkage [147] and vst (var- blue = Hoechst, yellow = h2b mRNA, green = EdU. iance stabilizing transformation) was used for visualiza- Additional file 2: S2. Differential expression of larval and juvenile tion [148]. transcriptomes, irradiated versus non-irradiated. A) Differential expression of larval transcriptomes with baseMean, log2FoldChange, lfcSE, pvalue, padj. B) Differential expression of juvenile transcriptomes with baseMean, In‑situ hybridization log2FoldChange, lfcSE, pvalue, padj. C) Differential expression of piwi, Samples were relaxed using 3.5% MgCl2 prior to fixation vasa, and nanos in larvae with baseMean, log2FoldChange, lfcSE, pvalue, and fixed in 3.7% formaldehyde in MOPS fix buffer for padj. D) Differential expression of piwi, vasa, and nanos in juveniles with baseMean, log2FoldChange, lfcSE, pvalue, padj. 1 h at room temperature (RT), washed in fix buffer, dehy - Additional file 3: S3. Additional characterization of TUNEL during larval drated in 100% ethanol and stored at − 20 °C. Genes were development and metamorphosis. All: blue = Hoechst, grey = TUNEL, amplified from stage specific cDNA with random hexam - scale bar is 100um. A) lateral of view of mid tornaria body plan. B) Late ers and cloned into pGEM-T Easy (Promega). Digoxy- larva with very few TUNEL + cells. C, Highlights regions of B) a few TUNEL + cells. C). D) Early in metamorphosis from Fig. 7C with an increase genin labeled antisense probes were synthesized using in TUNEL + cells. E) TUNEL + cells found in the mesodermal protocoel. SP6 or T7 RNA polymerase (Promega). In  situ hybridi- F) Ventral view during the middle of metamorphosis. G) Positive control zation was performed a combination of what has been of TUNEL labeling by artificially nicking the ends of DNA with DNAse-1 Bump  et al. EvoDevo (2022) 13:13 Page 17 of 20 2. Jäegersten G. Evolution of the metazoan life cycle; a comprehensive metamorphosis H) Inset of positive control with TUNEL detected in theory. New York: Academic Press; 1972. deeper tissue layers 3. Nielsen C, Nørrevang A. The trochaea theory: an example of life cycle phylogeny. In: Conway Morris S, George JD, Gibson R, Platt HM, editors. Additional file 4: S4. Gene Trees of HCR candidate Genes. Gene trees for The origins and relationships of lower invertebrates, vol. The systemat- A) Lbr-1 B) Fgfr C) Spne-2 ics association special volume; 28. Oxford: Oxford University Press; 1985. p. 28–41. Acknowledgements 4. Raff RA. Origins of metazoan body plans: the larval revolution. Anim We would like to thank the staff of the Hopkins Marine Station and the staff Evol. 2009;43:1473–9. of Morro Bay State Park in particular Vince Cicero, John Sayers, and Katie 5. Hyman LH. The invertebrates, vol. 4. New York: McGraw-Hill; 1955. Drexhage for facilitating our collections. We would like to thank David Rank, 6. Nielsen C. Animal evolution: interrelationships of the living Phyla. 3rd Paul Peluso, and Greg Conception from Pacific Biosciences, Dan Rokhsar from ed. Oxford: Oxford University Press; 2012. UC Berkeley, and Norma Neff from Biohub for supporting the development 7. Gilbert L, Frieden E, editors. Metamorphosis, a problem in developmen- of genomic resources for Schizocardium. We thank members of the Lowe tal biology. 2nd ed. New York: Plenum Press; 1981. 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Journal

EvoDevoSpringer Journals

Published: Jun 6, 2022

Keywords: Metamorphosis; Hemichordate; Tornaria; Cell proliferation; Cell death

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