Developmental Biology

In developing chordate embryos, the retinoic acid (RA) pathway is involved in many key developmental patterning systems. Recently, it has become clear that at least some components of the RA pathway are more ancient than chordates. However, the participation of these components in an RA pathway, and the role of such a pathway in developing non-chordate embryos remain unclear. Here, we demonstrate the presence of an extensive set of RA pathway components in the genome of the mollusc Tritia obsoleta, and examine their expression using in situ hybridization. We then examined the function of multiple RA pathway genes using RA treatments, drug treatments and morpholino (MO) knockdowns. These manipulations impacted a similar set of structures in development, especially the shell and the digestive tract, indicating that the retinoic acid pathway is functional in Tritia. Together, this is the most comprehensive evidence yet for an RA signaling pathway functioning in embryogenesis of a non-chordate.

Integration of positional information across body axes is likely critical for whole-body regeneration to define the territories of missing tissue in three dimensions with fidelity. The body-wall musculature in planarians expresses patterning factors regulating the anteroposterior, dorsoventral, and mediolateral axes, but how this information coordinates is not fully understood. We identify a previously described factor specifically expressed in dorsal midline muscle as a BMP/Activin decoy receptor bambi-2. Analysis of scRNAseq indicates bambi-2+ cells coexpress midline-specifying transcription factor pitx and longitudinal muscle-specifying factor myoD, and production of bambi-2+ cells requires these factors. In laterally amputated animals regenerating an entirely new midline, bambi-2+ cells are initially formed at the wound site, then dynamically spread, and ultimately reset to restore bilateral symmetry. We further identify a system of dorsoventral Netrin and Netrin receptor signals expressed from body-wall muscle that control midline identity and blastema morphology. Ventral and laterally expressed netrins -1, -4, and -5 signal via dorsally-enriched netrin repulsion receptors unc5-C, unc5-E, and dcc-2, which together limit mediolateral spread of bambi-2+ dorsal midline muscle and influence the architecture of the muscle system. Our results suggest a model in which ventral determinants dictate mediolateral information important for blastema morphology.

Understanding how cnidarians pattern their nervous systems can provide insight into the ancestral mechanisms of neurogenesis that are shared with bilaterians, shedding light on the evolution of nervous systems. While previous studies have revealed deeply conserved mechanisms for neural induction and progenitor selection between cnidarians and bilaterians, less is known about how distinct neuronal subtypes are specified over time in cnidarians. We utilized single-cell mRNA sequencing to profile NvashA-expressing cells across embryonic and planula-larva stages of Nematostella neurogenesis, and functional experiments identified a dynamic role for NvashA over time. Our analysis revealed that unique neuronal subtypes emerge at different developmental stages, providing evidence for temporal patterning in developing cnidarian nerve nets. This can provide a foundation to better our understanding of neurogenic gene regulatory networks, and to compare neurogenesis across cnidarians, and with bilaterians, to improve our knowledge of nervous system evolution.

A highly-conserved extracellular matrix protein, Tinagl1, modulates Wnt, integrin, TGF-{beta}, and EGF-R signaling in vitro, but its significance in vivo has remained in doubt. To bypass possible genetic compensation by an ortholog encoded exclusively in mammalian genomes, we examine Tinagl1 function in zebrafish embryos. In this model, tinagl1 mRNA is detected in the developing spinal cord and pronephros. Acute knockdown using either CRISPR/Cas9 somatic mutagenesis or splice-blocking morpholinos reveals left-right (LR) heart looping defects, pronephros dilatations, and ventral body curvature. This constellation of defects characteristically results from the loss of motile cilia function, and we confirm the presence of shortened and fewer cilia in the pronephric duct and in the Kupffers vesicle where LR asymmetry is established. A link to known Wnt3a/{beta}-catenin signaling that activates the motile cilia transcriptional program is supported by manipulation of Wnt3a and {beta}-catenin levels in tinagl1 knockdown embryos. In addition to ciliopathy-like defects, the tinagl1 knockdown shows disorganization of longitudinal axon tracts in the spinal cord and defects in motor neuron outgrowth. Together, these results provide evidence that Tinagl1 is important in development, and that zebrafish is an ideal model in which to explore its relationships to cilia and secreted signaling molecules.

The morphogenesis of complex vertebrate appendages requires precise regulation of growth, governed by distinct positional identities. The zebrafish caudal fin achieves a symmetrical, forked morphology through the regional specialization of the bony rays: peripheral rays are composed of relatively long, thick segments; while the central rays are made up of shorter, thinner segments, and their overall length is restricted. This length differential establishes the definitive forked shape of the organ. We asked whether these regional morphological differences reflect distinct underlying positional identities. Transcriptomic profiling of intact tissues from adult wild-type zebrafish suggested that central rays possess unique expression profiles, distinct from those of peripheral rays. We previously identified a treatment during embryogenesis that allows excess growth in the central rays, creating a truncate fin shape in adults-we asked whether this novel fin shape was caused by a peripheralization of the central rays. Indeed, the central rays of truncate fins were not only longer, but were composed of longer and thicker individual segments, reminiscent of peripheral rays. Further, gene expression in the central regions of truncate backgrounds showed signatures of peripheral identity. During development of the truncate phenotype, peripheral markers became expressed in more central domains of the growing truncate caudal fin, and in the supportive endoskeleton, the central hypural diastema was lost from the earliest stages. Ultimately, our results demonstrate how adult morphologies may be altered by shifts in positional identities. These findings clarify the anatomical patterning and molecular profiles that underlie regional specialization during caudal fin development.

Oriented cell division is essential for establishing the anterior-posterior body (A-P) axis in diverse species. However, in Xenopus laevis, blocking cell division during gastrulation does not impair the development of the neural tube and body elongation. Here, we demonstrate that neither neurulation nor dorsal mesoderm formation is dependent on cell division. On the other hand, neural plate elongation and A-P patterning are impacted in the absence of cell division, resulting in trunk defects and anterior defects that appear during tailbud stages. We also show that cell division is abundant around the ectoderm of the gastrulating embryo. Still, it is more intense in the dorsal ectoderm (presumptive neural plate), where there is a clear preference for A-P oriented cell divisions. Taken together, our results highlight a conserved mechanism of A-P oriented cell division that is present during neural plate elongation and patterning.

An animals ability to regrow lost tissues or structures can vary greatly during its life cycle. The annelid Capitella teleta exhibits posterior, but not anterior, regeneration as juveniles and adults. In contrast, embryos display only limited replacement of specific tissues. To investigate when during development C. teleta becomes capable of regeneration, we assessed the extent to which larvae can regenerate. We hypothesized that larvae exhibit intermediate regeneration potential and demonstrate some features of juvenile regeneration, but do not successfully replace all lost structures. Both anterior and posterior regeneration potential of larvae were evaluated following amputation. Wound sites were analyzed for re-epithelialization, cell proliferation by EdU incorporation, stem cell and differentiation marker expression by in situ hybridization, presence of neurites and muscle fibers by immunohistochemistry and phalloidin staining respectively, and regrowth of structures. Wound healing occurred within 6 hours of amputation for both anterior and posterior amputations. Cell proliferation at both wound sites was observed for up to 7 days following amputation. In addition, the stem cell marker vasa was expressed at anterior and posterior wound sites. However, growth of new tissue was observed only in posterior amputations. Neurites from the ventral nerve cord were also observed at posterior wound sites. De novo ash expression in the ectoderm of anterior wound sites indicated neuronal cell specification, although the absence of elav expression indicated an inability to progress to neuronal differentiation. In rare instances, cilia and eyes reformed. Both amputations induced expanded expression of the myogenesis gene MyoD in pre-existing tissues. Our results indicate that amputated larvae complete early, but not late, stages of regeneration, indicating a gradual acquisition of regenerative ability in C. teleta. Furthermore, amputated larvae can metamorphose into burrowing juveniles, including those missing brain and anterior sensory structures. To our knowledge, this is the first study to assess regenerative potential of annelid larvae.

The complexity of cell types and states revealed by single-cell RNAseq atlases presents a challenge for the systematic analysis of fate determinants using traditional screening methodologies. Differentiation in the planarian Schmidtea mediterranea exemplifies this problem, as these animals continuously produce over 100 differentiated cell types for homeostasis and regeneration using neoblast adult pluripotent stem cells. The signaling factors enabling neoblast self-renewal and selective differentiation of these many fates are still incompletely understood. We developed a method using high-throughput expression profiling by qPCR and whole-animal RNAseq to simultaneously assess numerous cell fate markers as the phenotypic readout in large-scale RNAi screens. Applying this method, we performed an RNAi screen of 400 kinases, receptors, and other regulatory molecules to reveal specific functions for 30 previously unknown factors in neoblast biology. 17 genes were required for neoblast maintenance, including factors likely involved in cell-cycle regulation, nutrient sensing, and chromatin modification. Multidimensional expression information additionally revealed several specific regulators of other neoblast activities, including a mink1 kinase regulating global neoblast differentiation, the energy responsive kinase adenylate kinase-2 regulating intestine specification within the neoblast population, an RNA acetyl transferase nat10 regulating epidermal differentiation, and a pak1 kinase restricting neoblast localization to prevent tissue outgrowths. These results identify several new regulators of neoblast activities and demonstrate the applicability of expression-based screening for systematic analysis of stem cell phenotypes in whole animals.

Increased brain size and its rostral bias are hallmarks of vertebrate evolution, but the underlying developmental and genetic basis remains poorly understood. To provide clues to understanding vertebrate brain evolution, we investigated the developmental mechanisms of brain enlargement observed in the offspring of a previously unrecognized, spontaneously occurring female variant line of Xenopus that appears to reflect a genetic variation. Brain enlargement in larvae from this line showed a pronounced rostral bias that could be traced back to the neural plate, the primordium of the brain. At the gastrula stage, the Spemann organizer, which is known to induce the neural plate from the adjacent dorsal ectoderm and give it the initial rostrocaudal patterning, was expanded from dorsal to ventral in a large proportion of the offspring of variant females. Consistently, siamois expression, which is required for Spemann organizer formation, was expanded laterally from dorsal to ventral at the blastula stage in variant offspring. This implies that the active region of the Wnt/{beta}-catenin signaling pathway was similarly expanded in advance on the dorsal side, as siamois is a target gene of this pathway. Notably, the earliest detectable change in variant offspring was in fertilized eggs, in which maternal wnt11b mRNA, a candidate dorsalizing factor that activates Wnt/{beta}-catenin signaling, had a wider distribution in the vegetal cortical cytoplasm. Since lateral spreading of wnt11b mRNA, and possibly that of other potential maternal dorsalizing factors in these eggs, is expected to facilitate lateral expansion of the active region of the Wnt/{beta}-catenin pathway during subsequent embryonic stages, we concluded that aberrant Wnt/{beta}-catenin signaling could cause rostral-biased brain enlargement via expansion of siamois expression and consequent expansion of the Spemann organizer in Xenopus. Our studies of spontaneously occurring variations in brain development in Xenopus would help uncover genetic mutations that drive analogous morphogenetic variations during vertebrate brain evolution.

We investigated the initial stages of head development using a new method to randomly label chicken epiblast cells with enhanced green fluorescent protein, and tracking the labeled cells. This analysis was combined with grafting mCherry-expressing quail nodes, or node-derived anterior mesendoderm (AME). These live imagings provided a new conception of the cellular mechanisms regulating brain and head ectoderm development. Virtually all anterior epiblast cells are bipotent for the development into the brain or head ectoderm. Their fate depends on the positioning after converging to the AME. When two AME tissues exist following the ectopic node graft, the epiblast cells converge to the two AME positions and develop into two brain tissues. The anterior epiblast cells bear gross regionalities that already correspond to the forebrain, midbrain, and hindbrain axial levels shortly after the node is formed. Therefore, brain portions that develop with the graft-derived AME are dependent on graft positioning.

Proper stem cell targeting and differentiation is necessary for regeneration to succeed. In organisms capable of whole body regeneration, considerable progress has been made identifying wound signals initiating this process, but the mechanisms that control the differentiation of progenitors into mature organs are not fully understood. Using the planarian as a model system, we identify a novel function for map3k1, a MAP3K family member possessing both kinase and ubiquitin ligase domains, to negatively regulate terminal differentiation of stem cells during eye regeneration. Inhibition of map3k1 caused the formation of multiple ectopic eyes within the head, but without controlling overall head, brain, or body patterning. By contrast, other known regulators of planarian eye patterning like WntA and notum also regulate head regionalization, suggesting map3k1 acts distinctly. Eye resection and regeneration experiments suggest that unlike Wnt signaling perturbation, map3k1 inhibition did not shift the target destination of eye formation in the animal. Instead, map3k1(RNAi) ectopic eyes emerge in the regions normally occupied by migratory eye progenitors, and the onset of ectopic eyes after map3k1 inhibition coincides with a reduction to eye progenitor numbers. Furthermore, RNAi dosing experiments indicate that progenitors closer to their normal target are relatively more sensitive to the effects of map3k1, implicating this factors in controlling the site of terminal differentiation. Eye phenotypes were also observed after inhibition of map2k4, map2k7, jnk, and p38, identifying a putative pathway through which map3k1 prevents differentiation. Together, these results suggest that map3k1 regulates a novel control point in the eye regeneration pathway which suppresses the terminal differentiation of progenitors during their migration to target destinations. Author SummaryDuring adult regeneration, progenitors must migrate and differentiate at the proper locations in order to successfully restore lost or damaged organs and tissues, yet the mechanisms underlying these abilities are not fully understood. The planarian eye is a model to study this problem, because this organ is regenerated using migratory progenitors that travel long distances through the body in an undifferentiated state prior to terminal differentiation upon their arrival at target destinations. We determined that a pathway involving the MAP kinase kinase kinase map3k1 holds planarian eye progenitors in an undifferentiated state during their transit. Inhibition of map3k1 caused a dramatic body transformation in which migratory progenitors differentiate inappropriately early, and in the wrong locations, into mature eyes. By analyzing this phenotype and measuring the change to eye progenitor abundance after map3k1 inhibition, we found that map3k1 prevents ectopic differentiation of eye cells rather than mediating body-wide patterning through the Wnt pathway. Our study argues that whole-body regeneration mechanisms involve separate steps to control patterning and progenitor differentiation.

Precisely regulated cell death plays a critical role in normal development and is controlled by the balance of pro-apoptotic and anti-apoptotic signals. In Drosophila, transcription of the clustered cell death activators grim and reaper is turned on in the developing nervous system to eliminate neural stem cells at the end of embryonic development. This transcription is activated by a pulse of the Hox gene abdominal-A. We show here that the Sox2 homologue Dichaete inhibits neural stem cell death when overexpressed, and loss of Dichaete promotes premature neural stem cell death. The anti-apoptotic activity of Dichaete opposes the pro-apoptotic factors abdominal-A, as well as the transcription factor grainyhead. The function of all three genes impinge on an enhancer that regulates the transcription of grim and reaper. Furthermore, we find that the balance between abdominal-A and Dichaete is likely to regulate the death of other cells during development, including cells in the developing midline, the developing hindgut, and in the early abdominal epidermis. Loss of Dichaete results in premature death in these tissues. This death can be rescued by the deletion of the enhancer region between grim and reaper. These data suggest that Dichaete functions to inhibit cell death activated by abdominal-A in multiple developmental contexts.

Knowledge about the genetic pathways that control renal cell lineage development is essential to better understand the basis of congenital malformations of the kidney and design regenerative medicine therapies. The embryonic zebrafish kidney, or pronephros, contains two nephrons that are conserved with humans. Recently, the transcription factors Osr1 and Hand2 were found to exert antagonistic influences to balance kidney specification (Perens et al., 2016). Here, we performed a forward genetic screen in zebrafish to identify nephrogenesis regulators, where whole genome sequencing of the novel oceanside (ocn) mutant revealed a nonsense mutation in osr1. ocn mutants evince severe pronephros defects including abrogation of podocytes and proximal tubule cells. Our studies reveal that osr1 is not needed to specify renal progenitors, but rather required to maintain their survival. Additionally, osr1 is requisite for expression of the canonical Wnt ligand wnt2ba, where wnt2ba is expressed in the intermediate mesoderm (IM) and later restricts to podocytes. Deficiency of wnt2ba reduced podocyte progenitors, where overexpression of wnt2ba was sufficient to rescue the podocyte lineage as well as osr1 loss of function. Finally, we demonstrate that reciprocal antagonism between osr1 and hand2 mediates podocyte development specifically by controlling wnt2ba expression in the IM. Together, our data show that Osr1 is essential for a sequence of temporal functions that mediate the survival and lineage decisions of IM progenitors, and subsequently the maintenance of podocytes and proximal tubule epithelium in the embryonic nephron.

The present study explored the significance of the microtubule-associated protein Tau in the morphogenesis and physiological processes of Malpighian tubules (MTs) in Drosophila melanogaster. Employing genetic manipulation techniques and microscopy, we established that Tau plays a crucial role in MTs development and function. Genetic ablation and RNAi-mediated suppression of Tau led to pronounced structural abnormalities, including cystic formations, non-uniform tubule diameters, and disorganized epithelial architecture. These aberrations are accompanied by perturbations in the cytoskeletal arrangement, compromised cellular polarity, and ionic imbalances. Functional analyses revealed deficiencies in salt homeostasis and fluid secretion within Tau-deficient tubules. Attenuation of Rho1 expression mitigated the structural defects observed in Tau mutants, highlighting the importance of Tau in modulating Rho1-mediated actin-microtubule dynamics suggesting a genetic interplay between Tau and Rho1 GTPase in orchestrating tubule morphogenesis The phenotypic parallels between Tau-deficient MTs and mammalian models of polycystic kidney disease implies an evolutionarily conserved function of Tau in tubular organ biology. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=111 SRC="FIGDIR/small/634261v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@1f4afc6org.highwire.dtl.DTLVardef@1402eb7org.highwire.dtl.DTLVardef@bb089dorg.highwire.dtl.DTLVardef@1328b2e_HPS_FORMAT_FIGEXP M_FIG C_FIG

Given the critical role of progenitor cells staying within the eye field transcription factor (EFTF) signaling niche for normal eye development, we hypothesized that retinal progenitor cells (RPCs) differentiate within their initial region of inception during eye development. To investigate this, we utilized EosFP, a photoconvertible protein, as a lineage tracer in the model organism Xenopus laevis. By employing confocal laser microscopy for photoconversion, we labeled cells within elongated rectangular regions that encompassed both the eye field and the adjacent tissues. In a separate set of embryos, we identified which portions of these rectangular regions harbored cells destined to become part of the mature eye versus those that would form the surrounding tissues, tracing their development from stage 15 to stage 35. This allowed us to create a fate map of the stage 15 embryo using EosFP to accurately locate and label the eye field to address our hypothesis. With the eye field delineated using our lineage tracer, we further employed EosFP to label RPCs within individual quadrants of the developing eye. Tracking these RPCs from stage 15 to stage 35, we observed the retinal cells organizing into three principal layers of cell bodies, mirroring the layered neuroanatomy characteristic of the mature retina. We observed the red-labeled RPCs proliferated but remained predominantly within their quadrant of inception, with no dispersion into other, unlabeled quadrants of the eye by stage 35. These findings corroborate our hypothesis that RPCs undergo differentiation within their initial locations in the eye field. Our study illuminates the cellular dynamics of eye development in Xenopus laevis and introduces a novel method for lineage tracing of stem cell populations during embryonic development.

Hox genes establish regional identity along the anterior-posterior axis in diverse animals. Changes in Hox expression can induce striking homeotic transformations, where one region of the body is transformed into another. Previous work in Drosophila has demonstrated that Hox cross-regulatory interactions are crucial for maintaining proper Hox expression. One major mechanism is the phenomenon of "posterior prevalence", wherein anterior Hox genes are repressed by more posterior Hox genes. Loss of posterior Hox expression under this model would predict posterior-to-anterior transformations, as is frequently observed in Drosophila. While posterior prevalence is thought to occur in many animals, studies of such Hox cross-regulation have focused on a limited number of organisms. In this paper, we examine the cross-regulatory interactions of three Hox genes, Ultrabithorax (Ubx), abdominal-A (abd-A), and Abdominal-B (Abd-B) in patterning thoracic and abdominal appendages in the amphipod crustacean Parhyale hawaiensis. Studies of Hox function in Parhyale have previously revealed two striking phenotypes which differed markedly from what a "posterior prevalence" model would predict, including non-contiguous and anterior-to-posterior transformations. We probe the logic of Parhyale Hox cross-regulation by using CRISPR/Cas9 to systematically examine all combinations of Ubx, abd-A, and Abd-B loss of function in Parhyale. By analyzing homeotic phenotypes and examining the expression of additional Hox genes, we reveal Hox cross-regulatory interactions in Parhyale. From these data, we also demonstrate that some Parhyale Hox genes function combinatorially to specify posterior limb identity, rather than abiding by a posterior prevalence mechanism. These results provide evidence that combinatorial Hox interactions may be responsible for the tremendous body plan diversity of crustaceans. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=192 SRC="FIGDIR/small/485717v1_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@16bfc85org.highwire.dtl.DTLVardef@8f859eorg.highwire.dtl.DTLVardef@8d7892org.highwire.dtl.DTLVardef@1e6c9f0_HPS_FORMAT_FIGEXP M_FIG C_FIG

Xenopus laevis tadpoles and newts regenerate a limb almost completely after amputation, with recapitulation of the patten formation of the limb. Metamorphosed Xenopus froglets form a cone-shaped regenerating blastema, similar to tadpoles and newts, but ultimately regenerate only a hypomorphic cartilaginous spike. Previous study suggested that excessive chondrogenesis, distinct from Xenopus tadpoles, may occur in the regenerating limb of a froglet and may prevent pattern formation during regeneration. However, it remains unclear whether excessive chondrogenesis actually occurs in froglet blastemas. If it does, when does it initiate and how does it progress in the blastemas? To answer these questions, we examined the extent of chondrogenesis in regenerating blastemas which have the common morphological shapes observed in newts (Pleurodeles waltl), Xenopus laevis tadpoles, and froglets. To evaluate excessive chondrogenesis, we developed a simplified procedure using immunofluorescence for cartilage markers (Sox9 or Col2a1) and quantitative image analysis. Our analysis revealed that signs of excessive chondrogenesis were detected not in newts and tadpoles but in froglets blastemas. During limb regeneration in froglets, the first sign of excessive chondrogenesis was detected in the cone-shaped blastema at the medium bud (MB) stage, and excessive chondrogenesis progressed to a more severe state as the blastema grew. These results indicate that excessive chondrogenesis initiates specifically in froglet blastemas at the MB stage at the latest and progresses in a definite spatio-temporal manner. Further elucidation of the mechanisms underlying froglet-specific excessive chondrogenesis in the blastema may lead to the recovery of patterned limb regeneration in froglets with adequate inhibition of chondrogenesis.

BackgroundSplit Hand/Foot Malformation (SHFM) is a congenital limb disorder presenting with limb anomalies, such as missing, hypoplastic, or fused digits, and often craniofacial defects, including a cleft lip/palate, microdontia, micrognathia, or maxillary hypoplasia. We previously identified three novel variants in the transcription factor, PRDM1, that are associated with SHFM phenotypes. One individual also presented with a high arch palate. Studies in vertebrates indicate that PRDM1 is important for development of the skull; however, prior to our study, human variants in PRDM1 had not been associated with craniofacial anomalies. MethodsUsing transient mRNA overexpression assays in prdm1a-/- mutant zebrafish, we tested whether the PRDM1 SHFM variants were functional and could lead to a rescue of the craniofacial defects observed in prdm1a-/- mutants. We also mined a CUT&RUN and RNA-seq dataset to examine Prdm1a binding and the effect of Prdm1a loss on craniofacial genes. Resultsprdm1a-/- mutants exhibit craniofacial defects including a hypoplastic neurocranium, a loss of posterior ceratobranchial arches, a shorter palatoquadrate, and an inverted ceratohyal. Injection of wildtype hPRDM1 in prdm1a-/- mutants partially rescues these structures. However, injection of each of the three SHFM variants fails to rescue the skeletal defects. Loss of prdm1a leads to a decreased expression of important craniofacial genes, such as dlx5a/dlx6a, hand2, sox9b, col2a1a, and hoxb genes. ConclusionThese data suggest that the three SHFM variants are not functional and may have led to the craniofacial defects observed in the humans. Finally, they demonstrate how Prdm1a can directly bind and regulate craniofacial gene expression.

A major gap in our understanding of animal development is how adult body plans arise in animals with indirect development, where adults emerge from the transformation of a distinct larval form during metamorphosis. We address this question by examining cellular changes in the enteropneust hemichordate Schizocardium californicum, a species with a complex lifecycle and dramatic metamorphosis. Employing whole-body single-cell RNA sequencing, we chart the cellular composition and transcriptional dynamics of larval, metamorphosis, and adult stages. Our tissue level atlas reveals that ectodermal and endodermal cell types in larvae and adults occupy distinct transcriptional spaces, showing greater similarity to other cell types within the same life stage than to their counterparts in the opposite stage. In contrast, mesodermal cell types from both larvae and adults cluster closely together, indicating conserved transcriptional profiles. These findings demonstrate that the extensive morphological reorganization during metamorphosis is paralleled by profound shifts in cell-type specific transcriptional programs, highlighting the complexity of the larva-to-adult transition.

In chordate embryos, placodes are ectodermal thickenings around the borders of the neural plate that give rise to various sensory organs and cell types. While generally thought to be a vertebrate-specific innovation, homologous placodes are proposed to exist in non-vertebrate chordates as well. In Ciona robusta, a solitary tunicate, the adult mouth (the oral siphon) is derived from one such "cranial-like" placode in the larva, which we term the oral siphon placode (OSP). At embryonic and larval stages, the OSP consists of a small rosette of cells that forms from the neuropore at the anteriormost extent of neural tube closure. While the morphogenesis of the OSP and its physical separation from other surface ectoderm structures have been described in detail, how this is regulated at the molecular level is currently unknown. Here we show the involvement of protocadherin-mediated cell-cell adhesion in the segregation and structural cohesiveness of the OSP. Protocadherin.e (Pcdhe.e) is expressed specifically in the OSP but not in other surface ectoderm cells. CRISPR/Cas9-mediated disruption of Pcdh.e in these cells results in loss of OSP structural integrity and ability to physically separate from other structures derived from the same cell lineage. Overexpression of Pcdh.e throughout the anterior surface ectoderm results in similar loss of a physically separate and distinct OSP territory. Furthermore, we show that Pcdh.e expession in the OSP depends on oral placode-specific transcription factors such as Six1/2 and Pitx. Our results suggest that OSP integrity and morphogenesis require precise regulation of a homotypic cell-cell adhesion molecule, which might reflect a conserved mechanism for placode formation in chordates.