MorphoSeq: Full Single-Cell Transcriptome Dynamics Up to Gastrulation in a Chordate.

Single-cell RNA sequencing (scRNA-seq) provides a leap forward in resolving cellular diversity and developmental trajectories but fails to comprehensively delineate the spatial organization and precise cellular makeup of individual embryos. Here, we reconstruct from scRNA-seq and light sheet imaging data a canonical digital embryo that captures the genome-wide gene expression trajectory of every single cell at every cell division in the 18 lineages up to gastrulation in the ascidian Phallusia mammillata. By using high-coverage scRNA-seq, we devise a computational framework that stratifies single cells of individual embryos into cell types without prior knowledge. Unbiased transcriptome data analysis mapped each cell's physical position and lineage history, yielding the complete history of gene expression at the genome-wide level for every single cell in a developing embryo. A comparison of individual embryos reveals both extensive reproducibility between symmetric embryo sides and a large inter-embryonic variability due to small differences in embryogenesis timing.

Extreme genome scrambling in marine planktonic Oikopleura dioica cryptic species.

Genome structural variations within species are rare. How selective constraints preserve gene order and chromosome structure is a central question in evolutionary biology that remains unsolved. Our sequencing of several genomes of the appendicularian tunicate Oikopleura dioica around the globe reveals extreme genome scrambling caused by thousands of chromosomal rearrangements, although showing no obvious morphological differences between these animals. The breakpoint accumulation rate is an order of magnitude higher than in ascidian tunicates, nematodes, Drosophila, or mammals. Chromosome arms and sex-specific regions appear to be the primary unit of macrosynteny conservation. At the microsyntenic level, scrambling did not preserve operon structures, suggesting an absence of selective pressure to maintain them. The uncoupling of the genome scrambling with morphological conservation in O. dioica suggests the presence of previously unnoticed cryptic species and provides a new biological system that challenges our previous vision of speciation in which similar animals always share similar genome structures.

Specification of distinct cell types in a sensory-adhesive organ important for metamorphosis in tunicate larvae.

The papillae of tunicate larvae contribute sensory, adhesive, and metamorphosis-regulating functions that are crucial for the biphasic lifestyle of these marine, non-vertebrate chordates. We have identified additional molecular markers for at least 5 distinct cell types in the papillae of the model tunicate Ciona, allowing us to further study the development of these organs. Using tissue-specific CRISPR/Cas9-mediated mutagenesis and other molecular perturbations, we reveal the roles of key transcription factors and signaling pathways that are important for patterning the papilla territory into a highly organized array of different cell types and shapes. We further test the contributions of different transcription factors and cell types to the production of the adhesive glue that allows for larval attachment during settlement, and to the processes of tail retraction and body rotation during metamorphosis. With this study, we continue working towards connecting gene regulation to cellular functions that control the developmental transition between the motile larva and sessile adult of Ciona.

Cardiopharyngeal deconstruction and ancestral tunicate sessility.

A central question in chordate evolution is the origin of sessility in adult ascidians, and whether the appendicularian complete free-living style represents a primitive or derived condition among tunicates1. According to the 'a new heart for a new head' hypothesis, the evolution of the cardiopharyngeal gene regulatory network appears as a pivotal aspect to understand the evolution of the lifestyles of chordates2-4. Here we show that appendicularians experienced massive ancestral losses of cardiopharyngeal genes and subfunctions, leading to the 'deconstruction' of two ancestral modules of the tunicate cardiopharyngeal gene regulatory network. In ascidians, these modules are related to early and late multipotency, which is involved in lineage cell-fate determination towards the first and second heart fields and siphon muscles. Our work shows that the deconstruction of the cardiopharyngeal gene regulatory network involved the regressive loss of the siphon muscle, supporting an evolutionary scenario in which ancestral tunicates had a sessile ascidian-like adult lifestyle. In agreement with this scenario, our findings also suggest that this deconstruction contributed to the acceleration of cardiogenesis and the redesign of the heart into an open-wide laminar structure in appendicularians as evolutionary adaptations during their transition to a complete pelagic free-living style upon the innovation of the food-filtering house5.

Regulation of anterior neurectoderm specification and differentiation by BMP signaling in ascidians.

The most anterior structure of the ascidian larva is made of three palps with sensory and adhesive functions essential for metamorphosis. They derive from the anterior neural border and their formation is regulated by FGF and Wnt. Given that they also share gene expression profiles with vertebrate anterior neural tissue and cranial placodes, their study should shed light on the emergence of the unique vertebrate telencephalon. We show that BMP signaling regulates two phases of palp formation in Ciona intestinalis. During gastrulation, the anterior neural border is specified in a domain of inactive BMP signaling, and activating BMP prevented its formation. During neurulation, BMP defines ventral palp identity and indirectly specifies the inter-papilla territory separating the ventral and dorsal palps. Finally, we show that BMP has similar functions in the ascidian Phallusia mammillata, for which we identified novel palp markers. Collectively, we provide a better molecular description of palp formation in ascidians that will be instrumental for comparative studies.

Revealing enigmatic mucus structures in the deep sea using DeepPIV.

Many animals build complex structures to aid in their survival, but very few are built exclusively from materials that animals create 1,2. In the midwaters of the ocean, mucoid structures are readily secreted by numerous animals, and serve many vital functions3,4. However, little is known about these mucoid structures owing to the challenges of observing them in the deep sea. Among these mucoid forms, the 'houses' of larvaceans are marvels of nature5, and in the ocean twilight zone giant larvaceans secrete and build mucus filtering structures that can reach diameters of more than 1 m6. Here we describe in situ laser-imaging technology7 that reconstructs three-dimensional models of mucus forms. The models provide high-resolution views of giant larvacean houses and elucidate the role that house structure has in food capture and predator avoidance. Now that tools exist to study mucus structures found throughout the ocean, we can shed light on some of nature's most complex forms.

Genome-wide analysis of early vascular tunic repair and regeneration for Botrylloides digenesis reveals striking similarities to human wound healing.

The early stages of regeneration after injury are similar to those of wound healing. The ascidian Botrylloides diegensis can regenerate an entire adult from a small fragment of vascular tunic following the removal of all zooids in an injury-induced regeneration model. We investigated the molecular and cellular changes following injury to determine the differences between the healing process and the initiation of whole-body regeneration (WBR). We conducted transcriptome analysis at specific time points during regeneration and wound healing to identify differentially expressed genes (DEGs) and the unique biological processes associated with each state. Our findings revealed 296 DEGs at 10 h post-injury (hpi), with 71 highly expressed in healed tissue and 225 expressed during the WBR process. These DEGs were predicted to play roles in tissue reorganization, integrin signaling, extracellular matrix organization, and the innate immune system. Pathway analysis of the upregulated genes in the healed tunic indicated functional enrichment related to tissue repair, as has been observed in other species. Additionally, we examined the cell types in the tunic and ampullae in both tissue states using histology and in situ hybridization for six genes identified by transcriptome analysis. We observed strong mRNA expression in cells within the WBR tunic, and in small RNA-positive granules near the tunic edge. We hypothesized that many of these genes function in the compaction of the ampullae tunic, which is a pivotal process for WBR and dormancy in B. diegensis, and in an immune response. These findings establish surprising similarities between ascidian regeneration and human wound healing, emphasizing the potential for future investigations into human regenerative and repair mechanisms. This study provides valuable insights into the gene sets specifically activated during regeneration compared to wound healing, shedding light on the divergent activities of these processes.

Regulators specifying cell fate activate cell cycle regulator genes to determine cell numbers in ascidian larval tissues.

In animal development, most cell types stop dividing before terminal differentiation; thus, cell cycle control is tightly linked to cell differentiation programmes. In ascidian embryos, cell lineages do not vary among individuals, and rounds of the cell cycle are determined according to cell lineages. Notochord and muscle cells stop dividing after eight or nine rounds of cell division depending on their lineages. In the present study, we showed that a Cdk inhibitor, Cdkn1.b, is responsible for stopping cell cycle progression in these lineages. Cdkn1.b is also necessary for epidermal cells to stop dividing. In contrast, mesenchymal and endodermal cells continue to divide even after hatching, and Myc is responsible for maintaining cell cycle progression in these tissues. Expression of Cdkn1.b in notochord and muscle is controlled by transcription factors that specify the developmental fate of notochord and muscle. Likewise, expression of Myc in mesenchyme and endoderm is under control of transcription factors that specify the developmental fate of mesenchyme and endoderm. Thus, cell fate specification and cell cycle control are linked by these transcription factors.

IFI16 phase separation via multi-phosphorylation drives innate immune signaling.

The interferon inducible protein 16 (IFI16) is a prominent sensor of nuclear pathogenic DNA, initiating innate immune signaling and suppressing viral transcription. However, little is known about mechanisms that initiate IFI16 antiviral functions or its regulation within the host DNA-filled nucleus. Here, we provide in vitro and in vivo evidence to establish that IFI16 undergoes liquid-liquid phase separation (LLPS) nucleated by DNA. IFI16 binding to viral DNA initiates LLPS and induction of cytokines during herpes simplex virus type 1 (HSV-1) infection. Multiple phosphorylation sites within an intrinsically disordered region (IDR) function combinatorially to activate IFI16 LLPS, facilitating filamentation. Regulated by CDK2 and GSK3ß, IDR phosphorylation provides a toggle between active and inactive IFI16 and the decoupling of IFI16-mediated cytokine expression from repression of viral transcription. These findings show how IFI16 switch-like phase transitions are achieved with temporal resolution for immune signaling and, more broadly, the multi-layered regulation of nuclear DNA sensors.

Two distinct evolutionary conserved neural degeneration pathways characterized in a colonial chordate.

Colonial tunicates are marine organisms that possess multiple brains simultaneously during their colonial phase. While the cyclical processes of neurogenesis and neurodegeneration characterizing their life cycle have been documented previously, the cellular and molecular changes associated with such processes and their relationship with variation in brain morphology and individual (zooid) behavior throughout adult life remains unknown. Here, we introduce Botryllus schlosseri as an invertebrate model for neurogenesis, neural degeneration, and evolutionary neuroscience. Our analysis reveals that during the weekly colony budding (i.e., asexual reproduction), prior to programmed cell death and removal by phagocytes, decreases in the number of neurons in the adult brain are associated with reduced behavioral response and significant change in the expression of 73 mammalian homologous genes associated with neurodegenerative disease. Similarly, when comparing young colonies (1 to 2 y of age) to those reared in a laboratory for ∼20 y, we found that older colonies contained significantly fewer neurons and exhibited reduced behavioral response alongside changes in the expression of 148 such genes (35 of which were differentially expressed across both timescales). The existence of two distinct yet apparently related neurodegenerative pathways represents a novel platform to study the gene products governing the relationship between aging, neural regeneration and degeneration, and loss of nervous system function. Indeed, as a member of an evolutionary clade considered to be a sister group of vertebrates, this organism may be a fundamental resource in understanding how evolution has shaped these processes across phylogeny and obtaining mechanistic insight.

Molecular control of cellulosic fin morphogenesis in ascidians.

BACKGROUND: The tunicates form a group of filter-feeding marine animals closely related to vertebrates. They share with them a number of features such as a notochord and a dorsal neural tube in the tadpole larvae of ascidians, one of the three groups that make tunicates. However, a number of typical chordate characters have been lost in different branches of tunicates, a diverse and fast-evolving phylum. Consequently, the tunic, a sort of exoskeleton made of extracellular material including cellulose secreted by the epidermis, is the unifying character defining the tunicate phylum. In the larva of ascidians, the tunic differentiates in the tail into a median fin (with dorsal and ventral extended blades) and a caudal fin. RESULTS: Here we have performed experiments in the ascidian Phallusia mammillata to address the molecular control of tunic 3D morphogenesis. We have demonstrated that the tail epidermis medio-lateral patterning essential for peripheral nervous system specification also controls tunic elongation into fins. More specifically, when tail epidermis midline identity was abolished by BMP signaling inhibition, or CRISPR/Cas9 inactivation of the transcription factor coding genes Msx or Klf1/2/4/17, median fin did not form. We postulated that this genetic program should regulate effectors of tunic secretion. We thus analyzed the expression and regulation in different ascidian species of two genes acquired by horizontal gene transfer (HGT) from bacteria, CesA coding for a cellulose synthase and Gh6 coding for a cellulase. We have uncovered an unexpected dynamic history of these genes in tunicates and high levels of variability in gene expression and regulation among ascidians. Although, in Phallusia, Gh6 has a regionalized expression in the epidermis compatible with an involvement in fin elongation, our functional studies indicate a minor function during caudal fin formation only. CONCLUSIONS: Our study constitutes an important step in the study of the integration of HGT-acquired genes into developmental networks and a cellulose-based morphogenesis of extracellular material in animals.

Transient impacts of UV-B irradiation on whole body regeneration in a colonial urochordate.

Within the chordates, only some colonial ascidians experience whole body regeneration (WBR), where amputated small colonial fragments containing blood-vessels have the capability to regenerate the entire functional adult zooid within 1-3 weeks. Studying WBR in small colonial fragments taken at different blastogenic stages (the weekly developmental process characteristic to botryllid ascidians) from the ascidian Botrylloides leachii, about half of the fragments were able to complete regeneration (cWBR) three weeks following separation, about half were still in uncomplete, running regeneration (rWBR), and only a small percentage died. cWBR significantly increased in fragments that originated from a late blastogenic stage compared to an early stage. Most B. leachii populations reside in shallow waters, under variable daily natural UV irradiation, and it is of interest to elucidate irradiation effects on development and regeneration. Here, we show that UV-B irradiation resulted in enhanced mortality, with abnormal morphological changes in surviving fragments, yet with non-significant cWBR vs. rWBRs. Further, UV-B irradiation influenced the proportion of blood cells (morula cells, hemoblasts) and of multinucleated cells, a new WBR-associated cell type. At 24-h post-amputation we observed enhanced expression of ß-catenin (a signaling pathway that plays indispensable roles in cell renewal and regeneration), H3 and PCNA in all cell types of non-irradiated as compared to irradiated fragments. These elevated levels were considerably reduced 9-days later. Since WBR is a highly complex phenomenon, the employment of specific experimental conditions, as UV-B irradiation, alongside blastogenesis (the weekly developmental process), elucidates undisclosed facets of this unique biological occurrence such as transient expression of signature genes.

Ascidian gastrulation and blebbing activity of isolated endoderm blastomeres.

Gastrulation is the first dynamic cell movement during embryogenesis. Endoderm and mesoderm cells are internalized into embryos during this process. Ascidian embryos provide a simple system for studying gastrulation in chordates. Gastrulation starts in spherical late 64-cell embryos with 10 endoderm blastomeres. The mechanisms of gastrulation in ascidians have been investigated, and a two-step model has been proposed. The first step involves apical constriction of endoderm cells, followed by apicobasal shortening in the second step. In this study, isolated ascidian endoderm progenitor cells displayed dynamic blebbing activity at the gastrula stage, although such a dynamic cell-shape change was not recognized in toto. Blebbing is often observed in migrating animal cells. In ascidians, endoderm cells displayed blebbing activity, while mesoderm and ectoderm cells did not. The timing of blebbing of isolated endoderm cells coincided with that of cell invagination. The constriction rate of apical surfaces correlated with the intensity of blebbing activity in each endoderm-lineage cell. Fibroblast growth factor (FGF) signaling was both necessary and sufficient for inducing blebbing activity, independent of cell fate specification. In contrast, the timing of initiation of blebbing and intensity of blebbing response to FGF signaling were controlled by intrinsic cellular factors. It is likely that the difference in intensity of blebbing activity between the anterior A-line and posterior B-line cells could account for the anteroposterior difference in the steepness of the archenteron wall. Inhibition of zygotic transcription, FGF signaling, and Rho kinase, all of which suppressed blebbing activity, resulted in incomplete apical constriction and failure of the eventual formation of cup-shaped gastrulae. Blebbing activity was involved in the progression and maintenance of apical constriction, but not in apicobasal shortening in whole embryos. Apical constriction is mediated by distinct blebbing-dependent and blebbing-independent mechanisms. Surface tension and consequent membrane contraction may not be the sole mechanical force for apical constriction and formation of cup-shaped gastrulae. The present study reveals the hidden cellular potential of endodermal cells during gastrulation and discusses the possible roles of blebbing in the invagination process.

Two distinct motifs for Zic-r.a drive specific gene expression in two cell lineages.

Zic-r.a, a maternal transcription factor, specifies posterior fate in ascidian embryos. However, its direct target, Tbx6-r.b, does not contain typical Zic-r.a-binding sites in its regulatory region. Using an in vitro selection assay, we found that Zic-r.a binds to sites dissimilar to the canonical motif, by which it activates Tbx6-r.b in a sub-lineage of muscle cells. These sites with non-canonical motifs have weak affinity for Zic-r.a; therefore, it activates Tbx6-r.b only in cells expressing Zic-r.a abundantly. Meanwhile, we found that Zic-r.a expressed zygotically in late embryos activates neural genes through canonical sites. Because different zinc-finger domains of Zic-r.a are important for driving reporters with canonical and non-canonical sites, it is likely that the non-canonical motif is not a divergent version of the canonical motif. In other words, our data indicate that the non-canonical motif represents a motif distinct from the canonical motif. Thus, Zic-r.a recognizes two distinct motifs to activate two sets of genes at two timepoints in development. This article has an associated 'The people behind the papers' interview.

Single-cell morphometrics reveals ancestral principles of notochord development.

Embryonic tissues are shaped by the dynamic behaviours of their constituent cells. To understand such cell behaviours and how they evolved, new approaches are needed to map out morphogenesis across different organisms. Here, we apply a quantitative approach to learn how the notochord forms during the development of amphioxus: a basally branching chordate. Using a single-cell morphometrics pipeline, we quantify the geometries of thousands of amphioxus notochord cells, and project them into a common mathematical space, termed morphospace. In morphospace, notochord cells disperse into branching trajectories of cell shape change, revealing a dynamic interplay between cell shape change and growth that collectively contributes to tissue elongation. By spatially mapping these trajectories, we identify conspicuous regional variation, both in developmental timing and trajectory topology. Finally, we show experimentally that, unlike ascidians but like vertebrates, posterior cell division is required in amphioxus to generate full notochord length, thereby suggesting this might be an ancestral chordate trait that is secondarily lost in ascidians. Altogether, our novel approach reveals that an unexpectedly complex scheme of notochord morphogenesis might have been present in the first chordates. This article has an associated 'The people behind the papers' interview.

FGF signaling acts on different levels of mesoderm development within Spiralia.

FGF signaling is involved in mesoderm induction in members of deuterostomes (e.g. tunicates, hemichordates), but not in flies and nematodes, in which it has a role in mesoderm patterning and migration. However, we need comparable studies in other protostome taxa in order to decipher whether this mesoderm-inducing function of FGF extends beyond the lineage of deuterostomes. Here, we investigated the role of FGF signaling in mesoderm development in three species of lophophorates, a clade within the protostome group Spiralia. Our gene expression analyses show that the mesodermal molecular patterning is conserved between brachiopods and phoronids, but the spatial and temporal recruitment of transcription factors differs significantly. Moreover, the use of the inhibitor SU5402 demonstrates that FGF signaling is involved in different steps of mesoderm development, as well as in morphogenetic movements of gastrulation and axial elongation. Our findings suggest that the mesoderm-inducing role of FGF extends beyond the group of deuterostomes.

Henri de Lacaze-Duthiers and the ascidian hypothesis.

In 1830, Cuvier and Geoffroy Saint-Hilaire confronted each other in a famous debate on the unity of the animal kingdom, which permeated the zoology of the 19th century. From that time, a growing number of naturalists attempted to understand the large-scale relationships among animals. And among all the questions, that of the origin of vertebrates was one of the most controversial. Analytical methods based on comparative anatomy, embryology and paleontology were developed to identify convincing homologies that would reveal a logical sequence of events for the evolution of an invertebrate into the first vertebrate. Within this context, several theories have clashed on the question of the identity of the ancestor of vertebrates. Among the proposals, a group of rather discrete organisms, the ascidians, played a central role. Because he had discovered an ascidian with a particularly atypical larval development, the Molgula, Henri de Lacaze-Duthiers, a rigorous and meticulous naturalist, became involved in the ascidian hypothesis. While the visionary mind of Lacaze-Duthiers led him to establish a particularly innovative methodology and the first marine biology station in Europe, at Roscoff, the tailless tadpole of the Molgula prevented him from recognizing the ancestor of vertebrates. This old 19th century story echoes the ever-present questions driving the field of Eco-Evo-Devo.

The appendicularian Oikopleura dioica can enhance carbon export in a high CO2 ocean.

Gelatinous zooplankton are increasingly recognized to play a key role in the ocean's biological carbon pump. Appendicularians, a class of pelagic tunicates, are among the most abundant gelatinous plankton in the ocean, but it is an open question how their contribution to carbon export might change in the future. Here, we conducted an experiment with large volume in situ mesocosms (~55-60 m3 and 21 m depth) to investigate how ocean acidification (OA) extreme events affect food web structure and carbon export in a natural plankton community, particularly focusing on the keystone species Oikopleura dioica, a globally abundant appendicularian. We found a profound influence of O. dioica on vertical carbon fluxes, particularly during a short but intense bloom period in the high CO2 treatment, during which carbon export was 42%-64% higher than under ambient conditions. This elevated flux was mostly driven by an almost twofold increase in O. dioica biomass under high CO2 . This rapid population increase was linked to enhanced fecundity (+20%) that likely resulted from physiological benefits of low pH conditions. The resulting competitive advantage of O. dioica resulted in enhanced grazing on phytoplankton and transfer of this consumed biomass into sinking particles. Using a simple carbon flux model for O. dioica, we estimate that high CO2 doubled the carbon flux of discarded mucous houses and fecal pellets, accounting for up to 39% of total carbon export from the ecosystem during the bloom. Considering the wide geographic distribution of O. dioica, our findings suggest that appendicularians may become an increasingly important vector of carbon export with ongoing OA.

Model of neural induction in the ascidian embryo.

How cell specification can be controlled in a reproducible manner is a fundamental question in developmental biology. In ascidians, a group of invertebrate chordates, geometry plays a key role in achieving this control. Here, we use mathematical modeling to demonstrate that geometry dictates the neural-epidermal cell fate choice in the 32-cell stage ascidian embryo by a two-step process involving first the modulation of ERK signaling and second, the expression of the neural marker gene, Otx. The model describes signal transduction by the ERK pathway that is stimulated by FGF and attenuated by ephrin, and ERK-mediated control of Otx gene expression, which involves both an activator and a repressor of ETS-family transcription factors. Considering the measured area of cell surface contacts with FGF- or ephrin-expressing cells as inputs, the solutions of the model reproduce the experimental observations about ERK activation and Otx expression in the different cells under normal and perturbed conditions. Sensitivity analyses and computations of Hill coefficients allow us to quantify the robustness of the specification mechanism controlled by cell surface area and to identify the respective role played by each signaling input. Simulations also predict in which conditions the dual control of gene expression by an activator and a repressor that are both under the control of ERK can induce a robust ON/OFF control of neural fate induction.

Dopamine-Derived Guanidine Alkaloids from a Didemnidae Tunicate: Isolation, Synthesis, and Biological Activities.

Mellpaladines A-C (1-3) and dopargimine (4) are dopamine-derived guanidine alkaloids isolated from a specimen of Palauan Didemnidae tunicate as possible modulators of neuronal receptors. In this study, we isolated the dopargimine derivative 1-carboxydopargimine (5), three additional mellpaladines D-F (6-8), and serotodopalgimine (9), along with a dimer of serotonin, 5,5'-dihydroxy-4,4'-bistryptamine (10). The structures of these compounds were determined based on spectrometric and spectroscopic analyses. Compound 4 and its congeners dopargine (11), nordopargimine (15), and 2-(6,7-dimethoxy-3,4-dihydroisoquinolin-1-yl)ethan-1-amine (16) were synthetically prepared for biological evaluations. The biological activities of all isolated compounds were evaluated in comparison with those of 1-4 using a mouse behavioral assay upon intracerebroventricular injection, revealing key functional groups in the dopargimines and mellpaladines for in vivo behavioral toxicity. Interestingly, these alkaloids also emerged during a screen of our marine natural product library aimed at identifying antiviral activities against dengue virus, SARS-CoV-2, and vesicular stomatitis Indiana virus (VSV) pseudotyped with Ebola virus glycoprotein (VSV-ZGP).