Hmx gene conservation identifies the origin of vertebrate cranial ganglia.

The evolutionary origin of vertebrates included innovations in sensory processing associated with the acquisition of a predatory lifestyle1. Vertebrates perceive external stimuli through sensory systems serviced by cranial sensory ganglia, whose neurons arise predominantly from cranial placodes; however, the understanding of the evolutionary origin of placodes and cranial sensory ganglia is hampered by the anatomical differences between living lineages and the difficulty in assigning homology between cell types and structures. Here we show that the homeobox transcription factor Hmx is a constitutive component of vertebrate sensory ganglion development and that in the tunicate Ciona intestinalis, Hmx is necessary and sufficient to drive the differentiation programme of bipolar tail neurons, cells previously thought to be homologues of neural crest2,3. Using Ciona and lamprey transgenesis, we demonstrate that a unique, tandemly duplicated enhancer pair regulated Hmx expression in the stem-vertebrate lineage. We also show notably robust vertebrate Hmx enhancer function in Ciona, demonstrating that deep conservation of the upstream regulatory network spans the evolutionary origin of vertebrates. These experiments demonstrate regulatory and functional conservation between Ciona and vertebrate Hmx, and point to bipolar tail neurons as homologues of cranial sensory ganglia.

Lhx3/4 initiates a cardiopharyngeal-specific transcriptional program in response to widespread FGF signaling.

Individual signaling pathways, such as fibroblast growth factors (FGFs), can regulate a plethora of inductive events. According to current paradigms, signal-dependent transcription factors (TFs), such as FGF/MapK-activated Ets family factors, partner with lineage-determining factors to achieve regulatory specificity. However, many aspects of this model have not been rigorously investigated. One key question relates to whether lineage-determining factors dictate lineage-specific responses to inductive signals or facilitate these responses in collaboration with other inputs. We utilize the chordate model Ciona robusta to investigate mechanisms generating lineage-specific induction. Previous studies in C. robusta have shown that cardiopharyngeal progenitor cells are specified through the combined activity of FGF-activated Ets1/2.b and an inferred ATTA-binding transcriptional cofactor. Here, we show that the homeobox TF Lhx3/4 serves as the lineage-determining TF that dictates cardiopharyngeal-specific transcription in response to pleiotropic FGF signaling. Targeted knockdown of Lhx3/4 leads to loss of cardiopharyngeal gene expression. Strikingly, ectopic expression of Lhx3/4 in a neuroectodermal lineage subject to FGF-dependent specification leads to ectopic cardiopharyngeal gene expression in this lineage. Furthermore, ectopic Lhx3/4 expression disrupts neural plate morphogenesis, generating aberrant cell behaviors associated with execution of incompatible morphogenetic programs. Based on these findings, we propose that combinatorial regulation by signal-dependent and lineage-determinant factors represents a generalizable, previously uncategorized regulatory subcircuit we term "cofactor-dependent induction." Integration of this subcircuit into theoretical models will facilitate accurate predictions regarding the impact of gene regulatory network rewiring on evolutionary diversification and disease ontogeny.

Cellular remodeling and JAK inhibition promote zygotic gene expression in the Ciona germline.

Transcription control is a major determinant of cell fate decisions in somatic tissues. By contrast, early germline fate specification in numerous vertebrate and invertebrate species relies extensively on RNA-level regulation, exerted on asymmetrically inherited maternal supplies, with little-to-no zygotic transcription. However delayed, a maternal-to-zygotic transition is nevertheless poised to complete the deployment of pre-gametic programs in the germline. Here, we focus on early germline specification in the tunicate Ciona to study zygotic genome activation. We first demonstrate that a peculiar cellular remodeling event excludes localized postplasmic Pem-1 mRNA, which encodes the general inhibitor of transcription. Subsequently, zygotic transcription begins in Pem-1-negative primordial germ cells (PGCs), as revealed by histochemical detection of elongating RNA Polymerase II, and nascent Mef2 transcripts. In addition, we uncover a provisional antagonism between JAK and MEK/BMPRI/GSK3 signaling, which controls the onset of zygotic gene expression, following cellular remodeling of PGCs. We propose a 2-step model for the onset of zygotic transcription in the Ciona germline and discuss the significance of germ plasm dislocation and remodeling in the context of developmental fate specification.

Metazoan operons accelerate recovery from growth-arrested states.

Existing theories explain why operons are advantageous in prokaryotes, but their occurrence in metazoans is an enigma. Nematode operon genes, typically consisting of growth genes, are significantly upregulated during recovery from growth-arrested states. This expression pattern is anticorrelated to nonoperon genes, consistent with a competition for transcriptional resources. We find that transcriptional resources are initially limiting during recovery and that recovering animals are highly sensitive to any additional decrease in transcriptional resources. We provide evidence that operons become advantageous because, by clustering growth genes into operons, fewer promoters compete for the limited transcriptional machinery, effectively increasing the concentration of transcriptional resources and accelerating recovery. Mathematical modeling reveals how a moderate increase in transcriptional resources can substantially enhance transcription rate and recovery. This design principle occurs in different nematodes and the chordate C. intestinalis. As transition from arrest to rapid growth is shared by many metazoans, operons could have evolved to facilitate these processes.

A digital twin reproducing gene regulatory network dynamics of early Ciona embryos indicates robust buffers in the network.

How gene regulatory networks (GRNs) encode gene expression dynamics and how GRNs evolve are not well understood, although these problems have been studied extensively. We created a digital twin that accurately reproduces expression dynamics of 13 genes that initiate expression in 32-cell ascidian embryos. We first showed that gene expression patterns can be manipulated according to predictions by this digital model. Next, to simulate GRN rewiring, we changed regulatory functions that represented their regulatory mechanisms in the digital twin, and found that in 55 of 100 cases, removal of a single regulator from a conjunctive clause of Boolean functions did not theoretically alter qualitative expression patterns of these genes. In other words, we found that more than half the regulators gave theoretically redundant temporal or spatial information to target genes. We experimentally substantiated that the expression pattern of Nodal was maintained without one of these factors, Zfpm, by changing the upstream regulatory sequence of Nodal. Such robust buffers of regulatory mechanisms may provide a basis of enabling developmental system drift, or rewiring of GRNs without changing expression patterns of downstream genes, during evolution.

Cereblon influences the timing of muscle differentiation in Ciona tadpoles.

Thalidomide has a dark history as a teratogen, but in recent years, its derivates have been shown to function as potent chemotherapeutic agents. These drugs bind cereblon (CRBN), the substrate receptor of an E3 ubiquitin ligase complex, and modify its degradation targets. Despite these insights, remarkably little is known about the normal function of cereblon in development. Here, we employ Ciona, a simple invertebrate chordate, to identify endogenous Crbn targets. In Ciona, Crbn is specifically expressed in developing muscles during tail elongation before they acquire contractile activity. Crbn expression is activated by Mrf, the ortholog of MYOD1, a transcription factor important for muscle differentiation. CRISPR/Cas9-mediated mutations of Crbn lead to precocious onset of muscle contractions. By contrast, overexpression of Crbn delays contractions and is associated with decreased expression of contractile protein genes such as troponin. This reduction is possibly due to reduced Mrf protein levels without altering Mrf mRNA levels. Our findings suggest that Mrf and Crbn form a negative feedback loop to control the precision of muscle differentiation during tail elongation.

Regulatory cocktail for dopaminergic neurons in a protovertebrate identified by whole-embryo single-cell transcriptomics.

The CNS of the protovertebrate Ciona intestinalis contains a single cluster of dopaminergic (DA) neurons, the coronet cells, which have been likened to the hypothalamus of vertebrates. Whole-embryo single-cell RNA sequencing (RNA-seq) assays identified Ptf1a as the most strongly expressed cell-specific transcription factor (TF) in DA/coronet cells. Knockdown of Ptf1a activity results in their loss, while misexpression results in the appearance of supernumerary DA/coronet cells. Photoreceptor cells and ependymal cells are the most susceptible to transformation, and both cell types express high levels of Meis Coexpression of both Ptf1a and Meis caused the wholesale transformation of the entire CNS into DA/coronet cells. We therefore suggest that the reiterative use of functional manipulations and single-cell RNA-seq assays is an effective means for the identification of regulatory cocktails underlying the specification of specific cell identities.

Optimizing CRISPR/Cas9 approaches in the polymorphic tunicate Ciona intestinalis.

CRISPR/Cas9 became a powerful tool for genetic engineering and in vivo knockout also in the invertebrate chordate Ciona intestinalis. Ciona (ascidians, tunicates) is an important model organism because it shares developmental features with the vertebrates, considered the sister group of tunicates, and offers outstanding experimental advantages: a compact genome and an invariant developmental cell lineage that, combined with electroporation mediated transgenesis allows for precise and cell type specific targeting in vivo. A high polymorphism and the mosaic expression of electroporated constructs, however, often hamper the efficient CRISPR knockout, and an optimization in Ciona is desirable. Furthermore, seasonality and artificial maintenance settings can profit from in vitro approaches that would save on animals. Here we present improvements for the CRISPR/Cas9 protocol in silico, in vitro and in vivo. Firstly, in designing sgRNAs, prior sequencing of target genomic regions from experimental animals and alignment with reference genomes of C. robusta and C. intestinalis render a correction possible of subspecies polymorphisms. Ideally, the screening for efficient and non-polymorphic sgRNAs will generate a database compatible for worldwide Ciona populations. Secondly, we challenged in vitro assays for sgRNA validation towards reduced in vivo experimentation and report their suitability but also overefficiency concerning mismatch tolerance. Thirdly, when comparing Cas9 with Cas9:Geminin, thought to synchronize editing and homology-direct repair, we could indeed increase the in vivo efficiency and notably the access to an early expressed gene. Finally, for in vivo CRISPR, genotyping by next generation sequencing (NGS) ex vivo streamlined the definition of efficient single guides. Double CRISPR then generates large deletions and reliable phenotypic excision effects. Overall, while these improvements render CRISPR more efficient in Ciona, they are useful when newly establishing the technique and very transferable to CRISPR in other organisms.

Comprehensive single-cell transcriptome lineages of a proto-vertebrate.

Ascidian embryos highlight the importance of cell lineages in animal development. As simple proto-vertebrates, they also provide insights into the evolutionary origins of cell types such as cranial placodes and neural crest cells. Here we have determined single-cell transcriptomes for more than 90,000 cells that span the entirety of development-from the onset of gastrulation to swimming tadpoles-in Ciona intestinalis. Owing to the small numbers of cells in ascidian embryos, this represents an average of over 12-fold coverage for every cell at every stage of development. We used single-cell transcriptome trajectories to construct virtual cell-lineage maps and provisional gene networks for 41 neural subtypes that comprise the larval nervous system. We summarize several applications of these datasets, including annotating the synaptome of swimming tadpoles and tracing the evolutionary origin of cell types such as the vertebrate telencephalon.

Neuronal identities derived by misexpression of the POU IV sensory determinant in a protovertebrate.

The protovertebrate Ciona intestinalis type A (sometimes called Ciona robusta) contains a series of sensory cell types distributed across the head-tail axis of swimming tadpoles. They arise from lateral regions of the neural plate that exhibit properties of vertebrate placodes and neural crest. The sensory determinant POU IV/Brn3 is known to work in concert with regional determinants, such as Foxg and Neurogenin, to produce palp sensory cells (PSCs) and bipolar tail neurons (BTNs), in head and tail regions, respectively. A combination of single-cell RNA-sequencing (scRNA-seq) assays, computational analysis, and experimental manipulations suggests that misexpression of POU IV results in variable transformations of epidermal cells into hybrid sensory cell types, including those exhibiting properties of both PSCs and BTNs. Hybrid properties are due to coexpression of Foxg and Neurogenin that is triggered by an unexpected POU IV feedback loop. Hybrid cells were also found to express a synthetic gene battery that is not coexpressed in any known cell type. We discuss these results with respect to the opportunities and challenges of reprogramming cell types through the targeted misexpression of cellular determinants.

Brachyury controls Ciona notochord fate as part of a feed-forward network.

The notochord is a defining feature of the chordates. The transcription factor Brachyury (Bra) is a key regulator of notochord fate but here we show that it is not a unitary master regulator in the model chordate Ciona Ectopic Bra expression only partially reprograms other cell types to a notochord-like transcriptional profile and a subset of notochord-enriched genes is unaffected by CRISPR Bra disruption. We identify Foxa.a and Mnx as potential co-regulators, and find that combinatorial cocktails are more effective at reprogramming other cell types than Bra alone. We reassess the network relationships between Bra, Foxa.a and other components of the notochord gene regulatory network, and find that Foxa.a expression in the notochord is regulated by vegetal FGF signaling. It is a direct activator of Bra expression and has a binding motif that is significantly enriched in the regulatory regions of notochord-enriched genes. These and other results indicate that Bra and Foxa.a act together in a regulatory network dominated by positive feed-forward interactions, with neither being a classically defined master regulator.

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.

Cyclin-dependent Kinase 1 and Aurora Kinase choreograph mitotic storage and redistribution of a growth factor receptor.

Endosomal trafficking of receptors and associated proteins plays a critical role in signal processing. Until recently, it was thought that trafficking was shut down during cell division. Thus, remarkably, the regulation of trafficking during division remains poorly characterized. Here we delineate the role of mitotic kinases in receptor trafficking during asymmetric division. Targeted perturbations reveal that Cyclin-dependent Kinase 1 (CDK1) and Aurora Kinase promote storage of Fibroblast Growth Factor Receptors (FGFRs) by suppressing endosomal degradation and recycling pathways. As cells progress through metaphase, loss of CDK1 activity permits differential degradation and targeted recycling of stored receptors, leading to asymmetric induction. Mitotic receptor storage, as delineated in this study, may facilitate rapid reestablishment of signaling competence in nascent daughter cells. However, mutations that limit or enhance the release of stored signaling components could alter daughter cell fate or behavior thereby promoting oncogenesis.

The Mitotic Checkpoint Complex Requires an Evolutionary Conserved Cassette to Bind and Inhibit Active APC/C.

The Spindle Assembly Checkpoint (SAC) ensures genomic stability by preventing sister chromatid separation until all chromosomes are attached to the spindle. It catalyzes the production of the Mitotic Checkpoint Complex (MCC), which inhibits Cdc20 to inactivate the Anaphase Promoting Complex/Cyclosome (APC/C). Here we show that two Cdc20-binding motifs in BubR1 of the recently identified ABBA motif class are crucial for the MCC to recognize active APC/C-Cdc20. Mutating these motifs eliminates MCC binding to the APC/C, thereby abolishing the SAC and preventing cells from arresting in response to microtubule poisons. These ABBA motifs flank a KEN box to form a cassette that is highly conserved through evolution, both in the arrangement and spacing of the ABBA-KEN-ABBA motifs, and association with the amino-terminal KEN box required to form the MCC. We propose that the ABBA-KEN-ABBA cassette holds the MCC onto the APC/C by binding the two Cdc20 molecules in the MCC-APC/C complex.

Ciona Brachyury proximal and distal enhancers have different FGF dose-response relationships.

Many genes are regulated by two or more enhancers that drive similar expression patterns. Evolutionary theory suggests that these seemingly redundant enhancers must have functionally important differences. In the simple ascidian chordate Ciona, the transcription factor Brachyury is induced exclusively in the presumptive notochord downstream of lineage specific regulators and FGF-responsive Ets family transcription factors. Here we exploit the ability to finely titrate FGF signaling activity via the MAPK pathway using the MEK inhibitor U0126 to quantify the dependence of transcription driven by different Brachyury reporter constructs on this direct upstream regulator. We find that the more powerful promoter-adjacent proximal enhancer and a weaker distal enhancer have fundamentally different dose-response relationships to MAPK inhibition. The Distal enhancer is more sensitive to MAPK inhibition but shows a less cooperative response, whereas the Proximal enhancer is less sensitive and more cooperative. A longer construct containing both enhancers has a complex dose-response curve that supports the idea that the proximal and distal enhancers are moderately super-additive. We show that the overall expression loss from intermediate doses of U0126 is not only a function of the fraction of cells expressing these reporters, but also involves graded decreases in expression at the single-cell level. Expression of the endogenous gene shows a comparable dose-response relationship to the full length reporter, and we find that different notochord founder cells are differentially sensitive to MAPK inhibition. Together, these results indicate that although the two Brachyury enhancers have qualitatively similar expression patterns, they respond to FGF in quantitatively different ways and act together to drive high levels of Brachyury expression with a characteristic input/output relationship. This indicates that they are fundamentally not equivalent genetic elements.

Differentially expressed chaperone genes reveal a stress response required for unidirectional regeneration in the basal chordate Ciona.

BACKGROUND: Unidirectional regeneration in the basal chordate Ciona intestinalis involves the proliferation of adult stem cells residing in the branchial sac vasculature and the migration of progenitor cells to the site of distal injury. However, after the Ciona body is bisected, regeneration occurs in the proximal but not in the distal fragments, even if the latter include a part of the branchial sac with stem cells. A transcriptome was sequenced and assembled from the isolated branchial sacs of regenerating animals, and the information was used to provide insights into the absence of regeneration in distal body fragments. RESULTS: We identified 1149 differentially expressed genes, which were separated into two major modules by weighted gene correlation network analysis, one consisting of mostly upregulated genes correlated with regeneration and the other consisting of only downregulated genes associated with metabolism and homeostatic processes. The hsp70, dnaJb4, and bag3 genes were among the highest upregulated genes and were predicted to interact in an HSP70 chaperone system. The upregulation of HSP70 chaperone genes was verified and their expression confirmed in BS vasculature cells previously identified as stem and progenitor cells. siRNA-mediated gene knockdown showed that hsp70 and dnaJb4, but not bag3, are required for progenitor cell targeting and distal regeneration. However, neither hsp70 nor dnaJb4 were strongly expressed in the branchial sac vasculature of distal fragments, implying the absence of a stress response. Heat shock treatment of distal body fragments activated hsp70 and dnaJb4 expression indicative of a stress response, induced cell proliferation in branchial sac vasculature cells, and promoted distal regeneration. CONCLUSIONS: The chaperone system genes hsp70, dnaJb4, and bag3 are significantly upregulated in the branchial sac vasculature following distal injury, defining a stress response that is essential for regeneration. The stress response is absent from distal fragments, but can be induced by a heat shock, which activates cell division in the branchial sac vasculature and promotes distal regeneration. This study demonstrates the importance of a stress response for stem cell activation and regeneration in a basal chordate, which may have implications for understanding the limited regenerative activities in other animals, including vertebrates.

A Novel Hemocyte-Derived Peptide and Its Possible Roles in Immune Response of Ciona intestinalis Type A.

A wide variety of bioactive peptides have been identified in the central nervous system and several peripheral tissues in the ascidian Ciona intestinalis type A (Ciona robusta). However, hemocyte endocrine peptides have yet to be explored. Here, we report a novel 14-amino-acid peptide, CiEMa, that is predominant in the granular hemocytes and unilocular refractile granulocytes of Ciona. RNA-seq and qRT-PCR revealed the high CiEma expression in the adult pharynx and stomach. Immunohistochemistry further revealed the highly concentrated CiEMa in the hemolymph of the pharynx and epithelial cells of the stomach, suggesting biological roles in the immune response. Notably, bacterial lipopolysaccharide stimulation of isolated hemocytes for 1-4 h resulted in 1.9- to 2.4-fold increased CiEMa secretion. Furthermore, CiEMa-stimulated pharynx exhibited mRNA upregulation of the growth factor (Fgf3/7/10/22), vanadium binding proteins (CiVanabin1 and CiVanabin3), and forkhead and homeobox transcription factors (Foxl2, Hox3, and Dbx) but not antimicrobial peptides (CrPap-a and CrMam-a) or immune-related genes (Tgfbtun3, Tnfa, and Il17-2). Collectively, these results suggest that CiEMa plays roles in signal transduction involving tissue development or repair in the immune response, rather than in the direct regulation of immune response genes. The present study identified a novel Ciona hemocyte peptide, CiEMa, which paves the way for research on the biological roles of hemocyte peptides in chordates.

Snail Transcriptionally Represses Brachyury to Promote the Mesenchymal-Epithelial Transition in Ascidian Notochord Cells.

Mesenchymal-epithelial transition (MET) is a widely spread and evolutionarily conserved process across species during development. In Ciona embryogenesis, the notochord cells undergo the transition from the non-polarized mesenchymal state into the polarized endothelial-like state to initiate the lumen formation between adjacent cells. Based on previously screened MET-related transcription factors by ATAC-seq and Smart-Seq of notochord cells, Ciona robusta Snail (Ci-Snail) was selected for its high-level expression during this period. Our current knockout results demonstrated that Ci-Snail was required for notochord cell MET. Importantly, overexpression of the transcription factor Brachyury in notochord cells resulted in a similar phenotype with failure of lumen formation and MET. More interestingly, expression of Ci-Snail in the notochord cells at the late tailbud stage could partially rescue the MET defect caused by Brachyury-overexpression. These results indicated an inverse relationship between Ci-Snail and Brachyury during notochord cell MET, which was verified by RT-qPCR analysis. Moreover, the overexpression of Ci-Snail could significantly inhibit the transcription of Brachyury, and the CUT&Tag-qPCR analysis demonstrated that Ci-Snail is directly bound to the upstream region of Brachyury. In summary, we revealed that Ci-Snail promoted the notochord cell MET and was essential for lumen formation via transcriptionally repressing Brachyury.

Proteomic identification of intracellular vesicle trafficking and protein glycosylation requirements for lumen inflation in Ciona notochord.

Lumen formation and inflation are crucial steps for tubular organ morphogenesis, yet the underling mechanism remains largely unrevealed. Here, we applied 4D proteomics to screen the lumenogenesis-related proteins and revealed the biological pathways potentially that are involved in lumen inflation during notochord lumen formation in the ascidian Ciona savignyi. In total, 910 differentiated expressed proteins (DEPs) were identified before and after notochord lumen formation utilizing Mfuzz analysis. Those DEPs were grouped into four upregulated clusters based on their quantitative expression patterns; the functions of these proteins were enriched in protein metabolic and biosynthetic process, the establishment of localization, and vesicle-mediated transport. We analyzed the vesicle trafficking cluster and focused on several vesicle transport hub proteins. In vivo function-deficient experiments showed that mutation of vesicle transport proteins resulted in an abnormal lumen in notochord development, demonstrating the crucial role of intracellular trafficking for lumen formation. Moreover, abundant extracellular matrix proteins were identified, the majority of which were predicted to be glycosylated proteins. Inhibition of glycosylation markedly reduced the lumen expansion rate in notochord cells, suggesting that protein glycosylation is essential for lumenogenesis. Overall, our study provides an invaluable resource and reveals the crucial mechanisms in lumen formation and expansion.

Repetitive and zonal expression profiles of absorption-related genes in the gastrointestinal tract of ascidian Ciona intestinalis type A.

Intestinal absorption is essential for heterotrophic bilaterians with a tubular gut. Although the fundamental features of the digestive system were shared among chordates with evolution, the gut morphologies of vertebrates diverged and adapted to different food habitats. The ascidian Ciona intestinalis type A, a genome-wide research model of basal chordates, is used to examine the functional morphology of the intestines because of its transparent juvenile body. In the present study, the characteristic gene expression patterns (GEP) of Ciona absorptive proteins, e.g., brush border membrane enzymes for terminal digestion (lactase, maltase, APA, and APN) and transporters (SGLT1, GLUT5, PEPT1, and B0AT1), were investigated in juveniles and young adults, with a special reference to the absorption of other nutrients by pinocytosis- and phagocytosis-related proteins (megalin, cubilin, amnionless, Dab2, Rab7, LAMP, cathepsins, and MRC1). Whole-mount in situ hybridization revealed that these GEP showed multi-regional and repetitive features along the Ciona gastrointestinal tract, mainly in the stomach and several regions of the intestines. In young adults, many absorption-related genes, including pinocytosis-/phagocytosis-related genes, were also expressed between the stomach and mid-intestine. In the gastrointestinal epithelium, absorption-related genes showed zonal GEP along the epithelial structure. Comparisons of GEP, including other intestinal functions, such as nutrient digestion and intestinal protection, indicated the repetitive assignment of a well-coordinated set of intestinal GEP in the Ciona gastrointestinal tract.