A resource of single-cell gene expression profiles in a planarian Dugesia japonica.

The freshwater planarian Dugesia japonica maintains an abundant heterogeneous cell population called neoblasts, which include adult pluripotent stem cells. Thus, it is an excellent model organism for stem cell and regeneration research. Recently, many single-cell RNA sequencing (scRNA-seq) databases of several model organisms, including other planarian species, have become publicly available; these are powerful and useful resources to search for gene expression in various tissues and cells. However, the only scRNA-seq dataset for D. japonica has been limited by the number of genes detected. Herein, we collected D. japonica cells, and conducted an scRNA-seq analysis. A novel, automatic, iterative cell clustering strategy produced a dataset of 3,404 cells, which could be classified into 63 cell types based on gene expression profiles. We introduced two examples for utilizing the scRNA-seq dataset in this study using D. japonica. First, the dataset provided results consistent with previous studies as well as novel functionally relevant insights, that is, the expression of DjMTA and DjP2X-A genes in neoblasts that give rise to differentiated cells. Second, we conducted an integrative analysis of the scRNA-seq dataset and time-course bulk RNA-seq of irradiated animals, demonstrating that the dataset can help interpret differentially expressed genes captured via bulk RNA-seq. Using the R package "Seurat" and GSE223927, researchers can easily access and utilize this dataset.

The complete dorsal structure is formed from only the blastocoel roof of Xenopus blastula: insight into the gastrulation movement evolutionarily conserved among chordates.

Gastrulation is a critical event whose molecular mechanisms are thought to be conserved among vertebrates. However, the morphological movement during gastrulation appears to be divergent across species, making it difficult to discuss the evolution of the process. Previously, we proposed a novel amphibian gastrulation model, the "subduction and zippering (S&Z) model". In this model, the organizer and the prospective neuroectoderm are originally localized in the blastula's blastocoel roof, and these embryonic regions move downward to make physical contact of their inner surfaces with each other at the dorsal marginal zone. The developmental stage when contact between the head organizer and the anterior-most neuroectoderm is established is called "anterior contact establishment (ACE)." After ACE, the A-P body axis elongates posteriorly. According to this model, the body axis is derived from limited regions of the dorsal marginal zone at ACE. To investigate this possibility, we conducted stepwise tissue deletions using Xenopus laevis embryos and revealed that the dorsal one-third of the marginal zone had the ability to form the complete dorsal structure by itself. Furthermore, a blastocoel roof explant of the blastula, which should contain the organizer and the prospective neuroectoderm in the S&Z model, autonomously underwent gastrulation and formed the complete dorsal structure. Collectively, these results are consistent with the S&Z gastrulation model and identify the embryonic region sufficient for construction of the complete dorsal structure. Finally, by comparing amphibian gastrulation to gastrulation of protochordates and amniotes, we discuss the gastrulation movement evolutionarily conserved among chordates.

Migratory regulation by MTA homologous genes is essential for the uniform distribution of planarian adult pluripotent stem cells.

The migration of adult stem cells in vivo is an important issue, but the complex tissue structures involved, and limited accessibility of the cells hinder a detailed investigation. To overcome these problems, the freshwater planarian Dugesia japonica was used because it has a simple body plan and abundant adult pluripotent stem cells (neoblasts) distributed uniformly throughout its body. To investigate the migratory mechanisms of neoblasts, two planarian homologous genes of metastatic tumor antigen (MTA-A and MTA-B), a protein involved in cancer metastasis that functions through histone deacetylation, were identified, and their function was analyzed using RNA interference (RNAi). MTA-A or MTA-B knockdown disrupted homeostatic tissue turnover and regeneration in planarians. Whereas neoblasts in MTA-A (RNAi) and MTA-B (RNAi) animals were maintained, neoblast differentiation was inhibited. Furthermore, the normal uniform neoblast distribution pattern changed to a branch-like pattern in MTA-A (RNAi) and MTA-B (RNAi) animals. To examine the neoblast migratory ability, a partial X-ray irradiation assay was performed in D. japonica. Using this assay system, the MTA-A knockdown neoblasts migrated collectively in a branch-like pattern, and the MTA-B knockdown neoblasts were not able to migrate. These results indicated that MTA-A was required for the exit of neoblasts from the branch-like region, and that MTA-B was required for neoblast migration. Thus, the migration mediated by MTA-A and MTA-B enabled uniform neoblast distribution and was required for neoblast differentiation to achieve tissue homeostasis and regeneration.

Proliferation maintains the undifferentiated status of stem cells: The role of the planarian cell cycle regulator Cdh1.

The coincidence of cell cycle exit and differentiation has been described in a wide variety of stem cells and organisms for decades, but the causal relationship is still unclear due to the complicated regulation of the cell cycle. Here, we used the planarian Dugesia japonica since they may possess a simple cell cycle regulation in which Cdh1 is one of the factors responsible for exiting the cell cycle. When cdh1 was functionally inhibited, the planarians could not maintain their tissue homeostasis and could not regenerate their missing body parts. While the knockdown of cdh1 caused pronounced accumulation of the stem cells, the progenitor and differentiated cells were decreased. Further analyses indicated that the stem cells with cdh1 knockdown did not undergo differentiation even though they received ERK signaling activation as an induction signal. These results suggested that stem cells could not acquire differentiation competence without cell cycle exit. Thus, we propose that cell cycle regulation determines the differentiation competence and that cell cycle exit to G0 enables stem cells to undergo differentiation.

Nucleotide receptor P2RY4 is required for head formation via induction and maintenance of head organizer in Xenopus laevis.

Vertebrates have unique head structures that are mainly composed of the central nervous system, the neural crest, and placode cells. These head structures are brought about initially by the neural induction between the organizer and the prospective neuroectoderm at early gastrula stage. Purinergic receptors are activated by nucleotides released from cells and influence intracellular signaling pathways, such as phospholipase C and adenylate cyclase signaling pathways. As P2Y receptor is vertebrate-specific and involved in head formation, we expect that its emergence may be related to the acquisition of vertebrate head during evolution. Here, we focused on the role of p2ry4 in early development in Xenopus laevis and found that p2ry4 was required for the establishment of the head organizer during neural induction and contributed to head formation. We showed that p2ry4 was expressed in the head organizer region and the prospective neuroectoderm at early gastrula stage, and was enriched in the head components. Disruption of p2ry4 function resulted in the small head phenotype and the reduced expression of marker genes specific for neuroectoderm and neural border at an early neurula stage. Furthermore, we examined the effect of p2ry4 disruption on the establishment of the head organizer and found that a reduction in the expression of head organizer genes, such as dkk1 and cerberus, and p2ry4 could also induce the ectopic expression of these marker genes. These results suggested that p2ry4 plays a key role in head organizer formation. Our study demonstrated a novel role of p2ry4 in early head development.

The Spemann organizer meets the anterior-most neuroectoderm at the equator of early gastrulae in amphibian species.

The dorsal blastopore lip (known as the Spemann organizer) is important for making the body plan in amphibian gastrulation. The organizer is believed to involute inward and migrate animally to make physical contact with the prospective head neuroectoderm at the blastocoel roof of mid- to late-gastrula. However, we found that this physical contact was already established at the equatorial region of very early gastrula in a wide variety of amphibian species. Here we propose a unified model of amphibian gastrulation movement. In the model, the organizer is present at the blastocoel roof of blastulae, moves vegetally to locate at the region that lies from the blastocoel floor to the dorsal lip at the onset of gastrulation. The organizer located at the blastocoel floor contributes to the anterior axial mesoderm including the prechordal plate, and the organizer at the dorsal lip ends up as the posterior axial mesoderm. During the early step of gastrulation, the anterior organizer moves to establish the physical contact with the prospective neuroectoderm through the "subduction and zippering" movements. Subduction makes a trench between the anterior organizer and the prospective neuroectoderm, and the tissues face each other via the trench. Zippering movement, with forming Brachet's cleft, gradually closes the gap to establish the contact between them. The contact is completed at the equator of early gastrulae and it continues throughout the gastrulation. After the contact is established, the dorsal axis is formed posteriorly, but not anteriorly. The model also implies the possibility of constructing a common model of gastrulation among chordate species.

Tail structure is formed when blastocoel roof contacts blastocoel floor in Xenopus laevis.

The tail organizer has been assessed by such transplantation methods as the Einsteck procedure. However, we found that simple wounding of blastocoel roof (BCR) made it possible to form secondary tails without any transplantation in Xenopus laevis. We revealed that the ectopic expression of Xbra was blocked by inhibiting the contact between BCR and blastocoel floor (BCF), and wounding per se seemed to be not directly related to the secondary tail formation. Therefore, the secondary tail might be induced by the contact between BCR and BCF due to the leak of blastocoel fluid from the wound. This secondary tail was similar to the original tail in the expression pattern of tail genes, and in the fact that the inhibition of fibroblast growth factor signaling prevented the secondary tail induction. Our results imply that the secondary tail formation reflects the developmental processes of the original tail, indicating that simple wounding of BCR is useful for the analysis of tail formation in normal development.

Xhairy2 functions in Xenopus lens development by regulating p27(xic1) expression.

Lens of vertebrate eyes is derived from competent pre-placodal ectoderm in response to signal(s) from retinal lineage. We herein report that the Xenopus Hes gene Xhairy2, which is expressed in pre-placodal ectoderm, is required for lens development from the initial stage. We show that Xhairy2 knockdown reduced the expression of lens marker genes at every step of lens determination, eventually resulting in ocular lens malformation. Interestingly, retina marker gene expression and retinal anlage morphology remained normal upon Xhairy2 knockdown. Furthermore, loss of lens field caused by Xhairy2 depletion was partially rescued by simultaneous knockdown of the cell cycle inhibitor gene p27(xic1). These results suggest that Xhairy2 is required for lens development through the regulation of p27(xic1) expression, independent of the known cascade of transcription factors. Based on these findings, we propose that Xhairy2 may maintain an intracellular environment in which inducing signal(s) can be accepted.

Xenopus hairy2 functions in neural crest formation by maintaining cells in a mitotic and undifferentiated state.

The neural crest is a population of mitotically active, multipotent progenitor cells that arise at the neural plate border. Neural crest progenitors must be maintained in a multipotent state until after neural tube closure. However, the molecular underpinnings of this process have yet to be fully elucidated. Here we show that the basic helix-loop-helix (bHLH) transcriptional repressor gene, Xenopus hairy2 (Xhairy2), is an essential early regulator of neural crest formation in Xenopus. During gastrulation, Xhairy2 is localized at the presumptive neural crest prior to the expression of such neural crest markers as Slug and FoxD3. Morpholino-mediated knockdown of Xhairy2 results in the repression of neural crest marker gene expression while inducing the ectopic expression of the cell cycle inhibitor p27(xic1) in the presumptive neural crest. We also found that ectopic p27(xic1) disturbs neural crest formation. Furthermore, the depletion of Xhairy2 leads to the apoptosis of mitotic cells. Our results suggest that Xhairy2 functions in neural crest specification by maintaining cells in the mitotic and undifferentiated state.

Two modes of action by which Xenopus hairy2b establishes tissue demarcation in the Spemann-Mangold organizer.

The Hairy and Enhancer-of-Split (HES) family of transcriptional repressors plays important roles in pattern formation during development throughout the animal kingdom. Generally, HES proteins repress the expression of genes specific for neighboring tissues to maintain the nature of cells expressing HES proteins, resulting in pattern formation. Xhairy2b, a Xenopus HES, establishes the prospective anterior prechordal mesoderm identity in the Spemann-Mangold organizer by both inducing specific genes and repressing the genes specific for neighboring tissues. Here we report that Xhairy2b has two modes of action, each of which corresponds to inductive and repressive functions. We show that the inductive function is independent of direct transcriptional regulation and is exhibited by the C-terminal WRPW tetrapeptide motif alone, although it induces the expression of a wide variety of the organizer genes that Xhairy2b represses. The transcriptional repression by Xhairy2b is responsible for only the repressive function. We propose that the activity of the WRPW motif intrinsically induces the expression of genes specific for the organizer in a rather non-specific manner to ensure the organizer environment. Then, the transcriptional repression selectively down-regulates the expression of some of these genes, resulting in the regionalization of the axial mesoderm. Our study provides new insight into how a region of the vertebrate embryo is demarcated by one dual-functional transcription factor in the early stages of development.

Xenopus hairy2b specifies anterior prechordal mesoderm identity within Spemann's organizer.

Spemann's organizer is a region of the gastrula stage embryo that contains future anterior endodermal and dorsal mesodermal tissues. During gastrulation, the dorsal mesoderm is divided into the prechordal mesoderm and the chordamesoderm. However, little is known regarding how this division is established. We analyzed the role of the anterior prechordal mesoderm-specific gene Xhairy2b in the regionalization of the organizer. We found that mesoderm-inducing transforming growth factor-beta signaling induced Xhairy2b expression. On the other hand, the ectopic expression of Xhairy2b induced the expression of organizer-specific genes and resulted in the formation of a secondary dorsal axis lacking head and notochord structures. We also showed that Xhairy2b down-regulated the expression of ventral mesodermal, anterior endodermal, and chordamesodermal genes. In Xhairy2b-depleted embryos, defects in the specification of anterior prechordal mesoderm identity were observed as the border between the prechordal mesoderm and the chordamesoderm was anteriorly shifted. These results suggest that Xhairy2b establishes the identity of the anterior prechordal mesoderm within Spemann's organizer by inhibiting the formation of neighboring tissues.

Choice of either beta-catenin or Groucho/TLE as a co-factor for Xtcf-3 determines dorsal-ventral cell fate of diencephalon during Xenopus development.

Co-repressor Groucho/Transducin-Like Enhancer of split (TLE) interacts with transcription factors that are expressed in the central nervous system (CNS), and regulates transcriptional activities. In this study, we examined the contribution of Groucho/TLE to CNS development in Xenopus. The functional inhibition of Groucho/TLE using the WRPW motif as a competitor resulted in the conversion of the ventral cell into the dorsal fate in the prospective diencephalon. We also found that the neural plate was expanded laterally without inhibiting neural crest development. In tailbud, the disturbance of trigeminal ganglion development was observed. These observations allow us to conclude that Groucho/TLE plays important roles in the induction and patterning of distinct CNS territories. We found that Xtcf-3 is involved in some of the patterning in these territories. We generated the variant of Xtcf-3, Xtcf-3BDN-, which is suspected to interfere with the interaction between endogenous Groucho/TLE and Xtcf-3. The transcriptional activation of the Xtcf-3-target genes in response to endogenous Wnt/beta-catenin signaling by the overexpression of Xtcf-3BDN- led to a reduction of the ventral diencephalon. This result indicates that transcriptional repression by the Groucho/TLE-Xtcf-3 complex is important for ventral diencephalon patterning. This idea is supported by the finding that the overexpression of the dominant-negative form of Xtcf-3 or axil causes the expansion of the ventral diencephalon. Based on these data, we propose that the localized activation of Wnt/beta-catenin signaling, which converts Tcf from a repressor to an activator, is required for the establishment of dorsal-ventral patterning in the prospective diencephalon.

Expression pattern of a basic helix-loop-helix transcription factor Xhairy2b during Xenopus laevis development.

We report on the temporal and spatial expression pattern of the Xenopus laevis hairy2b ( Xhairy2b) transcription factor. Xhairy2b transcripts are present maternally, and expressed throughout the prospective ectoderm prior to the gastrula stage. During gastrulation, Xhairy2b expression is restricted to the deep layer of the Spemann organizer and three distinct regions in the prospective neuroectoderm, neural plate border, notoplate and anterior neural plate. At later stages, Xhairy2b expression is localized to prechordal plate, presomitic mesoderm, neural tube, neural crest derivatives and several tissue territories of the central nervous system. The analyses of Xhairy2b and several other hairy-related genes suggest potential roles for Xhairy2b in the formation of boundaries in neural tissue territories.

When does the anterior endomesderm meet the anterior-most neuroectoderm during Xenopus gastrulation?

During amphibian gastrulation, the anterior endomesoderm is thought to move forward along the inner surface of the blastocoel roof toward the animal pole where it comes into physical contact with the anterior-most portion of the prospective head neuroectoderm (PHN), and it is also believed that this physical interaction occurs during the mid-gastrula stage. However, using Xenopus embryos we found that the interaction between the anterior endomesoderm and the PHN occurs as early as stage 10.25 and the blastocoel roof ectoderm at this stage contributed only to the epidermal tissue. We also found that once the interaction was established, these tissues continued to associate in register and ultimately became the head structures. From these findings, we propose a new model of Xenopus gastrulation. The anterior endomesoderm migrates only a short distance on the inner surface of the blastocoel roof during very early stages of gastrulation (by stage 10.25). Then, axial mesoderm formation occurs, beginning dorsally (anterior) and progressing ventrally (posterior) to complete gastrulation. This new view of Xenopus gastrulation makes it possible to directly compare vertebrate gastrulation movements.