regeneration factors expressed on myeloid expression in macrophage-like cells is required for tail regeneration in Xenopus laevis tadpoles.

Xenopus laevis tadpoles can regenerate whole tails after amputation. We have previously reported that interleukin 11 (il11) is required for tail regeneration. In this study, we have screened for genes that support tail regeneration under Il11 signaling in a certain cell type and have identified the previously uncharacterized genes Xetrov90002578m.L and Xetrov90002579m.S [referred to hereafter as regeneration factors expressed on myeloid.L (rfem.L) and rfem.S]. Knockdown (KD) of rfem.L and rfem.S causes defects of tail regeneration, indicating that rfem.L and/or rfem.S are required for tail regeneration. Single-cell RNA sequencing (scRNA-seq) revealed that rfem.L and rfem.S are expressed in a subset of leukocytes with a macrophage-like gene expression profile. KD of colony-stimulating factor 1 (csf1), which is essential for macrophage differentiation and survival, reduced rfem.L and rfem.S expression levels and the number of rfem.L- and rfem.S-expressing cells in the regeneration bud. Furthermore, forced expression of rfem.L under control of the mpeg1 promoter, which drives rfem.L in macrophage-like cells, rescues rfem.L and rfem.S KD-induced tail regeneration defects. Our findings suggest that rfem.L or rfem.S expression in macrophage-like cells is required for tail regeneration.

Effective enrichment of stem cells in regenerating Xenopus laevis tadpole tails using the side population method.

Xenopus laevis tadpoles have a strong regenerative ability and can regenerate their whole tails after tail amputation. Lineage-restricted tissue stem cells are thought to provide sources for the regenerating tissues by producing undifferentiated progenitor cells in response to tail amputation. However, elucidating the behavioral dynamics of tissue stem cells during tail regeneration is difficult because of their rarity, and there are few established methods of isolating these cells in amphibians. Here, to detect and analyze rare tissue stem cells, we attempted to enrich tissue stem cells from tail regeneration buds. High Hoechst dye efflux capacity is thought to be a common characteristic of several types of mammalian tissue stem cells; these stem cells, designated as the "side population (SP)," may be enriched by flow cytometry (SP method). To evaluate the effectiveness of stem cell enrichment using the SP method in regenerating X. laevis tadpole tails, we performed single-cell RNA sequencing (scRNA-seq) of SP cells from regeneration buds and analyzed the frequency of satellite cells, which are muscle stem/progenitor cells expressing pax7. The pax7-expressing cells were enriched in the SP compared with whole normal tails and regeneration buds. Furthermore, hes1-expressing cells, which are assumed to be neural stem/progenitor cells, were also enriched in the SP. Our findings suggest that the SP method is efficient for successfully enriching tissue stem cells in regenerating X. laevis tadpole tails, indicating that the combination of the SP method and scRNA-seq is useful for studying tissue stem cells that contribute to tail regeneration.

Cellular responses in the FGF10-mediated improvement of hindlimb regenerative capacity in Xenopus laevis revealed by single-cell transcriptomics.

Xenopus laevis tadpoles possess regenerative capacity in their hindlimb buds at early developmental stages (stages ~52-54); they can regenerate complete hindlimbs with digits after limb bud amputation. However, they gradually lose their regenerative capacity as metamorphosis proceeds. Tadpoles in late developmental stages regenerate fewer digits (stage ~56), or only form cartilaginous spike without digits or joints (stage ~58 or later) after amputation. Previous studies have shown that administration of fibroblast growth factor 10 (FGF10) in late-stage (stage 56) tadpole hindlimb buds after amputation can improve their regenerative capacity, which means that the cells responding to FGF10 signaling play an important role in limb bud regeneration. In this study, we performed single-cell RNA sequencing (scRNA-seq) of hindlimb buds that were amputated and administered FGF10 by implanting FGF10-soaked beads at a late stage (stage 56), and explored cell clusters exhibiting a differential gene expression pattern compared with that in controls treated with phosphate-buffered saline. The scRNA-seq data showed expansion of fgf8-expressing cells in the cluster of the apical epidermal cap of FGF10-treated hindlimb buds, which was reported previously, indicating that the administration of FGF10 was successful. On analysis, in addition to the epidermal cluster, a subset of myeloid cells and a newly identified cluster of steap4-expressing cells showed remarkable differences in their gene expression profiles between the FGF10- or phosphate-buffered saline-treatment conditions, suggesting a possible role of these clusters in improving the regenerative capacity of hindlimbs via FGF10 administration.

Xenopus laevis il11ra.L is an experimentally proven interleukin-11 receptor component that is required for tadpole tail regeneration.

Xenopus laevis tadpoles possess high regenerative ability and can regenerate functional tails after amputation. An early event in regeneration is the induction of undifferentiated cells that form the regenerated tail. We previously reported that interleukin-11 (il11) is upregulated immediately after tail amputation to induce undifferentiated cells of different cell lineages, indicating a key role of il11 in initiating tail regeneration. As Il11 is a secretory factor, Il11 receptor-expressing cells are thought to mediate its function. X. laevis has a gene annotated as interleukin 11 receptor subunit alpha on chromosome 1L (il11ra.L), a putative subunit of the Il11 receptor complex, but its function has not been investigated. Here, we show that nuclear localization of phosphorylated Stat3 induced by Il11 is abolished in il11ra.L knocked-out culture cells, strongly suggesting that il11ra.L encodes an Il11 receptor component. Moreover, knockdown of il11ra.L impaired tadpole tail regeneration, suggesting its indispensable role in tail regeneration. We also provide a model showing that Il11 functions via il11ra.L-expressing cells in a non-cell autonomous manner. These results highlight the importance of il11ra.L-expressing cells in tail regeneration.

Understanding and optimization of hard magnetic compounds from first principles.

First-principles calculation based on density functional theory is a powerful tool for understanding and designing magnetic materials. It enables us to quantitatively describe magnetic properties and structural stability, although further methodological developments for the treatment of strongly correlated 4f electrons and finite-temperature magnetism are needed. Here, we review recent developments of computational schemes for rare-earth magnet compounds, and summarize our theoretical studies on Nd2Fe14B and RFe12-type compounds. Effects of chemical substitution and interstitial dopants are clarified. We also discuss how data-driven approaches are used for studying multinary systems. Chemical composition can be optimized with fewer trials by the Bayesian optimization. We also present a data-assimilation method for predicting finite-temperature magnetization in wide composition space by integrating computational and experimental data.

Low-temperature incubation improves both knock-in and knock-down efficiencies by the CRISPR/Cas9 system in Xenopus laevis as revealed by quantitative analysis.

The recent development of the CRISPR/Cas9-mediated gene editing technique has provided various gene knock-down and knock-in methods for Xenopus laevis. Gene-edited F0 individuals created by these methods, however, are mosaics with both mutated/knocked-in and unedited wild-type cells, and therefore precise determination and higher efficiency of knock-down and knock-in methods are desirable, especially for analyses of F0 individuals. To clarify the ratio of cells that are gene-edited by CRISPR/Cas9 methods to the whole cells in F0 individuals, we subjected Inference of CRISPR Edits analysis for knock-down experiments and flow cytometry for knock-in experiments to the F0 individuals. With these quantitative methods, we showed that low-temperature incubation of X. laevis embryos after microinjection improved the mutation rate in the individuals. Moreover, we applied low-temperature incubation when using a knock-in method with long single-strand DNA and found improved knock-in efficiency. Our results provide a simple and useful way to evaluate and improve the efficiency of gene editing in X. laevis.

interleukin-11 induces and maintains progenitors of different cell lineages during Xenopus tadpole tail regeneration.

Unlike mammals, Xenopus laevis tadpoles possess high ability to regenerate their lost organs. In amphibians, the main source of regenerated tissues is lineage-restricted tissue stem cells, but the mechanisms underlying induction, maintenance and differentiation of these stem/progenitor cells in the regenerating organs are poorly understood. We previously reported that interleukin-11 (il-11) is highly expressed in the proliferating cells of regenerating Xenopus tadpole tails. Here, we show that il-11 knockdown (KD) shortens the regenerated tail length, and the phenotype is rescued by forced-il-11-expression in the KD tadpoles. Moreover, marker genes for undifferentiated notochord, muscle, and sensory neurons are downregulated in the KD tadpoles, and the forced-il-11-expression in intact tadpole tails induces expression of these marker genes. Our findings demonstrate that il-11 is necessary for organ regeneration, and suggest that IL-11 plays a key role in the induction and maintenance of undifferentiated progenitors across cell lineages during Xenopus tail regeneration. Xenopus laevis tadpoles have maintained their ability to regenerate various organs. Here, the authors show that interleukin-11 is necessary for organ regeneration, by inducing and maintaining undifferentiated progenitors across cell lineages during Xenopus tail regeneration.

Acute phase response in amputated tail stumps and neural tissue-preferential expression in tail bud embryos of the Xenopus neuronal pentraxin I gene.

Regeneration of lost organs involves complex processes, including host defense from infection and rebuilding of lost tissues. We previously reported that Xenopus neuronal pentraxin I (xNP1) is expressed preferentially in regenerating Xenopus laevis tadpole tails. To evaluate xNP1 function in tail regeneration, and also in tail development, we analyzed xNP1 expression in tailbud embryos and regenerating/healing tails following tail amputation in the 'regeneration' period, as well as in the 'refractory' period, when tadpoles lose their tail regenerative ability. Within 10 h after tail amputation, xNP1 was induced at the amputation site regardless of the tail regenerative ability, suggesting that xNP1 functions in acute phase responses. xNP1 was widely expressed in regenerating tails, but not in the tail buds of tailbud embryos, suggesting its possible role in the immune response/healing after an injury. xNP1 expression was also observed in neural tissues/primordia in tailbud embryos and in the spinal cord in regenerating/healing tails in both periods, implying its possible roles in neural development or function. Moreover, during the first 48 h after amputation, xNP1 expression was sustained at the spinal cord of tails in the 'regeneration' period tadpoles, but not in the 'refractory' period tadpoles, suggesting that xNP1 expression at the spinal cord correlates with regeneration. Our findings suggest that xNP1 is involved in both acute phase responses and neural development/functions, which is unique compared to mammalian pentraxins whose family members are specialized in either acute phase responses or neural functions.

Phyhd1, an XPhyH-like homologue, is induced in mouse T cells upon T cell stimulation.

We previously identified XPhyH-like as a gene whose expression is enhanced in Xenopus blood cells during the refractory period, in which Xenopus tadpoles transiently lose their tail regenerative ability. Although we hypothesized that some autoreactive immune cells attack tail blastemal cells during the refractory period and XPhyH-like expressing immune cells were involved in the process, the nature of cells expressing XPhyH-like remain unknown, partly due to the lack of leukocyte markers available in Xenopus. In the present study, we used mice to analyze the expression pattern of XPhyH-like homologues. When we used quantitative reverse transcription-polymerase chain reaction (RT--PCR) to analyze the expression of mouse Phyhd1, an XPhyH-like orthologue, and Phyh, a Phyhd1 paralogue, both Phyhd1 and Phyh showed similar tissue-specific expression patterns. The expression pattern in leukocytes, however, differed between Phyhd1 and Phyh; Phyhd1 was considerably expressed in T cells and B cells. Moreover, the expression of Phyhd1 in T cells was up-regulated for approximately 3- to 7-times by T cell stimulation 3-4 days after the stimulation, unlike Phyh. Our findings suggest that Phyhd1 and Phyh have distinct roles in mouse leukocytes and Phyhd1 is related to T cell differentiation and/or function of effector T cells.

Egress of mature murine regulatory T cells from the thymus requires RelA.

The mechanism of egress of mature regulatory T cells (Tregs) from the thymus to the periphery remains enigmatic, as does the nature of those factors expressed in the thymic environment. In this study, we examined the fate of thymic Tregs in TNF-α/RelA double-knockout (TA-KO) mice, because TA-KO mice retain a Treg population in the thymus but have only a small Treg population at the periphery. Transplantation of whole TA-KO thymus to under the kidney capsule of Rag1-null mice failed to induce the production of donor-derived splenic Tregs expressing neuropilin-1, which is reported to be a marker of naturally occurring Tregs, indicating that TA-KO thymic Tregs either do not leave the thymus or are lost at the periphery. We next transplanted enriched TA-KO thymic Tregs to the peripheries of TA-KO mice and traced mouse survival. Transplantation of TA-KO thymic Tregs rescued the lethality in TA-KO mice, demonstrating that TA-KO thymic Tregs remained functional at the periphery. The TA-KO thymic Treg population had highly demethylated CpG motifs in the foxp3 locus, indicating that the cells were arrested at a late mature stage. Also, the population included a large subpopulation of Tregs expressing IL-7Rα, which is a possible marker of late-stage mature Tregs. Finally, TA-KO fetal liver chimeric mice developed a neuropilin-1(+) splenic Treg population from TA-KO cells, suggesting that Treg arrest was caused by a lack of RelA in the thymic environment. Taken together, these results suggest that egress of mature Tregs from the thymus depends on RelA in the thymic environment.

Optimized effective potential method and application to static RPA correlation.

The optimized effective potential (OEP) method is a promising technique for calculating the ground state properties of a system within the density functional theory. However, it is not widely used as its computational cost is rather high and, also, some ambiguity remains in the theoretical framework. In order to overcome these problems, we first introduced a method that accelerates the OEP scheme in a static RPA-level correlation functional. Second, the Krieger-Li-Iafrate (KLI) approximation is exploited to solve the OEP equation. Although seemingly too crude, this approximation did not reduce the accuracy of the description of the magnetic transition metals (Fe, Co, and Ni) examined here, the magnetic properties of which are rather sensitive to correlation effects. Finally, we reformulated the OEP method to render it applicable to the direct RPA correlation functional and other, more precise, functionals. Emphasis is placed on the following three points of the discussion: (i) level-crossing at the Fermi surface is taken into account; (ii) eigenvalue variations in a Kohn-Sham functional are correctly treated; and (iii) the resultant OEP equation is different from those reported to date.

NF-κB RELA-deficient bone marrow macrophages fail to support bone formation and to maintain the hematopoietic niche after lethal irradiation and stem cell transplantation.

Bone remodeling and hematopoiesis are interrelated and bone marrow (BM) macrophages are considered to be important for both bone remodeling and maintenance of the hematopoietic niche. We found that NF-κB Rela-deficient chimeric mice, generated by transplanting Rela (-/-) fetal liver cells into lethally irradiated hosts, developed severe osteopenia, reduced lymphopoiesis and enhanced mobilization of hematopoietic stem and progenitor cells when BM cells were completely substituted by Rela-deficient cells. Rela (-/-) hematopoietic stem cells from fetal liver had normal hematopoietic ability, but those harvested from the BM of osteopenic Rela (-/-) chimeric mice had reduced repopulation ability, indicating impairment of the microenvironment for the hematopoietic niche. Osteopenia in Rela (-/-) chimeric mice was due to reduced bone formation, even though osteoblasts differentiated from host cells. This finding indicates impaired functional coupling between osteoblasts and hematopoietic stem cell-derived cells. Rela-deficient BM macrophages exhibited an aberrant inflammatory phenotype, and transplantation with wild-type F4/80(+) BM macrophages recovered bone formation and ameliorated lymphopoiesis in Rela (-/-) chimeric mice. Therefore, RELA in F4/80(+) macrophages is important both for bone homeostasis and for maintaining the hematopoietic niche after lethal irradiation and hematopoietic stem cell transplantation.

Expression analysis of XPhyH-like during development and tail regeneration in Xenopus tadpoles: possible role of XPhyH-like expressing immune cells in impaired tail regenerative ability.

Xenopus tadpoles have high regenerative ability of amputated tails except during the 'refractory period', when the ability is transiently lost. We previously demonstrated that distinct immune responses occur in tail stumps between the refractory and pre/post-refractory regeneration periods. Furthermore, treatment with an immunosuppressant, FK506, restores the tail regenerative ability during the refractory period. Based on these findings, we previously proposed that autoreactive immune cells infiltrate the tail stumps to attack blastema cells as 'non-self' during the refractory period, resulting in the impaired regenerative ability. The immune cells that attack the blastema cells, however, remained unclear. Here we screened for genes whose expression in the tail stumps was altered by FK506 treatment during the refractory period and identified a Xenopus homolog of phytanoyl-CoA dioxygenase (PhyH)-like. XPhyH-like expression transiently increased in tail stumps after amputation during the refractory period, and was reduced by FK506 treatment. XPhyH-like expression in the whole tadpole body specifically increased during the refractory period and was enriched in the blood cell fraction. These findings suggest that XPhyH-like is expressed in autoreactive immune cells that are distributed in the whole body during the refractory period and transiently infiltrate the tail stumps to attack the blastema cells as 'non-self'.

Suppression of the immune response potentiates tadpole tail regeneration during the refractory period.

Regenerative ability varies depending on animal species and developmental stage, but the factors that determine this variability remain unclear. Although Xenopus laevis tadpole tails possess high regenerative ability, this is transiently lost during the ;refractory period'. Here, we show that tail amputation evokes different immune responses in wound tail stumps between the ;refractory' and ;regeneration' periods: there was delayed or prolonged expression of some immune-related genes in the refractory period, whereas there was no obvious or transient expression of other immune-related genes in the regeneration periods. In addition, immune suppression induced by either immunosuppressant treatment or immune cell depletion by knockdown of PU.1 significantly restored regenerative ability during the refractory period. These findings indicate that immune responses have a crucial role in determining regenerative ability in Xenopus tadpole tails.

Early fin primordia of zebrafish larvae regenerate by a similar growth control mechanism with adult regeneration.

Some vertebrate species, including urodele amphibians and teleost fish, have the remarkable ability of regenerating lost body parts. Regeneration studies have been focused on adult tissues, because it is unclear whether or not the repairs of injured tissues during early developmental stages have the same molecular base as that of adult regeneration. Here, we present evidence that a similar cellular and molecular mechanism to adult regeneration operates in the repair process of early zebrafish fin primordia, which are composed of epithelial and mesenchymal cells. We show that larval fin repair occurs through the formation of wound epithelium and blastema-like proliferating cells. Cell proliferation is first induced in the distal-most region and propagates to more proximal regions, as in adult regeneration. We also show that fibroblast growth factor signaling helps induce cell division. Our results suggest that the regeneration machinery directing cell proliferation in response to injury may exist from the early developmental stages.