深澤 太郎

ABSTRACT 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.

Abstract 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.

Abstract 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.

Abstract Xenopus laevistadpoles 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 thatinterleukin-11(il11) is upregulated immediately after tail amputation to induce undifferentiated cells of different cell lineages, indicating a key role ofil11in initiating tail regeneration. As Il11 is a secretory factor, Il11 receptor-expressing cells are thought to mediate its function.X. laevishas a gene annotated asinterleukin 11 receptor subunit alphaon 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 inil11ra.Lknocked-out culture cells, strongly suggesting thatil11ra.Lencodes an Il11 receptor component. Moreover, knockdown ofil11ra.Limpaired tadpole tail regeneration, suggesting its indispensable role in tail regeneration. We also provide a model showing that Il11 functions viail11ra.L-expressing cells in a non-cell autonomous manner. These results highlight the importance ofil11ra.L-expressing cells in tail regeneration.

Abstract Unlike mammals,Xenopus laevistadpoles 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 thatinterleukin-11(il-11) is highly expressed in the proliferating cells of regeneratingXenopustadpole tails. Here, we show thatil-11knockdown (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 thatil-11is necessary for organ regeneration, and suggest that IL-11 plays a key role in the induction and maintenance of undifferentiated progenitors across cell lineages duringXenopustail regeneration.

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.

Abstract 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.

Abstract 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.

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.

Abstract 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. Developmental Dynamics 231:693–699, 2004. © 2004 Wiley‐Liss, Inc.