Depletion of Neural Crest-Derived Cells Leads to Plasma Noradrenaline Decrease and Alters T Cell Development.

The differentiation of neural crest (NC) cells into various cell lineages contributes to the formation of many organs, including the thymus. In this study, we explored the role of NC cells in thymic T cell development. In double-transgenic mice expressing NC-specific Cre and the Cre-driven diphtheria toxin receptor, plasma noradrenaline and adrenaline levels were significantly reduced, as were thymic T cell progenitors, when NC-derived cells were ablated with short-term administration of diphtheria toxin. Additionally, yellow fluorescent protein+ NC-derived mesenchymal cells, perivascular cells, and tyrosine hydroxylase+ sympathetic nerves in the thymus significantly decreased. Furthermore, i.p. administration of 6-hydroxydopamine, a known neurotoxin for noradrenergic neurons, resulted in a significant decrease in thymic tyrosine hydroxylase+ nerves, a phenotype similar to that of depleted NC-derived cells, whereas administration of a noradrenaline precursor for ablating NC-derived cells or sympathetic nerves rarely rescued this phenotype. To clarify the role of NC-derived cells in the adult thymus, we transplanted thymus into the renal capsules of wild-type mice and observed abnormal T cell development in lethally irradiated thymus with ablation of NC-derived cells or sympathetic nerves, suggesting that NC-derived cells inside and outside of the thymus contribute to T cell development. In particular, the ablation of NC-derived mesenchymal cells in the thymus decreases the number of thymocytes and T cell progenitors. Overall, ablation of NC-derived cells, including sympathetic nerves, in the thymus leads to abnormal T cell development in part by lowering plasma noradrenalin levels. This study reveals that NC-derived cells including mesenchymal cells and sympathetic nerves within thymus regulate T cell development.

Multiple cell populations generate macrophage progenitors in the early yolk sac.

Yolk sac (YS) CSF1 receptor positive (CSF1R+) cells are thought to be the progenitors for tissue-resident macrophages present in various tissues. The YS progenitors for tissue-resident macrophages are referred to as erythroid-myeloid progenitors (EMPs). However, diverse types of hematopoietic progenitors are present in the early YS, thus it is not precisely known which type of hematopoietic cell gives rise to the CSF1R+ lineage. In this study, an analysis was conducted to determine when CSF1R+ progenitors appeared in the early YS. It showed that CSF1R+ cells appeared in the YS as early as embryonic day 9 (E9) and that the earliest hematopoietic progenitors that differentiate into CSF1R+ cells were found in E8. Since these progenitors possessed the capability to generate primitive erythroid cells, it was likely that primitive erythroid lineages shared progenitors with the CSF1R+ lineage. Mutual antagonism appears to work between PU.1 and GATA1 when CSF1R+ cells appear in the early YS. One day later (E9), multiple progenitors, including myeloid-restricted progenitors and multipotent progenitors, in the YS could immediately generate CSF1R+ cells. These results suggest that EMPs are not an exclusive source for the CSF1R+ lineage; rather, multiple hematopoietic cell populations give rise to CSF1R+ lineage in the early YS.

Isolation of CD35+ follicular dendritic cells and its role in the differentiation from B cells to IgA+GL7+ cells.

Follicular dendritic cells (FDCs) are non-hematopoietic cells that are localized in the germinal centers (GCs) of lymph nodes (LNs) and are involved in humoral immunity. FDCs are a rare population that are sensitive to mechanical and chemical stimuli, making their isolation for analysis difficult. In Peyer's Patches, which are the main IgA-inductive sites, FDCs have been reported to be activated by retinoic acid receptor (RAR) and toll-like receptor (TLR) signals to induce IgA production. However, little is known about FDCs in mesenteric LNs (MLNs), although MLNs are also an IgA-inductive site. In this study, we efficiently isolated FDCs as CD35+ cells using anti-CD35 antibodies (Abs) and magnetic bead sorting. We found that CD35+ FDCs facilitated differentiation from B220+ B cells into IgA+GL7+ GC B-like cells but not IgA+CD138+ plasma cells. Furthermore, using CD35+ FDCs from LPS-resistant C3H/HeJ mice, the generation of IgA+GL7+ GC B-like cells was not altered significantly between wild-type and LPS-resistant mice. Moreover, the addition of RAR antagonists and agonists revealed that differentiation into IgA+GL7+ GC B-like cells required the activation of RAR, especially RAR-ß, in FDCs. The differentiation of IgA+GL7+ cells was promoted by FDCs in peripheral LNs as well as MLNs in our in vitro assay. Taken together, these results indicate that magnetic bead sorting with anti-CD35 Abs enable the efficient isolation of FDCs. Our data suggested that CD35+ FDCs can support differentiation of B cells into IgA+GL7+ GC B-like cells in environments that are not limited to MLNs, which can be stimulated by retinoic acid.

Inflammatory Skin-Derived Cytokines Accelerate Osteoporosis in Mice with Persistent Skin Inflammation.

Secondary osteoporosis can also be caused by chronic inflammatory skin disease as well as rheumatoid arthritis or inflammatory bowel disease. However, the exact role of osteoporosis in inflammatory skin conditions has not been elucidated. Using a mouse model of dermatitis, we investigated the pathophysiology of osteoporosis in inflammatory skin conditions and the therapeutic impact of osteoporosis medication on inflammatory skin disease. We employed model mice of spontaneous skin inflammation, specifically overexpressing human caspase-1 in the epidermis. Bone density and the expression of various mRNAs in the femur were examined by micro CT and RT-PCR. The effects of minodronate and anti-RANKL antibody on bone structure, histology, and femur blood flow were studied. The mouse model of skin inflammation showed a marked decrease in bone density compared to wild-type littermates with abnormalities in both bone resorption and formation. Minodronate improved bone density by decreasing osteoclasts, but anti-RANKL antibody did not improve. In the dermatitis model, the blood flow in the bone marrow was decreased, and minodronate restored this parameter. A model of persistent dermatitis exhibited marked osteoporosis, but the impact of chronic dermatitis on osteoporosis has not been thoroughly investigated. We should explore the pathogenesis of osteoporosis in skin inflammatory diseases.

Repression of Primitive Erythroid Program Is Critical for the Initiation of Multi-Lineage Hematopoiesis in Mouse Development.

Formation of the hematopoietic cells occurs in multiple steps. The first hematopoietic cells observed during ontogeny are primitive erythrocytes, which are produced in the early yolk sac within a limited temporal window. Multi-lineage hematopoiesis, which supplies almost the entire repertoire of blood cell lineages, lags behind primitive erythropoiesis in the tissue. However, molecular mechanisms regulating sequential generation of primitive erythrocytes and multipotent hematopoietic progenitors in the yolk sac are largely unknown. In this study, the transcription factors involved in the development of hematopoietic cells were examined in purified progenitor cell populations from pluripotent stem cell cultures and from the yolk sac of developing embryos. We found that the earliest committed hematopoietic progenitors highly expressed Gata1, Scl/tal1, and Klf1 genes. Expression of these transcription factors, which is known to form a core erythroid transcriptional network, explained the prompt generation of primitive erythrocytes from these earliest progenitors. Importantly, the multipotent hematopoietic cells, which lack the differentiation potential into primitive erythroid cells, down-regulated these genes during a transition from the earliest committed progenitors. In addition, we showed that Pu.1 is involved in the multipotent cell differentiation through the suppression of erythroid transcription program. We propose that these molecular mechanisms governed by transcription factors form sequential waves of primitive erythropoiesis and multi-lineage hematopoiesis in the early yolk sac of developing embryos. J. Cell. Physiol. 232: 323-330, 2017. © 2016 Wiley Periodicals, Inc.

Depletion of Neural Crest-Derived Cells Leads to Reduction in Plasma Noradrenaline and Alters B Lymphopoiesis.

Hematopoietic stem cells and their lymphoid progenitors are supported by the bone marrow (BM) microenvironmental niches composed of various stromal cells and Schwann cells and sympathetic nerve fibers. Although neural crest (NC) cells contribute to the development of all the three, their function in BM is not well understood. In this study, NC-derived cells were ablated with diphtheria toxin in double-transgenic mice expressing NC-specific Cre and Cre-driven diphtheria toxin receptor with yellow fluorescent protein reporter. We found that yellow fluorescent protein-expressing, NC-derived nonhematopoietic cells in BM expressed hematopoietic factors Cxcl12 and stem cell factor The ablation of NC-derived cells led to a significant decrease in B cell progenitors but not in hematopoietic stem cells or myeloid lineage cells in BM. Interestingly, plasma noradrenaline was markedly decreased in these mice. The i.p. administration of 6-hydroxydopamine, a known neurotoxin for noradrenergic neurons, led to a similar phenotype, whereas the administration of a noradrenaline precursor in NC-ablated mice partially rescued this phenotype. Additionally, the continuous administration of adrenergic receptor ß antagonists partially decreased the number of B cell progenitors while preserving B lymphopoiesis in vitro. Taken together, our results indicate that NC-derived cell depletion leads to abnormal B lymphopoiesis partially through decreased plasma noradrenaline, suggesting this as a novel mechanism regulated by molecules released by the sympathetic neurons.

Common developmental pathway for primitive erythrocytes and multipotent hematopoietic progenitors in early mouse development.

Development of the hematopoietic system proceeds in a multistep manner. Primitive erythrocytes are the first hematopoietic cells to be observed that were produced transiently in developing embryos. Multilineage lymphohematopoiesis occurs after the primitive erythropoiesis. However, the lineage relationship of cells that comprise embryonic hematopoietic system is not well characterized. To clarify this process, careful analyses of the embryonic cells that differentiate into these cell lineages are necessary. We identified the common precursors of primitive erythrocytes and multipotent hematopoietic cells in mouse embryonic stem cell cultures and mouse embryos. A subset defined as CD45(-)CD41(+)AA4.1(-) cells showed bipotential capability to produce primitive erythrocytes and lymphomyeloid cells at the single-cell level. The cell population was present in vivo before hematopoietic stem cells (HSCs) appeared. Our results show that primitive erythrocytes and lymphomyeloid cells are not completely separate cell lineages, and these precursors comprise the embryonic hematopoietic system before HSC emergence.

Earliest hematopoietic progenitors at embryonic day 9 preferentially generate B-1 B cells rather than follicular B or marginal zone B cells.

The lymphoid potential of the hematopoietic system is observed as early as embryonic day 9 (E9) before transplantable hematopoietic stem cells (HSCs) appear at E11 in mice. However, it is largely unknown as to which cell fraction is responsible for the initial wave of lymphopoiesis and whether these earliest lymphocytes make any contributions to the adult lymphoid system. We previously isolated the earliest hematolymphoid progenitors at E9 that had CD45(+)c-Kit(+)AA4.1(+) phenotypes. In this study, the differentiation potency into B cell subsets of the E9 hematolymphoid progenitors was examined in detail. In culture, E9 hematolymphoid progenitors produced B220(-/low) B cell progenitors in striking contrast to adult BM c-Kit(+)Sca-1(+)Lin(-) cells. Upon in vivo transplantation, B cell progenitors derived from E9 hematolymphoid progenitors preferentially differentiated into the B-1 B lymphocyte subset, whereas their differentiation into B-2 B lymphocyte subsets [follicular B (FoB), marginal zone B (MZB) cells] was inefficient. Of note, these donor B lymphocytes permanently repopulated in host mice, even if adult mice were used as recipients. These results suggest that B cell progenitors produced from an initial wave of definitive hematopoiesis before authentic HSCs appear could be a permanent source for, at least, the B-1 B lymphocyte subset.

Origins and properties of dental, thymic, and bone marrow mesenchymal cells and their stem cells.

Mesenchymal cells arise from the neural crest (NC) or mesoderm. However, it is difficult to distinguish NC-derived cells from mesoderm-derived cells. Using double-transgenic mouse systems encoding P0-Cre, Wnt1-Cre, Mesp1-Cre, and Rosa26EYFP, which enabled us to trace NC-derived or mesoderm-derived cells as YFP-expressing cells, we demonstrated for the first time that both NC-derived (P0- or Wnt1-labeled) and mesoderm-derived (Mesp1-labeled) cells contribute to the development of dental, thymic, and bone marrow (BM) mesenchyme from the fetal stage to the adult stage. Irrespective of the tissues involved, NC-derived and mesoderm-derived cells contributed mainly to perivascular cells and endothelial cells, respectively. Dental and thymic mesenchyme were composed of either NC-derived or mesoderm-derived cells, whereas half of the BM mesenchyme was composed of cells that were not derived from the NC or mesoderm. However, a colony-forming unit-fibroblast (CFU-F) assay indicated that CFU-Fs in the dental pulp, thymus, and BM were composed of NC-derived and mesoderm-derived cells. Secondary CFU-F assays were used to estimate the self-renewal potential, which showed that CFU-Fs in the teeth, thymus, and BM were entirely NC-derived cells, entirely mesoderm-derived cells, and mostly NC-derived cells, respectively. Colony formation was inhibited drastically by the addition of anti-platelet-derived growth factor receptor-ß antibody, regardless of the tissue and its origin. Furthermore, dental mesenchyme expressed genes encoding critical hematopoietic factors, such as interleukin-7, stem cell factor, and cysteine-X-cysteine (CXC) chemokine ligand 12, which supports the differentiation of B lymphocytes and osteoclasts. Therefore, the mesenchymal stem cells found in these tissues had different origins, but similar properties in each organ.

Expression of AA4.1 marks lymphohematopoietic progenitors in early mouse development.

The hematopoietic system of mice is established during the early to midgestational stage of development. However, the earliest lymphohematopoietic progenitors that appear during mouse development have been less well characterized compared with the hematopoietic stem cell compartment of fetal liver and bone marrow. We isolated the earliest lymphohematopoietic progenitors by using embryonic stem (ES) cell culture in vitro. Cells with the c-Kit(+)Lin(-) cell surface phenotype were present abundantly in ES cells cocultured with stromal cell lines. We further separated the cells into two distinct cell subsets based on AA4.1 expression. Although AA4.1(+) and AA4.1(-) cells had equivalent potency to generate myeloid cell lineages, the lymphoid potential in ES-cell-derived cells was largely restricted to the cells expressing AA4.1. The same cell type was present abundantly in the early yolk sac and in fewer numbers (approximately 5% of that in the yolk sac) in the caudal half of the developing embryos. These data suggest that AA4.1 is a cell surface marker that can identify the earliest lymphohematopoietic progenitors in mouse development.

Comparison of osteoclast precursors in peripheral blood mononuclear cells from rheumatoid arthritis and osteoporosis patients.

Osteolytic disorders cause serious problems for quality of life with aging. Osteolysis is performed by osteoclasts of the hematopoietic lineage that share some characteristics with monocytes and macrophages. As osteoclast precursors (pOCs) are present in peripheral blood, their characterization in osteolytic diseases may help us to understand risk factors. Although essential factors for osteoclastogenesis have been reported, the effective induction from pOCs in human peripheral blood mononuclear cells (PBMCs) to mature osteoclasts in culture requires further improvement. The aim of this study was development of an efficient culture system for human osteoclastogenesis and providing a simple system for the enrichment of pOCs from PBMCs. We employed coculturing of human PBMCs with a mouse stromal cell line. Significant numbers of tartrate-resistant acid phosphatase-positive (TRAP(+)) multinucleated osteoclasts (MNCs), which could resorb dentine slices, were efficiently induced in this culture condition. pOCs were enriched in an anti-CD16 antibody column-passed anti-CD14 antibody-bound cell population isolated by magnetic cell sorting. We compared the percentage of the CD14(high) CD16(dull) cell population, which mainly contained pOCs in PBMCs, from age-matched patients with rheumatoid arthritis (RA) and osteoporosis (OP), but it was comparable. However, the mean number of TRAP(+) MNCs generated in cultures from PBMCs of RA was higher. In contrast, the frequency of pOCs in PBMCs from OP was relatively higher. These results suggest the characteristics of pOCs from RA and OP may be different, because single pOCs from OP gave rise to lower numbers of osteoclasts than those from RA.

Analysis of capturing skin antigens in the steady state using milk fat globule EGF factor 8-deficient skin-hyperpigmented mice.

Dendritic cells (DCs) can capture apoptotic cells and present them to immune competent cells as self-antigens (Ags). Langerhans cells (LCs), DCs in the epidermis, are capable of presenting tissue-associated Ags in the steady state, suggesting that LCs may also capture apoptotic cells and transport them to skin regional lymph nodes (LNs). However, to what extent LCs utilize apoptotic cells as self-Ags in vivo is still unclear. To clarify this point, we examined the contribution of milk fat globule EGF factor 8 (MFG-E8), a secreted glycoprotein, to capturing skin Ags. MFG-E8 is expressed in several subsets of macrophages (M phi s) and DCs, including LCs, and crucial for recognizing and engulfing apoptotic cells. Using a skin-hyperpigmented KRT14-Kitl-Tg (Kitl-Tg) mouse system, we measured the accumulation of melanin granules (MGs), a marker of skin Ags, transported from the skin to regional LNs in Mfge8-deficient mice. Unexpectedly, their accumulation in Mfge8-deficient Kitl-Tg mice was comparable to that in Mfge8-heterozygous littermates. Mfge8-deficient DCs engulfed skin-derived MGs efficiently in vitro. The results indicate that MFG-E8 does not contribute critically or functions redundantly to capturing and trafficking of skin Ags in the steady state, and suggest a possibility that LCs may capture skin Ags in forms other than apoptotic cells in vivo.

Potential of dental mesenchymal cells in developing teeth.

The tooth, composed of dentin and enamel, develops through epithelium-mesenchyme interactions. Neural crest (NC) cells contribute to the dental mesenchyme in the developing tooth and differentiate into dentin-secreting odontoblasts. NC cells are known to differentiate into chondrocytes and osteoblasts in the craniofacial region. However, it is not clear whether the dental mesenchymal cells in the developing tooth possess the potential to differentiate into a lineage(s) other than the odontoblast lineage. In this study, we prepared mesenchymal cells from E13.5 tooth germ cells and assessed their potential for differentiation in culture. They differentiated into odontoblasts, chondrocyte-like cells, and osteoblast-like cells. Their derivation was confirmed by tracing NC-derived cells as LacZ(+) cells using P0-Cre/Rosa26R mice. Using the flow cytometry-fluorescent di-beta-D-galactosidase system, which makes it possible to detect LacZ(+) cells as living cells, cell surface molecules of dental mesenchymal cells were characterized. Large number of LacZ(+) NC-derived cells expressed platelet-derived growth factor receptor alpha and integrins. Taken together, these results suggest that NC-derived cells with the potential to differentiate into chondrocyte-like and osteoblast-like cells are present in the developing tooth, and these cells may contribute to tooth organogenesis.

Constant rate of steady-state self-antigen trafficking from skin to regional lymph nodes.

It is suggested that dendritic cells (DCs) capture and present both foreign antigens such as components of pathogens as well as endogenous self-antigens. However, the magnitude of self-antigen trafficking to secondary lymphoid organs is still unclear. Here we show constitutive trafficking of self-antigens from skin to regional lymph nodes (LNs) quantitatively using a KRT14-Kitl transgenic mouse. This mouse model expresses the Kit ligand in keratinocytes, shows hyperpigmentation of the epidermis and exhibits constitutive accumulation of melanin granules (MGs) in skin regional LNs transported by Langerhans cells. Using an MG-solubilization technique, we revealed that 128 microg per week of MGs, a marker of self-antigens, accumulated in skin regional LNs and that the rate of accumulation was constant from 3 to 50 weeks. Activation markers such as CD40, CD54 and CD86 did not increase in the LNs, and abrogation of CD40 signaling did not affect the accumulation. Additionally, the total amount of MGs did not increase significantly following stimulation with intravenous LPS injections. These results suggest that the accumulation is not caused by inflammatory stimuli, and the steady-state trafficking of self-antigens is intrinsically maintained at a constant rate. Because the levels of self-antigens as well as the phenotype of these DCs are thought to be important in the strength of immune responses, the results may imply that the constant rate of trafficking of self-antigens is required for maintaining homeostatic conditions, such as self-tolerance.

Ascorbic acid promotes osteoclastogenesis from embryonic stem cells.

Ascorbic acid (AA) is known to regulate cell differentiation; however, the effects of AA on osteoclastogenesis, especially on its early stages, remain unclear. To examine the effects of AA throughout the process of osteoclast development, we established a culture system in which tartrate-resistant acid phosphate (TRAP)-positive osteoclasts were induced from embryonic stem cells without stromal cell lines. In this culture system, the number of TRAP-positive cells was strongly increased by the addition of AA during the development of osteoclast precursors, and reducing agents, 2-mercaptoethanol, monothioglycerol, and dithiothreitol, failed to substitute for AA. The effect of AA was stronger when it was added during the initial 4 days during the development of mesodermal cells than when it was added during the last 4 days. On day 4 of the culture period, AA increased the total cell recovery and frequency of osteoclast precursors. Magnetic cell sorting using anti-Flk-1 antibody enriched osteoclast precursors on day 4, and the proportion of Flk-1-positive cells but not that of platelet-derived growth factor receptor alpha-positive cells was increased by the addition of AA. These results suggest that AA might promote osteoclastogenesis of ES cells through increasing Flk-1-positive cells, which then give rise to osteoclast precursors.

Suppression of progressive loss of coat color in microphthalmia-vitiligo mutant mice.

The coat color of C57BL/6-Mitfvit/vit mice whitens with age, because of a one-nucleotide mutation in the DNA-binding region of the microphthalmia-associated transcription factor (MITF), which plays an important role in melanocyte growth and differentiation. To investigate the signals regulating MITF function, we prepared transgenic mice expressing three of the external signals that are important for melanocyte development, i.e., hepatocyte growth factor (HGF), stem cell factor (SCF), and endothelin-3 (ET3), and crossed these mice with Mitfvit/vit mice. We found that the age-dependent coat color whitening of the Mitfvit/vit mice was completely suppressed by the overexpression of HGF or SCF in the skin, but not by that of ET3. Moreover, HGF, but not ET3, promoted the proliferation of Mitfvit/vit mice-derived melanocytes in culture. These results suggest that the signals from exogenous HGF and SCF rescued the mi-vitiligo mutation and also that ET3 does not stimulate the common signal transduction pathway for MITF activation shared by HGF and SCF.

Presence and distribution of neural crest-derived cells in the murine developing thymus and their potential for differentiation.

Neural crest (NC) cells are multipotent cells that can differentiate into melanocytes, neurons, glias and myofibroblasts. They migrate into the fetal thymus on embryonic day (E) 12 in mice and may participate in thymic organogenesis. Although the abnormality of migration and distribution of NC cells in the thymus results in immunodeficiency, the spatial and temporal presence of their progeny cells has not been defined in detail. In this study, we traced NC-derived cells based on the myelin protein zero gene promoter-Cre-mediated excision. We demonstrated that large numbers of NC-derived cells in the thymus were detected on E11.5 to E16.5 but rarely on E17.5. A colony formation assay of single thymic cells demonstrated that multipotent cells with the potential to differentiate into melanocytes, neurons and/or glias were present in the E14.5 and E15.5 but not in the E17.5 fetal thymus. Furthermore, we confirmed that these multipotent cells were NC-derived cells. Taken together, these findings imply that multipotent NC-derived cells are present in the developing thymus, but rarely in this organ at a later stage, suggesting that NC-derived cells may play roles in thymic organogenesis at an early embryonic stage.

Cooperative and indispensable roles of endothelin 3 and KIT signalings in melanocyte development.

The development of melanocytes from neural crest-derived precursor cells depends on signaling by the receptor tyrosine kinase KIT and the G protein-coupled endothelin receptor B (EDNRB) pathways. Loss-of-function mutations in either of these two signaling receptor molecules cause a loss or a marked reduction in the number of melanocyte precursors in the embryo and finally lead to loss of the coat color. Using cultures of embryonic stem (ES) cells to induce melanocyte differentiation in vitro, we investigated the requirement for EDNRB signaling during the entire developmental process of the melanocyte, in association with that for KIT signaling. During the 21-day period necessary for the induction of mature melanocytes from undifferentiated ES cells, endothelin 3 (EDN3), a ligand for EDNRB, increased the number of melanocytes in proportion to the period during which it was present. We tested the compensatory effect of EDNRB signaling on KIT signaling in vivo by using Kit(W-LacZ)/Kit(W-LacZ) ES cells and confirmed that the ectopic expression of EDN3 in the skin reduced the white spotting of Kit(W57)/Kit(W57)mice. KIT ligand (KITL) and EDN3 worked synergistically to induce melanocyte differentiation in vitro; however, the complete lack of EDNRB signaling attained by the use of EDN3-/- ES cells and an EDNRB antagonist, BQ788, revealed that the resulting failure of melanocyte development was not compensated by the further activation of KIT signaling by adding KITL. Simultaneous blockade of EDNRB and KIT signalings eliminated melanocyte precursors completely, suggesting that the maintenance or survival of early melanocyte precursors at least required the existence of either EDNRB or KIT signalings.