Sox10 Functions as an Inducer of the Direct Conversion of Keratinocytes Into Neural Crest Cells.

Neural crest cells (NCCs) are highly migratory multipotent cells that play critical roles in embryogenesis. The generation of NCCs is controlled by various transcription factors (TFs) that are regulated by each other and combine to form a regulatory network. We previously reported that the conversion of mouse fibroblasts into NCCs was achieved by the overexpression of only one TF, Sox10; therefore, Sox10 may be a powerful inducer of the conversion of NCCs. We herein investigated whether Sox10 functions in the direct conversion of other somatic cells into NCCs. Sox10 directly converted bone marrow-derived mesenchymal cells, but not keratinocytes, into P75+ NCCs. However, by the co-expression of four TFs (Snail1, Snail2, Twist1, and Tcfap2a) that are involved in NCC generation, but unable convert cells into NCCs, Sox10 converted keratinocytes into P75+ NCCs. P75+ NCCs mainly differentiated into glial cells, and to a lesser extent into neuronal cells. On the other hand, when Sox10 was expressed after the four TF expression, which mimicked the expression order in in vivo NCC generation, it converted keratinocytes into multipotent NCCs. These results demonstrate that Sox10 functions as an inducer of direct conversion into NCCs in cooperation with the TFs involved in NCC generation. The sequence of expression of the inducer and cooperative factors is important for the conversion of somatic cells into bona fide target cells.

Early Development of Resident Macrophages in the Mouse Cochlea Depends on Yolk Sac Hematopoiesis.

Resident macrophages reside in all tissues throughout the body and play a central role in both tissue homeostasis and inflammation. Although the inner ear was once believed to be "immune-privileged," recent studies have shown that macrophages are distributed in the cochlea and may play important roles in the immune system thereof. Resident macrophages have heterogeneous origins among tissues and throughout developmental stages. However, the origins of embryonic cochlear macrophages remain unknown. Here, we show that the early development of resident macrophages in the mouse cochlea depends on yolk sac hematopoiesis. Accordingly, our results found that macrophages emerging around the developing otocyst at E10.5 exhibited dynamic changes in distribution and in situ proliferative capacity during embryonic and neonatal stages. Cochlear examination in Csf1r-null mice revealed a substantial decrease in the number of Iba1-positive macrophages in the spiral ganglion and spiral ligament, whereas they were still observed in the cochlear mesenchyme or on the intraluminal surface of the perilymphatic space. Our results demonstrated that two subtypes of resident macrophages are present in the embryonic cochlea, one being Csf1r-dependent macrophages that originate from the yolk sac and the other being Csf1r-independent macrophages that appear to be derived from the fetal liver via systemic circulation. We consider the present study to be a starting point for elucidating the roles of embryonic cochlear resident macrophages. Furthermore, resident macrophages in the embryonic cochlea could be a novel target for the treatment of various inner ear disorders.

Direct Conversion of Mouse Embryonic Fibroblasts into Neural Crest Cells.

Neural crest cells (NCCs) are multipotent cells that emerge from the edges of the neural folds and extensively migrate throughout developing embryos. Dorsolaterally migrating NCCs colonize skin, differentiate into skin melanocytes, and lose their multipotency. Multipotent NCCs or NCCs derived multipotent stem cells (MSCs) were recently detected in their migrated locations, including skin, despite restrictions in cell fate acquisition following migration. Since many features of NCCs have yet to be revealed, the novel properties of NCCs represent an important and interesting field in stem cell biology. We previously reported the direct conversion of mouse embryonic fibroblasts (MEFs) into NCCs by the forced expression of the transcription factors C-MYC, KLF4, and SOX10. We herein describe the methods employed for direct conversion: retrovirus infection for the forced expression of transcription factors, a flow cytometry-sorting method for the isolation of converted NCCs, and culture methods for the maintenance and differentiation of the converted NCCs.

Melanoblasts as Multipotent Cells in Murine Skin.

Melanoblasts (MBs) are melanocyte precursors that are derived from neural crest cells (NCCs). We recently demonstrated the multipotency of MBs; they differentiate not only into pigmented melanocytes but also other NCC derivatives. We herein describe methods for the isolation of MBs from mouse skin by flow cytometry. Methods to culture isolated MBs that retain their multipotency and isolation methods for MBs using gene-modified mouse are also described.

Galectin-1 enhances the generation of neural crest cells.

Neural crest (NC) cells are multipotent cells that emerge from the dorsal region of the neural tube. After delaminating from the neural tube, NC cells migrate throughout the developing embryo and differentiate into various cells: neurons and glial cells of the peripheral nervous system, melanocytes of skin, and skeletal elements of the face and head. We previously analyzed the gene expression profile of a NC subpopulation isolated from Sox10-IRES-Venus mice and found that the carbohydrate-binding protein, Galectin-1 (Gal-1) was strongly expressed in generating NC cells. In the present study, we identified GAL-1 as a factor that promotes NC cell generation. Gal-1 was significantly expressed in NC cells generated in explanted neural tubes. The presence of GAL-1 enhanced the generation of NC-like cells from mouse embryonic stem (ES) cells. In the differentiation of ES cells into NC-like cells, GAL-1 enhanced neurogenesis in the early stages and facilitated NC-like cell generation in the later stages. GAL-1 also enhanced the generation of NC cells from explanted neural tubes. These results suggest that GAL-1 plays a facilitative role in NC cell generation.

Gene array analysis of neural crest cells identifies transcription factors necessary for direct conversion of embryonic fibroblasts into neural crest cells.

Neural crest cells (NC cells) are multipotent cells that emerge from the edge of the neural folds and migrate throughout the developing embryo. Although the gene regulatory network for generation of NC cells has been elucidated in detail, it has not been revealed which of the factors in the network are pivotal to directing NC identity. In this study we analyzed the gene expression profile of a pure NC subpopulation isolated from Sox10-IRES-Venus mice and investigated whether these genes played a key role in the direct conversion of Sox10-IRES-Venus mouse embryonic fibroblasts (MEFs) into NC cells. The comparative molecular profiles of NC cells and neural tube cells in 9.5-day embryos revealed genes including transcription factors selectively expressed in developing trunk NC cells. Among 25 NC cell-specific transcription factor genes tested, SOX10 and SOX9 were capable of converting MEFs into SOX10-positive (SOX10+) cells. The SOX10+ cells were then shown to differentiate into neurons, glial cells, smooth muscle cells, adipocytes and osteoblasts. These SOX10+ cells also showed limited self-renewal ability, suggesting that SOX10 and SOX9 directly converted MEFs into NC cells. Conversely, the remaining transcription factors, including well-known NC cell specifiers, were unable to convert MEFs into SOX10+ NC cells. These results suggest that SOX10 and SOX9 are the key factors necessary for the direct conversion of MEFs into NC cells.

Multipotency of melanoblasts isolated from murine skin depends on the Notch signal.

BACKGROUND: Melanoblasts (MBs), derived from neural crest cells, only differentiate into melanocytes (Ms) in vivo. We previously showed that MBs isolated from mouse skin were multipotent, generating neurons (Ns) and glial cells (Gs) together with Ms. Using Sox10-IRES-Venus mice and mouse embryonic stem cells, we investigated how MBs expressed their multipotency. RESULTS: MBs generated colonies containing Ns, Gs, and Ms in the presence of ST2 stromal cells, but they generated only M colonies when incubated with keratinocytes or ST2 culture supernatant, thus showing that MBs required contact with ST2 stromal cells for expression of their multipotency. Notch signaling was shown to be one of the important cues for the maintenance and differentiation of MBs through cell-cell contact. When Notch signaling was inhibited, MBs mainly generated colonies that contained just one type of cells, Ns, Gs, or Ms; the number of colonies containing two or three types of cells markedly decreased even on ST2 stromal cells, showing restriction of their differentiation potency. Whereas when Notch signaling was activated, the number of colonies containing two or three types of cells increased, indicating that their multipotency had been maintained. CONCLUSIONS: Our results demonstrate that Notch signaling played novel roles in MB multipotency.

Extended multipotency of neural crest cells and neural crest-derived cells.

Neural crest cells (NCC) are migratory multipotent cells that give rise to diverse derivatives. They generate various cell types during embryonic development, including neurons and glial cells of the peripheral sensory and autonomic ganglia, Schwann cells, melanocytes, endocrine cells, smooth muscle, and skeletal and connective tissue cells of the craniofacial complex. The multipotency of NCC is thought to be transient at the early stage of NCC generation; once NCC emerge from the neural tube, they change into lineage-restricted precursors. Although many studies have described the clear segregation of NCC lineages right after their delamination from the neural tube, recent reports suggest that multipotent neural crest stem cells (NCSC) are present not only in migrating NCC in the embryo, but also in their target tissues in the fetus and adult. Furthermore, fully differentiated NCC-derived cells such as glial cells and melanocytes have been shown to dedifferentiate or transdifferentiate into other NCC derivatives. The multipotency of migratory and postmigratory NCC-derived cells was found to be similar to that of NCSC. Collectively, these findings support the multipotency or plasticity of NCC and NCC-derived cells.

The stemness of neural crest cells and their derivatives.

Neural crest cells (NCCs) are unique to vertebrates and emerge from the border of the neural plate and subsequently migrate extensively throughout the embryo after which they differentiate into many types of cells. This multipotency is the main reason why NCCs are regarded as a versatile tool for stem cell biology and have been gathering attention for their potential use in stem cell based therapy. Multiple sets of networks comprised of signaling molecules and transcription factors regulate every developmental phase of NCCs, including maintenance of their multipotency. Pluripotent stem cell lines, such as embryonic stem cells and induced pluripotent stem (iPS) cells, facilitate the induction of NCCs in combination with sophisticated culture systems used for neural stem cells, although at present, clinical experiments for NCC-based cell therapy need to be improved. Unexpectedly, the multipotency of NCCs is maintained after they reach the target tissues as tissue neural crest stem cells (NCSCs) that may contribute to the establishment of NCC-derived multipotential stem cells. In addition, under specific culture conditions, fate-restricted unipotent descendants of NCCs, such as melanoblasts, show multipotency to differentiate into melanocytes, neurons, and glia cells. These properties contribute to the additional versatility of NCCs for therapeutic application and to better understand NCC development.

Preferential gene transcription of T helper 2 cytokines in peripheral CCR4(+) CD4(+) lymphocytes in dogs.

BACKGROUND: Previous studies reported the involvement of CC chemokine receptor 4 (CCR4)-positive CD4(+) cells in the pathogenesis of canine atopic dermatitis. In humans, CCR4 is selectively expressed on type 2 helper T (Th2) cells; however, a subset of canine CCR4(+) helper T cells has not been determined. HYPOTHESIS/OBJECTIVES: To characterize the transcription profile of CCR4(+) CD4(+) lymphocytes isolated from the peripheral blood of healthy dogs. ANIMALS: Three healthy dogs were used. METHODS: The transcription levels of type 1 helper T (Th1) and Th2 cytokines in CCR4(+) CD4(+) and CCR4(-) CD4(+) lymphocytes isolated from healthy dogs were quantified by real-time RT-PCR. RESULTS: The CCR4(+) CD4(+) lymphocytes preferentially transcribed Th2 cytokines, such as interleukin-4 and interleukin-13, but not Th1 cytokines, such as interferon-γ. CONCLUSIONS AND CLINICAL IMPORTANCE: CCR4 can be used as a specific marker of Th2 cells for elucidation of the pathogenesis or the establishment of novel therapeutics in canine Th2-associated diseases, such as canine atopic dermatitis.

Neural crest-derived cells sustain their multipotency even after entry into their target tissues.

BACKGROUND: Neural crest cells (NC cells) are highly migratory multipotent cells. Their multipotency is transient at the early stage of their generation; soon after emerging from the neural tube, these cells turn into lineage-restricted precursors. However, recent studies have disputed this conventionally believed paradigm. In this study, we analyzed the differentiation potency of NC-derived cells after their arrival at target tissues. RESULTS: Using Sox10-IRES-Venus mice, we found that the NC-derived cells in the skin, DRG, and inner ear could be divided into two populations: Sox10-positive/Kit-negative cells (Sox10+/Kit- cells) and Sox10- and Kit-positive cells (Sox10+/Kit+ cells). Only the Sox10+/Kit- cells were detected in the intestines. Unexpectedly, the Sox10+/Kit+ cells differentiated into neurons, glial cells, and melanocytes, showing that they had maintained their multipotency even after having entered the target tissues. The Sox10+/Kit+ cells in the DRG maintained their multipotency for a restricted period during the earlier embryonic stages, whereas those in the skin and inner ear were multipotent yet even in later embryonic stages. CONCLUSIONS: We showed that NC-derived Sox10+/Kit+ cells maintained their multipotency even after entry into the target tissues. This unexpected differentiation potency of these cells in tissues seems to have been strictly restricted by the tissue microenvironment.

Tracing Sox10-expressing cells elucidates the dynamic development of the mouse inner ear.

The inner ear is constituted by complicated cochlear and vestibular compartments, which are derived from the otic vesicle, an embryonic structure of ectodermal origin. Although the inner ear development has been analyzed using various techniques, the developmental events have not been fully elucidated because of the intricate structure. We previously developed a Sox10-IRES-Venus mouse designed to express green fluorescent protein under the control of the Sox10 promoter. In the present study, we showed that the Sox10-IRES-Venus mouse enabled the non-destructive visualization and understanding of the morphogenesis during the development of the inner ear. The expression of the transcription factor Sox10 was first observed in the invaginating otic placodal epithelium, and continued to be expressed in the mature inner ear epithelium except for the hair cells and mesenchymal cells. We found that Sox10 was expressed in immature hair cells in the developing inner ear, suggesting that hair cells were generated from the Sox10-expressing prosensory cells. Furthermore, we demonstrated that scattered Sox10-expressing cells existed around the developing inner ear, some of which differentiated into pigmented melanocytes in the stria vascularis, suggesting that they were neural crest cells. Further analyzing the Sox10-IRES-Venus mice would provide important information to better understand the development of the inner ear.

Keratinocyte stem cells but not melanocyte stem cells are the primary target for radiation-induced hair graying.

Ionizing radiation (IR)-induced hair graying is caused by the ectopic differentiation of melanocyte stem cells (MSCs) in their niche located at the bulge region of the hair follicle. Keratinocyte stem cells (KSCs) in the bulge region are an important component of that niche. However, little is known about the relationship between MSC differentiation and the KSC niche during IR-induced hair graying. We found that both follicular MSCs and KSCs were affected by IR by using immunohistochemical detection of γH2AX as a genotoxicity marker. We also found that KSCs prepared from irradiated mice were functionally affected by IR as indicated by their reduced colony-forming activity in culture and the delayed hair cycle in vivo. However, these effects of IR on KSCs were temporal. The MSC population, which proliferated and differentiated to melanocytes, was persistently maintained after irradiation. In addition to the loss of colony-forming activity, irradiated keratinocytes including KSCs suppressed the colony formation of MSCs in vitro. Furthermore, pigmented hairs were not reconstituted in vivo in the presence of irradiated KSCs or keratinocytes. These results provide a previously unreported insight that the primary target of IR during the induction of hair graying is follicular KSCs rather than MSCs.

Melanoblasts as multipotent cells in murine skin.

Melanoblasts are melanocyte precursors that are derived from neural crest cells (NCCs). Recently we showed that melanoblasts differentiate into not only pigmented melanocytes but also into other NCCs derivatives. Here, we describe methods for the isolation of melanoblasts from mouse skin by flow-cytometry. Methods for culturing the isolated melanoblasts, allowing them to express their multipotentiality, are also described.

Dual origin of melanocytes defined by Sox1 expression and their region-specific distribution in mammalian skin.

Melanocytes are pigment-producing cells generated from neural crest cells (NCCs) that delaminate from the dorsal neural tube. The widely accepted premise that NCCs migrating along the dorsolateral pathway are the main source of melanocytes in the skin was recently challenged by the finding that Schwann cell precursors are the major cellular source of melanocytes in the skin. Still, in a wide variety of vertebrate embryos, melanocytes are exclusively derived from NCCs. In this study, we show that a NCC population that is not derived from Sox1(+) dorsal neuroepithelial cells but are derived from Sox1(-) cells differentiate into a significant population of melanocytes in the skin of mice. Later, these Sox1(-) cells clearly segregate from cells that originated from Sox1(+) dorsal neuroepithelial cell-derived NCCs. The possible derivation of Sox1(-) cells from epidermal cells also strengthens their non-neuroepithelial origin.

Protective effect of Kit signaling for melanocyte stem cells against radiation-induced genotoxic stress.

Radiation-induced hair graying is caused by irreversible defects in the self-renewal and/or development of follicular melanocyte stem cells in the hair follicles. Kit signaling is an essential growth and differentiation signaling pathway for various cell lineages including melanocytes, and its radioprotective effects have been shown in hematopoietic cells. However, it is uncertain whether Kit signaling exerts a radioprotective effect for melanocytes. In this study, we found that various loss-of-function mutations of Kit facilitate radiation-induced hair graying. In contrast, transgenic mice expressing the ligand for Kit (Kitl) in the epidermis have significantly reduced levels of radiation-induced hair graying. The X-ray doses used did not show a systemic lethal effect, indicating that the in vivo radiosensitivity of Kit mutants is mainly caused by the damaged melanocyte stem cell population. X-ray-damaged melanocyte stem cells seemed to take the fate of ectopically pigmented melanocytes in the bulge regions of hair follicles in vivo. Endothelin 3, another growth and differentiation factor for melanocytes, showed a lesser radioprotective effect compared with Kitl. These results indicate the prevention of radiation-induced hair graying by Kit signaling.

Neural crest cells retain their capability for multipotential differentiation even after lineage-restricted stages.

Multipotency of neural crest cells (NC cells) is thought to be a transient phase at the early stage of their generation; after NC cells emerge from the neural tube, they are specified into the lineage-restricted precursors. We analyzed the differentiation of early-stage NC-like cells derived from Sox10-IRES-Venus ES cells, where the expression of Sox10 can be visualized with a fluorescent protein. Unexpectedly, both the Sox10+/Kit- cells and the Sox10+/Kit+ cells, which were restricted in vivo to the neuron (N)-glial cell (G) lineage and melanocyte (M) lineage, respectively, generated N, G, and M, showing that they retain multipotency. We generated mice from the Sox10-IRES-Venus ES cells and analyzed the differentiation of their NC cells. Both the Sox10+/Kit- cells and Sox10+/Kit+ cells isolated from these mice formed colonies containing N, G, and M, showing that they are also multipotent. These findings suggest that NC cells retain multipotency even after the initial lineage-restricted stages.

Functionally distinct melanocyte populations revealed by reconstitution of hair follicles in mice.

Hair follicle reconstitution analysis was used to test the contribution of melanocytes or their precursors to regenerated hair follicles. In this study, we first confirmed the process of chimeric hair follicle regeneration by both hair keratinocytes and follicular melanocytes. Then, as first suggested from the differential growth requirements of epidermal skin melanocytes and non-cutaneous or dermal melanocytes, we confirmed the inability of the latter to be involved as follicular melanocytes to regenerate hair follicles during the hair reconstitution assay. This clear functional discrimination between non-cutaneous or dermal melanocytes and epidermal melanocytes suggests the presence of two different melanocyte cell lineages, a finding that might be important in the pathogenesis of melanocyte-related diseases and melanomas.

Unexpected multipotency of melanoblasts isolated from murine skin.

Melanoblasts, precursor of melanocytes, are generated from the neural crest and differentiate into melanocytes during their migration throughout the entire body. The melanoblasts are thought to be progenitor cells that differentiate only into melanocyte. Here, we show that melanoblasts, even after they have already migrated throughout the skin, are multipotent, being able to generate neurons, glial cells, and smooth muscle cells in addition to melanocytes. We isolated Kit-positive and CD45-negative (Kit+/CD45-) cells from both embryonic and neonate skin by flow cytometry and cultured them on stromal cells. The Kit+/CD45- cells formed colonies containing neurons, glial cells, and smooth muscle cells, together with melanocytes. The Kit+/CD45- cells expressed Mitf-M, Sox10, and Trp-2, which are genes known to be expressed in melanoblasts. Even a single Kit+/CD45- cell formed colonies that contained neurons, glial cells, and melanocytes, confirming their multipotential cell fate. The colonies formed from Kit+/CD45- cells retained Kit+/CD45- cells even after 21 days in culture and these retained cells also differentiated into neurons, glial cells, and melanocytes, confirming their self-renewal capability. When the Kit signal was inhibited by the antagonist ACK2, the Kit+/CD45- cells did not form colonies that contained multidifferentiated cells. These results indicate that melanoblasts isolated from skin have multipotency and self-renewal capabilities.