Mutations at the W locus affect survival of neural crest-derived melanocytes in the mouse

The development of melanoblasts in normally pigmented and dominant spotting (W) embryos was followed by in situ hybridisation to TRP-2/DT mRNA, which labels migratory melanoblasts from 10 days post coitum. Numerous melanoblasts migrate to the inner ear around 11 days. In contrast, few migratory melanoblasts are associated with the eye or skin at this stage and melanoblast distribution within the trunk and tail is patchy. The distribution of melanoblasts in 10.5–11-day-old W^v/W^v, W^sh/W^sh and W⁴¹/W⁴¹ mutants was similar to that in controls but melanoblast density was lower and by 12 days was severely reduced. These results suggest that mutations of the c-kit receptor tyrosine kinase encoded at the W locus do not alter early migration or differentiation of melanoblasts but severely affect melanoblast survival.     

Expression of a novel secreted factor, Seraf indicates an early segregation of Schwann cell precursors from neural crest during avian development

The neural crest gives rise to glial cells in the peripheral nervous system. Among the peripheral glia, Schwann cells form the myelin often wrapping the peripheral axons. Compared to other crest-derived cell lineages such as neurons, the analysis of fate determination and subsequent differentiation of Schwann cells is not well advanced, partly due to the lack of early markers of this phenotype. In this study, we have identified a gene, uniquely expressed in avian embryo Schwann cell precursors, which encodes a novel secreted factor, designated Seraf (Schwann cell-specific EGF-like repeat autocrine factor). Expression of Seraf and P0 delineates the earliest phase of Schwann cell differentiation. Seraf binds to neural crest cells and Schwann cells, and affects the distribution of Schwann cells, when introduced to chicken embryos during neural crest migration. Our results suggest an autocrine function of Seraf and provide a significant step to understand the developmental processes of Schwann cell lineage.     

Fibronectin Combined with Stem Cell Factor Plays an Important Role in Melanocyte Proliferation,Differentiation and Migration in Cultured Mouse Neural Crest Cells

Stem cell factor (SCF) is essential to the migration and differentiation of melanocytes during embryogenesis because mutations in either the SCF gene, or its ligand, KIT, result in defects in coat pigmentation in mice. Using a neural crest cell (NCC) primary culture system from wild‐type mice, we previously demonstrated that KIT‐positive and/or L ‐3, 4‐dihydroxyphenylalanine (DOPA)‐positive melanocyte precursors proliferate following the addition of SCF to the culture medium. Extracellular matrix (ECM) proteins are considered to play a role in the migration and differentiation of various cells including melanocytes. We cultured mouse NCCs in the presence of SCF in individual wells coated with ECM; fibronectin (FN), collagen I (CLI), chondroitin sulphate, or dermatan sulphate. More KIT‐positive cells and DOPA‐positive cells were detected in the presence of SCF on ECM‐coated wells than on non‐coated wells. A statistically significant increase in DOPA‐positive cells was evident in FN and CLI wells. In contrast, in the absence of SCF, few DOPA‐positive cells and KIT‐positive cells were detected on either the ECM‐coated or non‐coated wells. We concluded that ECM affect melanocyte proliferation and development in the presence of SCF. To determine the key site of FN function, RGDS peptides in the FN sequence, which supports spreading of NCCs, were added to the NCC culture. The number of DOPA‐positive cells decreased with RGDS concentration in a dose‐dependent fashion. Immunohistochemical staining revealed the presence of integrin a5, a receptor of RGDS, in NCCs. These results suggest the RGDS domain of FN plays a contributory role as an active site in the induction of FN function in NCCs. In addition, we examined the effect of FN with SCF on the NCC migration by measuring cluster size, and found an increase in size following treatment with FN.     

Ectodermal-derived Endothelin1 is required for patterning the distal and intermediate domains of the mouse mandibular arch

Morphogenesis of the vertebrate head relies on proper dorsal-ventral (D-V) patterning of neural crest cells (NCC) within the pharyngeal arches. Endothelin-1 (Edn1)-induced signaling through the endothelin-A receptor (Ednra) is crucial for cranial NCC patterning within the mandibular portion of the first pharyngeal arch, from which the lower jaw arises. Deletion of Edn1, Ednra or endothelin-converting enzyme in mice causes perinatal lethality due to severe craniofacial birth defects. These include homeotic transformation of mandibular arch-derived structures into more maxillary-like structures, indicating a loss of NCC identity. All cranial NCCs express Ednra whereas Edn1 expression is limited to the overlying ectoderm, core paraxial mesoderm and pharyngeal pouch endoderm of the mandibular arch as well as more caudal arches. To define the developmental significance of Edn1 from each of these layers, we used Cre/loxP technology to inactivate Edn1 in a tissue-specific manner. We show that deletion of Edn1 in either the mesoderm or endoderm alone does not result in cellular or molecular changes in craniofacial development. However, ectodermal deletion of Edn1 results in craniofacial defects with concomitant changes in the expression of early mandibular arch patterning genes. Importantly, our results also both define for the first time in mice an intermediate mandibular arch domain similar to the one defined in zebrafish and show that this region is most sensitive to loss of Edn1. Together, our results illustrate an integral role for ectoderm-derived Edn1 in early arch morphogenesis, particularly in the intermediate domain.     

Isolation,expansion and neural differentiation of stem cells from human plucked hair: a further step towards autologous nerve recovery

Stem cells from the adult hair follicle bulge can differentiate into neurons and glia, which is advantageous for the development of an autologous cell-based therapy for neurological diseases. Consequently, bulge stem cells from plucked hair may increase opportunities for personalized neuroregenerative therapy. Hairs were plucked from the scalps of healthy donors, and the bulges were cultured without prior tissue treatment. Shortly after outgrowth from the bulge, cellular protein expression was established immunohistochemically. The doubling time was calculated upon expansion, and the viability of expanded, cryopreserved cells was assessed after shear stress. The neuroglial differentiation potential was assessed from cryopreserved cells. Shortly after outgrowth, the cells were immunopositive for nestin, SLUG, AP-2α and SOX9, and negative for SOX10. Each bulge yielded approximately 1 × 10⁴ cells after three passages. Doubling time was 3.3 (±1.5) days. Cellular viability did not differ significantly from control cells after shear stress. The cells expressed class III β-tubulin (TUBB3) and synapsin-1 after 3 weeks of neuronal differentiation. Glial differentiation yielded KROX20- and MPZ-immunopositive cells after 2 weeks. We demonstrated that human hair follicle bulge-derived stem cells can be cultivated easily, expanded efficiently and kept frozen until needed. After cryopreservation, the cells were viable and displayed both neuronal and glial differentiation potential.     

Cell-autonomous and nonautonomous actions of endothelin-A receptor signaling in craniofacial and cardiovascular development

Craniofacial and cardiac development relies on the proper patterning of the neural crest-derived ectomesenchyme of the pharyngeal arches, from which many craniofacial and great vessel structures arise. One of the intercellular signaling molecules that is involved in this process, endothelin-1 (ET-1), is expressed in the arch epithelium and influences arch development by binding to its cognate receptor, the endothelin A (ET(A)) receptor, found on ectomesenchymal cells. We have previously shown that absence of ET(A) signaling in ET(A)(-/-) mouse embryos disrupts neural crest cell development, resulting in craniofacial and cardiovascular defects similar in many aspects to those in mouse models of DiGeorge syndrome. These changes may reflect a cell-autonomous requirement for ET(A) signaling during crest cell development because the ET(A) receptor is an intracellular signaling molecule. However, it is also possible that some of the observed defects in ET(A)(-/-) embryos could arise from the absence of downstream signaling that act in a non-cell-autonomous manner. To address this question, we performed chimera analysis using ET(A)(-/-) embryonic stem cells. We observe that, in almost all early ET(A)(-/-) --> (+/+) chimeric embryos, ET(A)(-/-) cells are excluded from the caudoventral aspects of the pharyngeal arches, suggesting a cell-autonomous role for ET(A) signaling in crest cell migration and/or colonization. Interestingly, in the few embryos in which mutant cells do reach the ventral arch, structures derived from this area are either composed solely of wild type cells or are missing, suggesting a second cell-autonomous role for ET(A) signaling in postmigratory crest cell differentiation. In the cardiac outflow tract and great vessels, ET(A)(-/-) cells are excluded from the walls of the developing pharyngeal arch arteries, indicating that ET(A) signaling also acts cell-autonomously during cardiac neural crest cell development.     

Control of Melanoblast Differentiation in Amphibia by α-Melanocyte Stimulating Hormone,a Serum Melanization Factor,and a Melanization Inhibiting Factor

A ventrally localized melanization inhibiting factor (MIF) has been suggested to play an important role in the establishment of the dorsal-ventral pigment pattern in Xenopus laevis [Fukuzawa and Ide: Dev. Biol., 129:25–36, 1988]. To examine the possibility that melanoblast expression might be controlled by local putative MIF and melanogenic factors, the effects of α-melanocyte stimulating hormone (α-MSH), a serum melanization factor (SMF) from X. laevis or Rana pipiens, and MIF on the “outgrowth” and “melanization” of Xenopus neural crest cells were studied. Outgrowth represents the number of neural crest cells emigrating from cultured neural tubes, and melanization concerns the percentage of differentiated melanophores among the emigrated cells. MSH or SMF stimulate both outgrowth and melanization. The melanogenic effect of Xenopus serum in this system is more than twice that of Rana serum. The actions of MSH and Xenopus serum on melanization seem to be different: 1) Stronger melanization is induced by Xenopus serum than by MSH, and the onset of melanization occurs earlier with Xenopus serum; 2) MSH stimulates melanization only in the presence of added tyrosine; and 3) MSH causes young melanophores to assume a prominent state of melanophore dispersion during culture, while Xenopus serum (10%) had only a slight dispersing effect and not until day 3. A fraction of Xenopus serum presumably containing molecules of a smaller molecular weight (MW <30 kDa) than that of a pigment promoting factor reported in calf serum [Jerdan et al.: J. Cell Biol., 100:1493–1498, 1985] produces the same remarkable melanogenic effects as does intact serum. While this fraction stimulates outgrowth, another fraction presumably containing larger molecules (MW > 100 kDa) does not. MIF contained in Xenopus ventral skin conditioned medium (VCM) inhibits both outgrowth and melanization dose dependently. When VCM is used in combination with MSH, the stimulating effects of MSH on both outgrowth and melanization are completely inhibited. In contrast, the stimulatory effects of Xenopus serum are not completely inhibited when combined with VCM, although melanization is reduced to approximately 40% that of controls. MIF activity was also found to be present in ventral, but not in dorsal, skin conditioned media of R. pipiens when tested in the Xenopus neural crest system. We suggest that ventrally localized MIF plays an important role in amphibian pigment pattern formation and that the interacting effects of MIF and melanogenic factors influence melanoblast differentiation, migration, and/or proliferation of neural crest cells to effect the expression of pigmentary patterns.     

Both Notch1 and Notch2 contribute to the regulation of melanocyte homeostasis

Notch signaling affects a variety of mammalian stem cells, but there has been limited evidence that a specific Notch molecule regulates adult stem cells. Recently, it was reported that the reduced Notch signaling initiated at the embryonic stage results in a gradual hair graying phenotype after birth. Here we demonstrate that the oral administration of a gamma-secretase inhibitor (GSI) to wild-type adult C57/Bl6 mice led to a gradual increase in gray spots, which remained unchanged for at least 20 weeks after discontinuing the GSI. In GSI-treated mice, there was a severe decrease in unpigmented melanocytes in the bulge/subbulge region where melanocyte stem cells are located. While we confirmed that Notch1+/-Notch2+/- double heterozygous mice with a C57/Bl6 background were born with a normal hair color phenotype and gradually turned gray after the second hair cycle, in the c-kit mutant Wv background, Notch1+/- and Notch2+/- mice had larger white spots on the first appearance of hair than did the Wv/+ mice, which did not change throughout life. Notch1+/-Notch2+/-Wv/+ mice had white hair virtually all over the body at the first appearance of hair and the depigmentation continued to progress thereafter. Using a neural crest organ culture system, GSI blocked the generation of pigmented melanocytes when added to the culture during the period of melanoblast proliferation, but not during the period of differentiation. These observations imply roles of Notch signaling in both development of melanocyte during embryogenesis and maintenance of melanocyte stem cells in adulthood, while the degree of requirement is distinct in these settings: the latter is more sensitive than the former to the reduced Notch signaling. Furthermore, Notch1 and Notch2 cooperates with c-kit signaling during embryogenesis, and they cooperate with each other to regulate melanocyte homeostasis after birth.     

The mouse Dlx-2 (Tes-1) gene is expressed in spatially restricted domains of the forebrain, face and limbs in midgestation mouse embryos

The pattern of RNA expression of the murine Dlx-2 (Tes-1) homeobox gene is described in embryos ranging in age from E8.5 through E11.5. Dlx-2 is a vertebrate homologue of the Drosophila Distal-less (Dll) gene. Dll expression in the Drosophila embryo is principally limited to the primordia of the brain, head and limbs. Dlx-2 is also expressed principally in the primordia of the forebrain, head and limbs. Within these regions it is expressed in spatially restricted domains. These include two discontinuous regions of the forebrain (basal telencephalon and ventral diencephalon), the branchial arches, facial ectoderm, cranial ganglia and limb ectoderm. Several mouse and human disorders have phenotypes which potentially are the result of mutations in the Dlx genes.     

Development of Melanocyte Progenitors in Murine Steel Mutant Neural Crest Explants Cultured With Stem Cell Factor,Endothelin-3, or TPA

Stem cell factor (SCF) has been suggested to be indispensable for the development of neural crest cells into melanocytes because Steel mutant mice (i.e., Sl/Sf¹) have no pig-mented hairs. On the other hand, it has been demonstrated that the addition of endothelin 3 (ET-3) or TPA to neural crest cell cultures can induce melanocyte differentiation without addition of extrinsic SCF. In this study, we excluded the influence of intrinsic SCF by using SI/SI mouse embryos to study more precisely the effects of natural cytokines, such as extrinsic soluble SCF or ET-3, or chemical reagents, such as TPA or cholera toxin. We found that SCF is supplied within the wild-type neural crest explants and that ET-3 cannot induce melanocyte differentiation or proliferation without SCF. These results indicate that SCF plays a critical role in survival or G1/S entry of melanocyte progenitors and that SCF initially stimulates their proliferation and then ET-3 accelerates their proliferation and differentiation. TPA has the ability to elicit neural crest cell differentiation into melanocytes without exogenously added SCF but it is not as effective as SCF because many more melanocytes developed in the wild-type neural crest explants cultured with TPA.     

Ultrastructural and tissue-culture studies on the role of fibronectin,collagen and glycosaminoglycans in the migration of neural crest cells in the fowl embryo

Summary The initial migration of neural crest (NC) cells into cell-free space was studied by transmission electron microscopy at trunk levels of fowl embryos, some of which were fixed in the presence of ruthenium red. Migrating NC cells occurred in zones which contained fewer ruthenium-red stained 15–40 nm diameter granules than other regions. The ruthenium-red stained granules were linked by similarly stained thin ( 3 nm diameter) microfibrils. The granules resemble proteoglycan and the microfibrils may be hyaluronate. NC cells contacted thicker ( 10 nm diameter) fibrils and interstitial bodies, which did not require ruthenium red for visualization. Cytoplasmic microfilaments were sometimes aligned at the point of contact with the extracellular fibrils, which may be fibronectin and collagen.Phase-contrast time-lapse videotaping and scanning electron microscopy showed that NC cells of the fowl embryo in vitro migrated earlier and more extensively on glass coated with fibronectin-rich fibrous material and adsorbed fibronectin molecules than on glass coated with collagen type I (fibres and adsorbed molecules). NC cells became completely enmeshed in fibronectin-rich fibres, but generally remained on the surface of collagen-fibre gels. When given a choice, NC cells strongly preferred fibronectin coatings to plain glass, and plain glass to dried collagen gels. NC cells showed a slight preference for plain glass over glass to which collagen was adsorbed. Addition to the culture medium of hyaluronate (initial conc. 20 mg/ml), chondroitin (5 mg/ml) and fully sulphated chondroitin sulphate and dermatan sulphate (up to 10 mg/ml) did not drastically alter NC cell migration on fibronectin-rich fibrous substrates. However, partially desulphated chondroitin sulphate (5mg/ml) strongly retarded the migration of NC cells.The in vivo and in vitro studies suggest that fibronectin may dictate the pathways of NC cell migration by acting as a highly preferred physical substrate. However, the utilization of these pathways may be reduced by the presence of proteoglycans bearing undersulphated chondroitin sulphate.Abbreviations NC neural crest - ECM extracellular material - GAG glycosaminoglycan - FN fibronectin - CIG cold insoluble globulin - TEM transmission electron microscopy - SEM scanning electron microscopy - DMEM-H HEPES buffered Dulbecco's modified Eagle's medium - FCS foetal calf serum - CEE chick embryo extract - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis - PBS phosphate-buffered saline     

Cellular origin and developmental mechanisms during the formation of skin melanocytes

Melanocytes are derived from the neural crest (NC), which are transient multipotent cells arising by delamination from the developing dorsal neural tube. During recent years, signaling systems and molecular mechanisms of melanocyte development have been studied in detail, but the exact diversification of the NC into melanocytes and how they migrate, expand and disperse in the skin have not been fully understood. The recent finding that Schwann cell precursors (SCPs) of the growing nerve represents a stem cell niche from which various cell types, including Schwann cells, endoneural fibroblasts and melanocytes arise has exposed new knowledge on the cellular basis for melanocyte development. This opens for the identification of new factors and reinterpretation of old data on cell fate instructive, proliferative, survival and cell homing factors participating in melanocyte development.     

326 例眼睑肿物的临床病理分析

目的:总结眼睑肿物的临床病理类型及特点。方法:收集2000年1月至2011年6月到青岛大学医学院附属医院眼科住院部行手术治疗眼睑肿物患者326例的临床病理资料进行分析。结果:在326例眼睑肿物中,良性肿瘤156例,占47.9%,恶性肿瘤63例,占19.3%;炎性改变98例,占30.1%。良性肿瘤的前五位分别是色素痣、乳头状瘤、囊肿、疣、血管瘤;恶性肿瘤前三位分别是基底细胞癌、睑板腺癌、鳞状细胞癌;炎性改变以炎性肉芽肿最常见。儿童多发的眼睑肿物为皮样瘤、钙化上皮瘤。结论:眼睑病变以良性肿瘤多见,其次为炎性改变。良性肿瘤中以色素痣、乳头状瘤和囊肿多见;恶性肿瘤最常见的为基底细胞癌,儿童眼睑肿瘤以皮样瘤最多见。