A novel antigen is shared by retinal pigment epithelium and pigmented neural crest

 Pigment cells in vertebrate embryos are formed in both the central and peripheral nervous system. The neural crest, a largely pluripotent population of precursor cells derived from the embryonic neural tube, gives rise to pigment cells which migrate widely in head and trunk.The retinal pigment epithelium is derived from the optic cup, which arises from ectoderm of the neural tube. We have generated an antibody, ips6, which stains an antigen common to pigment cells of retinal pigment epithelium and neural crest. Ips6 stains retinal pigment epithelium and choroid as well as a subset of crest cells that migrate in pathways typical of melanoblasts. Immunoreactivity is seen first in the eye and later in a subset of migrating crest cells. Crest cells in the amphibian embryo migrate along specific, stereotyped routes; ips6 immunoreactive cells are found in some but not all of these pathways. In older wild-type embryos, cells expressing ips6 appear coincident with pigment-containing cells in the flank, head, eye and embryonic gut. In older animals, staining in the eye extends to the intraretinal segment of optic nerve and interstices between photoreceptors and cells at the retinal periphery. We suggest that the ips6 antibody defines an antigen common to pigment cells of central and peripheral origin. Received: 22 January 1996/Accepted: 15 July 1996     

Clonally cultured differentiated pigment cells can dedifferentiate and generate multipotent progenitors with self-renewing potential

The differentiation of a given cell should be irreversible in order to ensure cell-type-specific function and stability of resident tissue. However, under stimulation in vitro or during regeneration, differentiated cells may recover properties of immature cells. Yet the mechanisms whereby differentiated cells can change fate or reverse to precursor cells are poorly understood. We show here that neural crest (NC)-derived pigment cells that have differentiated in quail embryo, when isolated from the skin and clonally cultured in vitro, are able to generate glial and myofibroblastic cells. The phenotypic reprogramming involves dedifferentiation of dividing pigment cells into cells that re-express NC early marker genes Sox10, FoxD3, Pax3 and Slug. Single melanocytes generate multipotent progenitors able to self-renew along serial subcloning, thus exhibiting stem cell properties. The presence of endothelin 3 promotes the emergence and maintenance of multipotent progenitors in melanocyte progeny. These multipotent cells are heterogeneous with respect to marker identity, including pigmented cells and dedifferentiated cells that have reacquired expression of the early NC marker HNK1. These data provide evidence that, when removed from their niche and subjected to appropriate culture conditions, pigment cells are phenotypically unstable and can reverse to their NC-like ancestors endowed with self-renewal capacity.     

Quail neural crest cells cannot read positional values in the dorsal trunk feathers of the chicken embryo

Summary If quail neural crest cells are grafted to the chick, they migrate into the feathers of the host and produce melanin pigment. In one study, the dorsal trunk feathers of the chimaera were found to have quail-like pigment patterns. This was interpreted in terms of a positional information model. By contrast, in another study it was found that pigment patterns in the wing plumage of the chimaera bore little or no resemblance to the quail, showing instead a rather uniform, dark pigmentation. This was interpreted in terms of a prepattern in the ectoderm. This striking difference in results could be because the wing and trunk plumages have their pigment patterns specified in different ways. We have examined this possibility by making detailed maps of the dorsal trunk plumage of the normal quail and the quail-chick chimaera. Using this novel technique, we can accurately record the secondary pigment patterns in the embryonic down plumage. In the quail there are well-defined, longitudinal stripes running down the back, whereas the chimaera shows rather uniform, dark pigment in this area. There is little or no indication of stripes and some chimaerae develop asymmetric, mottled patterns. Grafts to the cephalic region also produce uniform pigmentation with no quail-like patterning. These findings indicate that neural crest cells cannot read positional values in the feathers of another species.     

Distinct roles for ERK1 and ERK2 in pathophysiology of CNS

Mitogen-activated protein kinases ERK1 and ERK2 have been implicated in various pathophysiological events of the CNS,but their specific roles in cell processes under physiologic and pathological condit...     

Genetic analysis of steel and the PG-M/versican-encoding gene AxPG as candidates for the white (d) pigmentation mutant in the salamander Ambystoma mexicanum

 Vertebrate non-retinal pigment cells are derived from neural crest (NC) cells, and several mutations have been identified in the Mexican axolotl Ambystoma mexicanum (Ambystomatidae) that affect the development of these cell lineages. In ”white” (d) mutant axolotls, premigratory NC cells differentiate as pigment cells, yet fail to disperse, survive, or both, and this leads to a nearly complete absence of pigment cells in the skin. Previous studies revealed that d affects pigment cell development non-autonomously, and have reported differences between white and wild-type axolotls in the structure and composition of the extracellular matrix through which NC and pigment cells migrate. Here we test the correspondence of d and two candidate genes: steel and AxPG. In amniotes, Steel encodes the cytokine Steel factor (mast cell growth factor; stem cell factor; kit ligand), which is expressed along the migratory pathways of melanocyte precursors and is required by these cells for their migration and survival; mammalian Steel mutants resemble white mutant axolotls in having a deficit or complete absence of pigment cells. In contrast, AxPG encodes aPG-M/versican-like proteoglycan that may promote the migration of A. mexicanum pigment cells, and AxPGexpression is reduced in white mutant axolotls. We cloned a salamander orthologue of steel and used a partial genetic linkage map of Ambystoma to determine the genomic locations of steel, AxPG, and d. We show that the three genes map to different linkage groups, excluding steel and AxPG as candidates for d. Received: 11 November 1998 / Accepted: 30 January 1999     

Brain induction in ascidian embryos is dependent on juxtaposition of FGF9/16/20-producing and -receiving cells

Coordinated regulation of inductive events, both spatially and temporally, during animal development ensures that tissues are induced at their specific positions within the embryo. The ascidian brain is induced in cells at the anterior edge of the animal hemisphere by fibroblast growth factor (FGF) signals secreted from vegetal cells. To clarify how this process is spatially regulated, we first identified the sources of the FGF signal by examining the expression of brain markers Hr-Otx and Hr-ETR-1 in embryos in which FGF signaling is locally inhibited by injecting individual blastomeres with morpholino oligonucleotide against Hr-FGF9/16/20, which encodes an endogenous brain inducer. The blastomeres identified as the inducing sources are A5.1 and A5.2 at the 16-cell stage and A6.2 and A6.4 at the 24-cell stage, which are juxtaposed with brain precursors at the anterior periphery of the embryo at the respective stages. We also showed that all the cells of the animal hemisphere are capable of expressing Hr-Otx in response to the FGF signal. These results suggest that the position of inducers, rather than competence, plays an important role in determining which animal cells are induced to become brain tissues during ascidian embryogenesis. This situation in brain induction contrasts with that in mesoderm induction, where the positions at which the notochord and mesenchyme are induced are determined mainly by intrinsic competence factors that are inherited by signal-receiving cells.     

In vivo characterization of neural crest-derived fibro/adipogenic progenitor cells as a likely cellular substrate for craniofacial fibrofatty infiltrating disorders

The cellular substrate underlying aberrant craniofacial connective tissue accumulation that occurs in disorders such as congenital infiltration of the face (CILF) remain elusive. Here we analyze the in vivo properties of a recently identified population of neural crest-derived CD31-:CD45-:alpha7-:Sca1+:PDGFRa+ fibro/adipogenic progenitors (NCFAPs). In serial transplantation experiments in which NCFAPs were prospectively purified and transplanted into wild type mice, NCFAPs were found to be capable of self-renewal while keeping their adipogenic potential. NCFAPs constitute the main responsive FAP fraction following acute masseter muscle damage, surpassing the number of mesoderm-derived FAPs (MFAPs) during the regenerative response. Lastly, NCFAPs differentiate into adipocytes during muscle regeneration in response to pro-adipogenic systemic cues. Altogether our data indicate that NCFAPs are a population of stem/primitive progenitor cells primarily involved in craniofacial muscle regeneration that can cause tissue degeneration when the damage co-occurs with an obesity inducing diet.     

Molecular Characterization of the Mouse Tyrosinase Gene:Pigment Cell-Specific Expression in Transgenic Mice

Tyrosinase is the key enzyme in melanin synthesis, and is expressed in the pigment epithelium of the retina, a cell layer derived from the optic cup; and in neural crest-derived melanocytes of skin, hair follicle, choroid, and iris. The tyrosinase gene has been cloned and shown to map to the well-characterized c-locus (albino locus) of the mouse. Subsequent studies demonstrated that a functional tyrosinase minigene was able to rescue the albino phenotype in transgenic mice. The transgene was expressed in a cell type-specific manner in skin and eye. During development of the mouse, the tyrosinase gene is expressed in the pigment epithelium of the retina as early as day 10.5 of gestation. In the hair follicle, tyrosinase gene expression is detected from day 16.5 onwards. This cell-type–specific expression is largely reproduced in transgenic mice. Our results suggest that sequences in the immediate vicinity of the mouse tyrosinase gene are sufficient to provide cell type-specificity and developmental regulation in melanocytes and the pigment epithelium.     

Neural crest cell-specific deletion of Rac1 results in defective cell-matrix interactions and severe craniofacial and cardiovascular malformations

The small GTP-binding protein Rac1, a member of the Rho family of small GTPases, has been implicated in regulation of many cellular processes including adhesion, migration and cytokinesis. These functions have largely been attributed to its ability to reorganize cytoskeleton. While the function of Rac1 is relatively well known in vitro, its role in vivo has been poorly understood. It has previously been shown that in neural crest cells (NCCs) Rac1 is required in a stage-specific manner to acquire responsiveness to mitogenic EGF signals. Here we demonstrate that mouse embryos lacking Rac1 in neural crest cells (Rac1/Wnt1-Cre) showed abnormal craniofacial development including regional ectodermal detachment associated with mesenchymal acellularity culminating in cleft face at E12. Rac1/Wnt1-Cre mutants also displayed inappropriate remodelling of pharyngeal arch arteries and defective outflow tract septation resulting in the formation of a common arterial trunk (‘persistent truncus arteriosus’ or PTA). The mesenchyme around the aortic sac also developed acellular regions, and the distal aortic sac became grossly dysmorphic, forming a pair of bilateral, highly dilated arterial structures connecting to the dorsal aortas. Smooth muscle cells lacking Rac1 failed to differentiate appropriately, and subpopulations of post-migratory NCCs demonstrated aberrant cell death and attenuated proliferation. These novel data demonstrate that while Rac1 is not required for normal NCC migration in vivo, it plays a critical cell-autonomous role in post-migratory NCCs during craniofacial and cardiac development by regulating the integrity of the craniofacial and pharyngeal mesenchyme.     

Involvement of the collagen I-binding motif in the anti-angiogenic activity of pigment epithelium-derived factor

Pigment epithelium-derived factor (PEDF) is the most potent endogenous inhibitor of angiogenesis in age-related macular degeneration and tumors. However, the molecular mechanism of the anti-angiogenic activity of PEDF is poorly understood. PEDF interacts with the extracellular matrix (ECM) in vitro. Here, we investigated the possible involvement of the motif for ECM interaction in the anti-angiogenic activity of PEDF. The growth rates of HeLa cells in culture were not affected by transfection of PEDF, indicating that PEDF did not suppress tumor cell growth directly. In tumor xenografts, the overexpression of wild-type PEDF significantly suppressed tumor growth, whereas a mutant of the collagen I-binding site of PEDF (Col-mut PEDF) did not inhibit tumor growth. A mutant of the heparin-binding site of PEDF (Hep-mut PEDF) suppressed tumor growth. Histological analysis showed that the density and area of microvasculatures in either PEDF or Hep-mut PEDF were suppressed when compared with those in either vector or Col-mut PEDF. Our data indicate that PEDF inhibits tumor growth via its anti-angiogenic activity, and the collagen I-binding motif of PEDF is involved in the biological activity.     

A novel vanadium transporter of the Nramp family expressed at the vacuole of vanadium-accumulating cells of the ascidian Ascidia sydneiensis samea

Background

Vanadium is an essential transition metal in biological systems. Several key proteins related to vanadium accumulation and its physiological function have been isolated, but no vanadium ion transporter has yet been identified.

Methods

We identified and cloned a member of the Nramp/DCT family of membrane metal transporters (AsNramp) from the ascidian Ascidia sydneiensis samea, which can accumulate extremely high levels of vanadium in the vacuoles of a type of blood cell called signet ring cells (also called vanadocytes). We performed immunological and biochemical experiments to examine its expression and transport function.

Results

Western blotting analysis showed that AsNramp was localized at the vacuolar membrane of vanadocytes. Using the Xenopus oocyte expression system, we showed that AsNramp transported VO²⁺ into the oocyte as pH-dependent manner above pH 6, while no significant activity was observed below pH 6. Kinetic parameters (K_m and V_max) of AsNramp-mediated VO²⁺ transport at pH 8.5 were 90 nM and 9.1 pmol/oocyte/h, respectively. A rat homolog, DCT1, did not transport VO²⁺ under the same conditions. Excess Fe²⁺, Cu²⁺, Mn²⁺, or Zn²⁺ inhibited the transport of VO²⁺. AsNramp was revealed to be a novel VO²⁺/H⁺ antiporter, and we propose that AsNramp mediates vanadium accumulation coupled with the electrochemical gradient generated by vacuolar H⁺-ATPase in vanadocytes.

General Significance

This is the first report of identification and functional analysis on a membrane transporter for vanadium ions.     

ERK1/2在高温致神经管畸形神经上皮细胞中的表达变化

目的 探讨高温致神经管畸形(NTDs)作用的分子机制,为防治NTDs的发生提供理论依据.方法 在高温致金黄地鼠NTDs模型的基础上,应用免疫荧光染色技术,观察NTDs发生过程中p-ERK1/2在鼠胚神经上皮细胞中的表达变化.结果 对照组和实验组孕鼠在高温水浴处理后16、24h,p-ERK1/2免疫阳性产物分布于鼠胚神经上皮细胞和周围间充质细胞的胞浆中;水浴后36、60h,p-ERK1/2表达部位出现了由细胞浆向细胞核的转移;高温处理后,p-ERK1/2在实验组各期胚胎神经上皮细胞内的表达均比对照组减弱.结论 ERK1/2参与胚胎神经管的发育过程,其表达降低在高温致神经管畸形的发生中起重要作用.     

Trunk lateral cells are neural crest-like cells in the ascidian Ciona intestinalis: insights into the ancestry and evolution of the neural crest

Neural crest-like cells (NCLC) that express the HNK-1 antigen and form body pigment cells were previously identified in diverse ascidian species. Here we investigate the embryonic origin, migratory activity, and neural crest related gene expression patterns of NCLC in the ascidian Ciona intestinalis. HNK-1 expression first appeared at about the time of larval hatching in dorsal cells of the posterior trunk. In swimming tadpoles, HNK-1 positive cells began to migrate, and after metamorphosis they were localized in the oral and atrial siphons, branchial gill slits, endostyle, and gut. Cleavage arrest experiments showed that NCLC are derived from the A7.6 cells, the precursors of trunk lateral cells (TLC), one of the three types of migratory mesenchymal cells in ascidian embryos. In cleavage arrested embryos, HNK-1 positive TLC were present on the lateral margins of the neural plate and later became localized adjacent to the posterior sensory vesicle, a staging zone for their migration after larval hatching. The Ciona orthologues of seven of sixteen genes that function in the vertebrate neural crest gene regulatory network are expressed in the A7.6/TLC lineage. The vertebrate counterparts of these genes function downstream of neural plate border specification in the regulatory network leading to neural crest development. The results suggest that NCLC and neural crest cells may be homologous cell types originating in the common ancestor of tunicates and vertebrates and support the possibility that a putative regulatory network governing NCLC development was co-opted to produce neural crest cells during vertebrate evolution.     

Nitric oxide inhibition of ERK1/2 activity in cells expressing neuronal nitric-oxide synthase

Neuronal nitric-oxide synthase (nNOS) is a constitutively expressed enzyme responsible for the production of nitric oxide (NO*) from l-arginine and O2. Nitric oxide is an intra- and intercellular messenger that mediates a diversity of signaling pathways in target cells. In the absence of l-arginine, nNOS has been shown to generate superoxide (O2*). Superoxide, either directly or through its self-dismutation to H2O2, is likewise believed to be a cell-signaling agent. Because nNOS can generate NO* and O2*, we examined the activation of cellular signal transduction pathways in nNOS-transfected cells grown in the presence or absence of l-arginine. Spin trapping/EPR spectroscopy confirmed that stimulated nNOS-transfected cells grown in an l-arginine environment secreted NO* into the surrounding milieu. Production of NO* blocked Ca2+ ionophore-induced activation of the ERK1/2 through a mechanism involving inhibition of the Ras G-protein and Raf-1 kinase. In contrast, ERK activation was largely unaffected in nNOS-transfected cells grown in l-arginine-free media. Inhibition of nNOS-generated NO* with the competitive NOS inhibitor, NG-nitro-l-arginine methyl ester, in cells grown in l-arginine restored ERK1/2 activation to levels similar to that found when nNOS was activated in l-arginine-free media. These findings indicate that nNOS can differentially regulate the ERK signal transduction pathway in a manner dependent on the presence of l-arginine and the production of NO*.