Migratory neural crest-like cells form body pigmentation in a urochordate embryo

The neural crest, a source of many different cell types in vertebrate embryos, has not been identified in other chordates. Current opinion therefore holds that neural crest cells were a vertebrate innovation. Here we describe a migratory cell population resembling neural crest cells in the ascidian urochordate Ecteinascidia turbinata. Labelling of embryos and larvae with the vital lipophilic dye DiI enabled us to detect cells that emerge from the neural tube, migrate into the body wall and siphon primordia, and subsequently differentiate as pigment cells. These cells express HNK-1 antigen and Zic gene markers of vertebrate neural crest cells. The results suggest that migratory cells with some of the features of neural crest cells are present in the urochordates. Thus, we propose a hypothesis for neural crest evolution beginning with the release of migratory cells from the CNS to produce body pigmentation in the common ancestor of the urochordates and vertebrates. These cells may have gained additional functions or were joined by other cell types to generate the variety of derivatives typical of the vertebrate neural crest.     

Two rat homologues of Drosophila achaete-scute specifically expressed in neuronal precursors

In vertebrates, the peripheral nervous system is embryologically derived from the neural crest. Although the earliest neural crest cells seem to be multipotent, the molecular mechanisms responsible for the restriction of these cells to different sublineages are not understood. We therefore searched for developmental control genes expressed in crest cells or their derivatives. One important class of regulatory molecules comprises proteins with common DNA-binding and dimerization domains, the basic helix-loop-helix (B-HLH) region. Members of this family include MyoD, a mammalian myogenic determination molecule, and proteins encoded by genes of the achaete-scute complex of Drosophila, which have an important role in neuronal determination. From a sympathetic neuronal precursor cell line derived from the neural crest we have now isolated two different mammalian genes that are homologous to genes of the achaete-scute complex. The sequence of the B-HLH-encoding region of these genes is more similar to that of the genes of the achaete-scute complex than it is to that of other, mammalian members of the B-HLH family. At least one of these genes is transiently expressed in the embryonic rat nervous system, is not detected in non-neuronal tissues or cell lines, and is induced by nerve growth factor in PC12 cells.     

Acetylcholine synthesis by mesencephalic neural crest cells in the process of migration in vivo

Specific to the vertebrate embryo, the neural crest is a transitory structure whose constituent cells migrate extensively through the developing animal and ultimately give rise to many distinct cell types, including the components of the peripheral nervous system. The earliest clear indices of their differentiation have so far been detected only when cells from the crest have reached their destination. This is exemplified by the acquisition of the ability to synthesise and store catecholamines; absent from crest cells before and during their dorso-ventral migration, this ability appears concomitantly with their aggregation into the primary sympathetic ganglia. The chronology of cholinergic maturation, however, is less well defined. Appropriate biochemical markers are demonstrable as soon as parasympathetic or enteric ganglia are formed, but the lack of a suitable cytochemical method is a major obstacle to the identification of any cholinergic cells before then. Although acetylcholinesterase (AChE) is present in migrating neural crest, choline acetyltransferase (CAT), the enzyme catalysing acetylcholine (ACh) synthesis, is a much more relevant correlate, and definitive evidence for cholinergic differentiation should include the demonstration of ACh-synthesising activity in intact cells or their extracts. We show here that neural crest, as soon as it begins migration, can synthesise ACh.     

鱼类色素细胞及其生态学意义概述

本文综述了鱼类色素细胞的起源、分类以及体色的生态学意义。作为脊椎动物最大的类群,鱼类具有6种起源于神经嵴的色素细胞,其中黑色素细胞、黄色素细胞、红色素细胞和蓝色素细胞含有相应色素物质,而彩虹细胞和白色素细胞是通过嘌呤类结晶体的反射作用呈色。色素细胞的数量、大小以及分布情况,加上不同发育时期的变化,决定了鱼类的体色多样性。鱼类的体色作为视觉信号系统,在其生活史关键过程中起到十分重要的作用,主要用于个体的生存和社会种群交流,具体可体现为模拟色、婚姻色等;而紫外体色大大拓展了鱼类视觉信号系统的范围,用于种群内部交流,具有较高的隐蔽性,是为躲避捕食者而形成的一种适应性策略。充分了解鱼类色素细胞的多样性以及体色形成的复杂性,可为深入探索鱼类行为学和种群交流提供科学基础,同时可为优质经济鱼类的人工繁育提供参考。     

Division and differentiation of isolated CNS blast cells in microculture

The mechanism of transformation of the overtly similar cells of the neural plate into the numerous and diverse cell types of the mature vertebrate central nervous system (CNS) can better be understood by studying the clonal development of isolated CNS precursor cells. Here I describe a culture system in which blast cells (cells capable of division) isolated from embryonic day 13.5-14.5 rat forebrain can divide and differentiate into a variety of clonal types. Most clones contain only neurons or glia; 22% contain both neurons and non-neuronal cells. For the division of blast cells, live conditioning cells need to be present indicating that environmental signals influence proliferation. Heterogeneous clones develop in homogeneous culture conditions, so factors intrinsic to the blast cells are probably important in determining the number and type of clonal progeny.     

Regulatory networks define phenotypic classes of human stem cell lines

Stem cells are defined as self-renewing cell populations that can differentiate into multiple distinct cell types. However, hundreds of different human cell lines from embryonic, fetal and adult sources have been called stem cells, even though they range from pluripotent cells-typified by embryonic stem cells, which are capable of virtually unlimited proliferation and differentiation-to adult stem cell lines, which can generate a far more limited repertoire of differentiated cell types. The rapid increase in reports of new sources of stem cells and their anticipated value to regenerative medicine has highlighted the need for a general, reproducible method for classification of these cells. We report here the creation and analysis of a database of global gene expression profiles (which we call the 'stem cell matrix') that enables the classification of cultured human stem cells in the context of a wide variety of pluripotent, multipotent and differentiated cell types. Using an unsupervised clustering method to categorize a collection of approximately 150 cell samples, we discovered that pluripotent stem cell lines group together, whereas other cell types, including brain-derived neural stem cell lines, are very diverse. Using further bioinformatic analysis we uncovered a protein-protein network (PluriNet) that is shared by the pluripotent cells (embryonic stem cells, embryonal carcinomas and induced pluripotent cells). Analysis of published data showed that the PluriNet seems to be a common characteristic of pluripotent cells, including mouse embryonic stem and induced pluripotent cells and human oocytes. Our results offer a new strategy for classifying stem cells and support the idea that pluripotency and self-renewal are under tight control by specific molecular networks.     

Wnt proteins are lipid-modified and can act as stem cell growth factors

Wnt signalling is involved in numerous events in animal development, including the proliferation of stem cells and the specification of the neural crest. Wnt proteins are potentially important reagents in expanding specific cell types, but in contrast to other developmental signalling molecules such as hedgehog proteins and the bone morphogenetic proteins, Wnt proteins have never been isolated in an active form. Although Wnt proteins are secreted from cells, secretion is usually inefficient and previous attempts to characterize Wnt proteins have been hampered by their high degree of insolubility. Here we have isolated active Wnt molecules, including the product of the mouse Wnt3a gene. By mass spectrometry, we found the proteins to be palmitoylated on a conserved cysteine. Enzymatic removal of the palmitate or site-directed and natural mutations of the modified cysteine result in loss of activity, and indicate that the lipid is important for signalling. The purified Wnt3a protein induces self-renewal of haematopoietic stem cells, signifying its potential use in tissue engineering.     

Effect of kinase-negativeEGFR gene on differentiation of embryonic germ cell line EG4

EG4 cells derived from primordial germ cells (PGCs) of 10.5 d post coitum 129/svJ mouse embryos can be used as a model system for in vitro differentiation study due to their pluripotential development ability. EG4 cell lines with stable expression of kinase-negative EGFR cDNA, designated EG4-EGFR^d, were generated by gene transfection. We found that: (ⅰ) EG4-EGFR^d cells share the similar morphology and growing character with wildtype cells that can maintain undifferentiated state in long term culture. (ⅱ) Treatment of EG4 cells with RA resulted in differentiation of adipocyte, while in mutant clones of EG4-EGFR^d, adipocytes were sparse or absent under the same condition, indicating the role of EGFR expressed during adipocyte development. (ⅲ) Histological analysis showed that predominant tissues in teratocarcinomas derived from EG4-EGFR^d cells and wildtype cells are different. A large amount of undifferentiated cells was present in those coming from mutant cell clones. In addition some cardiac and skeletal muscles are prominently differentiated cell types. EG4 wildtype cells produced multiple differentiated cell types of three primary germ layers such as cartilage, epithelia and neural tube. These studies suggested that EGFR-dependent differentiation was inhibited in kinase-negative EG4 cells.     

Conservation and elaboration of Hox gene regulation during evolution of the vertebrate head

The comparison of Hox genes between vertebrates and their closest invertebrate relatives (amphioxus and ascidia) highlights two derived features of Hox genes in vertebrates: duplication of the Hox gene cluster, and an elaboration of Hox expression patterns and roles compared with non-vertebrate chordates. We have investigated how new expression domains and their associated developmental functions evolved, by testing the cis-regulatory activity of genomic DNA fragments from the cephalochordate amphioxus Hox cluster in transgenic mouse and chick embryos. Here we present evidence for the conservation of cis-regulatory mechanisms controlling gene expression in the neural tube for half a billion years of evolution, including a dependence on retinoic acid signalling. We also identify amphioxus Hox gene regulatory elements that drive spatially localized expression in vertebrate neural crest cells, in derivatives of neurogenic placodes and in branchial arches, despite the fact that cephalochordates lack both neural crest and neurogenic placodes. This implies an elaboration of cis-regulatory elements in the Hox gene cluster of vertebrate ancestors during the evolution of craniofacial patterning.     

Induction of cranial and posterior trunk neural crest by exogenous retinoic acid in zebrafish

Retinoic acid (RA) plays an important role in development of vertebrate embryos. We demonstrate impacts of exogenous RA on the formation of neural crest cells in zebrafish using specific neural crest markers sox9b and crestin. Treatment with all -trans RA at 10₋₇ mmol/L at 50% epiboly induces sox9b expression in the forebrain and crestin expression in the forebrain and midbrain, resulting in significant increase of pigment cells in the head derived from the cranial neural crest. In addition, RA treatment induces expression of sox9b and crestin in the caudal marginal cells of the neuroectoderm during early segmentation. Earlier commitment of these cells to the neural crest fate in the posterior margins leads to abnormal development of the posterior body, probably by preventing mingling of ventral derived and dorsal-derived cells during the formation of the tailbud.     

Potential of stem-cell-based therapies for heart disease

The use of stem cells to generate replacement cells for damaged heart muscle, valves, vessels and conduction cells holds great potential. Recent identification of multipotent progenitor cells in the heart and improved understanding of developmental processes relevant to pluripotent embryonic stem cells may facilitate the generation of specific types of cell that can be used to treat human heart disease. Secreted factors from circulating progenitor cells that localize to sites of damage may also be useful for tissue protection or neovascularization. The exciting discoveries in basic science will require rigorous testing in animal models to determine those most worthy of future clinical trials.     

Cell lineages generating axial muscle in the zebrafish embryo

Cell lineage may contribute to determining the numbers, positions and types of cells formed during embryogenesis. In vitro clonal analyses show that vertebrate cells can autonomously maintain lineage commitments to single fates and that terminal development may include an invariant sequence of cell divisions. In addition, in vivo studies with Xenopus led to the proposal that clonal restrictions to spatial 'compartmental' domains arise during early development, analogous to what is observed in insects. In the zebrafish, individual gastrula cells generate clones of progeny that are confined within single tissues, but spatial restrictions have not been described. We now have examined the in vivo terminal cell lineages of zebrafish axial muscles. We obtained no evidence either for strict developmental regulation of division pattern or for spatial compartmentation within muscle lineages.     

Intrinsic segmental identity of segmental founder cells of the leech embryo

Segmentation occurs in several animal phyla, and the cellular mechanisms generating this structural periodicity vary considerably. In the leech, an annelid worm, segmental founder cells arise through a fixed cell lineage (Fig. 1), and come together in a longitudinally repeating array through a stereotyped pattern of morphogenesis. In this paper we demonstrate that founder cells forced to differentiate in a foreign segmental environment give rise to their normal, segment-specific clones of neuronal descendants, even in segments in which those neuronal phenotypes would not normally be observed. These findings indicate that the individual founder cells possess segmental identity at or shortly after the time of their birth, and further suggest that such identities are established by a mechanism in which the parent stem cell 'counts' mitotic cycles.     

Long-term haematopoietic reconstitution by Trp53-/-p16Ink4a-/-p19Arf-/- multipotent progenitors

Haematopoiesis is maintained by a hierarchical system where haematopoietic stem cells (HSCs) give rise to multipotent progenitors, which in turn differentiate into all types of mature blood cells. HSCs maintain themselves for the lifetime of the organism because of their ability to self-renew. However, multipotent progenitors lack the ability to self-renew, therefore their mitotic capacity and expansion potential are limited and they are destined to eventually stop proliferating after a finite number of cell divisions. The molecular mechanisms that limit the proliferation capacity of multipotent progenitors and other more mature progenitors are not fully understood. Here we show that bone marrow cells from mice deficient in three genes genetically downstream of Bmi1--p16Ink4a, p19Arf and Trp53 (triple mutant mice; p16Ink4a and p19Arf are alternative reading frames of the same gene (also called Cdkn2a) that encode different proteins)--have an approximately 10-fold increase in cells able to reconstitute the blood long term. This increase is associated with the acquisition of long-term reconstitution capacity by cells of the phenotype c-kit+Sca-1+Flt3+CD150-CD48-Lin-, which defines multipotent progenitors in wild-type mice. The pattern of triple mutant multipotent progenitor response to growth factors resembles that of wild-type multipotent progenitors but not wild-type HSCs. These results demonstrate that p16Ink4a/p19Arf and Trp53 have a central role in limiting the expansion potential of multipotent progenitors. These pathways are commonly repressed in cancer, suggesting a mechanism by which early progenitor cells could gain the ability to self-renew and become malignant with further oncogenic mutations.     

Dual epithelial origin of vertebrate oral teeth

The oral cavity of vertebrates is generally thought to arise as an ectodermal invagination. Consistent with this, oral teeth are proposed to arise exclusively from ectoderm, contributing to tooth enamel epithelium, and from neural crest derived mesenchyme, contributing to dentin and pulp. Yet in many vertebrate groups, teeth are not restricted only to the oral cavity, but extend posteriorly as pharyngeal teeth that could be derived either directly from the endodermal epithelium, or from the ectodermal epithelium that reached this location through the mouth or through the pharyngeal slits. However, when the oropharyngeal membrane, which forms a sharp ecto/endodermal border, is broken, the fate of these cells is poorly known. Here, using transgenic axolotls with a combination of fate-mapping approaches, we present reliable evidence of oral teeth derived from both the ectoderm and endoderm and, moreover, demonstrate teeth with a mixed ecto/endodermal origin. Despite the enamel epithelia having a different embryonic source, oral teeth in the axolotl display striking developmental uniformities and are otherwise identical. This suggests a dominant role for the neural crest mesenchyme over epithelia in tooth initiation and, from an evolutionary point of view, that an essential factor in teeth evolution was the odontogenic capacity of neural crest cells, regardless of possible 'outside-in' or 'inside-out' influx of the epithelium.     

A new secreted protein that binds to Wnt proteins and inhibits their activities

The Wnt proteins constitute a large family of extracellular signalling molecules that are found throughout the animal kingdom and are important for a wide variety of normal and pathological developmental processes. Here we describe Wnt-inhibitory factor-1 (WIF-1), a secreted protein that binds to Wnt proteins and inhibits their activities. WIF-1 is present in fish, amphibia and mammals, and is expressed during Xenopus and zebrafish development in a complex pattern that includes paraxial presomitic mesoderm, notochord, branchial arches and neural crest derivatives. We use Xenopus embryos to show that WIF-1 overexpression affects somitogenesis (the generation of trunk mesoderm segments), in agreement with its normal expression in paraxial mesoderm. In vitro, WIF-1 binds to Drosophila Wingless and Xenopus Wnt8 produced by Drosophila S2 cells. Together with earlier results obtained with the secreted Frizzled-related proteins, our results indicate that Wnt proteins interact with structurally diverse extracellular inhibitors, presumably to fine-tune the spatial and temporal patterns of Wnt activity.