The neural plate specifies somite size in the Xenopus laevis gastrula.

The organizer has traditionally been considered the major source of somite-inducing signals. We show here that signaling from the neural plate specifies somite tissue and regulates somite size in the Xenopus gastrula. Ectopic undifferentiated neural tissue induces massive somite expansion at the expense of intermediate and lateral plate mesoderm. Although the early expanded somite expresses muscle-specific markers, only a portion terminally differentiates, suggesting that myotome development requires additional signals. Explant assays demonstrate that neural tissue induces somite-specific marker expression even in the absence of the organizer. Finally, we demonstrate that neural tissue is required for proper somite development because elimination of neural precursors results in pronounced somite reduction. Thus, an important reciprocal interaction exists between somite and neural tissue that is mutually reinforcing and critical for normal embryonic patterning.     

Motile behavior and protrusive activity of migratory mesoderm cells from the Xenopus gastrula.

During Xenopus gastrulation, the mesoderm migrates across a fibronectin (FN)-containing substrate, the inner surface of the blastocoel roof (BCR). A possible role for FN is to promote the extension of cytoplasmic processes which serve as locomotory organelles for mesoderm cells. To test this idea, the interaction of prospective head mesoderm (HM) cells with FN was examined in vitro. Nonattached HM cells extend filiform processes from an active region of the cell surface. This spontaneous activity is modulated by cell attachment to FN. Additional active regions appear, and cytoplasmic lamellae extend from these sites, leading to cell spreading and translocation. Thus, although FN seems not to induce processes de novo, it modulates a spontaneous protrusive activity to yield the extension of lamellae along the substrate surface. As putative locomotory organelles, HM cell protrusions were characterized functionally. They adhere rapidly and selectively to in situ substrates, preferentially to FN, and retract upon attachment. During translocation, the passive cell body is moved by the activity of the protrusions. Lamellae continuously extend, retract, or split into parts. This leads to an intermittent, nonpersistent mode of translocation. The polarity of HM cells, as expressed in the arrangement of protrusions, bears no constant relationship to the orientation of the cell body, and a cell can change its direction of movement without a corresponding rotation of the cell body. This may be relevant with respect to the mechanism by which mesoderm cells translate guidance cues of the BCR into a polarized, oriented cell structure during directional migration in situ.     

Xenopus Tetraspanin-1 regulates gastrulation movements and neural differentiation in the early Xenopus embryo

The tetraspanin family of four-pass transmembrane proteins has been implicated in fundamental biological processes, including cell adhesion, migration, and proliferation. Tetraspanins interact with various transmembrane proteins, establishing a network of large multimolecular complexes that allows specific lateral secondary interactions. Here we report the identification and functional characterization of Xenopus Tetraspanin-1 (xTspan-1). At gastrula and neurula, xTspan-1 is expressed in the dorsal ectoderm and neural plate, respectively, and in the hatching gland, cement gland, and posterior neural tube at tailbud stages. The expression of xTspan-1 in the early embryo is negatively regulated by bone morphogenetic protein (BMP) and stimulated by Notch signals. Microinjection of xTspan-1 mRNA interfered with gastrulation movements and reduced ectodermal cell adhesion in a cadherin-dependent manner. Morpholino knock-down of endogenous xTspan-1 protein revealed a requirement of xTspan-1 for gastrulation movements and primary neurogenesis. Our data suggest that xTspan-1 could act as a molecular link between BMP signalling and the regulation of cellular interactions that are required for gastrulation movements and neural differentiation in the early Xenopus embryo.     

Dorsoventral differences in cell-cell interactions modulate the motile behaviour of cells from the Xenopus gastrula.

When groups of cells from the inner marginal zone (mesendoderm) of the early Xenopus gastrula are placed on a fibronectin-coated substratum, the explants of the dorsal region spread into monolayers whereas those from the ventral region, though they adhere to the substratum, do not show this spreading reaction. This different behaviour is not reflected in the in vitro behaviour of the respective cells kept in isolation. No difference between dorsal and ventral cells was observed, when they were tested for lamellipodia-driven spreading, movement over the substratum or properties of integrin- and cadherin-mediated adhesion. However, cell contacts between individual dorsal cells are significantly less stable than those between ventral cells. The higher flexibility of the cell-cell contacts seems to determine the spreading behaviour of the dorsal explants, which includes lamellipodia-driven outward movement of the peripheral cells, rearrangements of the cells, building up a horizontal tension within the aggregate and intercalation of cells from above into the bottom layer. Ventral explants lack these properties. Staining for F-actin revealed a decisive difference of the supracellular organisation of the cytoskeleton that underlies the morphology of the different types of explants. Evidence for a higher flexibility of cell-cell contacts in the dorsal mesendoderm was also obtained in SEM studies on gastrulating embryos. Dorsal mesendodermal cells show stronger protrusive activity as compared to ventral mesendodermal cells. The meaning of these observations for the mechanisms of morphogenetic movements during gastrulation is central to the discussion.     

Directed differentiation of pluripotent cells to neural lineages: homogeneous formation and differentiation of a neurectoderm population

During embryogenesis the central and peripheral nervous systems arise from a neural precursor population, neurectoderm, formed during gastrulation. We demonstrate the differentiation of mouse embryonic stem cells to neurectoderm in culture, in a manner which recapitulates embryogenesis, with the sequential and homogeneous formation of primitive ectoderm, neural plate and neural tube. Formation of neurectoderm occurs in the absence of extraembryonic endoderm or mesoderm and results in a stratified epithelium of cells with morphology, gene expression and differentiation potential consistent with positionally unspecified neural tube. Differentiation of this population to homogeneous populations of neural crest or glia was also achieved. Neurectoderm formation in culture allows elucidation of signals involved in neural specification and generation of implantable cell populations for therapeutic use.     

Tension-dependent collective cell movements in the early gastrula ectoderm of Xenopus laevis embryos

Ventral ectodermal explants taken from early gastrula embryos of Xenopus laevis were artificially stretched either by two opposite concentrated forces or by a distributed force applied to the internal explant’s layer. These modes of stretching reflect different mechanical situations taking place in the normal development. Two main types of kinematic response to the applied tensions were detected. First, by 15 min after the onset of concentrated stretching a substantial proportion of the explant’s cells exhibited a concerted movement towards the closest point of the applied stretching force. We define this movement as tensotaxis. Later, under both concentrated and distributed stretching, most of the cell’s trajectories became reoriented perpendicular to the stretching force, and the cells started to intercalate between each other, both horizontally and vertically. This was accompanied by extensive elongation of the outer ectodermal cells and reconstruction of cell-cell contacts. The intercalation movements led first to a considerable reduction in the stretch-induced tensions and then to the formation of peculiar bipolar ”embryoid” shapes. The type and intensity of the morphomechanical responses did not depend upon the orientation of a stretching force in relation to the embryonic axes. We discuss the interactions of the passive and active components in tension-dependent cell movements and their relations to normal morphogenetic events. Received: 26 April 1999 / Accepted: 30 August 1999     

The cell sorting process of Xenopus gastrula cells involves the acto-myosin system and TGF-β signaling

We have previously shown that the cell sorting process of animal pole cells (AC) and vegetal pole cells (VC) from Xenopus gastrulae is considered to involve two steps: concentrification and polarization. In this study, we addressed the question of what specified the spatial relationship of the AC and VC clusters during the process. First, we examined the inhibitory or facilitatory treatment for myosin 2 activity during each of the two steps. The aggregates treated with Y27632 or blebbistatin during the concentrification step showed a cluster random arrangement, suggesting the prevention of the cell sorting by inhibition of myosin 2. Meanwhile, the treatment with a Rac1 inhibitor, NSC23766, during the same step resulted in promotion of the fusion of the AC clusters and the progression of the cell sorting, presumably by an indirect activation of myosin 2. On the other hand, the treatments with any of the three drugs during the polarization step showed that the two clusters did not appose, and their array remained concentric. Thus, the modulation of cell contraction might be indispensable to each of the two steps. Next, the activin/nodal TGF-β signaling was perturbed by using a specific activin receptor-like kinase inhibitor, SB431542. The results revealed a bimodal participation of the activin/nodal TGF-β signaling, i.e., suppressive and promotive effects on the concentrification and the polarization, respectively. Thus, the present in vitro system, which permits not only the cell contraction-mediated cell sorting but also the TGF-β-directed mesodermal induction such as cartilage formation, may fairly reflect the embryogenesis in vivo.     

Mechanisms of mesendoderm internalization in the Xenopus gastrula: lessons from the ventral side.

Two main processes are involved in driving ventral mesendoderm internalization in the Xenopus gastrula. First, vegetal rotation, an active movement of the vegetal cell mass, initiates gastrulation by rolling the peripheral blastocoel floor against the blastocoel roof. In this way, the leading edge of the internalized mesendoderm is established, that remains separated from the blastocoel roof by Brachet's cleft. Second, in a process of active involution, blastopore lip cells translocate on arc-like trails around the tip of Brachet's cleft. Hereby the lower, Xbra-negative part of the lip moves toward the interior, to contribute mainly to endoderm. In contrast, the upper, Xbra-expressing part moves toward the blastocoel roof-apposed surface of the involuted mesoderm, and eventually becomes inserted into this surface. Vegetal rotation and active mesoderm surface insertion persist over much of gastrulation ventrally. Both processes are also active dorsally. In fact, internalization processes generally spread from dorsal to ventral, though at different rates, which suggests that they are independently controlled. Ventrally and laterally, mesoderm occurs not only in the marginal zone, but also in the adjacent blastocoel roof. Such blastocoel roof mesoderm shares properties with the remaining, ectodermal roof, that are related to its function as substratum for mesendoderm migration. It repels involuted mesoderm, thus contributing to separation of cell layers, and it assembles a fibronectin matrix. These properties change as the blastocoel roof mesoderm moves into the blastopore lip during gastrulation.     

FRL-1, a member of the EGF-CFC family,is essential for neural differentiation in Xenopus early development

Recent studies indicate an essential role for the EGF-CFC family in vertebrate development, particularly in the regulation of nodal signaling. Biochemical evidence suggests that EGF-CFC genes can also activate certain cellular responses independently of nodal signaling. Here, we show that FRL-1, a Xenopus EGF-CFC gene, suppresses BMP signaling to regulate an early step in neural induction. Overexpression of FRL-1 in animal caps induced the early neural markers zic3, soxD and Xngnr-1, but not the pan-mesodermal marker Xbra or the dorsal mesodermal marker chordin. Furthermore, overexpression of FRL-1 suppressed the expression of the BMP-responsive genes, Xvent-1 and Xmsx-1, which are expressed in animal caps and induced by overexpressed BMP-4. Conversely, loss of function analysis using morpholino-antisense oligonucleotides against FRL-1 (FRL-1MO) showed that FRL-1 is required for neural development. FRL-1MO-injected embryos lacked neural structures but contained mesodermal tissue. It was suggested previously that expression of early neural genes that mark the start of neuralization is activated in the presumptive neuroectoderm of gastrulae. FRL-1MO also inhibited the expression of these genes in dorsal ectoderm, but did not affect the expression of chordin, which acts as a neural inducer from dorsal mesoderm. FRL-1MO also inhibited the expression of neural markers that were induced by chordin in animal caps, suggesting that FRL-1 enables the response to neural inducing signals in ectoderm. Furthermore, we showed that the activation of mitogen-activated protein kinase by FRL-1 is required for neural induction and BMP inhibition. Together, these results suggest that FRL-1 is essential in the establishment of the neural induction response.     

Mapping of clonal lineages across developmental stages in human neural differentiation

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Localization of two IQGAPs in cultured cells and early embryos of Xenopus laevis

Mammalian IQGAP1 is considered to modulate organization of the actin cytoskeleton under regulation of signaling proteins Cdc42 or Rac and calmodulin [Bashour et al., 1997: J Cell Biol 137:1555-1566; Hart et al., 1996: EMBO J 15:2997-3005] and also to be involved in cadherin-based cell adhesion [Kuroda et al., 1998: Science 281:832-835]. However, its function in the cell has not been clear. In order to clarify the function of IQGAP, we investigated IQGAP in Xenopus laevis cells. We isolated two Xenopus cDNAs encoding homologues of mammalian IQGAP, XIQGAP1, and XIQGAP2, which show high homology with human IQGAP1 and IQGAP2, respectively. Immunofluorescent localization of XIQGAPs in Xenopus tissue cultured cells (XTC cells) and in developing embryos was examined. In XTC cells, XIQGAP1 was colocalized with F-actin at cell-to-cell contact sites, membrane ruffles in lamellipodia, and filopodia. During development of embryos, XIQGAP1 was concentrated in the borders of all embryonic cells. An intense staining for XIQGAP1 was found in regions undergoing active morphogenetic movements, such as the blastopore lip of gastrulae, and the neural plate, the notochord, and the somite of neurulae. These results suggest that XIQGAP1 is involved in both cell-to-cell adhesion and cell migration during Xenopus embryogenesis and in cultured cells. On the other hand, the localization of XIQGAP2 in XTC cells was distinct from that of XIQGAP1 although it was also seen in lamellipodia, filopodia, and borders between cells. In addition to these regions, strong nuclear staining was observed in both XTC cells and embryonic cells.     

Mammalian neuronal differentiation: early expression of a neuronal phenotype from mouse neural crest cells in a chemically defined culture medium

We show that mouse neural crest cells cultured in a serum-deprived chemically defined medium on appropriate culture substrata can be induced to express a neuronal phenotype. The uncommitted neural crest cells express a mesenchymal intermediate filament protein such as vimentin, but not the usual neuronal markers such as receptor sites for tetanus toxin or neurofilaments. In the chemically defined medium, receptor sites for tetanus toxin or neurofilaments can be characterized after a few hours in culture. Furthermore, these cells acquire tetrodotoxin-sensitive voltage-dependent Na+ channels and can generate action potentials. Such an in vitro system should allow us to analyze and manipulate early stages of neuronal differentiation in a mammalian embryo, at a level so far restricted to lower vertebrate embryos.     

Large-scale generation of human iPSC-derived neural stem cells/early neural progenitor cells and their neuronal differentiation

Induced pluripotent stem cell (iPSC)-based technologies offer an unprecedented opportunity to perform high-throughput screening of novel drugs for neurological and neurodegenerative diseases. Such screenings require a robust and scalable method for generating large numbers of mature, differentiated neuronal cells. Currently available methods based on differentiation of embryoid bodies (EBs) or directed differentiation of adherent culture systems are either expensive or are not scalable. We developed a protocol for large-scale generation of neuronal stem cells (NSCs)/early neural progenitor cells (eNPCs) and their differentiation into neurons. Our scalable protocol allows robust and cost-effective generation of NSCs/eNPCs from iPSCs. Following culture in neurobasal medium supplemented with B27 and BDNF, NSCs/eNPCs differentiate predominantly into vesicular glutamate transporter 1 (VGLUT1) positive neurons. Targeted mass spectrometry analysis demonstrates that iPSC-derived neurons express ligand-gated channels and other synaptic proteins and whole-cell patch-clamp experiments indicate that these channels are functional. The robust and cost-effective differentiation protocol described here for large-scale generation of NSCs/eNPCs and their differentiation into neurons paves the way for automated high-throughput screening of drugs for neurological and neurodegenerative diseases.     

A comparative evaluation of methods for isolation of RNA-directed DNA polymerase from cells in a reconstituted system.

We have compared the relative merits of several procedures for the isolation of RNA-directed DNA polymerase (EC 2.7.7.7.) from cells using a reconsituted model system consisting of a mixture of woolly monkey (simian) sarcoma virus and a cultured human lymphoblastoid cell line, NC-37. When the cell-virus mixture was gently disrupted and fractionated by differential centrifugation, most of the added polymerase was recovered associated with a particulate fraction obtained from the post-mitochondrial supernatant. Purification of the polymerase was best achieved starting from this fraction. The particulate fraction itself can be purified by gel filtration through a Sepharose 2 B column. This procedure did not significantly alter the composition of viral and cellular DNA polymerases. Whereas as little as 7.5 - 10(5) viral particles were sufficient for the detection of RNA-directed DNA polymerase activity, a minimum of about 10(11) particles were necessary for the isolation and unequivocal characterization of the enzyme from the cell-virus mixture by subcellular fractionation and chromatographic separation from cellular DNA polymerases. Purified RNA-directed DNA polymerase had the same primer-template characteristics, sedimentation properties, and immunological cross reactivity as the enzyme purified from density gradient-banded virions of simian sarcoma virus. Methods involving total extraction of the cell-virus mixture either by repeated freezing and thawing followed by detergent treatment or by Dounce homogenization and treatment with high salt and detergent failed to provide RNA-directed DNA polymerase free of cellular DNA polymerases. Because of this, low levels of cellular RNA-directed DNA polymerase may be missed when these approaches are used.     

In vitro proliferation and differentiation of myogenic cells from adult Xenopus

A technique is described for isolating amphibian myogenic cells from the muscle of adult Xenopus laevis (Dauchin). Muscles were dissociated with 0.2% collagenase and 0.1% trypsin. The resulting cell suspensions were separated from the remaining myofibres by filtration through nylon grids. Most of the cells remaining in the filtrate suspension were satellite cells or fibroblasts. When plated in Petri dishes, satellite cells adhered to the substrate, became spindle-shaped and proliferated activity in a culture medium supplemented with fetal calf serum. Mitotic waves lasted 4 days and consequently cell density markedly increased. Satellite cells came into contact and began to fuse into myotubes on day 8 of culture. Horse serum, which replaced fetal calf serum in the medium on day 12, accelerated cell fusions which were almost complete on day 18. However, under these conditions, some mononucleated cells continued to undergo mitosis. Cell proliferation with a high rate of mitosis was prolonged by repeated trypsinization and replating in medium supplemented with fetal calf serum. When myofibres from dissociated muscles were cultured under the same conditions, they never fragmented or divided.     

An assay system to study migratory behavior of cranial neural crest cells in Xenopus

An increasing number of genes are known to show expression in the cranial neural crest area. So far it is very difficult to analyze their effect on neural crest cell migration because of the lack of transplantation techniques. This paper presents a simple method to study the migratory behavior of cranial neural crest cells by homo- and heterotopic transplantations: Green fluorescent protein (GFP) RNA was injected into one blastomere of Xenopus laevis embryos at the 2-cell stage. The cranial neural crest area of stage 14 embryos was transplanted into the head or trunk region of an uninjected host embryo, and the migration was monitored by GFP fluorescence. The transplants were further examined by double immunostaining and confocal microscopy to trace migratory routes inside the embryo, and to exclude contaminations of grafts with foreign tissues. Our results demonstrate that we developed a highly efficient and reproducible technique to study the migratory ability of cranial neural crest cells. It offers the possibility to analyze genes involved in neural crest cell migration by coinjecting their RNA with that of GFP. Received: 28 September 1999 / Accepted: 17 November 1999     

In vitro differentiation of human processed lipoaspirate cells into early neural progenitors

Human processed lipoaspirate (PLA) cells are multipotent stem cells, capable of differentiating into multiple mesenchymal lineages (bone, cartilage, fat, and muscle). To date, differentiation to nonmesodermal fates has not been reported. This study demonstrates that PLA cells can be induced to differentiate into early neural progenitors, which are of an ectodermal origin. Undifferentiated cultures of human PLA cells expressed markers characteristic of neural cells such as neuron-specific enolase (NSE), vimentin, and neuron-specific nuclear protein (NeuN). After 2 weeks of treatment of PLA cells with isobutylmethylxanthine, indomethacin, and insulin, about 20 to 25 percent of the cells differentiated into cells with typical neural morphologic characteristics, accompanied by increased expression of NSE, vimentin, and the nerve-growth factor receptor trk-A. However, induced PLA cells did not express the mature neuronal marker, MAP, or the mature astrocyte marker, GFAP. It was also found that neurally induced PLA cells displayed a delayed-rectifier type K+ current (an early developmental ion channel) concomitantly with morphologic changes and increased expression of neural-specific markers. The authors concluded that human PLA cells might have the potential to differentiate in vitro into cells that represent early progenitors of neurons and/or glia.     

Identification and characterisation of the early differentiating cells in neural differentiation of human embryonic stem cells

One of the challenges in studying early differentiation of human embryonic stem cells (hESCs) is being able to discriminate the initial differentiated cells from the original pluripotent stem cells and their committed progenies. It remains unclear how a pluripotent stem cell becomes a lineage-specific cell type during early development, and how, or if, pluripotent genes, such as Oct4 and Sox2, play a role in this transition. Here, by studying the dynamic changes in the expression of embryonic surface antigens, we identified the sequential loss of Tra-1-81 and SSEA4 during hESC neural differentiation and isolated a transient Tra-1-81(-)/SSEA4(+) (TR-/S4+) cell population in the early stage of neural differentiation. These cells are distinct from both undifferentiated hESCs and their committed neural progenitor cells (NPCs) in their gene expression profiles and response to extracellular signalling; they co-express both the pluripotent gene Oct4 and the neural marker Pax6. Furthermore, these TR-/S4+ cells are able to produce cells of both neural and non-neural lineages, depending on their environmental cues. Our results demonstrate that expression of the pluripotent factor Oct4 is progressively downregulated and is accompanied by the gradual upregulation of neural genes, whereas the pluripotent factor Sox2 is consistently expressed at high levels, indicating that these pluripotent factors may play different roles in the regulation of neural differentiation. The identification of TR-S4+ cells provides a cell model for further elucidation of the molecular mechanisms underlying hESC neural differentiation.     

A fate map of superficial and deep circumblastoporal cells in the early gastrula of Pleurodeles waltl.

We have determined the fate of presumptive mesodermal cells in the early Pleurodeles waltl gastrula. We labeled all cells in a gastrula with RLDx cell lineage tracer and superficial cells with 125I and then grafted small pieces of the marginal zone orthotopically into unlabeled host embryos. Labeled progeny were identified in sectioned embryos at the tail bud stage. The use of double-labeled grafts allowed us to study the relative contributions by superficial and deep cells to different derivatives. We found that the presumptive regions are generally distributed according to classical fate maps for urodeles but that the boundaries between presumptive regions are indistinct, due to extensive intermingling between cells at the edges of grafted regions. We have shown that there is a high dorsal to low ventral gradient of mixing between superficial and deep cells.