Dll4, a novel Notch ligand expressed in arterial endothelium.

We report the cloning and characterization of a new member of the Delta family of Notch ligands, which we have named Dll4. Like other Delta genes, Dll4 is predicted to encode a membrane-bound ligand, characterized by an extracellular region containing several EGF-like domains and a DSL domain required for receptor binding. In situ analysis reveals a highly selective expression pattern of Dll4 within the vascular endothelium. The activity and expression of Dll4 and the known actions of other members of this family suggest a role for Dll4 in the control of endothelial cell biology.

A histone deacetylase corepressor complex regulates the Notch signal transduction pathway.

The Delta-Notch signal transduction pathway has widespread roles in animal development in which it appears to control cell fate. CBF1/RBP-Jkappa, the mammalian homolog of Drosophila Suppressor of Hairless [Su(H)], switches from a transcriptional repressor to an activator upon Notch activation. The mechanism whereby Notch regulates this switch is not clear. In this report we show that prior to induction CBF1/RBP-Jkappa interacts with a corepressor complex containing SMRT (silencing mediator of retinoid and thyroid hormone receptors) and the histone deacetylase HDAC-1. This complex binds via the CBF1 repression domain, and mutants defective in repression fail to interact with the complex. Activation by Notch disrupts the formation of the repressor complex, thus establishing a molecular basis for the Notch switch. Finally, ESR-1, a Xenopus gene activated by Notch and X-Su(H), is induced in animal caps treated with TSA, an inhibitor of HDAC-1. The functional role for the SMRT/HDAC-1 complex in CBF1/RBP-Jkappa regulation reveals a novel genetic switch in which extracellular ligands control the status of critical nuclear cofactor complexes.

Activin and its receptors during gastrulation and the later phases of mesoderm development in the chick embryo.

We have cloned chick homologues of the type-II activin receptor, which we have designated cActR-IIA and -IIB. Binding assays show that the two receptors are indistinguishable in their ability to bind activin-A, with comparable kds. Injection of mRNAs encoding these receptors into Xenopus embryos causes axial duplications. Expression of both receptors can first be detected in the primitive streak by in situ hybridization. This suggests that these genes may be activated in response to mesoderm induction. In agreement with this, we find that treatment of preprimitive streak chick embryos with activin-A leads to rapid induction of the expression of cActR-IIB. At later stages, cActR-IIA transcripts become localized mainly in the notochord and myotome and cActR-IIB in the dorsal neural tube, proximal-anterior part of the limb bud, sensory placodes, and specific regions of the fore- and midbrain. To test the response of early chick embryonic tissues to activin, we designed a new in vitro assay for differentiation. We find that explants of area opaca epiblast or posterior primitive streak from various stages can respond to activin treatment by differentiating into a variety of mesodermal cell types in a dose-dependent manner. These results suggest that the importance of activin-related signaling pathways is not confined to pregastrulation stages and that these receptors may be involved in mediating the effects of inducing signals during later stages of development of the mesoderm, limbs, and nervous system.

The transforming growth factor beta type II receptor can replace the activin type II receptor in inducing mesoderm.

The type II receptors for the polypeptide growth factors transforming growth factor beta (TGF-beta) and activin belong to a new family of predicted serine/threonine protein kinases. In Xenopus embryos, the biological effects of activin and TGF-beta 1 are strikingly different; activin induces a full range of mesodermal cell types in the animal cap assay, while TGF-beta 1 has no effects, presumably because of the lack of functional TGF-beta receptors. In order to assess the biological activities of exogenously added TGF-beta 1, RNA encoding the TGF-beta type II receptor was introduced into Xenopus embryos. In animal caps from these embryos, TGF-beta 1 and activin show similar potencies for induction of mesoderm-specific mRNAs, and both elicit the same types of mesodermal tissues. In addition, the response of animal caps to TGF-beta 1, as well as to activin, is blocked by a dominant inhibitory ras mutant, p21(Asn-17)Ha-ras. These results indicate that the activin and TGF-beta type II receptors can couple to similar signalling pathways and that the biological specificities of these growth factors lie in their different ligand-binding domains and in different competences of the responding cells.

Molecular genetics of neurulation.

The formation of the neural tube begins during gastrulation when ectoderm, an epithelial sheet on the outside of the embryo, is induced to form the neural plate. During the process of neural induction, the epithelium of the neural plate is regionalized along both the dorsoventral and anteroposterior axes of the embryo; this regionalization is likely to contribute to the cellular processes underlying neurulation. Genes whose expression marks the formation and regionalization of the neural plate and which encode cell adhesion molecules or putative transcription factors have been recently identified. The differential expression of these genes apparently subdivides the epithelium of the neural plate into small regions. Evidence from transgenic embryo experiments supports the idea that the differential expression of these genes in the neural plate plays a role in neural tube formation.

Expression of an extracellular deletion of Xotch diverts cell fate in Xenopus embryos.

Xotch is a Xenopus homolog of Notch, a receptor involved in cell fate decisions in Drosophila. Using an extracellular deletion construct, Xotch delta E, we show that Xotch has a similar role in Xenopus embryos. Broad expression causes the loss of dorsal structures and the expansion and disorganization of the brain. Single blastomere injections of Xotch delta E induce autonomous neural and mesodermal hypertrophy, even in the absence of cell division. Xotch delta E inhibits the early expression of epidermal and neural crest markers yet enhances and extends the response of animal caps to mesodermal and neural induction. Our data suggest a mechanism for the function of Notch homologs in which they delay differentiation and leave undetermined cells competent to respond to later inductive signals.

Cloning of a second type of activin receptor and functional characterization in Xenopus embryos.

A complementary DNA coding for a second type of activin receptor (ActRIIB) has been cloned from Xenopus laevis that fulfills the structural criteria of a transmembrane protein serine kinase. Ectodermal explants from embryos injected with activin receptor RNA show increased sensitivity to activin, as measured by the induction of muscle actin RNA. In addition, injected embryos display developmental defects characterized by inappropriate formation of dorsal mesodermal tissue. These results demonstrate that this receptor is involved in signal transduction and are consistent with the proposed role of activin in the induction and patterning of mesoderm in Xenopus embryos.

Hensen's node induces neural tissue in Xenopus ectoderm. Implications for the action of the organizer in neural induction.

The development of the vertebrate nervous system is initiated in amphibia by inductive interactions between ectoderm and a region of the embryo called the organizer. The organizer tissue in the dorsal lip of the blastopore of Xenopus and Hensen's node in chick embryos have similar neural inducing properties when transplanted into ectopic sites in their respective embryos. To begin to determine the nature of the inducing signals of the organizer and whether they are conserved across species we have examined the ability of Hensen's node to induce neural tissue in Xenopus ectoderm. We show that Hensen's node induces large amounts of neural tissue in Xenopus ectoderm. Neural induction proceeds in the absence of mesodermal differentiation and is accompanied by tissue movements which may reflect notoplate induction. The competence of the ectoderm to respond to Hensen's node extends much later in development than that to activin-A or to induction by vegetal cells, and parallels the extended competence to neural induction by axial mesoderm. The actions of activin-A and Hensen's node are further distinguished by their effects on lithium-treated ectoderm. These results suggest that neural induction can occur efficiently in response to inducing signals from organizer tissue arrested at a stage prior to gastrulation, and that such early interactions in the blastula may be an important component of neural induction in vertebrate embryos.

The effects of N-cadherin misexpression on morphogenesis in Xenopus embryos.

N-cadherin is a calcium-dependent, cell adhesion molecule that has been proposed to play a role in morphogenesis in vertebrate embryos. Throughout early neural development, N-cadherin is expressed during the morphogenetic changes that occur when ectoderm, in response to neural induction, forms a neural plate and tube. To study the role of N-cadherin in these processes, cDNA clones encoding Xenopus laevis N-cadherin were isolated and used to study the expression of N-cadherin in frog embryos. These studies showed that N-cadherin RNA is not expressed at detectable levels in early cleavage embryos or in isolated ectoderm in the absence of neural induction. However, N-cadherin RNA rapidly appeared in ectoderm exposed to a heterologous neural inducer, indicating that N-cadherin expression, as an early response to induction, precedes the morphogenetic events associated with early neural development. The role of N-cadherin in these morphogenetic events was studied by ectopically expressing N-cadherin in the ectoderm of embryos prior to induction. The ectopic expression of this protein in ectoderm led to the formation of cell boundaries and to severe morphological defects. These results are consistent with the hypothesis that the morphogenetic changes associated with early neural development are controlled, in part, by the induced expression of N-cadherin in the neural plate.

Primary structure and developmental expression of a large cytoplasmic domain form of Xenopus laevis neural cell adhesion molecule (NCAM).

The neural cell adhesion molecule, (NCAM), is involved in cell-cell interactions during development of the vertebrate nervous system. NCAM exists in multiple protein forms and these are selectively expressed in different cells and at different times during development. Here we report the complete amino acid sequence of the large cytoplasmic form of Xenopus laevis NCAM, derived from a full-length cDNA clone. Using specific probes the expression of different NCAM transcripts during Xenopus embryogenesis has been examined. We find that transcripts encoding the large cytoplasmic domain form of NCAM exist in maternal RNA and that these are the only significant NCAM transcripts present until late gastrula when transcripts encoding the small cytoplasmic domain form of NCAM are first detected. No RNA encoding the small surface domain form of NCAM is detected during early development. These results indicate that the expression of NCAM sequences during early development of Xenopus differs from that described in other species.

Cellular contacts required for neural induction in Xenopus embryos: evidence for two signals.

Neurogenesis begins in amphibian embryos around the time of gastrulation when a portion of the ectoderm receives an inducing signal from dorsal mesoderm. Two different proposals have been made for how ectoderm must come into contact with dorsal mesoderm in order for the inducing signal to pass between the two tissues. Induction in one proposal would require normal gastrulation movements to bring dorsal mesoderm underneath, and into apposition with, the overlying ectoderm. The inducing signal in this case would pass between dorsal mesoderm and ectoderm as apposed tissue layers. The other proposal is that induction requires only a small contact between ectoderm and dorsal mesoderm at the boundary they share before gastrulation. The inducing signal by this proposal would pass laterally across this small area of contact between mesoderm and ectoderm, perhaps before gastrulation, and spread within the ectodermal cell layer. Since it is not known to what extent neurogenesis depends on each of these proposed contacts between ectoderm and dorsal mesoderm, we have generated explants of embryonic tissue in which one or the other type of contact between mesoderm and ectoderm is favored. The amount of neural tissue formed under these various conditions was then assessed using a quantitative RNase protection assay to measure the levels of two neural-specific RNA transcripts. The results show that neural tissue forms efficiently when ectoderm and dorsal mesoderm only interact laterally within a plane of tissue. In contrast, neural tissue forms extremely poorly when ectoderm is placed experimentally in apposition with involuting, anterior-dorsal mesoderm.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of acetylcholine receptor transcript expression during development in Xenopus laevis.

The level of transcripts encoding the skeletal muscle acetylcholine receptor (AChR) was determined during embryonic development in Xenopus laevis. cDNAs encoding the alpha, gamma, and delta subunits of the Xenopus AChR were isolated from Xenopus embryo cDNA libraries using Torpedo AChR cDNAs as probes. The Xenopus AChR cDNAs have greater than 60% amino acid sequence homology to their Torpedo homologues and hybridize to transcripts that are restricted to the somites of developing embryos. Northern blot analysis demonstrates that a 2.3-kb transcript hybridizes to the alpha subunit cDNA, a 2.4-kb transcript hybridizes to the gamma subunit cDNA, and that two transcripts, of 1.9 and 2.5 kb, hybridize to the delta subunit cDNA. RNase protection assays demonstrate that transcripts encoding alpha, gamma, and delta subunits are coordinately expressed at late gastrula and that the amount of each transcript increases in parallel with muscle-specific actin mRNA during the ensuing 12 h. After the onset of muscle activity the level of actin mRNA per somite remains relatively constant, whereas the level of alpha subunit and delta subunit transcripts decrease fourfold per somite and the level of gamma subunit transcript decreases greater than 50-fold per somite. The decrease in amount of AChR transcripts per somite, however, occurs when embryos are paralyzed with local anaesthetic during their development. These results demonstrate that AChR transcripts in Xenopus are initially expressed coordinately, but that gamma subunit transcript levels are regulated differently than alpha and delta at later stages. Moreover, these results demonstrate that AChR transcript levels in Xenopus myotomal muscle cells are not responsive to electrical activity and suggest that AChR transcript levels are influenced by other regulatory controls.

Expression of Xenopus N-CAM RNA in ectoderm is an early response to neural induction.

We have isolated Xenopus laevis N-CAM cDNA clones and used these to study the expression of N-CAM RNA during neural induction. The results show that the first marked increase in N-CAM RNA levels occurs during gastrulation when mesoderm comes in contact with ectoderm and induces neural development. In situ hybridization results show that the early expression of N-CAM RNA is localized to the neural plate and its later expression is confined to the neural tube. Induction experiments with explanted germ layers show that N-CAM RNA is not expressed in ectoderm unless there is contact with inducing tissue. Together these results suggest an approach to studying how ectoderm is committed to form neural rather than epidermal tissue. Specifically, the data suggest that neural commitment is marked and perhaps mediated by the transcriptional activation of genes, like N-CAM, in the neural ectoderm.

Glial growth factor and nerve-dependent proliferation in the regeneration blastema of Urodele amphibians.

After amputation of a limb from Urodele amphibians, division of the blastemal cells (the progenitor cells of the regenerate) depends on one or more unidentified growth factors provided by the nerve supply. Here we show that glial growth factor (GGF), a mitogenic protein previously purified from the bovine pituitary, is present in newt nervous system extracts. It is also detectable in extracts of the forelimb regeneration blastema, and its level there decreases after denervation. We have previously shown that blastemal cells dependent on the nerve for division are marked by a monoclonal antibody called 22/18. When denervated blastemas are cultured in the presence of partially purified GGF from newt brain, or pure GGF from the bovine pituitary, the thymidine labeling index of blastemal cells that are 22/18-positive is increased as much as sevenfold. These data indicate that GGF plays a role in nerve-dependent proliferation in the blastema.

Monoclonal antibodies to the cells of a regenerating limb.

Monoclonal antibodies were raised against differentiated cells, and blastemal cells from regenerating limbs of adult newts (Notophthalmus viridescens) and screened for specific staining by immunocytochemistry. In addition to antibodies that identify muscle, Schwann cells and cartilage, two reagents were specific for subpopulations of blastemal cells. One of these latter antibodies, termed 22/18, has provided new evidence about the origin of blastemal cells from Schwann cells and myofibres, and also identifies blastemal cells whose division is persistently dependent on the nerve supply.

Monoclonal antibodies identify blastemal cells derived from dedifferentiating limb regeneration.

Blastemal cells arise after the amputation of limbs or tails in urodele amphibians. These histologically undifferentiated mesenchymal cells divide and subsequently differentiate to regenerate a new appendage. Various studies (reviewed in ref. 1) indicate that blastemal cells arise from tissues near the site of amputation, including muscle, cartilage, nerve and dermis. The multinucleated myofibre, however, is a controversial source of blastemal cells. The suggestion that myofibres can dedifferentiate is based on their histological appearance during the early stages of limb regeneration. This is contrary to the widely accepted view of muscle regeneration in higher vertebrates which attributes it to satellite cells. One prediction of the dedifferentiation hypothesis is that a population with properties of both myofibres and blastema cells should be present during the early stages of regeneration. Here we described the isolation of two monoclonal antibodies, one that recognizes an antigen found only in myofibres and another that recognizes an antigen restricted to blastemal cells. By using these antibodies as cell markers, we can detect a small population of cells in the regenerating limbs of adult newts that bear both the myofibre and blastemal cell antigens. The time and location of these double-labelled cells supports the idea that blastemal cells originate, in part, by dedifferentiation of myofibres.

The structure of the termini of the DNA of Epstein-Barr virus.

We have studied the DNA of Epstein-Barr virus (EBV) isolated from the B95-8 strain of that virus (Miller and Lipman, 1973). When EBV DNA is partially digested with lambda-exonuclease and allowed to reanneal, up to 50% of the full-length molecules circularize. The arrangements of nucleotide sequences containing the terminal repeats identified in this circularization experiment have been determined. Those fragments of viral DNA generated by digestion with restriction endonucleases which are terminal and contain the terminal repeats have been identified by their sensitivity to digestion of full-length DNA by lambda-exonuclease and by virtue of their being partially homologous to one another. The population of DNA molecules in the B95-8 strain of EBV was found to be nonuniform. The nonuniformity results from different molecules having different numbers of a 0.37 megadalton terminal repeat at each end. About 70% of molecules have four terminal repeats at one end, while four equal classes, each comprising approximately 25% of the population, have one, two, three or four repeats at the other end. The arrangements of nucleotide sequences identified as being terminal in virion DNA were studied in the intracellular circular viral DNA of cells transformed by a single particle on EBV. All fragments produced by digestion with endonucleases and scored as being terminal in virion DNA were absent from intracellular circular DNA. An additional fragment was identified in the digests of intracellular DNA of each transformed clone. The molecular weights of the new fragments equal the sum of the molecular weights of two terminal fragments which are joined upon intracellular circularization of viral DNA.