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J M Slack

Publications and source records attributed to J M Slack.

At least 19 recordsLinked to original sources
PubMed2001

The role of BMP signaling in outgrowth and patterning of the Xenopus tail bud.

Tail bud formation in Xenopus depends on interaction between a dorsal domain (dorsal roof) expressing lunatic fringe and Notch, and a ventral domain (posterior wall) expressing the Notch ligand Delta. Ectopic expression of an activated form of Notch, Notch ICD, by means of an animal cap graft into the posterior neural plate, results in the formation of an ectopic tail-like structure containing a neural tube and fin. However, somites are never formed in these tails. Here, we show that BMP signaling is activated in the posterior wall of the tail bud and is involved in the formation of tail somites from this region. Grafts into the posterior neural plate, in which BMP signaling is activated, will form tail-like outgrowths. Unlike the Notch ICD tails, the BMP tails contain well-organized somites as well as neural tube and fin, with the graft contributing to both somites and neural tube. Through a variety of epistasis-type experiments, we show that the most likely model involves a requirement for BMP signaling upstream of Notch activation, resulting in formation of the secondary neural tube, as well as a Notch-independent pathway leading to the formation of tail somites from the posterior wall.

Animals↗
PubMed2001

An amphibian with ambition: a new role for Xenopus in the 21st century.

Much of our knowledge about the mechanisms of vertebrate early development comes from studies using Xenopus laevis. The recent development of a remarkably efficient method for generating transgenic embryos is now allowing study of late development and organogenesis in Xenopus embryos. Possibilities are also emerging for genomic studies using the closely related diploid frog Xenopus tropicalis.

Animals↗
PubMed2001

Endoderm specification and differentiation in Xenopus embryos.

It is known from work with amniote embryos that regional specification of the gut requires cell-cell signalling between the mesoderm and the endoderm. In recent years, much of the interest in Xenopus endoderm development has focused on events that occur before gastrulation and this work has led to a different model whereby regional specification of the endoderm is autonomous. In this paper, we examine the specification and differentiation of the endoderm in Xenopus using neurula and tail-bud-stage embryos and we show that the current hypothesis of stable autonomous regional specification is not correct. When the endoderm is isolated alone from neurula and tail bud stages, it remains fully viable but will not express markers of regional specification or differentiation. If mesoderm is present, regional markers are expressed. If recombinations are made between mesoderm and endoderm, then the endodermal markers expressed have the regional character of the mesoderm. Previous results with vegetal explants had shown that endodermal differentiation occurs cell-autonomously, in the absence of mesoderm. We have repeated these experiments and have found that the explants do in fact show some expression of mesoderm markers associated with lateral plate derivatives. We believe that the formation of mesoderm cells by the vegetal explants accounts for the apparent autonomous development of the endoderm. Since the fate map of the Xenopus gut shows that the mesoderm and endoderm of each level do not come together until tail bud stages, we conclude that stable regional specification of the endoderm must occur quite late, and as a result of inductive signals from the mesoderm.

Animals↗
PubMed2001

Abdominal B-type Hox gene expression in Xenopus laevis.

We have studied the expression of the Abdominal B-type Hox genes in Xenopus embryos and tadpoles. The probes used represent all paralog groups and are designated Xhoxa9, Xhoxd9, Xhoxd10, Xhoxa11, Xhoxc12 and Xhoxa13 on the basis of comparison to other vertebrates. Three of these genes are novel while the others have previously been detected but expression patterns were only partially described. We find a typical nested pattern of expression in the main body axis, in both central nervous system and mesoderm. All the genes, except Xhoxc12, are also expressed in the mesenchyme of the large intestine and in the limb buds.

Animals↗
PubMed2001

Transdifferentiation and metaplasia--switching cell types.

States of developmental commitment are encoded as combinations of transcription factors and changes in their expression can bring about transdifferentiation or metaplasia. For example, ectopic expressions of Vestigial can convert Drosophila leg to wing; of C/EBPbeta can convert pancreatic exocrine cells to hepatocytes; and expression of C/EBPalpha and PPARgamma can convert myoblasts to adipocytes.

Adipocytes↗
PubMed2001

Measurement of membrane fusion activity from viral membrane fusion proteins based on a fusion-dependent promoter induction system in insect cells.

A number of viral membrane fusion proteins can be expressed alone on the surface of host cells, and then triggered to induce cell-to-cell fusion or syncytium formation. Although rapid and easily observed, syncytium formation is not easily quantified and differences in fusion activity are not easily distinguished or measured. To address this problem, we developed a rapid and quantitative cell-to-cell fusion system that is useful for comparative analysis and may be suitable for high throughput screening. In this system, expression of a reporter protein, enhanced green fluorescent protein (EGFP), is dependent on cell-to-cell fusion. Spodoptera frugiperda (Sf9) insect cells expressing a chimeric Lac repressor-IE1 protein were fused to Sf9 cells containing an EGFP reporter construct under the control of a responsive lac operator-containing promoter. Membrane fusion efficiency was measured from the resulting EGFP fluorescence activity. Sf9 cells expressing the Orgyia pseudotsugata multicapsid nucleopolyhedrovirus (OpMNPV) GP64 envelope fusion protein were used as a model to test this fusion assay. Subtle changes in fusion activities of GP64 proteins containing single amino acid substitutions in a putative membrane fusion domain were distinguished, and decreases in EGFP fluorescence corresponded to decreases in the hydrophobicity in the small putative membrane fusion domain.

Animals↗
PubMed2000

Stem cells in epithelial tissues.

Most, if not all, epithelial tissues contain stem cells. They are responsible for normal tissue renewal or for regeneration following damage. Our present knowledge of their properties is limited and is mainly derived from studies of cell kinetics and from clonal analysis.

Animals↗
PubMed2000

Regional gene expression in the epithelia of the Xenopus tadpole gut.

In recent years much progress has been made in the understanding of the genes and mechanisms involved in specification of the cells of the endoderm, which give rise to the epithelium of the gut and respiratory system. However, little is known about the way in which the gut becomes patterned along its anterior-posterior axis, that is, how boundaries are established between the different epithelia of the gut tube. Here we show that the expression patterns of five genes divide the Xenopus tadpole gut epithelium into at least four regions along this axis in the undifferentiated, 3-day-old gut (stage 41), and that these divisions are maintained until at least 7 days, when cell differentiation is well under way. In addition, the restricted expression patterns of these genes clearly mark the anterior and posterior boundaries of the intestine. Xsox2 is expressed in the anterior gut, spanning the oesophagus and stomach but terminating at the stomach/intestine boundary. Xcad1 and Xcad2, two caudal-type homeobox genes, are expressed in a region with an anterior limit at this boundary and a posterior limit between the colon and proctodeum, therefore covering the whole of the small and large intestines. Intestinal fatty acid binding protein (IFABP) is expressed only in the anterior small intestine, and the even-skipped homeobox gene Xhox3 is expressed in the most posterior part of the gut, the proctodeum.

Animals↗
PubMed2000

Transfection of Lymantria dispar insect cell lines.

Lepidopteran cell lines derived from the gypsy moth, Lymantria dispar, have not been widely used in protein expression studies or systems because they are weakly adherent, have specific growth requirements and characteristics, and are generally difficult to transfect. Using lipid-mediated transfection of a reporter plasmid, we modify the standard method for transfection of L. dispar-derived embryonic cell lines IPLB-LdEp and -LdEIta, obtaining transfection efficiencies of 34% and 30%, respectively, as determined by image analysis assays. Using the standard lipid-mediated method, we obtain transfection efficiencies for L. dispar-derived cell line IPLB-Ld652Y of at least 40% with high mean expression levels, indicating the IPLB-Ld652Y cell line may be a superior choice for expression studies or systems requiring L. dispar-derived cells.

Animals↗
PubMed2000

Molecular basis of transdifferentiation of pancreas to liver.

The appearance of hepatic foci in the pancreas has been described in animal experiments and in human pathology. Here we show that pancreatic cells can be converted into hepatocytes by treatment with a synthetic glucocorticoid, dexamethasone. This occurs both in a pancreatic cell line, AR42J-B13, and in organ cultures of pancreatic buds from mouse embryos. We have established several features of the mechanism behind this transdifferentiation. We show that a proportion of the hepatocytes arises directly from differentiated exocrine-like cells, with no intervening cell division. This conversion is associated with induction of the transcription factor C/EBPbeta and the activation of differentiated hepatic products. Transfection of C/EBPbeta into the cells can provoke transdifferentiation; conversely, a dominant-negative form of C/EBPbeta can inhibit the process. These results indicate that C/EBPbeta is a key component that distinguishes the liver and pancreatic programmes of differentiation.

Animals↗
PubMed2000

The Xenopus tadpole gut: fate maps and morphogenetic movements.

We have produced a comprehensive fate map showing where the organs of the gut and respiratory system are derived from in the early Xenopus laevis endoderm. We also show the origin of the associated smooth muscle layer on a separate fate map. Comparison of the two maps shows that for most organs of the gut the prospective epithelium and smooth muscle do not overlie each other in the early embryo but come together at a later stage. These fate maps should be useful for future studies into endoderm specification. It was not previously known how the elongation of the endoderm occurs, how the single-layered dorsal and many-layered ventral endoderm gives rise to the single layered epithelium, and whether or not the archenteron cavity actually gives rise to the gut lumen. Using a variety of labelling procedures we show firstly, that radial intercalation occurs in the gut transforming a short thick tube into a long thin tube; secondly, that the archenteron lining does not become the definitive gut lumen. Instead the archenteron cavity almost closes at tailbud stages before providing a nucleus for the definitive gut cavity, which opens up during elongation. Based on this work we present a model explaining the morphogenesis of the gut.

Animals↗
PubMed2000

Role of cell division in branching morphogenesis and differentiation of the embryonic pancreas.

A new culture system for the embryonic pancreas enables the formation of a branched organ in vitro. In such cultures, each terminal branch originates as a small bud and the number of buds and of terminal branches increases progressively with the expansion of the culture. However buds can also be resorbed during growth. The normal labelling index of cells in incipient buds ("tips") is greater than between buds ("dips") suggesting that budding may be driven by a local increase of cell division. Consistent with this, treatments that reduce cell division repress the formation of buds and branches. It is not possible to initiate budding in isolated endodermal epithelium by treatment with fibroblast growth factor, although this does increase the degree of differentiation of exocrine cells. Cultures in which cell division is completely inhibited by aphidicolin treatment will produce more endocrine cells than usual and inhibit the differentiation of exocrine cells. Consistent with this it is found that in untreated cultures the division of endocrine precursors cannot be detected by BrdU labelling whereas the division of exocrine precursors is frequent. It is concluded that cell division is necessary for bud formation in the embryonic pancreas and that the growth factors required for this normally come from the mesenchyme. Cell division is also necessary for exocrine differentiation. Endocrine cells, however, can arise from undifferentiated progenitors without cell division.

Animals↗
PubMed1999

Analysis of pancreatic development using a cell lineage label.

We have devised a new culture system for in vitro culture of pancreatic buds from mouse embryos which enables the organ to grow as a flat branched structure suitable for wholemount immunostaining. This system has been used to analyze pancreatic development. We have also used the ROSA-26 gene trap mouse strain as a source of tissue which expresses lacZ in a stable manner, in all cell types, during in vitro culture. Combinations of lacZ epithelium and unlabeled mesenchyme show that both exocrine and endocrine cells arise from the epithelium, and smooth muscle cells from the mesenchyme. Although previously suspected, this is the first formal proof that both exocrine and endocrine cells are of endodermal origin. Combinations of lacZ epithelium with unlabeled stomach mesenchyme give similar results and show that stomach mesenchyme has the same trophic effect as pancreatic mesenchyme. When a lacZ and an unlabeled epithelium are combined with an unlabeled mesenchyme, both acini and islets in the resulting culture can be of mixed cell composition. This shows that neither of the chief structural units of the pancreas is formed by clonal growth from a single cell.

Animals↗
PubMed1999

The first pure embryonic inducing factor.

This is a personal account of the discovery of the mesoderm-inducing activity of fibroblast growth factors. The background is my work on the dorsalising signal in early amphibian embryos that was done at the imperial Cancer Research Fund in London. I became interested in mesoderm induction because of the embryologic work of Nieuwkoop and the partial purification of a "vegetalising factor" by Tiedemann. Although, initially, we expected inducing factors to be novel substances, it gradually became clear that the impure preparations we were studying had properties in common with growth factors. This opened the way to the testing of candidate factors, resulting in the conclusion that some inducing factors and growth factors,in fact, were the same molecules despite being discovered through different routes and assayed using different methods. After a slow start looking at the molecular biology of fibroblast growth factors in Xenopus, we eventually found that their most interesting functions in early development lay not at the stage of mesoderm induction but, rather, in the anteroposterior patterning of the body, which occurs during gastrulation.

Animals↗
PubMed1999

Gut specific expression using mammalian promoters in transgenic Xenopus laevis.

The recent development of transgenic methods for the frog Xenopus laevis provides the opportunity to study later developmental events, such as organogenesis, at the molecular level. Our studies have focused on the development of the tadpole gut, where tissue specific promoters have yet to be identified. We have used mammalian promoters, for the genes elastase, pancreatic duodenal homeobox-1, transthyretin, and intestinal fatty acid binding protein to drive green fluorescent protein expression in live tadpoles. All of these were shown to drive appropriate tissue specific expression, suggesting that the molecular mechanisms organising the gut are similar in amphibians and mammals. Furthermore, expression from the elastase promoter is initiated in the pancreatic buds before morphological definition becomes possible, making it a powerful tool for the study of pancreatic determination.

Animals↗