Search PubMed⌕ Search

Biomedical subjects

J M Slack

Publications and source records attributed to J M Slack.

At least 37 records · Page 2Linked to original sources
PubMed1999

Spatial response to fibroblast growth factor signalling in Xenopus embryos.

We have examined the spatial pattern of activation of the extracellular signal-regulated protein kinase (ERK) during Xenopus development, and show that it closely resembles the expression of various fibroblast growth factors (FGFs). Until the tailbud stage of development, all ERK activation domains are sensitive to the dominant negative FGF receptor, showing that activation is generated by endogenous FGF signalling. ERK is not activated by application of other growth factors like BMP4 or activin, nor is endogenous activation blocked by the respective dominant negative receptors. This shows that various domains of FGF expression, including the periblastoporal region and the midbrain-hindbrain boundary, are also sites of FGF signalling in vivo. Wounding induces a transient (<60 minutes) activation of ERK which is not significantly reduced by the dominant negative FGF receptor. An artificial FGF source, created by injection of eFGF mRNA into cleavage stage embryos, provokes ERK activation outside of its injection site over a range of several cell diameters. The range and extent of ERK activation outside the source region is unchanged by co-injection of a dominant negative form of Ras, which blocks ERK-activation within the source. This suggests that FGF protein can diffuse over several cell diameters.

Actins↗
PubMed1999

A developmental pathway controlling outgrowth of the Xenopus tail bud.

We have developed a new assay to identify factors promoting formation and outgrowth of the tail bud. A piece of animal cap filled with the test mRNAs is grafted into the posterior region of the neural plate of a host embryo. With this assay we show that expression of a constitutively active Notch (Notch ICD) in the posterior neural plate is sufficient to produce an ectopic tail consisting of neural tube and fin. The ectopic tails express the evenskipped homologue Xhox3, a marker for the distal tail tip. Xhox3 will also induce formation of an ectopic tail in our assay. We show that an antimorphic version of Xhox3, Xhox3VP16, will prevent tail formation by Notch ICD, showing that Xhox3 is downstream of Notch signalling. An inducible version of this reagent, Xhox3VP16GR, specifically blocks tail formation when induced in tailbud stage embryos, comfirming the importance of Xhox3 for tail bud outgrowth in normal development. Grafts containing Notch ICD will only form tails if placed in the posterior part of the neural plate. However, if Xwnt3a is also present in the grafts they can form tails at any anteroposterior level. Since Xwnt3a expression is localised appropriately in the posterior at the time of tail bud formation it is likely to be responsible for restricting tail forming competence to the posterior neural plate in our assay. Combined expression of Xwnt3a and active Notch in animal cap explants is sufficient to induce Xhox3, provoke elongation and form neural tubes. Conservation of gene expression in the tail bud of other vertebrates suggests that this pathway may describe a general mechanism controlling tail outgrowth and secondary neurulation.

Animals↗
PubMed1998

Regulation of Hox gene expression and posterior development by the Xenopus caudal homologue Xcad3.

The caudal gene codes for a homeodomain transcription factor that is required for normal posterior development in Drosophila. In this study the biological activities of the Xenopus caudal (Cdx) family member Xcad3 are examined. A series of domain-swapping experiments demonstrate that the N-terminus of Xcad3 is necessary for it to activate Hox gene expression and that this function can be replaced by the activation domain from the viral protein VP16. In addition, experiments using an Xcad3 repressor mutant (XcadEn-R), which potently blocks the activity of wild-type Xcad3, are reported. Overexpression of XcadEn-R in embryos inhibits the activation of the same subset of Hox genes that are activated by wild-type Xcad3 and leads to a dramatic disruption of posterior development. We show that Xcad3 is an immediate early target of the FGF signalling pathway and that Xcad3 posteriorizes anterior neural tissue in a similar way to FGF. Furthermore, Xcad3 is required for the activation of Hox genes by FGFs. These data provide strong evidence that Xcad3 is required for normal posterior development and that it regulates the expression of the Hox genes downstream of FGF signalling.

Animals↗
PubMed1998

Two phases of Hox gene regulation during early Xenopus development.

We have shown previously that fibroblast growth factor (FGF) signalling in posterior regions of the Xenopus embryo is required for the development of the trunk and tail via a molecular pathway that includes the caudal-related gene Xcad3 and the posterior Hox genes [1]. These results have been contested by the work of Kroll and Amaya [2], which shows that Xenopus embryos transgenic for a dominant-negative form of the FGF receptor (FGF-RI) express posterior Hox genes normally, leading these authors to suggest that the FGFs are not required for anteroposterior (A-P) patterning of the dorsal axis. In order to investigate the apparent discrepancy between these studies, we have produced Xenopus embryos transgenic for two inhibitors of the FGF/Caudal pathway: a kinase-deficient dominant-negative FGF receptor (XFD) [3]; and a domain-swapped form of Xcad3 (Xcad-EnR) in which the activation domain of Xcad3 is replaced by the repression domain of the Drosophila Engrailed protein. Both of these were introduced as fusions with the green fluorescent protein (GFP), which allows identification of non-mosaic transgenic embryos at early gastrula stages by simply looking for GFP fluorescence. Analysis of gene expression in embryos transgenic for these constructs indicated that the activation of posterior Hox genes during early neurula stages absolutely requires FGF signalling and transcriptional activation by Xcad3, while the maintenance of Hox gene expression in the trunk and tail during later development is independent of both FGF and Xcad.

Animals↗
PubMed1998

Postgastrulation effects of fibroblast growth factor on Xenopus development.

We have investigated postgastrulation functions of FGFs in Xenopus development by the implantation of heparin beads soaked in FGF2 to various positions at various stages. Anterior implantations show different effects depending on whether they are made to early neurulae or to later stages. At stage 13-14 there is a total or partial suppression of anterior structures including the forebrain, eyes, and midbrain. From stage 15 onwards there is no loss of anterior parts but there is a change in the structure of the eye such that the neural retina remains continuous with the wall of the diencephalon and the territories normally forming the optic stalk and pigment epithelium instead become neural retina. Posterior implantations cause a disruption of somite segmentation without affecting the differentiation of muscle cells. This is associated with a prolongation of the uniform expression of X-Delta-2 during the phase of segmental determination. There is also an induction of ectopic otocysts, which can lie either ipsilateral or contralateral to the FGF-bead. The results are discussed in terms of the known late expression domains of the various Xenopus FGFs, and of the late functions of FGFs in higher vertebrates. They provide new evidence for a role of endogenous FGFs in the development of the eye, somites, and otocysts.

Animals↗
PubMed1998

Development of the gut in Xenopus laevis.

The lining of the gut, together with the pancreas, liver, gall bladder, and respiratory system, is formed from the endoderm. The gut also contains smooth muscle and connective tissue of mesodermal origin. The amphibian Xenopus laevis is potentially an excellent model organism for studying how the cells of the endoderm and mesoderm become programmed to produce these internal organs. However, the anatomical complexity of the coiled gut presents a problem in studying its development. In order to overcome this problem we here present a comprehensive guide to the anatomy and histology of the developing Xenopus gut. We use a simple dissection to display its anatomy and the expression of four endodermal markers (alkaline phosphatase, IFABP, XlHbox8, and endodermin). We present schematic diagrams that show how the gut is arranged in three dimensions and how this organisation changes during development. We also present drawings of histological sections of the gut which allow any region to be identified and so represent an atlas for working with sections. Finally, we describe the histology of the cells of the various organs of the gut. This histological identification may be necessary for the identification of parts following experiments in which the normal pattern is disturbed.

Alkaline Phosphatase↗
PubMed1998

Analysis of the developing Xenopus tail bud reveals separate phases of gene expression during determination and outgrowth.

We have studied Xenopus tail development from the end of gastrulation to the commencement of outgrowth at the tail bud stage. We show that an early group of genes are expressed at the stage of tail bud determination, at the end of gastrulation, and a late group are expressed at around stage 27 just before tail bud outgrowth. Together, these genes define seven distinct regions of the tail bud as outgrowth commences. We have previously shown that formation of a tail bud depends on the interaction of three tissue regions, called N, M and C, at stage 13. Here we show that expression of the late group of genes is dependent on this NMC interaction. We describe molecular correlates of two of these regions, M and C, which were formerly unobservable and whose existence was inferred from embryological experiments.

Animals↗
PubMed1998

Expression and functions of FGF-3 in Xenopus development.

We have analyzed the expression pattern of the Xenopus FGF-3 gene during early development and examined its biological activity in three different bioassays using Xenopus embryos. We show that from the early gastrula stage there is a domain of expression around the blastopore which becomes a posterior domain as the blastopore closes. An anterior ectodermal domain becomes detectable from mid-gastrula stages in the prospective hind-brain, and there are several later domains of expression: the midbrain-hindbrain junction, the otocyst, the pharyngeal pouches and the tailbud region. By using double whole-mount in situ hybridizations we show that the XFGF-3 expression in the brain is dynamically regulated both in time and space during development. The anterior domain of early neurula stage embryos corresponds to the prospective rhombomeres 3-5. By the time the neural tube is closed, XFGF-3 expression is restricted to r4 and later a new domain of expression is established at the midbrain/hindbrain junction. In addition, we show that, despite its difference in receptor specificity, XFGF-3 can induce the formation of mesoderm from animal caps similarly to other FGFs. It also displays a posteriorizing activity on whole embryos similar to other FGFs. Although the absence of maternal expression makes it unlikely that XFGF-3 is involved in mesoderm induction in vivo, its posterior domain of expression during gastrulation and its posteriorizing activity suggests that it participates in the maintenance of mesodermal gene expression and in the FGF mediated patterning of the anteroposterior axis during gastrulation.

Animals↗
PubMed1997

FGF-8 is associated with anteroposterior patterning and limb regeneration in Xenopus.

FGF-8 has attracted attention particularly because of its importance for limb development in the chick and mouse, although it also has a number of earlier expression domains in these species. We have now cloned an FGF-8 homologue from Xenopus in which it is easier to do functional studies on early development. There is no maternal expression, while zygotic expression is highest in the gastrula and neurula stages. XFGF-8 is expressed as a ring around the blastopore and subsequently in the tail bud. There are several domains in the head including the hatching gland, the branchial clefts, and the midbrain-hindbrain border. At later stages there is a prominent band of expression in the limb bud epidermis. Although there is no morphological apical ridge, this band of expression suggests that the Xenopus limb bud contains a cryptic region with a similar ability to stimulate mesenchymal outgrowth. The mesoderm-inducing activity of XFGF-8 is somewhat lower than that of other FGFs, while the posteriorizing activity is similar. These differences are probably due to the different receptor specificity. The posterior expression and high posteriorizing activity suggest that XFGF-8 contributes to the patterning of the anterior-posterior axis by FGF family members during gastrulation. In contrast to the amniotes, Xenopus limb buds can regenerate following damage. We show that regeneration is correlated with the reexpression of XFGF-8 in the distal epidermis, suggesting that this ability is critical for successful limb regeneration.

Amino Acid Sequence↗
PubMed1997

Growth control: action mouse.

A recently described knockout mouse has abnormally large muscles. The phenotype suggests that the ablated product, growth differentiation factor 8 or 'myostatin', may be one of the long sought inhibitors that control the growth of individual tissues and organs.

Animals↗
PubMed1997

Inhibition of eFGF expression in Xenopus embryos by antisense mRNA.

We studied the effects in Xenopus embryos of overexpression of antisense RNA complementary to the messenger RNA of eFGF. We show that the expression of sense RNA can be severely depressed in the presence of an excess of antisense RNA. This occurs by both partial destruction of the message and by a depression of translation of the residual message. The diminution of inducing activity of eFGF, measured in animal cap assays either by activation of the Brachyury gene or by morphology, parallels the reduction of translation. Endogenous eFGF expression is reduced to a similar extent, again by a combination of mRNA destruction and inhibition of translation. This shows that the overexpression of antisense RNA is, contrary to general opinion, a potentially useful technique for studying gene function in Xenopus embryos. However, in the case of eFGF, there is little or no overall phenotypic effect on whole embryos. This is probably because of the presence of several other FGFs with overlapping expression domains in the early embryo.

Animals↗
PubMed1997

Identification of two independent transcriptional activation domains in the Autographa californica multicapsid nuclear polyhedrosis virus IE1 protein.

The Autographa californica multicapsid nuclear polyhedrosis virus immediate-early protein, IE1, is a 582-amino-acid phosphoprotein that regulates the transcription of early viral genes. Deletion of N-terminal regions of IE1 in previous studies (G. R. Kovacs, J. Choi, L. A. Guarino, and M. D. Summers, J. Virol. 66:7429-7437, 1992) resulted in the loss of transcriptional activation, suggesting that this region may contain an acidic activation domain. To identify independently functional transcriptional activation domains, we developed a heterologous system in which potential regulatory domains were fused with a modified Escherichia coli Lac repressor protein that contains a nuclear localization signal (NLacR). Transcriptional activation by the resulting NLacR-IE1 chimeras was measured with a basal baculovirus early promoter containing optimized Lac repressor binding sites (lac operators). Chimeras containing IE1 peptides dramatically activated transcription of the basal promoter only when lac operator sequences were present. In addition, transcriptional activation by NLacR-IE1 chimeras was allosterically regulated by the lactose analog, isopropyl-beta-D-thiogalactopyranoside (IPTG). For a more detailed analysis of IE1 regulatory domains, the M1 to T266 N-terminal portion of IE1 was subdivided (on the basis of average amino acid charge) into five smaller regions which were fused in various combinations to NLacR. Regions M1 to N125 and A168 to G222 were identified as independent transcriptional activation domains. Some NLacR-IE1 chimeras exhibited retarded migration in sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels. As with wild-type IE1, this aberrant gel mobility was associated with phosphorylation. Mapping studies with the NLacR-IE1 chimeras indicate that the M1 to A168 region of IE1 is necessary for this phosphorylation-associated effect.

Amino Acid Sequence↗
PubMed1996

Actin binding and proteolysis by the baculovirus AcMNPV: the role of virion-associated V-CATH.

Infection of larvae by Autographa californica M nuclear polyhedrosis virus (AcMNPV) results in liquefaction of susceptible hosts, presumably due to the breakdown of cells and extracellular matrices. In Spodoptera frugiperda tissue culture cells, infection leads to dramatic rearrangement and eventual destruction of the actin cytoskeleton. The first of these rearrangements is the formation of actin cables in the cytoplasm of the cell. Cable formation requires release of the budded virus (BV) nucleocapsid from the endosome, but does not require new protein synthesis, suggesting that the nucleocapsid contains the activity necessary to induce cable formation. We have identified two distinct BV-associated actin-targeting activities. The first, a nucleocapsid-associated actin-binding activity, enabled actin copelleting and may also induce actin polymerization and cable formation. The second activity, associated with the nucleocapsid and envelope fractions of BV, was a protease that specifically degraded actin. This protease was identified as V-CATH, a cathepsin L-like protease that is a product of the AcMNPV v-cath gene.

Actins↗
PubMed1996

XFGF-9: a new fibroblast growth factor from Xenopus embryos.

We have identified the Xenopus homologue of mammalian FGF-9 (XFGF-9). Sequence comparison between Xenopus and mammals shows that they share 93% identity at the amino acid level, making FGF-9 the most highly conserved member within the family. The sequence shows that there is no N-terminal signal sequence but that there is an internal hydrophobic sequence resembling a transmembrane domain. By using an in vitro translation system, we demonstrate that XFGF-9 can be glycosylated by microsomes but shows no signal peptide cleavage. This suggests that it can be secreted using the internal hydrophobic domain to cross the endoplasmic reticulum membrane. Expression studies using RNAse protections and in situ hybridization show that XFGF-9 is expressed both maternally and zygotically. The maternal mRNA is detected at a higher level than other forms (XFGF-2 and eFGF), mainly in the animal hemisphere. A proportion of the maternal transcript persists until the early gastrula stage when it is joined by zygotic expression around the blastopore region, and thereafter the mRNA content shows some increase during further development. Zygotic XFGF-9 is expressed uniformly along the dorsal axis, as well as in the head region. We have expressed recombinant XFGF-9 protein in bacteria, and show that it has a mesoderm-inducing activity in the animal cap assay, with a similar specific activity to other fibroblast growth factor (FGFs). We have injected a synthetic mRNA into eggs, and show that it has both mesoderm-inducing activity in animal caps and also a posteriorizing activity in whole embryos. The levels of biological activity shown by the XFGF-9 mRNA injections compared to XFGF-2 and eFGF show that there is at least some extracellular function. This supports the biochemical results, suggesting that the protein has at least some capacity to be secreted.

Amino Acid Sequence↗
PubMed1996

eFGF, Xcad3 and Hox genes form a molecular pathway that establishes the anteroposterior axis in Xenopus.

Classical embryological experiments suggest that a posterior signal is required for patterning the developing anteroposterior axis. In this paper, we investigate a potential role for FGF signalling in this process. During normal development, embryonic fibroblast growth factor (eFGF) is expressed in the posterior of the Xenopus embryo. We have previously shown that overexpression of eFGF from the start of gastrulation results in a posteriorised phenotype of reduced head and enlarged proctodaeum. We have now determined the molecular basis of this phenotype and we propose a role for eFGF in normal anteroposterior patterning. In this study, we show that the overexpression of eFGF causes the up-regulation of a number of posteriorly expressed genes, and prominent among these are Xcad3, a caudal homologue, and the Hox genes, in particular HoxA7. There is both an increase of expression within the normal domains and an extension of expression towards the anterior. Application of eFGF-loaded beads to specific regions of gastrulae reveals that anterior truncations arise from an effect on the developing dorsal axis. Similar anterior truncations are caused by the dorsal overexpression of Xcad3 or HoxA7. This suggests that this aspect of the eFGF overexpression phenotype is caused by the ectopic activation of posterior genes in anterior regions. Further results using the dominant negative FGF receptor show that the normal expression of posterior Hox genes is dependent on FGF signalling and that this regulation is likely mediated by the activation of Xcad3. The biological activity of eFGF, together with its expression in the posterior of the embryo, make it a good candidate to fulfil the role of the 'transforming' activity proposed by Nieuwkoop in his 'activation and transformation' model for neural patterning.

Animals↗