harry v isaacs

Abstract: We have previously shown that the Gsx family homeobox gene Gsh2 is part of the regulatory network specifying dorsoventral pattern of primary neurons in the developing amphibian embryo. Here, we investigate the role of Gsx transcription factors in regulating the transcription of Iroquois family homeobox genes in the amphibian neural plate. Iroquois genes are key regulators of neural patterning and their expression is coincident with that of the Gsx genes during open neural plate stages. We show that Gsx proteins repress Iroquois expression in the embryo and conversely, inhibition of Gsx activity with either antisense morpholino oligos or an anti-morphic Gsx protein up-regulates Iroquois expression. These data indicate that Gsx factors act as negative regulators of Iroquois gene expression in the amphibian neural plate and support a model in which the Gsx proteins promote neuronal differentiation by repressing the expression of known inhibitors of neuronal differentiation such as Iro3.

Abstract: The Gsx genes encode members of the ParaHox family of homeodomain transcription factors, which are expressed in the developing central nervous system in members of all major groups of bilaterians. The Gsx genes in Xenopus show similar patterns of expression to their mammalian homologues during late development. However, they are also expressed from early neurula stages in an intermediate region of the open neural plate where primary interneurons form. The Gsx homologue in the protostome Drosophila is expressed in a corresponding intermediate region of the embryonic neuroectoderm, and is essential for the correct specification of the neuroblasts that arise from it, suggesting that Gsx genes may have played a role in intermediate neural specification in the last common bilaterian ancestor. Here, we show that manipulation of Gsx function disrupts the differentiation of primary interneurons. We demonstrate that, despite their similar expression patterns, the uni-directional system of interactions between homeodomain transcription factors from the Msx, Nkx and Gsx families in the Drosophila neuroectoderm is not conserved between their homologues in the Xenopus open neural plate. Finally, we report the identification of Dbx1 as a direct target of Gsh2-mediated transcriptional repression, and show that a series of cross-repressive interactions, reminiscent of those that exist in the amniote neural tube, act between Gsx, Dbx and Nkx transcription factors to pattern the medial aspect of the central nervous system at open neural plate stages in Xenopus.

Abstract: BACKGROUND INFORMATION: Developmental cell signals co-operate in the processes of cell specification and tissue patterning during embryogenesis. Interactions between the FGF (fibroblast growth factor) and Wnt signalling pathways have been demonstrated in a number of developmental processes, including mesoderm formation in amphibian embryos. However, the mechanism underlying the interactions between these key signalling pathways remains unclear. RESULTS: In the present study, we find that the ability of TLE4/Xgrg4 (transducin-like enhancer of split 4/Xenopus groucho-related gene 4) to inhibit a transcriptional target of canonical Wnt signalling is reduced in the presence of FGF and that this is partially dependent on a consensus site for MAPK (mitogen-activated protein kinase) phosphorylation in TLE4/Xgrg4. CONCLUSIONS: These data suggest to us a novel molecular mechanism where FGF and Wnt signalling pathways interact at the level of the co-repressor TLE4/Xgrg4: the weakening of TLE4/Xgrg4 repression by FGF signalling, combined with the stabilization of beta-catenin by Wnt signals, enhances expression of Wnt target genes.

Abstract: The osmotic pressure of plant phloem sap is generally higher than that of insect body fluids. Water cycling from the distal to proximal regions of the gut is believed to contribute to the osmoregulation of aphids and other phloem-feeding insects, with the high flux of water mediated by a membrane-associated aquaporin. A putative aquaporin referred to as ApAQP1 was identified by RT-PCR of RNA isolated from the guts of pea aphids Acyrthosiphon pisum. The ApAQP1 protein has a predicted molecular mass 28.94kDa. Molecular modeling suggests that ApAQP1 has the general aquaporin topology and possesses the conserved pore properties of water-specific aquaporins. When expressed in Xenopus oocytes, ApAQP1 showed the hallmarks of aquaporin-mediated water transport, including an 18-fold increase in the osmotic water permeability of the oolemma, a reduced activation energy, and inhibition of elevated water transport activity by Hg ions. The ApAQP1 transcript was localised to the stomach and distal intestine, and RNAi-mediated knockdown of its expression resulted in elevated osmotic pressure of the haemolymph. Taken together, these data suggest that ApAQP1 contributes to the molecular basis of water cycling in the aphid gut.

Abstract: Gsx class proteins are members of the ParaHox homeodomain transcription factor family with conserved roles in specification and patterning of the nervous system. We report the cloning of two Gsx genes, Gsh1 and Gsh2, from the frog Xenopus tropicalis. We demonstrate the existence of a single, intact Xenopus ParaHox cluster, containing Gsh1, Pdx, and Cdx2, plus three degenerate clusters containing Gsh2, Cdx1, and Cdx4. Anterior expression boundaries of genes from the intact ParaHox cluster are co-linear with respect to their genomic organization. We show that Gsh1 and Gsh2 exhibit complex, overlapping patterns of expression within the anterior nervous system from open neural plate stages. We also find that expression of Gsh2, Nkx6, and Msx1 across the medio-lateral axis of the amphibian neural plate is strikingly similar to that of related genes in the Drosophila neuroectoderm. These findings provide further evidence for a conserved pathway regulating dorso-ventral patterning in the Bilateria.

Abstract: BACKGROUND: FGF signaling has multiple roles in regulating processes in animal development, including the specification and patterning of the mesoderm. In addition, FGF signaling supports self renewal of human embryonic stem cells and is required for differentiation of murine embryonic stem cells into a number of lineages. METHODOLOGY/PRINCIPAL FINDINGS: Given the importance of FGF signaling in regulating development and stem cell behaviour, we aimed to identify the transcriptional targets of FGF signalling during early development in the vertebrate model Xenopus laevis. We analysed the effects on gene expression in embryos in which FGF signaling was inhibited by dominant negative FGF receptors. 67 genes positively regulated by FGF signaling and 16 genes negatively regulated by FGF signaling were identified. FGF target genes are expressed in distinct waves during the late blastula to early gastrula phase. Many of these genes are expressed in the early mesoderm and dorsal ectoderm. A widespread requirement for FGF in regulating genes expressed in the Spemann organizer is revealed. The FGF targets MKP1 and DUSP5 are shown to be negative regulators of FGF signaling in early Xenopus tissues. FoxD3 and Lin28, which are involved in regulating pluripotency in ES cells are shown to be down regulated when FGF signaling is blocked. CONCLUSIONS: We have undertaken a detailed analysis of FGF target genes which has generated a robust, well validated data set. We have found a widespread role for FGF signaling in regulating the expression of genes mediating the function of the Spemann organizer. In addition, we have found that the FGF targets MKP1 and DUSP5 are likely to contribute to the complex feedback loops involved in modulating responses to FGF signaling. We also find a link between FGF signaling and the expression of known regulators of pluripotency.

Abstract: Using Xenopus tropicalis, we present the first analysis of the developmental effects that result from knocking down the function of the three Cdx genes present in the typical vertebrate genome. Knockdowns of individual Cdx genes lead to a similar range of posterior defects; compound Cdx knockdowns result in increasingly severe posterior truncations, accompanied by posterior shifts and reduction of 5' Hox gene expression. We provide evidence that Cdx and Wnt3A genes are components of a positive feedback loop operating in the posterior axis. We show that Cdx function is required during later, but not early stages of development, for correct regional specification of the endoderm and morphogenesis of the gut. Our results support the hypothesis that during amphibian development the overall landscape of Cdx activity in the embryo is more important than the specific function of individual Cdx proteins.

Abstract: BACKGROUND INFORMATION: FGF (fibroblast growth factor) signalling is known to be required for many aspects of mesoderm formation and patterning during Xenopus development and has been implicated in regulating genes required for the specification of both blood and skeletal muscle lineages. RESULTS: In the present study, we have specifically knocked down the expression of FGF4 using AMO (antisense morpholino oligonucleotide)-mediated inhibition and demonstrate that FGF4 acts in the dorsal marginal zone to restrict blood development and promote the development of skeletal muscle. In addition, we used a drug inhibitor of FGF signalling and an inducible form of FGFR1 (FGF receptor 1) to identify a period of competence during late blastula and gastrula stages when FGF signalling acts to regulate blood versus muscle specification. Notably, we found that it is the dorsal activity of FGF that is required to restrict the expression of SCL (stem cell leukaemia) to the ventral blood island. CONCLUSIONS: Our data indicate that FGF4 is a key organizer-derived signal involved in the process of dorsoventral patterning of the mesoderm.

Abstract: BACKGROUND INFORMATION: There are significant indications that amphibians require TH (thyroid hormones) prior to their involvement in the regulation of metamorphosis and before the development of a functional thyroid. RESULTS: In order to investigate the potential role for TH in pre-metamorphic Xenopus tropicalis we have cloned cDNAs for, and analysed the expression of, TPO (thyroid peroxidase), 5'DII (type II iodothyronine deiodinase) and 5DIII (type III iodothyronine deiodinase), enzymes involved in TH metabolism. Zygotic expression of TPO was detected in neurula stage embryos. Expression was observed in the notochord and later in the thyroid. The notochord was also a common site of expression for 5'DII and 5DIII. Other sites of 5'DII expression are the otic vesicles, retina, liver, blood-forming region, branchial arches and brain. 5DIII is also expressed in the brain, retina, liver, developing pro-nephros, blood-forming region and branchial arches. Embryos exposed to the TPO inhibitor methimazole showed a distinctive dose-dependent phenotype of a crimped notochord and shortened axis, together with alterations in (125)I(-) uptake. CONCLUSIONS: These data suggest a novel extrathyroidal role for TH during early development, and support the proposal that embryos require thyroid signalling for normal development prior to metamorphosis.

Abstract: As a consequence of comprehensive transcriptome analysis followed by sequencing and draft assembly of the genome, the emphasis of schistosome research is shifting from the identification of genes to the characterization of their functions and interactions. Developmental biologists have long used whole mount in situ hybridization (WISH) to determine gene expression patterns, as a vital tool for formulating and testing hypotheses about function. This paper describes the application of WISH to the study of gene expression in larval and adult schistosomes. Fixed worms were permeablized by proteinase K treatment for hybridization with digoxygenin-labelled RNA probes, with binding being detected by alkaline phosphatase-coupled anti-digoxygenin antibodies, and BM Purple substrate. Discrete staining patterns for the transcripts of the molecules Sm29, cathepsin L, antigen 10.3 and chorion were observed in the tegument cell bodies, gut epithelium, oesophageal gland and vitelline lobules, respectively, of adult worms. Transcripts of the molecules SGTP4, GP18-22 and cathepsin L were localized to tegument cell bodies and embryonic gut, respectively, of lung schistosomula. We also showed that Fast Red TR fluorescent substrate can refine the pattern of localization permitting use of confocal microscopy. We believe that method of WISH will find broad application, in synergy with other emerging post-genomic techniques, such as RNA interference, to studies focused at increasing our molecular understanding of schistosomes.

Abstract: The hydrolysis of sucrose, the principal dietary source of carbon for aphids, is catalysed by a gut alpha-glucosidase/transglucosidase activity. An alpha-glucosidase, referred to as APS1, was identified in both a gut-specific cDNA library and a sucrase-enriched membrane preparation from guts of the pea aphid Acyrthosiphon pisum by a combination of genomic and proteomic techniques. APS1 contains a predicted signal peptide, and has a predicted molecular mass of 68 kDa (unprocessed) or 66.4 kDa (mature protein). It has amino acid sequence similarity to alpha-glucosidases (EC 3.2.1.20) of glycoside hydrolase family 13 in other insects. The predicted APS1 protein contains two domains: an N-terminal catalytic domain, and a C-terminal hydrophobic domain. In situ localisation and RT-PCR studies revealed that APS1 mRNA was expressed in the gut distal to the stomach, the same localisation as sucrase activity. When expressed heterologously in Xenopus embryos, APS1 was membrane-bound and had sucrase activity. It is concluded that APS1 is a dominant, and possibly sole, protein mediating sucrase activity in the aphid gut.

Abstract: Cdx homeodomain transcription factors play important roles in the development of the vertebrate body axis and gut epithelium. Signaling involving FGF, wnt and retinoic acid ligands has been implicated in the regulation of individual Cdx genes. In this study we examine the requirement for FGF-dependent signal transduction pathways in the regulation of Cdx gene expression. In the amphibian Xenopus laevis the earliest expression of Cdx1, Cdx2 and Cdx4 is within the developing mesoderm. We show that a functional FGF signaling pathway is required for the normal expression of all three amphibian Cdx genes during gastrula stages. We show that FGF stimulation activates signaling through both the MAP kinase pathway and the PI-3 kinase pathway in Xenopus tissue explants. However, our analysis of these pathways in gastrula stage embryos indicates that the MAP kinase pathway is required for Cdx gene expression, whereas the PI-3 kinase pathway is not. We show that FGF and wnt signaling can interact in the regulation of Cdx genes and during gastrula stages the normal expression of the Cdx genes requires the activity of both pathways. Furthermore, we show that wnt mediated Cdx regulation is independent of the MAP kinase pathway.

Abstract: Cdx homeodomain transcription factors have multiple roles in early vertebrate development. Furthermore, mis-regulation of Cdx expression has been demonstrated in metaplasias and cancers of the gut epithelium. Given the importance of Cdx genes in development and disease, the mechanisms underlying their expression are of considerable interest. We report an analysis of the upstream regulatory regions from the amphibian Xenopus laevis Cdx4 gene. We show that a GFP reporter containing 2.8 kb upstream of the transcription start site is expressed in the posterior of transgenic embryos. Deletion analysis of the upstream sequence reveals that a 247-bp proximal promoter fragment will drive posterior expression in transgenic embryos. We show that 63 bp of upstream sequence, that includes a consensus site for POU-domain octamer-binding proteins, retains significant promoter activity. Co-expression of the octamer-binding protein Oct1 induces expression from a Cdx4 reporter and mutation of the octamer site abolishes activity of the same reporter. We show that the octamer site is highly conserved in the promoters of the human, mouse, chicken, and zebrafish Cdx4 genes and within the promoters of amphibian Cdx1 and Cdx2. These data suggest a conserved function for octamer-binding proteins in the regulation of Cdx family members.

Abstract: Peroxidasin, originally identified in Drosophila, is a member of the myeloperoxidase family with a novel domain structure. It is proposed that peroxidasin is secreted and has functions associated with stabilization of the extracellular matrix. We report the identification of the Xenopus tropicalis orthologue of the peroxidasin gene. We show that the predicted protein sequence of Xenopus peroxidasin shows high sequence identity with the human orthologue and that the exon structure is highly conserved between the two species. We describe the first detailed developmental expression pattern for peroxidasin in a vertebrate species. Maternal expression of Xtpxn is localized to the animal hemisphere where it persists through early cleavage stages. Initial zygotic Xtpxn expression is detected in the developing neural tube and becomes localized to the hindbrain and midbrain. Xtpxn is expressed in the primordium of the pronephric kidney and expression persists in the pronephric tubules and duct throughout development. Potential roles for peroxidasin during early vertebrate development are discussed.

Abstract: The use of a novel inducible FGF signalling system in the frog Xenopus laevis is reported. We show that the lipophilic, synthetic, dimerizing agent AP20187 is able to rapidly activate signalling through an ectopically expressed mutant form of FGFR1 (iFGFR1) in Xenopus embryos. iFGFR1 lacks an extracellular ligand binding domain and contains an AP20187 binding domain fused to the intracellular domain of mouse FGFR1. Induction of signalling by AP20187 is possible until at least early neurula stages, and we demonstrate that ectopically expressed iFGFR1 protein persists until late neurula stages. We show that activation of signalling through iFGFR1 can mimic a number of previously reported FGF activities, including mesoderm induction, repression of anterior development, and neural posteriorization. We show that competence to morphological posteriorization of the anteroposterior axis by FGF signalling only extends until about stage 10.5. We demonstrate that the competence of neural tissue to express the posterior markers Hoxa7 and Xcad3, in response to FGF signalling, is lost by the end of gastrula stages. We also show that activation of FGF signalling stimulates morphogenetic movements in neural tissue until at least the end of the gastrula stage.

Abstract: The caudal-related (Cdx) homeodomain transcription factors have a conserved role in the development of posterior structures in both vertebrates and invertebrates. A particularly interesting finding is that Cdx proteins have an important function in the regulation of expression from a subset of Hox genes. In this study, we report the cloning of cDNAs from the Cdx genes of the amphibian Xenopus tropicalis. Xenopus tropicalis is a diploid species, related to the commonly used laboratory animal Xenopus laevis, and has attracted attention recently as a potential genetic model for animal development. The Xenopus tropicalis cDNAs, Xtcad1, Xtcad2, and Xtcad3, show between 88 and 94% sequence identity with their Xenopus laevis orthologues. This finding corresponds to between 90 and 95% identity at the level of derived amino acid sequence. We also present a detailed description of Xtcad1, Xtcad2, and Xtcad3 expression during normal development. In common with the Cdx genes of other vertebrates, the Xenopus tropicalis Cdx genes show overlapping and dynamic patterns of expression in posterior regions of the embryo through the early stages of development.

Abstract: This paper addresses the molecular mechanisms that regulate the transcriptional activation of the myogenic regulatory factor XmyoD in the skeletal muscle lineage of Xenopus laevis. Using antisense morpholino oligonucleotide-mediated inhibition, we show that the signalling molecule embryonic fibroblast growth factor (eFGF), which is the amphibian homologue of FGF4, is necessary for the initial activation of XmyoD transcription in myogenic cells. We demonstrate that eFGF can activate the expression of XmyoD in the absence of protein synthesis, indicating that this regulation is direct. Our data suggest that regulation of XmyoD expression may involve a labile transcriptional repressor. In addition, we show that eFGF is itself an immediate early response to activin, a molecule that mimics the endogenous mesoderm-inducing signal. We propose a model for the regulation of XmyoD within the early mesoderm, and discuss the relevance that these findings have for the understanding of myogenic specification in higher vertebrates.

Abstract: Members of the Otx (orthodenticle) and Cdx (caudal) families of homeodomain transcription factors are expressed in similar embryonic regions in all animal groups and have been shown to be directly involved in anteroposterior patterning in a number of species. In the amphibian Xenopus laevis, the Otx family gene Xotx2 and the Cdx family gene Xcad3 are both expressed within the early dorsal organizer. We show that they have mutually repressive activities, suggesting that they play a crucial role in the early regionalization of the organizer into anterior and posterior territories. Xotx2 can act both as an activator and repressor of gene expression depending on context. A form of Xotx2 that acts exclusively as a repressor (OtxEn-R) was made by fusing the Xotx2 homeodomain to the Drosophila melanogaster engrailed transcriptional repressor domain. Overexpression of this protein in vivo indicates that OtxEn-R antagonizes the activating function of endogenous Xotx2 for anterior marker genes such as XCG and goosecoid but retains the ability to repress the expression of posterior markers such as Xcad3 and Xbra. OtxEn-R overexpression causes a severe derangement of anterior development, resulting in the loss of cement gland, eyes, stomodeal opening, and pharynx. The specification and development of anterior neural structures is dramatically abnormal up to and including the isthmic signaling center at the midbrain/hindbrain junction. This study provides good evidence that Xenopus Otx2 is required for normal head patterning and the process of anterior neural specification. We propose that a mutually antagonistic relationship between Otx and Cdx factors is a basic aspect of anteroposterior patterning in all vertebrates.

Abstract: 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.

Abstract: 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.

Abstract: 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.

Abstract: It has been known for several years that the fibroblast growth factors (FGFs) have potent mesoderm-inducing activity. As a result they have been considered good candidates for one of the endogenous vegetally localized mesoderm-inducing signals in the amphibian Xenopus laevis. In this review the properties of the FGFs and their expression patterns in Xenopus are described. Recent work is discussed which reveals a close link between FGF signalling and regulation of the Xenopus brachyury (Xbra) gene. These data are used to build a model of FGF function which is quite different from what was originally conceived. Present evidence supports the view that during blastula stages the FGFs do not act as vegetally localized inducing signals. Instead, they are required in the animal hemisphere as competence factors, which provide a low level stimulation of the tyrosine kinase signal transduction pathway. FGF activity is necessary for the full range of responses to the vegetal inducing signals, including the activation of Xbra transcription in the marginal zone of the late blastula. Xbra is able to activate the zygotic transcription of eFGF, which suggests that there is a period of autocatalytic activation of eFGF and Xbra transcription within the forming mesoderm of the marginal zone. FGF activity continues to be required to maintain the expression of a sub-set of mesodermal genes, including Xbra, in the blastopore region and possibly also in the notochord through gastrula and neurula stages. In addition a role for the FGFs in anteroposterior specification and development of the myogenic lineages is discussed.

Abstract: In recent years we and others have been attempting to identify the molecular nature of the inducing signals in early Xenopus development. We have found that most members of the fibroblast growth factor (FGF) family are biologically active as mesoderm-inducing factors when applied to ectoderm from blastulae. In addition to this, they will support continued expression of the pan-mesodermal transcription factor Xbra in the mesoderm of gastrula stage embryos. We have studied the expression pattern of four types of FGF in early embryos. Two types (FGF-2 and FGF-9) are expressed maternally and are thus present at the time of natural mesoderm induction. The expression of two other types (FGF-3 and FGF-4) is activated in the newly formed mesoderm of the gastrula. If the activity of the FGF family is inhibited by overexpression of a dominant-negative FGF receptor, there is a reduction in mesoderm formation, there are abnormalities arising from an inhibition of normal gastrulation movements and there is a defect in formation of the posterior parts. We believe that the mesoderm formation and cell movement effects are attributable to loss of Xbra expression, and the posterior defects to lack of posterior HOX gene activity. Overexpression of eFGF gives rise to a posteriorized phenotype, in which posterior HOX genes are expressed in a more anterior position. We conclude that the FGF system has multiple functions in early development, including mesoderm formation, gastrulation movements and anteroposterior patterning.

Abstract: 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.

Abstract: A detailed study of the expression pattern of embryonic fibroblast growth factor (eFGF) during early Xenopus development has been undertaken using whole-mount DIG in situ hybridization. We show that the zygotic expression of eFGF is activated in the mesoderm of the early gastrula and is first visualized as a ring around the blastopore, with significantly higher levels of expression on the dorsal side of the embryo. As gastrulation proceeds, eFGF transcripts become increasingly abundant in the dorsal blastopore lip. In the early neurula eFGF expression can be detected in the extreme posterior of the embryo around the closed blastopore and in the cells of the notochord. This latter result is significant and represents the first report of a Xenopus FGF that is expressed in the notochord. In addition, we show that during gastrula and neurula stages, expression of eFGF closely follows the expression of the Xenopus brachyury (Xbra) gene. During later development eFGF expression is localized to the tail-bud region and a stripe at the mid-brain/hind-brain junction. These data provide further evidence that FGFs play an important role in regulating the expression of brachyury in the developing mesoderm.

Abstract: The behavior of colored beads and of various living tissues after implantation into the Xenopus blastocoel is investigated. It is confirmed that the location of a graft along the anteroposterior axis depends on its intrinsic anteroposterior character. Comparison with the behavior of the beads suggests that the final position can be achieved by movement of the graft around the dorsoventral circumference of the inner marginal zone during gastrulation. Ventral marginal explants, or animal caps treated with fibroblast growth factor, both form ventral vesicles if cultured in isolation, but in the implantation experiments they often yield projections containing segmented muscle blocks. This behavior does not occur when the axis of the host has been suppressed by ultraviolet irradiation and so it is concluded that it represents dorsalization of the graft by the host. The tail-like structures formed as a result of ventral-type tissue implantations do not contain neural tissue, while the mesodermal parts are typically of mixed graft and host origin.

Abstract: We show that, in addition to a role in mesoderm induction during blastula stages, FGF signalling plays an important role in maintaining the properties of the mesoderm in the gastrula of Xenopus laevis. eFGF is a maternally expressed secreted Xenopus FGF with potent mesoderm-inducing activity. However, it is most highly expressed in the mesoderm during gastrulation, suggesting a role after the period of mesoderm induction. eFGF is inhibited by the dominant negative FGF receptor. Embryos overexpressing the dominant negative receptor show a change of behaviour of the dorsal mesoderm such that it moves around the blastopore lip instead of elongating in an antero-posterior direction. In such embryos there is a reduction in Xbra expression during gastrulation. We show that during blastula stages eFGF and Xbra are able to activate the expression of each other, suggesting that they are components of an autocatalytic regulatory loop. Moreover, we show that Xbra expression in isolated gastrula mesoderm cells is maintained by eFGF, suggesting that eFGF continues to regulate the expression of Xbra in the blastopore region. In addition, overexpression of eFGF after the mid-blastula transition results in the up-regulation of Xbra expression during gastrula stages and causes suppression of the head and enlargement of the proctodeum, which is the converse of the posterior reductions of the FGF dominant negative receptor phenotype. These data suggest an important role for eFGF in regulating the expression of Xbra and for the eFGF-Xbra regulatory pathway in the control of mesodermal cell behaviour during gastrula stages.

Abstract:

Abstract: Although the mesoderm itself is induced at the blastula stage, its subdivision mainly occurs in response to further inductive signals during gastrulation. In the late blastula, most of the mesoderm has a ventral-type commitment except for the small organizer region which extends about 30 degrees on each side of the dorsal midline. During gastrulation, dorsal convergence movements bring the cells of the lateroventral marginal zone up near the dorsal midline and into the range of the dorsalizing signal emitted by the organizer. This dorsalizing signal operates throughout gastrulation, can cross a Nuclepore membrane, and is not mimicked by lithium, FGFs or activin. Anteroposterior specification also takes place during gastrulation and is probably controlled by a dominant region at the posterior end of the forming axis. We have studied the expression patterns in Xenopus of three members of the FGF family: bFGF, int-2 and a newly discovered species, eFGF. These all have mesoderm inducing activity on isolated animal caps, but are likely also to be involved with the later interactions. RNAase protections and in situ hybridizations show that the int-2 and eFGF mRNAs are concentrated at the posterior end, while bFGF is expressed as a posterior to anterior gradient from tailbud to head. Studies of embryos in which bFGF is overexpressed from synthetic mRNA show that biological activity is far greater when a functional signal sequence is provided. This suggests that int-2 and eFGF, which possess signal sequences, are better candidates for inducing factors in vivo than is bFGF.

Abstract: We have used a probe specific for the Xenopus homologue of the mammalian proto-oncogene int-2 (FGF-3) to examine the temporal and spatial expression pattern of the gene during Xenopus development. int-2 is expressed from just before the onset of gastrulation through to prelarval stages. In the early gastrula, it is expressed around the blastopore lip. This is maintained in the posterior third of the prospective mesoderm and neuroectoderm in the neurula. A second expression domain in the anterior third of the neuroectoderm alone appears in the late gastrula, which later resolves into the optic vesicles, hypothalamus and midbrain-hindbrain junction region. Further domains of expression arise in tailbud to prelarval embryos, including the stomodeal mesenchyme, the endoderm of the pharyngeal pouches and the cranial ganglia flanking the otocyst. It is shown, by treatment of blastula ectoderm with bFGF and activin, that int-2 can be expressed in response to mesoderm induction. By heterotypic grafting of gastrula ectoderm into axolotl neural plate, we have also demonstrated that int-2 can be expressed in response to neural induction. These results suggest that int-2 has multiple functions in development, including an early role in patterning of the anteroposterior body axis and a later role in the development of the tail, brain-derived structures and other epithelia.

Abstract: We have cloned and sequenced a new member of the fibroblast growth factor family from Xenopus laevis embryo cDNA. It is most closely related to both mammalian kFGF (FGF-4) and FGF-6 but as it is not clear whether it is a true homologue of either of these genes we provisionally refer to it as XeFGF (Xenopus embryonic FGF). Two sequences were obtained, differing by 11% in derived amino acid sequence, which probably represent pseudotetraploid variants. Both the sequence and the behaviour of in vitro translated protein indicates that, unlike bFGF (FGF-2), XeFGF is a secreted molecule. Recombinant XeFGF protein has mesoderm-inducing activity with a specific activity similar to bFGF. XeFGF mRNA is expressed maternally and zygotically with a peak during the gastrula stage. Both probe protection and in situ hybridization showed that the zygotic expression is concentrated in the posterior of the body axis and later in the tailbud. Later domains of expression were found near the midbrain/hindbrain boundary and at low levels in the myotomes. Because of its biological properties and expression pattern, XeFGF is a good candidate for an inducing factor with possible roles both in mesoderm induction at the blastula stage and in the formation of the anteroposterior axis at the gastrula stage.

Abstract: The first inductive interaction in amphibian development is mesoderm induction, during which a signal from the vegetal hemisphere of the blastula-staged embryo induces mesoderm from overlying equatorial cells. Recently, a number of 'mesoderm-inducing factors' (MIFs), which may be responsible for this interaction, have been discovered. Examples of these MIFs include members of the fibroblast growth factor family as well as members of the TGF-beta superfamily such as TGF-beta 2. In addition to these purified factors, several new sources of mesoderm-inducing activity have been described. One of the most potent of these is the murine myelomonocytic leukemia cell line WEHI-3. Even at high dilutions, conditioned medium from WEHI-3 cells induces isolated Xenopus animal pole regions to form a variety of mesodermal cell types. In this paper we show by several criteria, including N-terminal amino acid sequencing, Northern blotting and various functional assays, that the WEHI-MIF is activin A. Activins are known to modulate the release of follicle-stimulating hormone from cultured anterior pituitary cells and to cause the differentiation of two erythroleukemia cell lines. Our results, along with recent data from other laboratories, indicate that these molecules may also act in early development in the formation of the mesoderm.

Abstract: In early amphibian development the mesoderm is formed around the equator of the blastula in response to inductive signals from the endoderm. At the time of its formation the mesoderm consists of a large 'ventral type' zone and a small 'organizer' zone. A screen of candidate substances showed that a small group of heparin binding growth factors (HBGFs) were active as mesoderm inducing agents in vitro. The fibroblast growth factors (aFGF and bFGF) and embryonal carcinoma derived growth factor (ECDGF) all show similar potency and can produce ventral inductions at concentrations above about 100 pm. Single blastula ectoderm cells can be induced and will differentiate in a defined medium to form mesodermal tissues and all inner blastula cells are competent to respond to the factors. Inducing activity can be extracted from Xenopus blastulae and can be purified by heparin affinity chromatography. Antibody neutralization and Western blotting experiments identify this activity as bFGF. The amounts present are small but would be sufficient to evoke ventral inductions in vivo. It is not yet known whether the bFGF is localized to the endoderm, although it is known that inducing activity secreted by endodermal cells can be neutralized by heparin. The competence of ectoderm to respond to FGF rises from about the 128-cell-stage and falls again by the onset of gastrulation. This change is paralleled by a rise and fall of binding of 125I-labelled aFGF. Chemical cross-linking reveals that this binding is attributable to a receptor of molecular mass about 130 kilodaltons (kDa). The receptor is present both in the marginal zone, which responds to the signal in vivo, and in the animal pole region, which is not induced in vivo but which will respond to HBGFs in vitro. In intact embryos we believe that the ventral type mesoderm forms the somites, kidney and other intermediate structures as well as the blood islands of the ventral midline. These intermediate structures are induced as a function of distance from the organizer in a process called 'dorsalization'. Lithium salts have a dorsalizing effect on whole embryos and also on explants from the ventral marginal zone, causing them to form large blocks of muscle. Lithium will also cause large muscle blocks to form when applied to ectoderm explants together with FGF. It is difficult to extend these results directly to mammalian embryos, but we have shown that the products of the murine int-2 gene and of the human k-fgf genes are active as mesoderm inducing factors.

Abstract: In early amphibian development, the mesoderm is formed around the equator of the blastula in response to an inductive signal from the endoderm. A screen of candidate substances showed that a small group of heparin-binding growth factors (HBGFs) were active as mesoderm-inducing agents in vitro. The factors aFGF, bFGF, kFGF and ECDGF all show similar potency and can produce inductions at concentrations above about 100 pM. The product of the murine int-2 gene is also active, but with a lower specific activity. Above the induction threshold there is a progressive increase of muscle formation with dose. Single blastula ectoderm cells can be induced and will differentiate in a defined medium to form mesodermal tissues. All inner blastula cells are competent to respond to the factors but outer cells, bearing oocyte-derived membrane, are not. Inducing activity can be extracted from Xenopus blastulae and binds to heparin like the previously described HBGFs. Antibody neutralization and Western blotting experiments identify this activity as bFGF. The amounts present are small but would be sufficient to evoke inductions in vivo. It is not yet known whether the bFGF is localized to the endoderm, although it is known that inducing activity secreted by endodermal cells can be neutralized by heparin. The competence of ectoderm to respond to HBGFs rises from about the 128-cell stage and falls again by the onset of gastrulation. This change is paralleled by a rise and fall of binding of 125I-aFGF. Chemical cross-linking reveals that this binding is attributable to a receptor of relative molecular mass about 130 x 10(3).(ABSTRACT TRUNCATED AT 250 WORDS)

Abstract: We have studied the response of Xenopus blastula ectoderm to fibroblast growth factor and to lithium ion. The properties of acidic and basic FGF are very similar showing a 50% induction level at 1-2 ng ml-1 and a progressive increase of muscle formation up to concentrations of 100-200 ng ml-1. The elongation of explants also shows a dose-response relationship. The minimum contact requirement for induction of ectoderm explants is about 90 min and the stage range of ectodermal competence extends from midblastula to early gastrula, both these figures resembling those obtained in embryological experiments with vegetal tissue as the inducer. Lithium chloride concentrations which produce anteriorization of whole embryos have no effect on isolated ectoderms unless accompanied by FGF. Simultaneous treatment with FGF and Li lead to a marked enhancement of both elongation and muscle formation over that produced by FGF alone. By contrast, ventral marginal explants show increased elongation and muscle formation if treated with lithium alone suggesting that they have already received a low-dose FGF treatment within the embryo. It is concluded that endogenous FGF may be solely responsible for inducing the ventral mesoderm and that dorsalization of ventral mesoderm to the level of somitic muscle might be achieved either by a very high local concentration of FGF in the dorsal region, or by the action of a second, synergistic, agent in the dorsal region.

Abstract: Mesoderm-inducing factors (MIF's) from chick embryos, XTC cells and WEHI-3 cells were studied using various procedures. The object was to find whether they are similar to heparin-binding growth factors (HBGFs-the only known pure mesoderm-inducing substances) and, if not, whether they are similar to each other. The major active components from all three MIF sources behave as somewhat hydrophobic, acid-stable molecules and do not bind to heparin. They all have relative molecular masses of about 13,000 measured by HPLC size exclusion chromatography. The isoelectric points measured by chromatofocusing were 6.7 (WEHI) and 7.7 (XTC). The chick MIF seemed somewhat heterogeneous by chromatofocusing and a portion of its activity bound to heparin sepharose. All three MIFs have similar effects on explants of Xenopus blastula ectoderm to the heparin-binding growth factors, causing an elongation at the time of gastrulation followed by the development of mesenchyme, mesothelium and muscle cells, the proportion of muscle increasing with dose. Unlike the HBGFs they all also induce notochord if sufficiently high concentrations are used. Our study shows that the MIFs examined here form a small group of potent agents distinct from the HBGFs and from other known growth and differentiations factors. Their occurrence in various tissues and cell lines suggests that they have functions in the adult organism as well as during early development.