A water-specific aquaporin involved in aphid osmoregulation.

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.

Patterns of gene expression in schistosomes: localization by whole mount in situ hybridization.

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.

Molecular characterisation of a candidate gut sucrase in the pea aphid, Acyrthosiphon pisum.

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.

Anteroposterior patterning by mutual repression of orthodenticle and caudal-type transcription factors.

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.

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.

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.

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.

New perspectives on the role of the fibroblast growth factor family in amphibian development.

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.

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.

The role of fibroblast growth factors in early Xenopus development.

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.

eFGF is expressed in the dorsal midline of Xenopus laevis.

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.

eFGF regulates Xbra expression during Xenopus gastrulation.

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.

The Einsteck-method: position and structure of projections formed by implants of a ventral character.

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.

Developmental expression of the Xenopus int-2 (FGF-3) gene: activation by mesodermal and neural induction.

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.

Expression of a novel FGF in the Xenopus embryo. A new candidate inducing factor for mesoderm formation and anteroposterior specification.

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.

Specification of the body plan during Xenopus gastrulation: dorsoventral and anteroposterior patterning of the mesoderm.

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.

A mesoderm-inducing factor produced by WEHI-3 murine myelomonocytic leukemia cells is activin A.

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.

Mesoderm induction by fibroblast growth factor in early Xenopus development.

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.

The role of fibroblast growth factor in early Xenopus development.

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)