Harry V. Isaacs

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.

A novel role for a nodal-related protein; Xnr3 regulates convergent extension movements via the FGF receptor

Convergent extension behaviour is critical for the formation of the vertebrate body axis. In Xenopus, components of the Wnt signaling pathway have been shown to be required for convergent extension movements but the relationship between cell fate and morphogenesis is little understood. We show by loss of function analysis that Xnr3 activates Xbra expression through FGFR1. We show that eFGF activity is not essential in the pathway, and that dishevelled acts downstream of Xnr3 and not in a parallel pathway. We provide evidence for the involvement of the EGF-CFC protein FRL1, and suggest that the pro-domain of Xnr3 may be required for its activity. Since Xnr3 is a direct target of the maternal βcatenin/XTcf3 signaling pathway, it provides the link between the initial, maternally controlled, allocation of cell fate, and the morphogenetic movements of cells derived from the organizer.

Overlapping functions of Cdx1, Cdx2, and Cdx4 in the development of the amphibian Xenopus tropicalis

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. Developmental Dynamics 238:835–852, 2009.

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.

An inducible system for the study of FGF signalling in early amphibian development

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.

Characterisation of the fibroblast growth factor dependent transcriptome in early development.

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.

Expression of enzymes involved in thyroid hormone metabolism during the early development of Xenopus tropicalis.

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.

Cloning and expression of the Cdx family from the frog Xenopus tropicalis

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.

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

SUMMARY 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.

Lin28 proteins are required for germ layer specification in Xenopus.

Lin28 family proteins share a unique structure, with both zinc knuckle and cold shock RNA-binding domains, and were originally identified as regulators of developmental timing in Caenorhabditis elegans. They have since been implicated as regulators of pluripotency in mammalian stem cells in culture. Using Xenopus tropicalis, we have undertaken the first analysis of the effects on the early development of a vertebrate embryo resulting from global inhibition of the Lin28 family. The Xenopus genome contains two Lin28-related genes, lin28a and lin28b. lin28a is expressed zygotically, whereas lin28b is expressed both zygotically and maternally. Both lin28a and lin28b are expressed in pluripotent cells of the Xenopus embryo and are enriched in cells that respond to mesoderm-inducing signals. The development of axial and paraxial mesoderm is severely abnormal in lin28 knockdown (morphant) embryos. In culture, the ability of pluripotent cells from the embryo to respond to the FGF and activin/nodal-like mesoderm-inducing pathways is compromised following inhibition of lin28 function. Furthermore, there are complex effects on the temporal regulation of, and the responses to, mesoderm-inducing signals in lin28 morphant embryos. We provide evidence that Xenopus lin28 proteins play a key role in choreographing the responses of pluripotent cells in the early embryo to the signals that regulate germ layer specification, and that this early function is probably independent of the recognised role of Lin28 proteins in negatively regulating let-7 miRNA biogenesis.