Structural and functional evidences for a type 1 TGF-beta sensu stricto receptor in the lophotrochozoan Crassostrea gigas suggest conserved molecular mechanisms controlling mesodermal patterning across bilateria.

The transforming growth factor beta (TGFbeta) superfamily includes bone morphogenetic proteins, activins and TGF-betasensu stricto (s.s.). These ligands have been shown to play a key role in numerous biological processes including early embryonic development and immune regulation. They transduce their signal through a hetromeric complex of type I and type II receptors. Such receptors have been identified in ecdysozoans but none have been found as yet in the other major protostomal clade, the lophotrochozoans. Here, we report the identification of the first lophotrochozoan TGFbetas.s. type I receptor (Cg-TGFbetaRI) from the mollusk Crassostrea gigas. The phylogenetic and structural analyses as well as the expression pattern during early development suggest Cg-TGFbetaRI to belong to the TGFbetas.s./activin type I receptor clade and functional studies corroborate these deductions. The use of the zebrafish embryo as a reporter organism reveals that either Cg-TGFbetaRI or its dominant negative acting truncated form, when overexpressed during gastrulation, resulted in a range of phenotypes displaying severe disturbance of anterioposterior patterning due to a strong modulation of ventrolateral mesoderm patterning. Finally, a Cg-TGFbetaRI cytokine activity during immune regulation in C. gigas has been investigated by real-time PCR in haemocytes and mantle edge during an in vivo bacterial LPS challenge. One piece of evidence from this study suggests that the molecular mechanisms controlling mesodermal patterning and some immune regulations across all bilateria could be conserved through a functional TGF-beta s.s. pathway in lophotrochozoans.

Biomphalaria tenagophila/Schistosoma mansoni interaction: premises for a new approach to biological control of schistosomiasis.

Biomphalaria tenagophila is very important for schistosomiasis transmission in Brazil. However its mechanisms of interaction with Schistosoma mansoni are still scantly studied. Since this snail displays strains highly susceptible or completely resistant to the parasite infection, the knowledge of that would be a useful tool to understand the mechanism of snail resistance. Particularly, the Taim strain consistently shows absolute resistance against the trematode, and this resistance is a dominant character. A multidisciplinary research group was created aiming at studying B. tenagophila/S. mansoni interaction. The possibility for applying the knowledge acquired to obtain a biological model for the control of S. mansoni transmission in endemic areas is discussed.

Biomphalaria tenagophila/Schistosoma mansoni interaction: premises for a new approach to biological control of schistosomiasis

Biomphalaria tenagophila is very important for schistosomiasis transmission in Brazil. However its mechanisms of interaction with Schistosoma mansoni are still scantly studied. Since this snail displays strains highly susceptible or completely resistant to the parasite infection, the knowledge of that would be a useful tool to understand the mechanism of snail resistance. Particularly, the Taim strain consistently shows absolute resistance against the trematode, and this resistance is a dominant character. A multidisciplinary research group was created aiming at studying B. tenagophila/S. mansoni interaction. The possibility for applying the knowledge acquired to obtain a biological model for the control of S. mansoni transmission in endemic areas is discussed.

Cell autonomous commitment to an endodermal fate and behaviour by activation of Nodal signalling.

In vertebrates the endoderm germ layer gives rise to most tissues of the digestive tract and controls head and heart morphogenesis. The induction of endoderm development relies on extracellular signals related to Nodals and propagated intracellularly by TGFbeta type I receptors ALK4/Taram-A. It is unclear, however, whether Nodal/ALK4/Taram-A signalling is involved only in the specification of endodermal precursors or plays a more comprehensive role in the activation of the endodermal program leading to the irreversible commitment of cells to the endodermal fate. Using cell transplantation experiments in zebrafish, we show that marginal cells become committed to endoderm at the onset of gastrulation and that commitment to endoderm can be reached by intracellular activation of the Nodal pathway induced by expression of an activated form of the taram-A receptor, Tar*. In a manner similar to endoderm progenitors, Tar*-activated blastomeres translocate from their initial site of implantation in the blastoderm to reach the surface of their migration substratum, the yolk syncitial layer, where they join endogenous endodermal derivatives during gastrulation and differentiate according to their anteroposterior position. We demonstrate that Nodal/Tar*-induced commitment does not rely on a secondary signal released by Tar*-expressing cells or a signal released by endogenous endoderm since Tar*-expressing wild-type cells can restore endoderm derivatives when transplanted into the endoderm-deficient mutant casanova. Likewise, the YSL does not appear essential for the maintenance of endodermal identity during gastrulation once the Nodal pathway has been activated. Thus, our results demonstrate that the activation of Nodal signalling is sufficient to commit cells both to an endodermal fate and behaviour. Wild-type endoderm implantation into casanova embryos rescues, in a non-autonomous fashion, the defective fusion of the two heart primordia in the midline, highlighting the importance of endoderm for normal heart morphogenesis.

The BMP-related protein radar: a maintenance factor for dorsal neuroectoderm cells?

We have previously cloned several members of the TGF-beta superfamily of growth factors in zebrafish, one of which, Radar, belongs to the Dpp-Vg1-related (DVR) subgroup, with highest homology to GDF6. The pattern of expression of Radar suggested a possible involvement in several induction steps during embryogenesis including in the dorsal neural tube, red blood cells, the dorsal fin and the retina. We have analyzed the pattern of expression of Radar in comparison with that of a marker of dorsal neural tube structures, msxC and show that Radar and msxC are expressed in similar and/or adjacent tissues throughout embryogenesis. In order to demonstrate a functional relationship between these two proteins, we have generated a full-length cDNA for Radar and shown that Radar overexpression by DNA injection maintains expression of msxC in tissues where it is normally expressed then turned off, in particular in the dorsal neurectoderm. Study of the phenotype of a mutant carrying a deletion of Radar shows a loss of identity and death of the cells of the dorsal neural tube. Taken together these results suggest that Radar could be involved in maintaining the identity of cells of the dorsal-most neural tube and of at least a subset of neural crest cells.

Expression of contact, a new zebrafish DVR member, marks mesenchymal cell lineages in the developing pectoral fins and head and is regulated by retinoic acid.

Contact, a new zebrafish transforming growth factor-beta (TGF-beta) member is most closely related to mouse GDF5 and to human CDMP-1 responsible, when mutated, for limb brachypodism phenotype and Hunter-Thompson syndrome, respectively. Contact exhibits a dynamic spatial expression pattern in the pharyngeal arches and the pectoral fin buds that much prefigures cartilage formation. Within the fin buds, contact expression is detected in the proximal mesenchyme from which the endoskeleton will develop. Exogeneously applied retinoic acid (RA) induces duplication of the pectoral fin rudiment in zebrafish embryos as well as contact expression along the proximal margin of the fin mesenchyme showing that both endoskeleton and exoskeleton can be duplicated.

Dynamo, a new zebrafish DVR member of the TGF-beta superfamily is expressed in the posterior neural tube and is up-regulated by Sonic hedgehog.

Dynamo, a new zebrafish DVR detected from late gastrula on in the posterior neural plate, becomes restricted to the ventral region of the trunk neural tube, with the exclusion of floor plate and adjacent cells. Analysis of dynamo expression in zebrafish axial mutants indicated that dynamo expression in the ventral region of the central nervous system (CNS) is induced by axial mesoderm and maintained by notochord, but is independent of a differentiated floor plate. Ectopic Sonic hedgehog (shh) expression can up-regulate dynamo expression in the posterior neural tube providing evidence that cells of the posterior neural tube are competent to respond to shh signalling and that the close relationship between DVR members and hedgehog-related genes might also apply to vertebrate CNS development.

Zebrafish Radar: a new member of the TGF-beta superfamily defines dorsal regions of the neural plate and the embryonic retina.

Proper development of metazoan embryos requires cell to cell communications. In many instances, these communications involve diffusible molecules, particularly members of the Transforming Growth Factor beta superfamily. In an effort to identify new members of this superfamily involved in the control of early zebrafish embryogenesis, we have isolated a gene, Radar, which appears to be conserved throughout vertebrate evolution and defines a new subfamily within the superfamily. Its pattern of expression suggests that Radar plays a role in the dorso-ventral polarity of the neural plate, blood islands formation, blood cells differentiation, the establishment of retinal dorso-ventral polarity and/or proper axonal retinotectal projections. Radar expression in ntl homozygous mutants indicates that notochord and hypochord development are intimately linked.

Disruption of mesoderm and axis formation in fish by ectopic expression of activin variants: the role of maternal activin.

Formation of mesoderm in Xenopus embryos is the result of an induction event in which peptides such as FGF or activins have been implicated. It was recently demonstrated, by the ectopic expression of a truncated activin receptor, that activin receptor signaling pathways are involved in the processes of mesoderm and axis formation in vivo. However, this approach does not directly address the role of activin itself nor the involvement of activins in the formation of mesoderm in embryos from other vertebrates. In addition, activins are expressed maternally as a protein component of the egg as well as transcribed zygotically, and it is not clear which of the two forms is involved in mesoderm formation. To address those three issues, we analyzed the role of activins in the development of fish embryos by generating two activin dominant-negative variants. One of the variants behaves as an inhibitor of activin protein. The second variant was found to deplete the activin pool when cotranslated with wild-type activin. Injection of RNA encoding these variants into the two-cell embryo of the small teleost fish Oryzias latipes (Japanese medaka) demonstrates that only the maternally provided activin protein is required for mesoderm and axis formation in fish in vivo.

Mix.1, a homeobox mRNA inducible by mesoderm inducers, is expressed mostly in the presumptive endodermal cells of Xenopus embryos.

In frogs, mesoderm presumably derives from presumptive ectoderm by induction under the control of diffusible substances produced by the endoderm. To analyze the early phase of mesoderm induction, I have isolated cDNA copies of mRNAs induced in presumptive ectoderm by mesoderm inducing factor secreted by the XTC cell line. One of the inducible mRNAs encodes a homeodomain-containing protein that is likely to play a regulatory role in development. Mix.1 behaves as an immediate early response to induction, and its kinetics of expression suggest a major role for MBT in the control of inducible gene expression. Unexpectedly, Mix.1 is expressed mostly in the future endoderm, suggesting that endoderm may be formed by induction in a similar way as mesoderm.

Regulation of HLA-DR gene by IFN-gamma. Transcriptional and post-transcriptional control.

IFN-gamma increases the synthesis and level of mRNA of the HLA class I and II genes, in human cells such as melanomas which normally express both classes of molecules. It also induces the surface expression and mRNA synthesis of HLA-DR genes on cells which normally do not express HLA class II genes such as skin fibroblasts. We have investigated the mechanism by which IFN-gamma increases mRNA levels for class II MHC antigens in human cells. For this purpose, we have studied the effect of IFN-gamma on HLA-DR-alpha transcription rate in two different human cell types: VAL melanoma and JDA2 skin fibroblasts. HLA-DR-alpha mRNA is spontaneously produced in VAL cells and its level is enhanced upon IFN-gamma treatment. We demonstrate here that IFN-gamma increases the transcription of HLA-DR-alpha gene in this cell line. However, the discrepancy observed between HLA-DR-alpha mRNA and transcriptional rates led us to postulate that IFN-gamma also regulates the HLA-DR-alpha gene post-transcriptionally. In the course of these experiments, we found also that human skin fibroblasts, which do not contain detectable amounts of HLA-DR-alpha mRNA, spontaneously transcribe the HLA-DR-alpha gene.