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Graded distributions of netrin and semaphorin guidance cues convey instructive polarity information to migrating cells and growth cones, but also have permissive (i.e. non-polarity determining) functions in mammalian development and repair. The permissive functions of these cues are largely uncharacterised at a molecular level. We found previously that UNC-6 (netrin) signals permissively through UNC-40 (DCC) and UNC-5 receptors to prevent anterior displacement of the ray 1 sensillum in the C. elegans male tail. UNC-6/UNC-40 signalling functions in parallel with SMP-1 (semaporin 1)/PLX-1 (plexin) signalling to prevent this defect. Here, we report that a deletion allele of pvf-1, which encodes a VEGF-related protein, causes no ray 1 defects, but enhances ray 1 defects of a plx-1 mutant, and unexpectedly also suppresses unc-6(ev400)-null mutant ray 1 defects. These mutant ray 1 inductive and suppressive effects are mimicked by the ability of unc-40(+) and ced-10(gain-of-function) multi-copy transgene arrays to induce ray 1 defects or suppress unc-6 mutant ray 1 defects, depending on their dosage, suggesting the pvf-1 mutation causes UNC-40 overactivity that interferes with signalling but is partially sensitive to UNC-6. Additional data suggest PVF-1 functions through four VEGF receptor-related proteins and inhibits only CED-10 (a GTPase), but not MIG-2-dependent UNC-40 activity, even though UNC-40 functions through both GTPases to position ray 1. pvf-1 and receptor mutant ray 1 defects are rescued by transgenes expressing mouse VEGF164 and human VEGF receptors, respectively. These data report the first case of VEGF-induced inhibition of the netrin signalling and a molecular conservation of VEGF function from worms to humans.

Members of the platelet-derived growth factor/vascular endothelial growth factor (PDGF/VEGF) family have been implicated in a variety of functions in vertebrates, especially angiogenesis. Here we identify and characterize a PDGF/VEGF-like factor (named PVF-1) from the nematode C. elegans. We show that PVF-1 has biochemical properties similar to vertebrate PDGF/VEGF growth factors. More important, PVF-1 binds to the human receptors VEGFR-1 (Flt-1) and VEGFR-2 (KDR) and is able to induce angiogenesis in two model systems derived from vertebrates. Our results highlight the widespread evolutionary conservation of this important class of growth factors and raise the possibility that C. elegans can provide a simple experimental system in which to investigate how these factors function.—Tarsitano, M., De Falco, S., Colonna, V., McGhee, J. D., Persico, M. G. The C. elegans pvf-1 gene encodes a PDGF/VEGF-like factor able to bind mammalian VEGF receptors and to induce angiogenesis.

The EGF-CFC proteins have been recently recognized as a novel family of extracellular factors required during early vertebrate development. Cripto is the founder member of the EGF-CFC family initially related to the epidermal growth factor (EGF); its expression is increased in human colon, gastric, pancreatic and lung carcinoma and in different types of both mouse and human breast carcinomas. Genetic studies in the mouse have established an essential role of cripto in the formation and correct positioning of the anterior-posterior axis. Furthermore, the absence of cripto results in a defective precardiac mesoderm, unable to differentiate into functional cardiomyocytes. Although mouse and human Cripto have been shown to activate the ras/raf/MAP kinase pathway in mammary epithelial cell lines, genetic evidence in Zebrafish has been provided for a relationship between the EGF-CFC proteins and Nodal, a member of the transforming growth factor β family. Here we review the biological role of cripto in development and differentiation, pointing out recent findings on the biochemical interactions of Cripto, Nodal and Activin-like receptors.

Genetic isolates have been successfully used in the study of complex traits, mainly because due to their features, they allow a reduction in the complexity of the genetic models underlying the trait. The aim of the present study is to describe the population of Campora, a village in the South of Italy, highlighting its properties of a genetic isolate. Both historical evidence and multi-locus genetic data (genomic and mitochondrial DNA polymorphisms) have been taken into account in the analyses. The extension of linkage disequilibrium (LD) regions has been evaluated on autosomes and on a region of the X chromosome. We defined a study sample population on the basis of the genealogy and exogamy data. We found in this population a few different mitochondrial and Y chromosome haplotypes and we ascertained that, similarly to other isolated populations, in Campora LD extends over wider region compared to large and genetically heterogeneous populations. These findings indicate a conspicuous genetic homogeneity in the genome. Finally, we found evidence for a recent population bottleneck that we propose to interpret as a demographic crisis determined by the plague of the 17th century. Overall our findings demonstrate that Campora displays the genetic characteristics of a young isolate.

Essential hypertension (EH) affects a large proportion of the adult population in Western countries and is a major risk factor for cardiovascular diseases. EH is a multifactorial disease with a complex genetic component. To tackle the complexity of this genetic component, we have initiated a study of Campora, an isolated village in South Italy. A random sample of 389 adults was genotyped for a very dense microsatellite genome scan and phenotyped for EH. Of this sample, 173 affected individuals were all related through a 2,180-member pedigree and could be integrated within a linkage analysis. The complexity of the pedigree prevented its direct use for a non-parametric linkage (NPL) analysis. Therefore, the method proposed by Falchi et al. [2004, Am. J. Hum. Genet., 75, 1015-1031] was used for automatic pedigree-breaking. We identified a new locus for EH on chromosome 8q22-23 and detected linkage with two known loci for EH: 1q42-43 and 4p16. Simulations showed that the linkage with 8q22-23 is highly genome-wide significant, even when accounting for the breaking of the pedigree. An extension to qualitative traits of another pedigree-breaking approach [Pankratz et al., 2001, Genet. Epidemiol., 21 (Suppl. 1), S258-S263] also detected a significant linkage on 8q22-23 using a remarkably different set of sub-pedigrees and helped to refine the location of the linkage signal. This work both identifies a new locus strongly linked to hypertension and shows that the power of linkage analysis can be improved by the appropriate use of efficient pedigree-breaking strategies.

The discovery of a new gene with unknown functions and encoding a protein with novel characteristics left us with the puzzling question of a suitable name for it. The name cripto [1] was chosen to indicate that the main characteristic of this novel gene was the di¤culty in ¢nding its function. Nearly 10 years passed before we started to have a clue to some of the possible functions of cripto. This review focuses on the di¡erent studies that have ¢nally provided these clues.

Nodal proteins have crucial roles in mesendoderm formation and left–right patterning during vertebrate development. The molecular mechanisms of signal transduction by Nodal and related ligands, however, are not fully understood. In this paper, we present biochemical and functional evidence that the orphan type I serine/threonine kinase receptor ALK7 acts as a receptor for mouse Nodal and Xenopus Nodal-related 1 (Xnr1). Receptor reconstitution experiments indicate that ALK7 collaborates with ActRIIB to confer responsiveness to Xnr1 and Nodal. Both receptors can independently bind Xnr1. In addition, Cripto, an extracellular protein genetically implicated in Nodal signaling, can independently interact with both Xnr1 and ALK7, and its expression greatly enhances the ability of ALK7 and ActRIIB to respond to Nodal ligands. The Activin receptor ALK4 is also able to mediate Nodal signaling but only in the presence of Cripto, with which it can also interact directly. A constitutively activated form of ALK7 mimics the mesendoderm-inducing activity of Xnr1 in Xenopus embryos, whereas a dominant-negative ALK7 specifically blocks the activities of Nodal and Xnr1 but has little effect on other related ligands. In contrast, a dominant-negative ALK4 blocks all mesoderm-inducing ligands tested, including Nodal, Xnr1, Xnr2, Xnr4, and Activin. In agreement with a role in Nodal signaling, ALK7 mRNA is localized to the ectodermal and organizer regions of Xenopus gastrula embryos and is expressed during early stages of mouse embryonic development. Therefore, our results indicate that both ALK4 and ALK7 can mediate signal transduction by Nodal proteins, although ALK7 appears to be a receptor more specifically dedicated to Nodal signaling.

cripto is the founding member of the family of EGF-CFC genes, a class of extracellular factors essential for early vertebrate development. In this study we show that injection of Cripto recombinant protein in mid to late zebrafish Maternal-Zygotic one-eyed pinhead (MZoep) blastulae was able to fully rescue the mutant phenotype, thus providing the first direct evidence that Cripto activity can be added extracellularly to recover oep-encoded function in zebrafish early embryos. Moreover, 15 point mutations and two deletion mutants were generated to assess in vivo their functional relevance by comparing the ability of cripto wild-type and mutant RNAs to rescue the zebrafish MZoep mutant. From this study we concluded that the EGF-CFC domain is sufficient for Cripto biological activity and identified ten point mutations with a functional defective phenotype, two of which, located in the EGF-like domain, correspond to loss-of-function mutations. Finally, we have developed a three-dimensional structural model of Cripto protein and used it as a guide to predict amino acid residues potentially implicated in protein-protein interaction.

Vascular endothelial growth factor A (VEGF) is a potent mitogen for endothelial cells in vitro and promotes neo-angiogenesis in vivo. VEGF overexpression occurs in most human malignancies including thyroid carcinomas in which elevated VEGF expression is associated with a high tumorigenic potential. To investigate the role of VEGF in angiogenesis associated with development of thyroid carcinomas, we constitutively expressed VEGF121 into a poorly tumorigenic cell line (NPA) expressing minimal levels of endogenous VEGF. Here we report that VEGF overexpressing NPA cells showed the same growth potential as untransfected NPA in vitro but formed well-vascularized tumors when injected subcutaneously into nude mice with markedly reduced latency compared to parental cells. A complementary approach was to suppress VEGF expression in a highly tumorigenic anaplastic cell line (ARO) by the transfection of an antisense construct. Antisense-transfected ARO cells expressed reduced constitutive levels of VEGF, showed the same growth potential as untransfected ARO cells in vitro and formed small tumors characterized by minimal vascularization, extensive necrosis and longer latency compared to parental or vector-transfected ARO cells in vivo. Finally, we investigated the expression of both VEGF tyrosine kinase receptors (Flt-1 and Flk-1/KDR) in tumor specimens by RT - PCR. Expression of (Flt-1 and Flk-1/KDR) was low in tissue specimens derived from NPA tumors, but was found enhanced in NPA VEGF tumors; conversely, the expression of VEGF receptors was high in tissue specimens derived from ARO tumors but was decreased in tumors derived from VEGF depleted ARO cells. These results clearly demonstrate that VEGF indirectly promotes the growth of thyroid tumors by stimulating angiogenesis.

Placenta growth factor (PlGF) and vascular endothelial growth factor (VEGF) represent two closely related angiogenic growth factors active as homodimers or heterodimers. Since goiters of the thyroid gland are extremely hypervascular, we investigated the expression of PlGF, VEGF and their receptors, Flt-1 and Flk-1/KDR, in a small panel of human goiters from patients with Graves's disease, in an animal model of thyroid goitrogenesis and in in vitro cultured thyroid cells. Here we report that the mRNA expression of PlGF, VEGF and their receptors is markedly enhanced in biopsies of goiters resected from Graves's patients. in vivo studies demonstrated that in the thyroid gland of thiouracil-fed rats, increased mRNA and protein expression of PlGF, VEGF, Flt-1 and Flk-1/KDR occurred subsequent to the rise in the serum thyroid stimulating hormone (TSH) levels and in parallel with thyroid capillary proliferation. In vitro studies confirmed the existence of such TSH-dependent paracrine communication between thyroid epithelial cells and endothelium since the conditioned medium collected from TSH-stimulated thyrocytes acquired mitogenic activity for human umbilical vein endothelial (HUVE) cells, Altogether, these data suggest that PlGF and VEGF, released by thyrocytes in response to the chronic activation of the TSH receptor pathway, may act through a paracrine mechanism on thyroid endothelium.

Kallmann's syndrome (clinically characterized by hypogonadotropic hypogonadism and inability to smell) is caused by a defect in the migration of olfactory neurons, and neurons producing hypothalamic gonadotropin-releasing hormone. A gene has now been isolated from the critical region on Xp22.3 to which the syndrome locus has been assigned: this gene escapes X inactivation, has a homologue on the Y chromosome, and shows an unusual pattern of conservation across species. The predicted protein has significant similarities with proteins involved in neural cell adhesion and axonal pathfinding, as well as with protein kinases and phosphatases, which suggests that this gene could have a specific role in neuronal migration.

The complete coding sequence for human glucose-6-phosphate-dehydrogenase (G6PD) was inserted downstream from the tac promoter of a plasmid, pJF118EH, which also carries the lacI q repressor gene. When Escherichia coli strains (that are unable to grow on glucose due to the absence of functional zwf (G6PD − ) and pgi genes) were transformed with this plasmid (pAC1), they were able to grow on glucose as sole carbon source. The rate of growth on glucose was faster in the presence of the inducer of the tac promoter, isopropyl-β- d --thiogalactopyranoside (IPTG). Extracts of the transformed cells contained a G6PD activity that was not detectable in the parental strains and that was inducible by IPTG. The G6PD activities from normal E. coli and from pAC1-transformed cells comigrated with human G6PD when subjected to electrophoresis on agarose gels. However, when denatured, the G6PD produced by pAC1 was, like the human enzyme, distinguishable from the E. coli -encoded enzyme on the basis of its immunoreactivity with antibody specific for human G6PD. Therefore, human G6PD can be expressed in E. coli and can function to complement the bacterial enzyme deficiency.