The molecular mechanisms controlling inductive events leading to the specification and terminal differentiation of cardiomyocytes are still largely unknown. We have investigated the role of Cripto, an EGF-CFC factor, in the earliest stages of cardiomyogenesis. We find that both the timing of initiation and the duration of Cripto signaling are crucial for priming differentiation of embryonic stem (ES) cells into cardiomyocytes, indicating that Cripto acts early to determine the cardiac fate. Furthermore, we show that failure to activate Cripto signaling in this early window of time results in a direct conversion of ES cells into a neural fate. Moreover, the induction of Cripto activates the Smad2 pathway, and overexpression of activated forms of type I receptor ActRIB compensates for the lack of Cripto signaling in promoting cardiomyogenesis. Finally, we show that Nodal antagonists inhibit Cripto-regulated cardiomyocyte induction and differentiation in ES cells. All together our findings provide evidence for a novel role of the Nodal/Cripto/Alk4 pathway in this process.

cripto is the original member of the family of EGF-CFC genes, recently recognized as novel extracellular factors essential for vertebrate development. During the early stages of mouse gastrulation, cripto mRNA is detected in mesodermal cells; later, cripto mRNA is detected only in the truncus arteriosus of the developing heart. Here we describe the in vivo distribution of Cripto protein throughout mouse embryo development and show that cripto mRNA and protein colocalize. By means of immunofluorescence analysis and biochemical characterization, we show that Cripto is a membrane-bound protein anchored to the lipid bilayer by a glycosylphosphatidylinositol (GPI) moiety. We suggest that presentation of Cripto on the cell surface via a GPI-linkage is important in determining the spatial specificity of cell-cell interactions that play a critical role in the early patterning of the embryo.

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

Placenta growth factor (PlGF) and vascular endothelial growth factor (VEGF) are angiogenic factors containing the 8-cysteine motif of platelet-derived growth factor (PDGF). Both PlGF and VEGF are mitogens for endothelial cells in vitro and promote neoangiogenesis in vivo. In addition, PlGF strongly potentiates the proliferative and the permeabilization effects exerted by VEGF on the vascular endothelium. We have now isolated the cDNA coding for mouse Plgf by screening a mouse heart cDNA library with the human PlGF sequence as probe. The human PlGF protein has two forms, PlGF-1 and PlGF-2, that arise from alternative splicing of a single gene mapping on Chromosome (Chr) 14; the isolated mouse Plgf cDNA encodes the longer of these two forms (PlGF-2). We show that the mouse Plgf-2 mRNA is the only transcript present in the normal tissues analyzed. Mouse Plgf-2 is a 158-amino-acid-long protein that shows 78% similarity (65% identity) to the human PlGF-2. Computer analysis reveals a putative signal peptide and three probable N-glycosylation sites, two of which are also conserved in human PlGF. The mouse Plgf gene was isolated and characterized; the gene is encoded by 7 exons spanning a 13-kb DNA interval. Finally, we have mapped the mouse Plgf gene to Chr 12, one cM from D12Mit5, and the human PlGF gene to 14q24, using both FISH and genetic crosses.

Lrp, a major regulatory protein in Escherichia coli, controls the expression of numerous operons, including ilvIH. Lrp binds to six sites upstream of ilvIH, and Lrp binding is required for ilvIH expression. We show here that an Lrp-like protein is also present in Salmonella typhimurium. This protein can bind both E. coli and S. typhimurium ilvIH DNA, as can E. coli Lrp. Methidiumpropyl-EDTA footprinting studies were performed with purified E. coli Lrp and S. typhimurium ilvIH DNA. Six binding sites were defined, three of them being similar to corresponding sites in E. coli, and three being organized differently. A consensus derived from six S. typhimurium sites is compatible with that derived from a similar analysis of E. coli sequences.