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.

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During early mouse development, the TGFbeta-related protein Nodal specifies the organizing centers that control the formation of the anterior-posterior (A-P) axis. EGF-CFC proteins are important components of the Nodal signaling pathway, most likely by acting as Nodal coreceptors. However, the extent to which Nodal activity depends on EGF-CFC proteins is still debated. Cripto is the earliest EGF-CFC gene expressed during mouse embryogenesis and is involved in both A-P axis orientation and mesoderm formation. To investigate the relation between Cripto and Nodal in the early mouse embryo, we removed the Nodal antagonist Cerberus 1 (Cer1) and simultaneously Cripto, by generating Cer1;Cripto double mouse mutants. We observed that two thirds of the Cer1;Cripto double mutants are rescued in processes that are severely compromised in Cripto(-/-) embryos, namely A-P axis orientation, anterior mesendoderm and posterior neuroectoderm formation. The observed rescue is strongly reduced in Cer1;Cripto;Nodal triple mutants, suggesting that Nodal can signal extensively in the absence of Cripto, if Cer1 is also inhibited. This signaling activity drives A-P axis positioning. Our results provide evidence for the existence of Cripto-independent signaling mechanisms, by which Nodal controls axis specification in the early mouse embryo.

The EGF-CFC gene cripto governs anterior–posterior (A–P) axis specification in the vertebrate embryo. Existing models suggest that Cripto facilitates binding of Nodal to an ActRII–activin-like kinase (ALK) 4 receptor complex. Cripto also has a crucial function in cellular transformation that is independent of Nodal and ALK4. However, how ALK4-independent Cripto pathways function in vivo has remained unclear. We have generated cripto mutants carrying the amino acid substitution F78A, which blocks the Nodal–ALK4–Smad2 signaling both in embryonic stem cells and cell-based assays. In criptoF78A/F78A mouse embryos, Nodal fails to expand its own expression domain and that of cripto, indicating that F78 is essential in vivo to stimulate Smad-dependent Nodal autoinduction. In sharp contrast to cripto-null mutants, criptoF78A/F78A embryos establish an A–P axis and initiate gastrulation movements. Our findings provide in vivo evidence that Cripto is required in the Nodal–Smad2 pathway to activate an autoinductive feedback loop, whereas it can promote A–P axis formation and initiate gastrulation movements independently of its stimulatory effect on the canonical Nodal–ALK4–Smad2 signaling pathway.

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Oxidative stress and mitochondrial dysfunction play an important role in the onset of neurodegenerative diseases; thus, the antioxidant use could be a promising preventive or therapeutic intervention. The food and agricultural industries generate huge quantities of by-products rich in biophenolic compounds. If, on the one hand, they represent a serious environmental problem, on the other hand, are valuable natural sources of antioxidants. In the present work, the total phenolic content and the antioxidant power of the wastewater derived from the production of four Sicilian extra virgin olive oils are shown. Four biophenols, hydroxytyrosol and tyrosol alcohols and caffeic acid (CA) and p-coumaric acids, were identified and quantified and their antioxidant ability determined. Their effects in counteracting Abeta 1-42 oligomer oxidative damage in LAN5 cell culture were investigated. Results indicated that CA was the most effective compound in counteracting the cell damages induced by Abeta 1-42 oligomers. El contenido valioso de aguas residuales producidas por almazaras: el efecto protector de la fracción antioxidante en cultivos celulares RESUMEN El estrés oxidativo y la disfunción mitocondrial juegan un rol importante en el comienzo de ciertas enfermedades neurodegenerativas; en estos casos, el uso de antioxidantes puede ser una intervención preventiva o terapéutica prometedora. Las industrias alimentarias y agrícolas producen enormes cantidades de subproductos ricos en compuestos biofenólicos. Si bien por un lado representan un grave problema ambiental, por el otro constituyen valiosas fuentes naturales de antioxidantes. En el presente estudio se analizan el contenido fenólico total y el poder antioxidante de las aguas residuales (WW) derivadas de la producción de cuatro aceites de oliva virgen extra (AOVE) de Sicilia. Para el efecto, Se identificaron y cuantificaron cuatro biofenoles, alcoholes hidroxitirosol (HT) y tirosol (TY), además de ácidos cafeico (CA) y p-cumárico (CU), y se determinó su capacidad antioxidante. Asimismo, se investigaron sus efectos para contrarrestar el daño oxidativo provocado por el oligómero Abeta 1-42 en cultivos celulares LAN5, constatándose que el CA fue el más efectivo para contrarrestar el daño celular inducido por oligómeros Abeta 1-42 .

During development of the mammalian embryo, there is a complex relation between formation of the mesoderm and the neuroectoderm. In mouse, for example, the role of the node and its mesendoderm derivatives in anterior neural specification is still debated. Mouse Cripto -/-embryos could potentially help settle this debate because they lack almost all embryonic endoderm and mesoderm, including the node and its derivatives. In the present paper, we show that Cripto -/-embryos can still form functional neural stem cells that are able to differentiate and maintain a neural phenotype both in vivo and in vitro. These data suggest that signals emanating from the mesoderm and endoderm might not be essential for the formation and differentiation of neural stem cells. However, we use grafting experiments to show that the Cripto -/-isthmus (the secondary organizer located at the midbrain-hindbrain boundary) loses its inductive ability. We further show that the Cripto -/-isthmus expresses lower amounts of the isthmic signalling molecule, Fgf8. Since nearby tissues remain competent to respond to exogenously added Fgf8, this reduction in Fgf8 levels in the Cripto -/-isthmus is the potential cause of the loss of patterning ability in graft experiments. Overall, we interpret our data to suggest that the mammalian node and primitive streak are essential for the development of the regional identities that control the specification and formation of the secondary organizers within the developing brain.