INDENTIFICATION OF SIGNALING MOLECULES THAT INDUCE HEART FORMATION

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(Adapted from the Applicant's Abstract) The broad aim of this project is to understand how the cardiac myocyte differentiation program is activated during vertebrate development. In previous work the investigators have identified a cardiac-inducing activity in the anterior lateral plate endoderm of the gastrula stage avian embryos, which can induce cells from the posterior primitive streak (which normally gives rise to extra- embryonic tissues and to blood) to give rise to cardiac myocytes. Recently, the investigators have extended these studies to demonstrate that in addition to a cardiac inducing signal from the anterior endoderm, Bone Morphogenetic Proteins (BMPs) expressed in both endoderm and ectoderm also play an important role in heart induction. The result suggest that there are at least two stages in vertebrate cardiac myocyte determination Cells emerging from the primitive streak are still plastic with respect to their ability to form heart. Those mesodermal cells which contact anterior endoderm seem to lie in a developmental field in which cells display a potential to become heart. BMP-2 developmental field in which cells display a potential to become heart. BMP-2 and/or BMP-4 then appear to be capable of inducing cells within this field to initiated the cardiac differentiation program. Thus, a combination of two distinct signals is necessary for the induction of cardiac myocytes: a BMP signal present in the lateral regions of the embryo collaborates with an as yet to be identified signal secreted by the anterior endoderm to induce heart formation. The goal of this proposal will be to identify the signal secreted by cells in the anterior endoderm that induces the formation of cardiac myocytes in collaboration with a BMP signal. By employing subtractive hybridization technology the investigations have recently identified a signaling molecule, termed "crescent", which is a putative Wnt inhibitor, and is specifically expressed in the anterior endoderm at the time when cardiac myocytes are being induced in the chick embryos. During early chick development, crescent is expressed in a domain that precisely overlaps the location where the endodermal heart inducing activity lies. In this proposal the investigators intend to firstly investigate where crescent is the heart inducing activity in the anterior endoderm. If such is the case, the investigators will focus on understanding how this such is the case, the investigators will focus on understanding on understanding how this signaling molecule collaborates with BMP signals to induce heart formation in the various assay systems that the investigators have developed, they will employ an alternative approach to isolate the heart inducing activity in the anterior endoderm. Specific Aim 1 will investigate whether crescent, a putative anti-Wnt molecule secreted by the anterior endoderm collaborates with BMP signals to induce the formation of cardiac myocytes. Specific Aim 2 if crescent can induce the formation of cardiac myocytes in collaboration with a BMP signal, the investigators will determine whether crescent displays an anti-Wnt activity in this induction. Specific Aim 3 will determine whether mice genetically engineered to lack crescent display a defect in heart formation. Specific Aim 4 if the investigators find that crescent fails to display any heart inducing activity either in vitro or in vivo, they will develop develop a functional screen to identify the cardiac inducing molecule secreted by the anterior endoderm.