Formation of the outflow tract (OFT) is an essential aspect of cardiogenesis: the dimensions, orientation, and subdivision of the OFT are crucial for effective transport of blood from the heart to the periphery. OFT development initiates with the assembly of a small myocardial tube, which subsequently provides a vital foundation for OFT remodeling. Given the importance of establishing the OFT myocardium, the embryonic origins of OFT cardiomyocytes (CMs) have been of great interest. A series of studies in mouse and chick embryos have illuminated two major sources of cardiac progenitor cells, termed the first heart field (FHF) and the second heart field (SHF). Notably, the initial foundation of the OFT is built by SHF-derived CMs that are appended to the arterial pole of the heart. Although several signaling pathways have been implicated in regulating SHF differentiation, little is known about which genes function downstream of these key signals to execute OFT assembly or how the multiple relevant pathways interact to set the dimensions of the OFT. Here, we exploit the utility of the zebrafish as a model organism in order to identify novel regulators of OFT formation. Preliminary studies suggest that the zebrafish OFT, like the amniote OFT, is constructed from a population of SHF-derived CMs. Furthermore, in zebrafish, as in amniotes, Fgf signaling is required to promote the production of OFT CMs. However, it is unclear which genes act downstream of Fgf signaling to recruit the appropriate number of CMs into the OFT. Our preliminary data reveal an interesting set of genes - cell adhesion molecule 4 (cadm4), cadm3, and cadm2a - that are repressed by Fgf signaling and play essential roles in restricting the formation of OFT myocardium. These data suggest an intriguing model in which Fgf signaling drives the recruitment of OFT CMs by limiting the expression of cadm genes and thereby altering critical extracellular interactions of SHF-derived progenitor cells. In this proposal, we will test this model in detail by establishing the origins of the zebrafish OFT, deciphering the mechanisms of Cadm function, and integrating the Fgf-Cadm pathway into the context of the multiple influences that converge to define the size of the OFT.
In Aim 1, we will employ fate mapping, time-lapse tracking, and assays for the timing of myocardial differentiation to determine whether the zebrafish OFT myocardium is derived from a SHF equivalent.
In Aim 2, we will use loss-of-function, gain-of-function, structure-function, and biochemical analyses to test if Cadms mediate extracellular interactions that inhibit recruitment of OFT CMs.
In Aim 3, we will identify signals that counterbalance the impact of the Fgf-Cadm pathway on OFT size, focusing on the roles played by Notch, Bmp, and retinoic acid signaling in limiting the dimensions of the zebrafish OFT. Together, these experiments are likely to reveal new mediators of OFT CM recruitment, to uncover a novel mechanism for regulating OFT size through modulation of extracellular interactions, and to shed light on the network of pathways that collaborate to insure an appropriate myocardial foundation for the embryonic OFT.
Cardiac defects are found in as many as 1 in 100 live births and 1 in 10 still births and frequently include problems with the formation of the cardiac outflow tract. Outflow tract development initiates with the assembly of a small tube of muscle, the precise dimensions of which are essential for its subsequent remodeling into a mature structure. Therefore, a better comprehension of the mechanisms controlling the initial investment of muscle into the outflow tract is likely to illuminate the causes of cardiac birth defects and may also suggest strategies for directing multipotent cells to become cardiac muscle.
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