The heart is a specialized organ, responsible for distributing oxygenated blood through long distances of systemic vasculature. When fully formed it is composed of diverse cells types from several embryonic origins that must coordinate in order to function properly. During mammalian development the heart is the first organ to form and must become fully functional prior to the completion of organogenesis. The migratory cardiac progenitor population known as the second heart field is responsible for contributing not only additional cardiomyocytes to the nascent heart, but also endothelial and smooth muscle cells that will develop into specialized structures. Although the fates of the SHF have been well described, many potential mechanisms regulating the development of tissue derived from these progenitors remain to be examined.

This thesis is composed of five chapters including three original research articles (one of which has been published) and one that describes a recently established project. Chapter One will serve as an introduction and overview of heart development and cardiac-stem cell biology, including the cell fates of FHF and SHF progenitors. The contribution SHF-progenitors to the ascending aorta and the anatomical boundaries of the tissue derived from this source will be discussed in Chapter Two: "Nkx2-5 lineage tracing visualizes the distribution of second heart field-derived aortic smooth muscle". The role of the Serum Response Factor (SRF) co-factor Myocardin (Myocd) within the embryonic heart and SHF-derived smooth muscle will be described in Chapter Three: "The Role of Myocardin in Cardiogenesis and the Development SHF-derived Smooth Muscle". Finally, the role of inhibitory Smad (iSmad) proteins in the formation of SHF-derived cardiac structures will be discussed in Chapter Four: "The Role of Inhibitory Smads in the Embryonic Heart". The last chapter will discuss conclusions, implications of this work and future directions.

Muscle contractions, among other cellular functions, are driven by highly ordered structures composed of actin and myosin. For cells to properly execute these functions, actin filaments must be specifically organized, assembled, and maintained throughout the cell. The precise timing of actin assembly required for these functions is helped by actin nucleators, such as formins. Formins assemble many actin-based structures through their formin homology (FH) domains, FH1 and FH2. The FH2 domain helps form new filaments in a process called nucleation, while the FH1 domain helps to elongate actin filaments in a processive manner while the FH2 domain “walks” along the growing barbed ends of filaments. The formin homology domain-containing protein (Fhod) family of formins are important for building several contractile actin structures, including sarcomeres in muscle cells and stress fibers in various non-muscle cells. Despite Fhod family formins being required for structures in vivo, mammalian Fhods were initially reported to instead inhibit actin assembly in vitro. Here, we establish that mammalian formin FHOD3 (both isoforms FHOD3S and FHOD3L) nucleate and elongate actin filaments in vitro. Human FHOD3S/L elongate actin filaments quite differently than other formins, where we observe brief, rapid moments of elongation after elongation is paused. We performed rescue experiments for FHOD3L in neonatal rat ventricular myocytes (NRVMs) with mutants that separated its actin assembly activities to better understand whether nucleation or elongation is more important for sarcomere formation. We found that elongation activity by FHOD3L is necessary and sufficient for proper sarcomere formation and maintenance, whereas reducing its nucleation or bundling activity is tolerated in NRVMs. Further, mutations in FHOD3 have been implicated in 1-2% of cases of hypertrophic cardiomyopathy (HCM), a heart disease which results in the thickening of the septal muscle, eventually leading to arrhythmias and heart failure. Interestingly, the likely pathogenic R1386Q mutation for HCM results in a 37% increase in nucleation ability in vitro, leading to thinner sarcomeres in NRVM rescue experiments. We establish NRVMs as a model system to observe sarcomere structure and function and to understand which actin assembly activities are most crucial to the formation and maintenance of the sarcomere.

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