Understanding the mechanisms directing progressive specification of heart cells from multipotent cardiovascular progenitors is essential for the development of regenerative therapies using induced pluripotent stem (iPS) cells. A simple chordate model system, the tunicate Ciona intestinalis, will be used to analyze the cellular and molecular mechanisms that determine muscle-type specification in the cardiogenic lineage. In Ciona embryos, the bilateral pairs of precardiac cells, called trunk ventral cells (TVCs), undergo stereotyped asymmetric cell divisions that distinguish the heart from the atrial siphon muscle (ASM) precursors. The latter then migrate toward the dorso-lateral atrial siphon placode. Following asymmetric divisions of the TVCs, the genes encoding the transcription factors COE and Islet are specifically up- regulated in the ASMs. In addition, COE is necessary and sufficient to block heart specification and promote the ASM fate, including expression of an ASM-specific Islet enhancer and cell migration toward the dorsal side of the larva. Finally, targeted expression of the constitutively active Notch intracellular domain using a TVC-specific enhancer is sufficient to inhibit ASM- specific expression of COE, Islet and cell migration. These observations led to the hypothesis that the initial asymmetric divisions result in heart-specific Notch signaling, which blocks ASM fate specification, possibly by inhibiting the expression of COE. In order to test this hypothesis, the cis-regulatory sequences that control ASM-specific expression of COE will be isolated and characterized, and the function of Notch signaling upstream of COE will be determined. The expression and localization patterns of endogenous regulators and effectors of Notch signaling will be documented in order to gain insight into the mechanisms that polarize the Notch signal during asymmetric TVC divisions. The effects of Notch signaling, COE and Islet on heart vs. ASM fate specification and cell migration will be analyzed using previously established assays in order to begin to characterize the epistatic relationships between these regulators. Finally, whole genome gene expression changes underlying heart vs. ASM fate specification will be documented by obtaining heart and ASM-specific transcription profiles using fluorescence activated cell sorting and microarrays. The results obtained upon completion of this project will characterize the regulation and function of COE, a novel negative regulator of heart fate specification, and illuminate the cellular and molecular mechanisms controlling muscle fate specification and cell migration in the cardiogenic lineage.
Personalized medicine utilizing induced pluripotent stem cells based regenerative medicine represents a novel promising avenue for the treatment of cardiovascular diseases and will require a thorough understanding of the cellular and molecular mechanisms whereby naive cells are determined to form functional heart tissue. While studying these basic mechanisms using a simple invertebrate model, the tunicate Ciona intestinalis, we recently identified the transcription factor COE as a novel negative regulator of heart-fate specification. Here, we propose to conduct an in depth analysis of the molecular and cellular mechanisms that control COE regulation and function and heart-fate specification with potential novel implications for cardiovascular development and medicine.
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