Cross inhibition between NK4 and TBX1 transcription factors specifies heart versus pharyngeal muscle fates by promoting the activation of tissue-specific regulators in distinct precursors within the cardiopharyngeal lineage of the ascidian, Ciona intestinalis.
The heart and head muscles share common developmental origins and genetic underpinnings in vertebrates, including humans. Parts of the heart and cranio-facial musculature derive from common mesodermal progenitors that express NKX2-5, ISL1, and TBX1. This ontogenetic kinship is dramatically reflected in the DiGeorge/Cardio-Velo-Facial syndrome (DGS/CVFS), where mutations of TBX1 cause malformations in the pharyngeal apparatus and cardiac outflow tract. Cardiac progenitors of the first heart field (FHF) do not require TBX1 and segregate precociously from common progenitors of the second heart field (SHF) and pharyngeal muscles. However, the cellular and molecular mechanisms that govern heart versus pharyngeal muscle specification within this lineage remain elusive. Here, we harness the simplicity of the ascidian larva to show that, following asymmetric cell division of common progenitors, NK4/NKX2-5 promotes GATAa/GATA4/5/6 expression and cardiac specification in the second heart precursors by antagonizing Tbx1/10-mediated inhibition of GATAa and activation of Collier/Olf/EBF (COE), the determinant of atrial siphon muscle (ASM) specification. Our results uncover essential regulatory connections between the conserved cardio-pharyngeal factor Tbx1/10 and muscle determinant COE, as well as a mutual antagonism between NK4 and Tbx1/10 activities upstream of GATAa and COE. The latter cross-antagonism underlies a fundamental heart versus pharyngeal muscle fate choice that occurs in a conserved lineage of cardio-pharyngeal progenitors. We propose that this basic ontogenetic motif underlies cardiac and pharyngeal muscle development and evolution in chordates.
Mutations in the regulatory genes encoding the transcription factors NKX2-5 and TBX1, which govern heart and head muscle development, cause prevalent congenital defects. Recent studies using vertebrate models have shown that the heart and pharyngeal head muscle cells derive from common progenitors in the early embryo. To better understand the genetic mechanisms by which these progenitors select one of the two developmental trajectories, we studied the activity of these transcription factors in a simple invertebrate chordate model, the sea squirt Ciona intestinalis. We show that the sea squirt homolog of NKX2-5 promotes early heart specification by inhibiting the formation of pharyngeal muscles. Conversely, the TBX1 homolog determines pharyngeal muscle fate by inhibiting GATAa and thereby the heart program it instructs, as well as promoting the pharyngeal muscle program through activation of COE (Collier/Olf-1/EBF), a recently identified regulator of skeletal muscle differentiation. Finally, we show that the NKX2-5 homolog protein directly binds to the COE gene to repress its activity. Notably, these antagonistic interactions occur in heart and pharyngeal precursors immediately following the division of their pluripotent mother cells, thus contributing to their respective fate choice. These mechanistic insights into the process of early heart versus head muscle specification in this simple chordate provide the grounds for establishing the etiology of human congenital cardio-craniofacial defects.