Functional studies of the Ciona intestinalis myogenic regulatory factor reveal conserved features of chordate myogenesis

Abstract

Ci-MRF is the sole myogenic regulatory factor (MRF) of the ascidian Ciona intestinalis, an invertebrate chordate. In order to investigate its properties we developed a simple in vivo assay based on misexpressing Ci-MRF in the notochord of Ciona embryos. We used this assay to examine the roles of three structural motifs that are conserved among MRFs: an alanine-threonine (Ala-Thr) dipeptide of the basic domain that is known in vertebrates as the myogenic code, a cysteine/histidine-rich (C/H) domain found just N-terminal to the basic domain, and a carboxy-terminal amphipathic α-helix referred to as Helix III. We show that the Ala-Thr dipeptide is necessary for normal Ci-MRF function, and that while eliminating the C/H domain or Helix III individually has no demonstrable effect on Ci-MRF, simultaneous loss of both motifs significantly reduces its activity. Our studies also indicate that direct interaction between CiMRF and an essential E-box of Ciona Troponin I is required for the expression of this muscle-specific gene and that multiple classes of MRF-regulated genes exist in Ciona. These findings are consistent with substantial conservation of MRF-directed myogenesis in chordates and demonstrate for the first time that the Ala/Thr dipeptide of the basic domain of an invertebrate MRF behaves as a myogenic code.

Rewiring of an ancestral Tbx1/10-Ebf-Mrf network for pharyngeal muscle specification in distinct embryonic lineages

“…Mrf is necessary for differentiation of Ciona tail muscles, and is sufficient to activate expression of muscle-specific genes in the entire vegetal half of the embryo, where maternal Zic-r1.a activity determines the competence for muscle differentiation (Yagi et al, 2004;Meedel et al, 2007;Izzi et al, 2013). However, the function of Mrf in development of the ASMs and OSMs has not been characterized.…”

“…In the two secondary muscle lineages, differentiation begins later in development and depends on Nodal signals from neighboring cells (Hudson and Yasuo, 2005). Despite different regulatory inputs, Mrf regulates gene expression during differentiation of tail muscles (Kusakabe et al, 2004;Brown et al, 2007;Meedel et al, 2007;Izzi et al, 2013). The mechanisms of differentiation in the OSM and ASM, as well as the role of Mrf in myogenesis in those contexts, have not been explored.…”

More Than One-to-Four via 2R: Evidence of an Independent Amphioxus Expansion and Two-Gene Ancestral Vertebrate State for MyoD-Related Myogenic Regulatory Factors (MRFs)

“…The invertebrate chordate tunicates Ciona spp. have one MRF which activates expression of muscle genes in the tailbud ( Izzi et al. 2013 ), whereas the earliest-branching chordate lineage, the cephalochordates (amphioxus or lancelets), have been reported to have varying numbers of MRFs, from one to three and with confusing nomenclature reflecting ambiguous orthology relationships among these amphioxus MRFs ( supplementary information 1, Supplementary Material online; Schubert et al.…”

Tunicata

“…Macho1 protein in turn activates its targets, among them the Tbx6related family of genes ). (Johnson et al 2004(Johnson et al , 2005Izzi et al 2013 ). Mrf, Tbx6, and other transcription factors then cooperate to activate the expression of a battery of "differentiation" genes such as myosins, muscle actins, troponins, tropomyosins, etc.…”