Comprehensive gene networks in Ciona intestinalis embryos provide a foundation for characterizing complex developmental processes, such as the initial phases of chordate heart development. The basic helix-loop-helix regulatory gene Ci-Mesp is required for activation of cardiac transcription factors. Evidence is presented that Ci-Ets1/2, a transcriptional effector of receptor tyrosine kinase (RTK) signaling, acts downstream from Mesp to establish the heart field. Asymmetric activation of Ets1/2, possibly through localized expression of FGF9, drives heart specification within this field. During gastrulation, Ets1/2 is expressed in a group of four cells descended from two Mesp-expressing founder cells (the B7.5 cells). After gastrulation, these cells divide asymmetrically; the smaller rostral daughters exhibit RTK activation (phosphorylation of ERK) and form the heart lineage while the larger caudal daughters form the anterior tail muscle lineage. Inhibition of RTK signaling prevents heart specification. Targeted inhibition of Ets1/2 activity or FGF receptor function also blocks heart specification. Conversely, application of FGF or targeted expression of constitutively active Ets1/2 (EtsVp16) cause both rostral and caudal B7.5 lineages to form heart cells. This expansion produces an unexpected phenotype: transformation of a single-compartment heart into a functional multicompartment organ. We discuss these results with regard to the development and evolution of the multichambered vertebrate heart.[Keywords: Ets; heart; Mesp; development; evolution] Supplemental material is available at http://www.genesdev.org.

Heart development requires precise coordination of morphogenetic movements with progressive cell fate specification and differentiation. In ascidian embryos, FGF/MAPK-mediated activation of the transcription factor Ets1/2 is required for heart tissue specification and cell migration. We found that FoxF is one of the first genes to be activated in heart precursors in response to FGF signaling. We identified the FoxF minimal heart enhancer and used a cis-trans complementation test to show that Ets1/2 can interact with the FoxF enhancer in vivo. Next, we found that FoxF function is required downstream and in parallel to the FGF/MAPK/Ets cascade for cell migration. In addition, we demonstrated that targeted expression of a dominant-negative form of FoxF inhibits cell migration but not heart differentiation, resulting in a striking phenotype: a beating heart at an ectopic location within the body cavity of juveniles. Taken together, our results indicate that FoxF is a direct target of FGF signaling and is predominantly involved in the regulation of heart cell migration.

MicroRNAs (miRNAs) have been implicated in various cellular processes. They are thought to function primarily as inhibitors of gene activity by attenuating translation or promoting mRNA degradation. A typical miRNA gene produces a predominant ∼21-nucleotide (nt) RNA (the miRNA) along with a less abundant miRNA* product. We sought to identify miRNAs from the simple chordate Ciona intestinalis through comprehensive sequencing of small RNA libraries created from different developmental stages. Unexpectedly, half of the identified miRNA loci encode up to four distinct, stable small RNAs. The additional RNAs, miRNA-offset RNAs (moRs), are generated from sequences immediately adjacent to the predicted ∼60-nt pre-miRNA. moRs seem to be produced by RNAse III-like processing, are ∼20 nt long and, like miRNAs, are observed at specific developmental stages. We present evidence suggesting that the biogenesis of moRs results from an intrinsic property of the miRNA processing machinery in C. intestinalis.

The bHLH transcription factor Mesp has an essential but ambiguous role in early chordate heart development. Here, we employ the genetic and morphological simplicity of the basal chordate Ciona intestinalis to elucidate Mesp regulation and function. Characterization of a minimal cardiac enhancer for the Ciona Mesp gene demonstrated direct activation by the T-box transcription factor Tbx6c. The Mespenhancer was fused to GFP, permitting high-resolution visualization of heart cells as they migrate and divide. The enhancer was also used to drive targeted expression of an activator form of Mesp, which induces heart formation without migration. We discuss the implications of Tbx6-Mespinteractions for the evolution of cardiac mesoderm in invertebrates and vertebrates.