In the ascidian embryo, a fibroblast growth factor (FGF)-like signal from presumptive endoderm blastomeres between the 32-cell and early 64-cell stages induces the formation of notochord and mesenchyme cells. However, it has not been known whether endogenous FGF signaling is involved in the process. Here it is shown that 64-cell embryos exhibit a marked increase in endogenous extracellular signal-regulated kinase (ERK/MAPK) activity. The increase in ERK activity was reduced by treatment with an FGF receptor 1 inhibitor, SU5402, and a MEK (ERK kinase/MAPKK) inhibitor, U0126. Both drugs blocked the formation of notochord and mesenchyme when embryos were treated at the 32-cell stage, but not at the 2-or 110-cell stages. The dominant-negative form of Ras also suppressed notochord and mesenchyme formation. Both inhibitors suppressed induction by exogenous basic FGF. These results suggest that the FGF signaling cascade is indeed necessary for the formation of notochord and mesenchyme cells during ascidian embryogenesis. It is also shown that FGF signaling is required for formation of the secondary notochord, secondary muscle and neural tissues, and at least ERK activity is necessary for the formation of trunk lateral cells and posterior endoderm. Therefore, FGF and MEK signaling are required for the formation of various tissues in the ascidian embryo.

The tadpole larva of the ascidian Halocynthia roretzi has several hundred mesenchyme cells in its trunk. Mesenchyme cells are exclusively derived from the B8.5 and B7.7 blastomere pairs of the 110-cell embryo. It has been believed that specification of mesenchyme cells is an autonomous process. In the present study, we have demonstrated that presumptive-mesenchyme blastomeres isolated from early 32-cell embryos did not express mesenchyme-specific features, whereas those isolated after the late 64-cell stage developed mesenchyme markers autonomously. Results of experiments involving coisolation and recombination of blastomeres showed that cellular interaction with adjacent presumptive-endoderm blastomeres during the late 32- and early 64-cell stages is required for mesenchyme formation. When such interaction was absent, the presumptive-mesenchyme blastomeres developed into muscle cells. Therefore, a signal from endoderm precursor blastomeres promotes mesenchyme fate, suppressing the muscle fate that is specified by ooplasmic muscle determinants. In Halocynthia, the muscle actin gene was precociously activated in mesenchyme-muscle precursor blastomeres at the 32-cell stage, and the mesenchyme and muscle fates were separated into two daughter blastomeres at the next cleavage. In presumptive-mesenchyme blastomeres at the 64-cell stage, expression of the muscle actin gene was immediately down-regulated by the signal from the neighboring endoderm precursor blastomeres. Thus, mesenchyme formation involves a novel mechanism of fate specification in ascidians, where formation of mesenchyme cells requires cellular interaction that suppresses muscle fate in the mesenchyme precursor blastomeres.

SUMMARYA forward genetic screen in the ascidian Ciona intestinalis identified a mutant line (frimousse) with a profound disruption in neural plate development. In embryos with the frimousse mutation, the anteriormost neural plate cells, which are products of an FGF induction at the blastula and gastrula stages, initially express neural plate-specific genes but fail to maintain the induced state and ultimately default to epidermis. The genetic lesion in the frimousse mutant lies within a connexin gene (cx-11) that is transiently expressed in the developing neural plate in a temporal window corresponding to the period of a-lineage neural induction. Using a genetically encoded calcium indicator we observed multiple calcium transients throughout the developing neural plate in wild-type embryos, but not in mutant embryos. A series of treatments at the gastrula and neurula stages that block the calcium transients, including gap junction inhibition and calcium depletion, were also found to disrupt the development of the anterior neural plate in a similar way to the frimousse mutation. The requirement for cx-11 for anterior neural fate points to a crucial role for intercellular communication via gap junctions, probably through mediation of Ca 2+ transients, in Ciona intestinalis neural induction.

Asymmetric cell division plays a fundamental role in generating various types of embryonic cell. In ascidian embryos, asymmetric cell divisions occur in the vegetal hemisphere in a manner similar to those found in Caenorhabditis elegans. Early divisions in embryos of both species involve inductive events on a single mother cell that result in production of daughters with different cell fates. Here we show in the ascidian Halocynthia roretzi that polarity of muscle/mesenchyme mother precursors is determined solely by the direction from which the FGF9/16/20 signal is presented, a role similar to that of Wnt signaling in the EMS and T cell divisions in C. elegans. However, polarity of nerve cord/notochord mother precursors is determined by possible antagonistic action between the FGF signal and a signal from anterior ectoderm, providing a new mechanism underlying asymmetric cell division. The ectoderm signal suppresses MAPK activation and expression of Hr-FoxA, which encodes an intrinsic competence factor for notochord induction, in the nerve cord lineage.