Identification of downstream genes of the ascidian muscle determinant gene Ci-macho1

AbstractAutonomous differentiation of primary muscle cells in ascidian embryos is triggered by a maternal determinant recently identified as the macho-1 gene. macho-1 encodes a transcription factor of the Zic family with five C2H2 zinc-finger motifs. In the present study, we firstly performed a screen, using a quantitative PCR method, of genes encoding transcription factors and components in major signaling pathways to identify those regulated downstream of Ci-macho1 in early embryos of Ciona intestinalis. The amount of transcripts for a total of 64 genes was altered at the 32-cell stage depending on the Ci-macho1 activity level. Whole-mount in situ hybridization assays revealed that the alteration of expression for at least 13 of them was adequately visualized to confirm the results of quantitative PCR. Second, we determined a possible binding sequence of Ciona macho1. macho1 recombinant proteins of both C. intestinalis and Ciona savignyi recognized a sequence, 5′-GCCCCCCGCTG-3′, that resembles the mammalian Zic binding site. In addition, most of the genes identified as potential Ci-macho1 downstream genes, in particular Ci-Tbx6b and Ci-snail, possessed plausible Ci-macho1-binding sequences in their 5′ upstream region, suggesting their direct activation by Ci-macho1. Furthermore, some of the genes including three Wnt genes noted in the quantitative analyses implied that Ci-macho1 is involved in the differentiation of endoderm and mesenchyme via intracellular communications