Identification of downstream genes of the ascidian muscle determinant gene Ci-macho1.
Autonomous 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.
Yagi
Kasumi
K
Department of Zoology, Graduate School of Science, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan. kasumi@ascidian.zool.kyoto-u.ac.jp
Satoh
Nori
N
Satou
Yutaka
Y
D000818:Animals; D001483:Base Sequence; D002454:Cell Differentiation; D002938:Ciona intestinalis / Q000033:anatomy & histology / Q000196:embryology* / Q000235:genetics* / Q000378:metabolism; D020869:Gene Expression Profiling; D018507:Gene Expression Regulation, Developmental; D017403:In Situ Hybridization; D009132:Muscles / Q000196:embryology* / Q000502:physiology; D016376:Oligonucleotides, Antisense / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D016335:Zinc Fingers
D016376:Oligonucleotides, Antisense; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors