Project description:Fertilized eggs are plulipotent and during early embryogenesis, embryonic cells gradually restrict their developmental potential and are eventually destined to give rise to one type of cells. Molecular mechanism underlying developmental fate restriction is one of the major research subjects of developmental biology. Here we address this question by combining a classical technique of blastomere isolation with a modern technique of microarray analysis. During the 6th cleavage of the Ciona intestinalis embryo, from the 32-cell to the 64-cell stage, four mother cells divide into daughter cells with two distinct fates, one giving rise to notochord and the other to nerve-cord precursor cells. Nearly 500 notochord or nerve-cord precursor cells each were isolated, and difference in the quality of mRNAs between them was compared by microarray. This analysis identified 111 and 69 genes that are differentially expressed in the notochord and nerve-cord precursor cells, respectively. These included not only genes for transcription factors and signalling molecules but also those with functions that many kinds of cells used. In addition, whole-mount in situ hybridization showed complex and dynamic profiles of spatial expression of these genes during the segregation of the two fates. The process is accomplished by that transcripts that have been already present in the mother cell are properly partitioned into either type of cells and that new and preferential expression of genes in either type of cells. Two kinds of sample, Dye Swap design with 2 arrays
Project description:Fertilized eggs are plulipotent and during early embryogenesis, embryonic cells gradually restrict their developmental potential and are eventually destined to give rise to one type of cells. Molecular mechanism underlying developmental fate restriction is one of the major research subjects of developmental biology. Here we address this question by combining a classical technique of blastomere isolation with a modern technique of microarray analysis. During the 6th cleavage of the Ciona intestinalis embryo, from the 32-cell to the 64-cell stage, four mother cells divide into daughter cells with two distinct fates, one giving rise to notochord and the other to nerve-cord precursor cells. Nearly 500 notochord or nerve-cord precursor cells each were isolated, and difference in the quality of mRNAs between them was compared by microarray. This analysis identified 111 and 69 genes that are differentially expressed in the notochord and nerve-cord precursor cells, respectively. These included not only genes for transcription factors and signalling molecules but also those with functions that many kinds of cells used. In addition, whole-mount in situ hybridization showed complex and dynamic profiles of spatial expression of these genes during the segregation of the two fates. The process is accomplished by that transcripts that have been already present in the mother cell are properly partitioned into either type of cells and that new and preferential expression of genes in either type of cells.
Project description:We recently showed HMX is expressed in bipolar tail neurons (BTN) in early embryos of Ciona intestinalis. In order to assess the function of the homeobox transcription factor in this cell fate, we used an overexpression strategy. ciHMX was overexpressed in the epidermis, followed by RNAseq of experimental and control embryos. We then looked for differential expression of BTN fate markers, testing if HMX is able to regulate BTN fate determination.