Mesodermal determinants in the Tubifex embryo

Tubifex胚胎中D象限的第二(2d)和第四(4d)微体分别被指定为外胚层和中胚层胚胎干细胞的前体。为了深入了解这一干细胞规范的机制，我们使用2d和4d的cDNA之间的消减杂交，对特定血统的母体mRNAs进行了系统的筛选。我们初步鉴定了16个基因，包括IMPACT、HMGB、核仁素、TERE1、ELAV和RNA结合蛋白。与由这些基因合成的核探针进行整体原位杂交显示，母体提供的转录本的“行为”类似于极质(缺乏卵黄的细胞质)。也就是说，在第一次有丝分裂结束时，这些转录本首先被分离到动物极和植物极，然后被分割到D象限，最后定位到2d和4d细胞。在这项研究的过程中，我们没有遇到这样的基因，这些基因将专门分离到2d或4d。除了上述差减实验外，我们还分离并鉴定了Tubifex的几个发育基因(背部、有毛、十五麻性尾部和血管)。除了尾部合子表达外，这些基因都是母体表达的，储存在卵母细胞中的转录本的行为与减法实验中发现的基因转录本非常相似。

The second (2d) and fourth (4d) micromeres of the D quadrant in the Tubifex embryo are specified as precursors of ectodermal and mesodermal embryonic stem cells, respectively. To gain an insight into the mechanisms of this stem cell specification, we performed a systematic screen of lineage specific maternal mRNAs, using subtraction hybridization between cDNAs from 2d and 4d. We initially identified 16 genes, which include Impact, HMGB, Nucleolin, TERE1, ELAV and RNA binding proteins. Whole-mount in situ hybridization with riboprobes synthesized from these genes revealed that maternally supplied transcripts "behave" in a similar way to the pole plasms (yolk-deficient cytoplasm). That is, these transcripts are first segregated to the animal pole and the vegetal pole at the end of the first mitosis, partitioned to D quadrants, and finally localized to 2d and 4d cells. During the course of this study, we did not come across such genes as would be segregated exclusively to either 2d or 4d. Besides the above-mentioned subtraction experiments, we isolated and characterized several development genes (dorsal, hairy, decapentaplegic caudal and vasa) in Tubifex. Except caudal that is expressed zygotically, these genes are expressed maternally and transcripts stored in oocytes are found to behave in a very similar fashion to the gene transcripts discovered in the subtraction experiments.