Kenny A P, Kozlowski D, Oleksyn D W, Angerer L M, Angerer R C
Department of Biology, University of Rochester, Rochester, NY 14627, USA.
Development. 1999 Dec;126(23):5473-83. doi: 10.1242/dev.126.23.5473.
We have identified a Sox family transcription factor, SpSoxB1, that is asymmetrically distributed among blastomeres of the sea urchin embryo during cleavage, beginning at 4th cleavage. SpSoxB1 interacts with a cis element that is essential for transcription of SpAN, a gene that is activated cell autonomously and expressed asymmetrically along the animal-vegetal axis. In vitro translated SpSoxB1 forms a specific complex with this cis element whose mobility is identical to that formed by a protein in nuclear extracts. An anti-SpSoxB1 rabbit polyclonal antiserum specifically supershifts this DNA-protein complex and recognizes a single protein on immunoblots of nuclear proteins that comigrates with in vitro translated SpSoxB1. Developmental immunoblots of total proteins at selected early developmental stages, as well as EMSA of egg and 16-cell stage proteins, show that SpSoxB1 is present at low levels in unfertilized eggs and progressively accumulates during cleavage. SpSoxB1 maternal transcripts are uniformly distributed in the unfertilized egg and the protein accumulates to similar, high concentrations in all nuclei of 4- and 8-cell embryos. However, at fourth cleavage, the micromeres, which are partitioned by asymmetric division of the vegetal 4 blastomeres, have reduced nuclear levels of the protein, while high levels persist in their sister macromeres and in the mesomeres. During cleavage, the uniform maternal SpSoxB1 transcript distribution is replaced by a zygotic nonvegetal pattern that reinforces the asymmetric SpSoxB1 protein distribution and reflects the corresponding domain of SpAN mRNA accumulation at early blastula stage ( approximately 150 cells). The vegetal region lacking nuclear SpSoxB1 gradually expands so that, after blastula stage, only cells in differentiating ectoderm accumulate this protein in their nuclei. The results reported here support a model in which SpSoxB1 is a major regulator of the initial phase of asymmetric transcription of SpAN in the nonvegetal domain by virtue of its distribution at 4th cleavage and is subsequently an important spatial determinant of expression in the early blastula. This factor is the earliest known spatially restricted regulator of transcription along the animal-vegetal axis of the sea urchin embryo.
我们鉴定出一种Sox家族转录因子SpSoxB1，从第四次卵裂开始，它在海胆胚胎卵裂球中呈不对称分布。SpSoxB1与一个顺式元件相互作用，该顺式元件对于SpAN基因的转录至关重要，SpAN基因是一个细胞自主激活且沿动物 - 植物轴不对称表达的基因。体外翻译的SpSoxB1与这个顺式元件形成一种特异性复合物，其迁移率与核提取物中的一种蛋白质形成的复合物相同。抗SpSoxB1兔多克隆抗血清特异性地使这种DNA - 蛋白质复合物发生超迁移，并在核蛋白的免疫印迹上识别出一种与体外翻译的SpSoxB1共迁移的单一蛋白质。在选定的早期发育阶段对总蛋白进行的发育免疫印迹分析，以及对卵和16细胞期蛋白进行的电泳迁移率变动分析（EMSA）表明，SpSoxB1在未受精卵中含量较低，并在卵裂过程中逐渐积累。SpSoxB1母源转录本在未受精卵中均匀分布，并且该蛋白在4细胞和8细胞胚胎的所有细胞核中积累到相似的高浓度。然而，在第四次卵裂时，通过植物极4个卵裂球的不对称分裂产生的小分裂球中该蛋白的核水平降低，而在其姐妹大分裂球和中分裂球中仍保持高水平。在卵裂过程中，母源SpSoxB1转录本的均匀分布被合子非植物极模式所取代，这种模式强化了SpSoxB1蛋白的不对称分布，并反映了囊胚早期阶段（约150个细胞）SpAN mRNA积累的相应区域。缺乏核SpSoxB1的植物极区域逐渐扩大，因此在囊胚期之后，只有分化外胚层中的细胞在其细胞核中积累这种蛋白质。本文报道的结果支持这样一种模型：SpSoxB1凭借其在第四次卵裂时的分布，是非植物极区域SpAN不对称转录初始阶段的主要调节因子，并且随后是早期囊胚中基因表达的重要空间决定因素。该因子是已知最早沿海胆胚胎动物 - 植物轴在空间上受到限制的转录调节因子。
