β-catenin and Early Development in the Gastropod, Crepidula fornicata
β-catenin and Early Development in the Gastropod, Crepidula fornicata
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DOI：
10.1093/icb/icq076
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发表时间：
2010-11-01
影响因子：
2.6
通讯作者：
Martindale, Mark Q.
中科院分区：
文献类型：
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作者：
Henry, Jonathan Q.;Perry, Kimberly J.;Martindale, Mark Q.
本研究描述了β-连环蛋白的早期表达和功能的腹足类，Crepidula fornicata。在其他两侧体动物中，β-连环蛋白在细胞粘附、原肠胚形成和细胞信号传导中起作用，这与背腹轴和中内胚层的建立有关。本实验研究了β-catenin mRNA和蛋白在C.通过整体原位杂交和体内表达GFP标记的β-连环蛋白，在早期卵裂过程中，β-连环蛋白mRNA和蛋白质似乎广泛地定位于早期胚胎的所有细胞。在这些细胞中，mRNA倾向于集中在间期中心体。在后期阶段，mRNA主要是在植物性的大单体，随后在后肠，口道，和软腭叶的雏形。全长GFP标记蛋白的表达表明，在25细胞期之前的早期胚胎细胞内没有降解β-连环蛋白的活性机制。然而，到发育的第二天，当第四个四联体微粒形成时，β-连环蛋白在4d成间充质细胞的后代中变得选择性稳定（例如，ML和MR及其子代），并且在大多数其他卵裂球中缺失，包括植物性大粒细胞。在接下来的2天的发展，在随后的分裂4d，β-连环蛋白蛋白成为逐步降解，沿着近端-远端轴，在后代的配对中内胚层带。位于中胚层带尖端的细胞（2 mL（2）和2 mR（2））是最后含有该蛋白的细胞，在发育4天后不再检测到该蛋白。在像C. fornicata，其经历螺旋卵裂程序（例如，软体动物、环节动物、纽形动物和polyclad扁形虫），间充质细胞或4D细胞代表内中胚层的祖先（形成后肠、内部和外部肾脏以及各种肌肉）。因此，在C. fornicata的发现与β-连环蛋白的基本祖先作用在于形成内中胚层命运的论点一致。使用截短的β-连环蛋白克隆的实验表明，位于C-末端的区域，远离第11个犰狳重复序列，是胚胎内β-连环蛋白蛋白正常稳定/降解所需的。将翻译阻断剂吗啉代注射到受精卵中导致β-连环蛋白表达下调。这导致随后的原肠胚形成失败，但不干扰4d的形成和早期卵裂，尽管在这些有缺陷的胚胎中没有可辨别的分化细胞命运。这些结果进行了比较与其他后生动物。
This study describes the early expression and function of beta-catenin in the gastropod, Crepidula fornicata. In other bilaterians beta-catenin functions in cell adhesion, gastrulation, and cell signaling, which is related to the establishment of the dorso-ventral axis and mesendoderm. Here, we studied the distribution of beta-catenin mRNA and protein in C. fornicata via whole mount in situ hybridization and by expressing GFP-tagged beta-catenin in vivo. During early cleavage, beta-catenin mRNA and protein appear to be broadly localized to all cells in the early embryo. The mRNA tends to be concentrated at inter-phase centrosomes in these cells. At later stages, the mRNA is predominantly in the vegetal macromeres, and subsequently in the rudiment of the hindgut, stomodeum, and velar lobes. Expression of full-length GFP-tagged protein suggests that there is no active mechanism to degrade beta-catenin within cells of the early embryos prior to the 25-cell stage. However, by the second day of development, when the fourth quartet micromeres have formed, beta-catenin becomes selectively stabilized in the progeny of the 4d mesentoblast (e.g., ML and MR and their daughters) and is missing from most other blastomeres, including vegetal macromeres. Over the next 2 days of development, during subsequent divisions of 4d, beta-catenin protein becomes progressively degraded, along the proximo-distal axes, within the progeny of the paired mesendodermal bands. The cells located at the tips of the mesodermal bands (2 mL(2) and 2 mR(2)) are the last to contain this protein, which is no longer detected after 4 days of development. In animals like C. fornicata, which undergo a spiral cleavage program (e.g., molluscs, annelids, nemerteans, and polyclad flatworms), the mesentoblast or 4d cell represents the progenitor of endomesoderm (forming hindgut, internal and external kidneys, and various muscles). Therefore, the selective stabilization of beta-catenin in the progeny of 4d in C. fornicata is consistent with arguments that a basic, ancestral role of beta-catenin lies in the formation of endomesodermal fates. Experiments using a truncated beta-catenin clone show that the regions located in the C-terminus, distal to the 11th armadillo repeat, are required for normal stabilization/degradation of beta-catenin protein within the embryo. Microinjection of translation blocking beta-catenin morpholinos into zygotes led to the down-regulation of beta-catenin expression. This resulted in the subsequent failure of gastrulation, but did not interfere with the formation and early cleavage of 4d, although there were no discernable differentiated cell fates in these defective embryos. These results are compared with those obtained in other metazoans.