Gao Fei, Shi Heidi Y, Daughty Cathy, Cella Nathalie, Zhang Ming
Baylor College of Medicine, Department of Molecular and Cellular Biology, One Baylor Plaza, Houston, TX 77030, USA.
Development. 2004 Apr;131(7):1479-89. doi: 10.1242/dev.01048. Epub 2004 Feb 25.
Maspin (Mp) is a member of the serpin family with inhibitory functions against cell migration, metastasis and angiogenesis. To identify its role in embryonic development in vivo, we generated maspin knockout mice by gene targeting. In this study, we showed that homozygous loss of maspin expression was lethal at the peri-implantation stage. Maspin was specifically expressed in the visceral endoderm after implantation; deletion of maspin interfered with the formation of the endodermal cell layer, thereby disrupting the morphogenesis of the epiblast. In vitro, the ICM of the Mp(-/-) blastocysts failed to grow out appropriately. Data from embryoid body formation studies indicated that the Mp(-/-) EBs had a disorganized, endodermal cell mass and lacked a basement membrane layer. We showed that the embryonic ectoderm lineage was lost in the Mp(-/-) EBs, compared with that of the Mp(+/+) EBs. Re-expression of maspin partially rescued the defects observed in the Mp(-/-) EBs, as evidenced by the appearance of ectoderm cells and a layer of endoderm cells surrounding the ectoderm. In addition, a maspin antibody specifically blocked normal EB formation, indicating that maspin controls the process through a cell surface event. Furthermore, we showed that maspin directly increased endodermal cell adhesion to laminin matrix but not to fibronectin. Mp(+/-) endodermal cells grew significantly slower than Mp(+/+) endodermal cells on laminin substrate. We conclude that deletion of maspin affects VE function by reducing cell proliferation and adhesion, thereby controlling early embryonic development.
乳腺丝抑蛋白（Mp）是丝氨酸蛋白酶抑制剂家族的一员，具有抑制细胞迁移、转移和血管生成的功能。为了确定其在体内胚胎发育中的作用，我们通过基因靶向技术构建了乳腺丝抑蛋白基因敲除小鼠。在本研究中，我们发现纯合缺失乳腺丝抑蛋白表达在植入前期是致死的。植入后，乳腺丝抑蛋白在内脏内胚层中特异性表达；乳腺丝抑蛋白的缺失干扰了内胚层细胞层的形成，从而破坏了外胚层的形态发生。在体外，Mp（-/-）囊胚的内细胞团无法正常生长。胚胎体形成研究的数据表明，Mp（-/-）胚胎体具有紊乱的内胚层细胞团，且缺乏基底膜层。我们发现，与Mp（+/+）胚胎体相比，Mp（-/-）胚胎体中胚胎外胚层谱系缺失。乳腺丝抑蛋白的重新表达部分挽救了在Mp（-/-）胚胎体中观察到的缺陷，外胚层细胞的出现以及围绕外胚层的一层内胚层细胞的出现证明了这一点。此外，一种乳腺丝抑蛋白抗体特异性地阻断了正常胚胎体的形成，表明乳腺丝抑蛋白通过细胞表面事件控制这一过程。此外，我们发现乳腺丝抑蛋白直接增加内胚层细胞与层粘连蛋白基质的粘附，但不增加与纤连蛋白的粘附。在层粘连蛋白底物上，Mp（+/-）内胚层细胞的生长明显慢于Mp（+/+）内胚层细胞。我们得出结论，乳腺丝抑蛋白的缺失通过减少细胞增殖和粘附来影响内脏内胚层功能，从而控制早期胚胎发育。
