Behrendtsen O, Alexander C M, Werb Z
Department of Anatomy and Program in Developmental Biology, University of California, San Francisco 94143-0750, USA.
Development. 1995 Dec;121(12):4137-48. doi: 10.1242/dev.121.12.4137.
The outgrowth of parietal endoderm (PE) cells from precursor endodermal cells is one of the first differentiation events that occur in mouse embryos. We have analyzed the molecular determinants of this process by placing isolated inner cell masses (ICMs) on defined extracellular matrix substrata in microdrop cultures. Differentiation and outgrowth of PE required a fibronectin substratum. Laminin supported the adhesion and outgrowth of visceral endoderm (VE) and actively suppressed the differentiation of PE in mixtures of fibronectin and laminin. Collagen type IV, gelatin, vitronectin or entactin supported little or no endodermal outgrowth. Trophectoderm (TE) cells have been implied to be important in PE induction in vivo. We found that recombination of ICMs in culture with TE cells, or with medium conditioned by TE cells, greatly increased the differentiation of PE. TE cells stimulated PE outgrowth on substrata other than fibronectin. One cytokine secreted by trophoblast and endodermal cells, parathyroid hormone-related peptide (PTHrP), was critical for outgrowth on any substratum. A function-perturbing antibody to PTHrP reduced the number of PE cells, whereas the addition of PTHrP increased that number. Furthermore, addition of PTHrP changed the substratum requirements for outgrowth, making laminin, vitronectin and low concentrations of fibronectin permissive for PE outgrowth. Immunostaining with anti-integrin antibodies showed that fully differentiated PE cells outgrowing on fibronectin expressed alpha 5, alpha 6 and alpha v beta 3 integrins. However, analysis of outgrowths in the presence of function-perturbing antibodies to alpha 5, alpha 6 and alpha v beta 3 integrins showed that these integrins directed PE outgrowth only on fibronectin, laminin and vitronectin substrata, respectively. We have shown that there is a cooperative interplay of extracellular matrix, integrins and PTHrP that modulates PE outgrowth.
壁内胚层（PE）细胞从前体内胚层细胞中生长出来是小鼠胚胎中最早发生的分化事件之一。我们通过将分离的内细胞团（ICM）置于微滴培养中特定的细胞外基质底物上，分析了这一过程的分子决定因素。PE的分化和生长需要纤连蛋白底物。层粘连蛋白支持内脏内胚层（VE）的黏附和生长，并在纤连蛋白和层粘连蛋白的混合物中积极抑制PE的分化。IV型胶原、明胶、玻连蛋白或巢蛋白支持很少或不支持内胚层生长。滋养外胚层（TE）细胞在体内PE诱导中被认为很重要。我们发现，培养中的ICM与TE细胞或与TE细胞条件培养基重组，可大大增加PE的分化。TE细胞在纤连蛋白以外的底物上刺激PE生长。滋养层细胞和内胚层细胞分泌的一种细胞因子，甲状旁腺激素相关肽（PTHrP），对在任何底物上的生长都至关重要。针对PTHrP的功能干扰抗体减少了PE细胞的数量，而添加PTHrP则增加了该数量。此外，添加PTHrP改变了生长所需的底物条件，使层粘连蛋白、玻连蛋白和低浓度的纤连蛋白允许PE生长。用抗整合素抗体进行免疫染色显示，在纤连蛋白上生长的完全分化的PE细胞表达α5、α6和αvβ3整合素。然而，在存在针对α5、α6和αvβ3整合素的功能干扰抗体的情况下对生长进行分析表明，这些整合素分别仅在纤连蛋白、层粘连蛋白和玻连蛋白底物上指导PE生长。我们已经表明，细胞外基质、整合素和PTHrP之间存在协同相互作用，调节PE的生长。
