Rogers M B, Watkins S C, Gudas L J
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts.
J Cell Biol. 1990 May;110(5):1767-77. doi: 10.1083/jcb.110.5.1767.
We have examined the abundance and cell specificity of several mRNAs that are regulated during the retinoic acid (RA)-induced differentiation of F9 embryonal carcinoma cells to visceral endoderm. The experiments confirmed the multistep nature of this process by demonstrating the expression of the ERA-1/Hox 1.6 message within 6 h after RA addition; the expression of messages specific for the extracellular matrix proteins laminin B1 and B2, and collagen IV(alpha 1) between days 4 and 12; and the expression of two visceral endoderm markers, alpha-fetoprotein (AFP) and H19, by days 8-15. In situ hybridization experiments revealed that the collagen IV(alpha 1) mRNA is restricted to the outer cell layer of F9 cell aggregates regardless of the presence or absence of RA. Laminin B1 and B2 mRNAs are concentrated in the outer cell layer of RA-treated aggregates although significant levels of message are also observed within the interior cells of the aggregates. Unexpectedly, AFP mRNA is detectable in only a subset of the outer cells of F9 cell aggregates grown 15 d in the presence of RA. The results obtained from wild-type F9 cells were compared with those from a mutant F9 cell line, RA-5-1, which was previously shown to synthesize collagen IV containing six- to ninefold less 4-hydroxyproline than that in wild-type F9 cells. RA-5-1 cells exhibit four- to sixfold less of the mRNAs encoding two visceral endoderm proteins, AFP and H19, than wild-type F9 cells after RA treatment of RA-5-1 aggregates. RA-5-1 cells, however, do exhibit an RA-associated increase in the level of ERA-1/Hox 1.6 mRNA within 6 h after adding RA. Although the collagen IV protein level is similar in wild-type F9 and RA-5-1 aggregates, the collagen IV(alpha 1) message level is 6-20-fold greater in aggregates of mutant cells than in aggregates of wild-type cells. Moreover, in situ hybridizations showed that this message is evenly distributed throughout the RA-5-1 aggregates rather than restricted to the outer cell layers as it is in wild-type F9 aggregates. These results suggest that abnormal collagen IV expression and localization are associated with decreased expression of the visceral endoderm markers, AFP and H19, in RA-5-1 cell aggregates.
我们研究了几种mRNA的丰度和细胞特异性，这些mRNA在视黄酸（RA）诱导F9胚胎癌细胞分化为内脏内胚层的过程中受到调控。实验通过以下方式证实了这一过程的多步骤性质：在添加RA后6小时内显示ERA-1/Hox 1.6信息的表达；在第4天至第12天之间显示细胞外基质蛋白层粘连蛋白B1和B2以及胶原蛋白IV（α1）特异性信息的表达；在第8 - 15天显示两种内脏内胚层标志物甲胎蛋白（AFP）和H19的表达。原位杂交实验表明，无论有无RA，胶原蛋白IV（α1）mRNA都局限于F9细胞聚集体的外层细胞。层粘连蛋白B1和B2 mRNA集中在经RA处理的聚集体的外层细胞中，尽管在聚集体内部细胞中也观察到显著水平的信息。出乎意料的是，在有RA存在的情况下培养15天的F9细胞聚集体中，仅在外层细胞的一个子集中可检测到AFP mRNA。将野生型F9细胞的结果与来自突变F9细胞系RA - 5 - 1的结果进行比较，先前显示该细胞系合成的胶原蛋白IV含有的4 - 羟脯氨酸比野生型F9细胞少6至9倍。在对RA - 5 - 1聚集体进行RA处理后，RA - 5 - 1细胞中编码两种内脏内胚层蛋白AFP和H19的mRNA比野生型F9细胞少4至6倍。然而，RA - 5 - 1细胞在添加RA后6小时内确实显示出ERA - 1/Hox 1.6 mRNA水平与RA相关的增加。尽管野生型F9和RA - 5 - 1聚集体中的胶原蛋白IV蛋白水平相似，但突变细胞聚集体中的胶原蛋白IV（α1）信息水平比野生型细胞聚集体中的高6至20倍。此外，原位杂交显示该信息均匀分布于整个RA - 5 - 1聚集体，而不像在野生型F9聚集体中那样局限于外层细胞。这些结果表明，胶原蛋白IV表达和定位异常与RA - 5 - 1细胞聚集体中内脏内胚层标志物AFP和H19的表达降低有关。
