Smolich B D, Papkoff J
SUGEN, Inc., Redwood City, California 94063.
Dev Biol. 1994 Nov;166(1):300-10. doi: 10.1006/dbio.1994.1316.
The P19 embryonal carcinoma (EC) cell line represents a useful model system for analysis of neural development and differentiation processes that are difficult to study in mammalian embryos. Since many members of the Wnt family of signaling molecules are expressed in the developing as well as adult nervous system, we have examined expression of these genes in P19 cells. Analysis of the mRNA accumulation profiles for Wnt genes during retinoic acid (RA)-induced neural differentiation of P19 cells showed that nine Wnt family members were expressed in a regulated manner during this process. Most were induced by RA treatment, and some were also expressed in undifferentiated P19 cells. Since Wnt-1 is not expressed in undifferentiated P19 cells but is induced during neuroectodermal differentiation we have generated P19 cell lines that overexpress Wnt-1 in the absence of RA treatment, in order to address the role of Wnt-1 in P19 differentiation. In the presence of ectopic Wnt-1, expression of other endogenous Wnt genes, which serve as early differentiation markers in this system, were induced without RA, which is normally required for appearance of these gene products. Furthermore, ectopic expression of Wnt-1 resulted in a loss of SSEA-1 antigen expression, a marker of undifferentiated P19 cells. Similarly to the parental cell line, addition of RA to P19 cells overexpressing Wnt-1 induced the neuroectodermal pathway, but expression of cell type-specific markers such as MASH-1, HNK-1, and GAP-43 was diminished and the morphology of neuronal processes, stained with an antibody to neurofilament, was abnormal. These data suggest that Wnt-1 itself can induce some aspects of early neuroectodermal differentiation and, furthermore, that the correct timing of Wnt-1 expression is necessary for proper RA-induced expression of the neural phenotype.
P19胚胎癌细胞系是一个有用的模型系统，可用于分析在哺乳动物胚胎中难以研究的神经发育和分化过程。由于Wnt信号分子家族的许多成员在发育中的以及成年神经系统中都有表达，我们检测了这些基因在P19细胞中的表达。对P19细胞视黄酸（RA）诱导的神经分化过程中Wnt基因的mRNA积累谱分析表明，在此过程中有9个Wnt家族成员以受调控的方式表达。大多数是由RA处理诱导的，有些在未分化的P19细胞中也有表达。由于Wnt-1在未分化的P19细胞中不表达，但在神经外胚层分化过程中被诱导，我们构建了在无RA处理时过表达Wnt-1的P19细胞系，以研究Wnt-1在P19分化中的作用。在异位Wnt-1存在的情况下，作为该系统早期分化标志物的其他内源性Wnt基因的表达在无RA时被诱导，而这些基因产物的出现通常需要RA。此外，Wnt-1的异位表达导致未分化P19细胞标志物SSEA-1抗原表达丧失。与亲代细胞系类似，向过表达Wnt-1的P19细胞中添加RA可诱导神经外胚层途径，但细胞类型特异性标志物如MASH-1、HNK-1和GAP-43的表达减少，用神经丝抗体染色的神经元突起形态异常。这些数据表明，Wnt-1自身可诱导早期神经外胚层分化的某些方面，此外，Wnt-1表达的正确时机对于RA诱导神经表型的正确表达是必要的。
