1365 STEM CELLS AND DEVELOPMENT Volume 19, Number 9, 2010 © Mary Ann Liebert, Inc. DOI: 10.1089/scd.2009.0386 Transcription factor FoxA1 plays a critical role during embryonic development and is activated during retinoic acid (RA)-induced neural differentiation of pluripotent P19 embryonal carcinoma cells at the early stage, which is marked by decreased expression of Nanog and increased expression of neural stem cell marker Nestin. To further understand how FoxA1 mediates neural differentiation, we have overexpressed FoxA1 through an ad- enovirus vector in P19 cells and identiied that early neurogenesis-related sonic hedgehog (Shh) gene is activated directly by FoxA1. Knockdown of FoxA1 expression during P19 cell neural differentiation results in prevention of Shh and Nestin induction. FoxA1 binds to Shh promoter at −486 to −462 bp region and activates the pro- moter in cotransfection assays. Furthermore, overexpression of FoxA1 alone in P19 cells stimulates expression of Nestin and results in decreased protein levels of Nanog. During RA-induced P19 cell differentiation, ele- vated levels of FoxA1 increase the population of neurons, evidenced by stimulated expression of neuron-speciic Neuroilament-1 and Tubulin βIII. Together, our results suggest a critical involvement of FoxA1 in stimulating neural differentiation of pluripotent stem cells at early stages. Introduction T ranscription factor FoxA1 (previously known as HNF-3α) belongs to the fork head/winged-helix family of transcription factors that play important roles in cellular proliferation and differentiation during embryonic devel- opment [1–4] and also play emerging roles in cancer [5]. Expression of FoxA1 initiates during gastrulation of mouse embryogenesis in notochord, ventral loor plate of neural tube, and gut endoderm, and spreads to midbrain and spinal cord regions and to liver primordium [6–9], suggest- ing that FoxA1 plays a role in the early development of cen- tral nervous system and endoderm-derived organs like liver and pancreas. The range of FoxA1 expression in the adult includes tissues derived from endoderm (liver, lung, pan- creas, stomach, intestine, prostate, and bladder), mesoderm (kidney, vagina and uterus, mammary glands, and seminal and coagulating glands), and neuroectoderm (brain and olfactory epithelium) [10], indicating multiple functions of FoxA1 in different adult organs. The expression pattern of FoxA1 in developing neural tube and adult brain structures implicates its important roles in neurogenesis and brain functions. This is supported by a recent discovery in which FoxA1 was found to regulate multiple phases of midbrain dopaminergic neuron development by stimulating expres- sion of multiple neural differentiation-related genes such as Ngn2, Nurr1, and engrailed 1, at different stages of the neu- ronal differentiation [11]. Mouse embryonal carcinoma (EC) cell lines are derived from teratocarcinomas and have been well characterized as pluripotent cell lines that can be maintained as undif- ferentiated cells and induced under controlled conditions to differentiate in vitro to any cell type of all 3 germ layers [12], providing an attractive cell model system for studying differentiation of pluripotent stem cells [ 13 ]. The P19 EC cell line was derived from a teratocarcinoma in C3H/He mice, produced by grafting an embryo at 7 days of gestation to testes of an adult male mouse [ 14 ]. The cells contain a nor- mal karyotype, predicting the cells do not possess any gross genetic abnormalities. When injected into mouse blastocysts, P19 cells differentiate into a broad range of cell types in the Increased Levels of FoxA1 Transcription Factor in Pluripotent P19 Embryonal Carcinoma Cells Stimulate Neural Differentiation Yongjun Tan, Zhongqiu Xie, Miao Ding, Zhendong Wang, Qiqi Yu, Lei Meng, Hong Zhu, Xiaoqin Huang, Li Yu, Xiangxian Meng, and Yan Chen Biomedical Engineering Center and State Key Laboratory of Chemo/Biosensing and Chemometrics, Hunan University, Changsha, Hunan, China.