The signals of FGFs on the neurogenesis of embryonic stem cells

Journal of Biomedical Science RTehseaerchsignals of FGFs on the neurogenesis of embryonic stem cells Ching-Wen Chen 0 Chin-San Liu 2 Ing-Ming Chiu 1 Shih-Cheng Shen 0 Hung-Chuan Pan 6 Kun-Hsiung Lee 5 Shinn- Zong Lin 4 Hong-Lin Su 0 3 0 Department of Life Sciences, National Chung-Hsing University , Taichung , Taiwan 1 Institute of Cellular and Systems Medicine, National Health Research Institutes; Miaoli , Taiwan 2 Department of Medical Research, Changhua Christian Hospital , Changhua , Taiwan 3 Department of Physical Therapy, China Medical University , Taichung , Taiwan 4 Center for Neuropsychiatry, China Medical University and Hospital , Taichung , Taiwan; China Medical University Beigang Hospital , Yunlin , Taiwan ; Department of Immunology, China Medical University , Taichung , Taiwan 5 Animal Technology Institute Taiwan; Miaoli , Taiwan 6 Department of Neurosurgery, Taichung Veterans General Hospital; Taichung , Taiwan Background: Neural induction is a complex process and the detailed mechanism of FGF-induced neurogenesis remains unclear. Methods: By using a serum-free neural induction method, we showed that FGF1 dose-dependently promoted the induction of Sox1/N-cadherin/nestin triple positive cells, which represent primitive neuroblasts, from mouse embryonic stem (ES) cells. Results: We demonstrated that FGF1, FGF2, and FGF4, but not FGF8b, enhanced this neurogenesis. Especially, FGFenhanced neurogenesis is not mediated through the rescue of the apoptosis or the enhancement of the proliferation of Sox1+ cells. We further indicated that the inactivation of c-Jun N-terminal kinase-1 (JNK-1) and extracellular signalrelated kinase-2 (ERK-2), but not p38 mitogen-activated protein kinase (MAPK), inhibited the neural formation through the inhibition of ES differentiation, but not through the formation of endomesodermal cells. Conclusions: These lines of evidence delineated the roles of FGF downstream signals in the early neural differentiation of ES cells. - Background In the early gastrula of the chicken, temporary treatment of the primitive ectoderm with Hensen's node for 5 hours steers the ectoderm to become the neural fate [1,2]. FGF was shown to be responsible for this instructive ability of node and for the maintenance of later neural instructive signals [3,4]. FGF first activates ERNI during early gastrulation and consequently triggers the zinc-finger transcriptional activator, Churchill, and its downstream target Sip1 in late gastrulation [4]. In Xenopus, the study of neural induction has revealed the essential role of Ras/MAPK activation for neurogenesis in uncommitted ectoderm and in dissociated animal cap cells, suggesting that the requirement of FGF signals in neural induction is conserved in chordates [5]. ES cells, which resemble epiblast cells in the blastocyst, provide an alternative approach to the study of early development in mammals [6,7]. Several one-step neural induction models have been established. Trans-retinoic acid (RA), a pro-neural inducer, enriches the neural population in a serum-containing embryoid bodies (EBs) system [8,9]. However, RA treatment has several drawbacks, including the caudalization of the neural fate, blockage of forebrain induction, and the disruption of normal embryogenesis [9-11]. Co-culture of ES cells with mouse skull-derived stromal cells, such as PA6 cells, or bone marrow-derived cells, such as MS5 cells, efficiently induces the ES cells to become neuron lineages [8,12]. However, the factors contributing to this stromal-derived inducing activity are still uncharacterized. ES cells cultured in serum-free Neurobasal medium with N2B27 supplement efficiently differentiate into Sox1+ neural precursors, which represent the earliest committed neuroblast cells in the developing embryo [13,14]. Specific neuronal subtypes, such as dopaminergic and serotoninergic neurons, are derived from the Sox1 neuroblasts by the addition of defined patterning factors. Although the Neurobasal/N2B27 model provides a simple monoculture differentiation system for ES cells, these cells often undergo apoptosis on days 3 to 5. Recently, an efficient neural-induction monoculture system with a high survival rate for differentiating ES cells was developed and termed as serum-free embryoid bodies formation (SFEB) method [15]. This simple and reproducible system consists of defined components and is suitable for the exploration of downstream FGF signals in the early neurogenesis of mammals. Reagents Human recombinant FGF2, FGF4 and FGF8b were all from R&D Systems. Recombinant human FGF1 was prepared from Prof. Chiu in Institute of Cell and Systems Medicine, the National Health Research Institutes, Taiwan [17]. Synthetic inhibitors of FGF signaling, including SU5402, LY294002, SB203580, and SP600125, were from Calbiochem; U0126 was purchased from Tocris. Stable cell establishment The plasmid Flag-DsRedT4-NLS was a gift from Tim Shroeder at Helmholtz Center Munich, Institute of Stem Cell Research, Germany. The genes of JNK dominant negative mutants, Flag-JNK1a1apf and Flag-JNK2a2apf [18,19], were obtained from Addgene http:// www.addgene.org and fused with a IRES-DsRed as a reporter. The plasmids were transfected into ES cells with lipofectamine 2000 (Invitrogen). After selection with 0.4 mg/ml G418 for two weeks, stable clones with red fluorescence were picked up and maintained with 0.2 mg/ml G418. The selected ES cells showed normal ES cell morphology and pluripotent gene expression (data not shown). Immunocytochemistry Cells were fixed in 4% cold paraformaldehyde and permeabilized with 0.3% Triton-X 100. Immunocytochemistry was performed with the following primary antibodies: OCT3/4 (1:500, Santa Cruz), Nanog (1:100, Cosmo Bio, Japan), Sox2 (1:4000, Chemicon), N-cadherin (1:100, DSHB, Iowa), FGF receptor 1 (FGFR1) and FGFR3 (both 1:100, Santa Cruz), FGFR2 (1:500, Abcam) and GFP (1:1000, Aves Labs). Images of immunostaining were captured usinga fluorescent microscope (Nikon ECLIPSE 80I) or confocal microscope (LSM510 Meta, Zeiss). Flow cytometry Sox1-GFP ES cells were fully dissociated and analyzed with flow cytometry (FC500, Beckman Coulter). Apoptosis was measured by staining for Annexin V (AbD Serotec) at room temperature for 10 min in the dark. RT-PCR analysis Total RNA was isolated from ES cells using REzol C&T reagent (Protech technology, Taiwan). Primers were applied to detect the expression of FGFR1 (5'-CAC ACT GCC TTC TCC TCC TC-3', 5'-CTC TGC CTC CCT GTC TTC TG-3'), FGFR2 (5'-GGG GAT GTG GAG TTT GTC TG-3', 5'-GCT TCT TGG TCG TGG TCT TC-3'), FGFR3 (5'-CGG CTA CCT GTG AAG TGG AT-3', 5'GCT TGG TCT GTG GGA CTG TT-3'), FGFR4 (5'-AGG AAA TGT GGC TGC TCT TG-3', 5'-GGT GTG TCC AGT AGG GTG CT-3'), Sox1 (5'-CCT CGG ATC TCT GGT CAA GT-3', 5'-TAC AGA GCC GGC AGT CAT AC-3'), and G3PDH (5'-GTG AAG GTC GGT GTG AAC G-3', 5'-GGT GAA GAC ACC AGT AGA CAC TC-3'). Western blot analysis ES cells were lysed in RIPA buffer (50 mM Tris pH7.5, 150 mM NaC (...truncated)