QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

doi: 10.1242/10.1242/dev.00476

This Article Summary Full Text Full Text (PDF) Data Supplement Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Shimozaki, K. Articles by Taga, T. Search for Related Content PubMed PubMed Citation Articles by Shimozaki, K. Articles by Taga, T. Social Bookmarking                         What's this?

Involvement of Oct3/4 in the enhancement of neuronal differentiation of ES cells in neurogenesis-inducing cultures

¹ Department of Cell Fate Modulation, Institute of Molecular Embryology and Genetics, Kumamoto University, Kumamoto, 860-0811, Japan
² Laboratory of Pluripotent Cell Studies, RIKEN Center for Developmental Biology, Kobe, 650-0047, Japan

View larger version (46K): [in a new window]   Fig. 1. Efficient neurogenesis of ES cells under serum-free LIF-deficient conditions. ZHTc6 ES cells were established (Niwa et al., 2000) in which expression constructs for a tetracycline (Tc)-regulated transactivator (tTA) transgene and a tTA-responsive Oct3/4 transgene were introduced. The ZHTc6 cells with no transgene expression in the presence of Tc showed induced Oct3/4 expression the level of which is comparable to that from the endogenous allele. Withdrawal of Tc triggers the transgene expression, which induces Oct3/4 expression at a level 50% above that of Oct3/4 expression in normal ES cells. (A) ZHTc6 cells were cultured in serum-free N2-supplemented DMEM/F-12 without LIF for 8 days on poly-L-ornithine/fibronectin (O/F)-coated dishes in the presence (a) or absence (b) of Tc. Cells were stained with an anti-MAP2 antibody (green) and Hoechst 33258 (blue). Upregulation of Oct3/4 induced by withdrawal of Tc promoted neurogenesis of ES cells. Scale bar, 50 µm. (B) ZHTc6 cells were cultured as in A with or without Tc for 10 days. The frequency of MAP2-positive cells with respect to the total number of cells was analyzed every 2 days. Vertical bars indicate the s.d.

View larger version (34K): [in a new window]   Fig. 2. Upregulation of Oct3/4 expression in ES cells induces neural marker gene expression under serum-free LIF-deficient conditions. (A) ZHTc6 cells were cultured for 8 days in the presence or absence of Tc. Total RNA was extracted from each culture and subjected to RT-PCR analysis for the genes indicated on the right. (B) RNA was extracted from each ZHTc6 cell culture on days 2, 4 and 8, and subjected to RT-PCR analysis for Fgf5 expression.

View larger version (101K): [in a new window]   Fig. 3. Neurogenic function of Oct3/4 is not explained by the inhibition of cell autonomic Bmp signaling. MAP2 staining (green) of ZHTc6 cells cultured for 8 days with (A,C,E,G) or without (B,D,F,H) Tc. In some cultures, Bmp2 (10 ng/ml; C,D), BmpßR-Fc (150 ng/ml; E,F), or 10% serum (G,H) were added. Nuclei were stained with Hoechst 33258 (blue). Scale bar, 50 µm.

View larger version (64K): [in a new window]   Fig. 4. Effective SDIA-induced neurogenesis of ES cells accompanied by maintenance of Oct3/4. Expression EB5 ES cells were cultured on O/F-coated dishes (A,C,E) or a monolayer of PA6 cells (B,D,F). Cells were stained with anti-nestin (red) and anti-MAP2 (green) antibodies on each day indicated in the figure. Scale bar, 50 µm. (G) RT-PCR analysis of the expression of Oct3/4 and a mesencephalic dopaminergic neuron marker, Nurr1, in EB5 ES cells cultured as in A-F.

View larger version (54K): [in a new window]   Fig. 5. Requirement of Oct3/4 for SDIA-induced neurogenesis. (A,B) ZHBTc4 and (C,D) ZHTc6 cells were cultured on a PA6 monolayer in serum-free LIF-deficient medium with (B,C) or without (A,D) Tc for 8 days. The cells were stained with an anti-MAP2 antibody (green) and Hoechst 33258 (blue). In ZHBTc4 Oct3/4 expression can be shut off by adding Tc. (E) MAP2-positive colonies were counted. Scale bar, 100 µm.

View larger version (20K): [in a new window]   Fig. 6. Enhancement of SDIA-induced neurogenesis by upregulation of Oct3/4 expression. ZHTc6 cells were cultured with (open symbols) or without (closed symbols) Tc on a PA6 monolayer. The frequency of nestin-positive (circles) and MAP2-positive (triangles) colonies with respect to the total number of colonies on each day, as indicated, was calculated.

View larger version (51K): [in a new window]   Fig. 7. Temporal requirement of Oct3/4 for SDIA-induced neurogenesis. ZHBTc4 cells were cultured on a PA6 monolayer for 8 days and stained with anti-nestin and anti-MAP2 antibodies. (A) Tc was added to the medium on and after the indicated days to shut off the Oct3/4 expression until the end of culture. (B) Frequencies of nestin- and MAP2-positive colonies with respect to the total number of colonies are indicated. Vertical bars indicate the s.d. (C) Representative views of cultured ZHBTc4 cells immunostained on indicated days (anti-nestin, red; anti-MAP2, green). Scale bar, 100 µm.

View larger version (22K): [in a new window]   Fig. 8. Suppression of neural differentiation by Bmp2. ZHBTc4 cells were cultured on a PA6 monolayer for 8 days. Bmp2 (10 ng/ml) was added to the medium from the indicated days until the end of culture. On day 8, cells were stained for nestin and MAP2, and positive colonies were counted.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter