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The BMP/BMPR/Smad pathway directs expression of the erythroid-specific EKLF and GATA1 transcription factors during embryoid body differentiation in serum-free media

Department of Biochemistry and Molecular Biology, Mount Sinai School of Medicine, New York, NY 10029, USA

View larger version (28K): [in a new window]   Fig. 1. Formation and expression pattern of embryoid bodies (EBs). (A) EBs were differentiated for 10 days in methylcellulose with serum (FBS) or with two serum-free substitutes, knockout SR (SR1) or BIT 9500 (SR2). A single representative EB is shown for each case, as is the plating efficiency. (B) Total RNA from day 8 EBs differentiated in FBS, SR1 or SR2 (as indicated) was monitored by semi-quantitative RT/PCR for expression of EKLF, GATA1, adult ßmaj globin, embryonic ßh1 globin and HPRT.

View larger version (23K): [in a new window]   Fig. 2. Temporal pathway of erythroid commitment during development and expression of the markers used for analysis of EBs (based on that described by Orkin and Zon (Orkin and Zon, 1997). (A) ‘Cellular status’ denotes stages in lineage determination from uncommitted mesoderm to the terminally differentiated erythroid cell that are useful as a working model for analysis. Below each stage are their corresponding expression markers. The sets in brackets serve as endothelial, rather than hematopoietic, markers for hemangioblast cells. (B) Total RNA from day 8 EBs differentiated with 15% serum (+) or serum-substitute SR1 (–) was monitored for expression of the indicated hematopoietic markers by semi-quantitative RT/PCR.

View larger version (32K): [in a new window]   Fig. 3. EKLF and GATA1 gene expression in the presence of selected cytokines. Gene expression was monitored (by semi-quantitative RT/PCR) in EBs differentiated for 8 days in SR1 in the absence (–) or in the presence (+) of a cytokine cocktail that included BMP4, SCF, VEGF, T3 and erythropoietin. Pooled EBs from a single dish are shown in each lane.

View larger version (44K): [in a new window]   Fig. 4. Cytokine requirements for EKLF and GATA1 expression. EBs were differentiated with SR1 and various combinations of cytokines, focusing on VEGF, T3 and erythropoietin (A), or BMP4, SCF and VEGF (B). Total RNA was analyzed by semi-quantitative RT/PCR at day 8 of differentiation. For comparison, samples from EBs differentiated in serum (FBS) or in SR1 and all five cytokines were also analyzed. ‘Water’ indicates a no-RNA negative control. (C) EKLF and GATA1 gene expression in embryoid bodies differentiated for 8 days with in SR1 and the indicated cytokines (top), and quantitation/average of three experiments (bottom) after normalization to HPRT levels from the same samples. A previously determined, optimal concentration of BMP4 was used for these analyses. Signal with FBS was given an arbitrary level of ‘1’ (lane 8). ‘Water’ indicates a no-RNA negative control (lane 9).

View larger version (30K): [in a new window]   Fig. 5. Expression of endogenous BMP4 in differentiating EBs. EBs were differentiated for eight days in SR1 and the indicated cytokines and total RNA was monitored for expression of BMP4. Expression after differentiation in the presence of serum was included as a positive control, and ‘water’ indicates a no-RNA negative control.

View larger version (33K): [in a new window]   Fig. 6. Kinetics of EKLF and GATA1 expression during EB differentiation. (A) EBs were differentiated for varying lengths of time (days, as indicated) in SR1 before addition of BMP4. All samples were harvested at day 8 and total RNA was analyzed for EKLF, GATA1 or HPRT expression. Expression in the presence of serum was included as a positive control, and ‘water’ indicates a no-RNA negative control. (B) The onset of EKLF expression was monitored in EBs differentiating in FBS or in SR1 and BMP4, as indicated for varying lengths of time (days). Asterisk indicates a no-RNA negative control.

View larger version (52K): [in a new window]   Fig. 7. Tests of the BMP receptor/Smad pathway in EKLF and GATA1 expression. (A) EBs were differentiated for eight days in SR1 + the indicated cytokines, and total RNA was monitored for expression of Smad1, Smad5, Smad8 or HPRT. Expression after differentiation in the presence of serum was included as a positive control, and ‘water’ indicates a no-RNA negative control. (B,C) EBs derived from stable ES cell lines expressing wild-type BMPR-1B (wild-type line 648-3) or a dominant negative BMPR-1B (K231R line 649-6) in (B), or a mock transfected (mock) or Smad6-expressing ES lines (Smad6-4 and Smad6-5) in (C), were differentiated in FBS for 8 days before the harvest of total RNA and expression analysis for hematopoietic markers described in Fig. 2. ‘Water’ indicates a no-RNA negative control.

View larger version (42K): [in a new window]   Fig. 8. Kinetics of EKLF and GATA1 induction by BMP4. EBs were differentiated in SR1 until day 2 or day 3 as indicted before the addition of BMP4. After an additional incubation for the indicated lengths of time in BMP4, total RNA was harvested and analyzed for EKLF, GATA1 and HPRT expression.