Figure 4.
Figure 4. c-mybKD/KD CFU-E stage progenitors display aberrant expression of hematopoietic regulators and abnormal differentiation ex vivo. (A) CD71+c-kit+TER119–CD41– progenitors (Figure 2Biii) corresponding to the CFU-E stage were sorted by FACS from the fetal liver of c-myb+/+ and c-mybKD/KD E14 embryos. RT-PCR analysis was performed on RNA extracted from sorted cells. Ethidium bromide–stained agarose gels of PCR products from 2-cycle increments are shown. Normalization by β-actin was confirmed by real time PCR analysis (not shown). (B) Real-time PCR was performed on the cDNA samples described for panel A using SYBR green, primers specific for the indicated genes, and β-actin (for normalization). Reactions were repeated at least 6 times. Average fold change–normalized expression values are shown. Error bars represent the SEM. (C) Fetal liver cells were stained with α-CD71 (FITC), α-CD45 (APC) and α-TER119 (APC) (top panel) or α-CD71 (FITC), α-TER119 (PE), and α-c-Kit (PE-Cy5) (bottom panel). Histograms represent fluorescence intensities (log-scale) of cells gated as CD71+TER119– (CFU-E stage). (D) Fetal liver cells were stained and CFU-E stage cells were sorted as for panel A and were cultured for 48 hours, as described in Figure 2. Cells were stained with α-c-Kit (PE) and were analyzed by flow cytometry. Log-scale of PE-fluorescence intensity is shown. (E) CD71+c-Kit+TER119–CD41– (Figure 2Biii) CFU-E stage progenitors (top panels) and CD71– c-Kit+TER119–CD41– (Figure 2Bi) less mature progenitors (bottom panels) from E14 fetal liver were sorted by FACS and cultured in SP34 for 2 days. Cells were permeabilized with 0.1% NP-40 and were stained with 25 μg/mL propidium iodide for flow cytometric analysis of DNA content. Histograms represent linear fluorescence intensities. (F) Lower c-Myb expression limits the size of CFU-E colonies. Fetal liver cells were stained and CFU-E stage cells were sorted as for panel A and then plated in methylcellulose containing SCF and EPO for 3 days. Colonies were observed using an Olympus CKX41 microscope (Olympus, London, United Kingdom) and a 20 ×/0.40 numeric aperture Php objective under phase contrast. Images were acquired using an Olympus Camedia C3030 camera and were processed using Adobe Photoshop version 4.0 (Adobe Systems, San Jose, CA).

c-mybKD/KD CFU-E stage progenitors display aberrant expression of hematopoietic regulators and abnormal differentiation ex vivo. (A) CD71+c-kit+TER119–CD41– progenitors (Figure 2Biii) corresponding to the CFU-E stage were sorted by FACS from the fetal liver of c-myb+/+ and c-mybKD/KD E14 embryos. RT-PCR analysis was performed on RNA extracted from sorted cells. Ethidium bromide–stained agarose gels of PCR products from 2-cycle increments are shown. Normalization by β-actin was confirmed by real time PCR analysis (not shown). (B) Real-time PCR was performed on the cDNA samples described for panel A using SYBR green, primers specific for the indicated genes, and β-actin (for normalization). Reactions were repeated at least 6 times. Average fold change–normalized expression values are shown. Error bars represent the SEM. (C) Fetal liver cells were stained with α-CD71 (FITC), α-CD45 (APC) and α-TER119 (APC) (top panel) or α-CD71 (FITC), α-TER119 (PE), and α-c-Kit (PE-Cy5) (bottom panel). Histograms represent fluorescence intensities (log-scale) of cells gated as CD71+TER119– (CFU-E stage). (D) Fetal liver cells were stained and CFU-E stage cells were sorted as for panel A and were cultured for 48 hours, as described in Figure 2. Cells were stained with α-c-Kit (PE) and were analyzed by flow cytometry. Log-scale of PE-fluorescence intensity is shown. (E) CD71+c-Kit+TER119–CD41– (Figure 2Biii) CFU-E stage progenitors (top panels) and CD71– c-Kit+TER119–CD41– (Figure 2Bi) less mature progenitors (bottom panels) from E14 fetal liver were sorted by FACS and cultured in SP34 for 2 days. Cells were permeabilized with 0.1% NP-40 and were stained with 25 μg/mL propidium iodide for flow cytometric analysis of DNA content. Histograms represent linear fluorescence intensities. (F) Lower c-Myb expression limits the size of CFU-E colonies. Fetal liver cells were stained and CFU-E stage cells were sorted as for panel A and then plated in methylcellulose containing SCF and EPO for 3 days. Colonies were observed using an Olympus CKX41 microscope (Olympus, London, United Kingdom) and a 20 ×/0.40 numeric aperture Php objective under phase contrast. Images were acquired using an Olympus Camedia C3030 camera and were processed using Adobe Photoshop version 4.0 (Adobe Systems, San Jose, CA).

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