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First published online 18 July 2007
doi: 10.1242/dev.002907

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Endoglin is required for hemangioblast and early hematopoietic development

Department of Developmental Biology, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, TX 75390-9133, USA.

View larger version (29K): [in this window] [in a new window]   Fig. 1. Defective blast-colony formation in the absence of endoglin. (A) Number of BL-CFCs in EBs from Eng+/+, Eng+/- and Eng-/- ES cells on day 3 of differentiation. Error bars indicate standard deviations from three independent experiments performed in duplicate. *, P<0.001 versus control (+/+ and +/-). (B) Morphologic aspect of EBs and resulting BL-CFCs in Eng+/- (upper panels) and Eng-/- (lower panels) ES cells. (C) Evaluation of the hematopoietic and endothelial potentials of BL-CFCs: individual BL-CFCs were picked, disrupted and replated under the hematopoietic (methylcellulose supplemented with IL-3, IL-6, SCF and EPO) or endothelial (gelatin-coated dishes with VEGF, bFGF, IGF-1 and ECGS) conditions to determine their potential to generate secondary hematopoietic colonies and to form adherent endothelial cells, respectively. The number of colonies that yielded secondary CFCs or adherent endothelial cells and the total number of replated colonies are indicated in parentheses.

View larger version (31K): [in this window] [in a new window]   Fig. 2. Endoglin marks the blast colony-forming cell. (A) A representative FACS profile of a time-course analysis for endoglin and FLK-1 expression in Eng+/+ and Eng-/- ES cells (day 0, d0) and EBs differentiated for 2, 3, 4 and 6 days. Fluorescence intensity for endoglin is indicated on the y axis and for FLK-1 on the x axis. (B) Gene expression analysis of Eng+/+, Eng+/- and Eng-/- ES cells during EB differentiation. (C) Wild-type day 3 EBs were sorted based on expression of endoglin and FLK-1, according to represented gates, as follows: ENG+FLK-1+, ENG+FLK-1-, ENG-FLK-1+ and ENG-FLK-1-. Resulting cell fractions were plated immediately for blast colonies (BL-CFCs). Error bars indicate standard deviations from two independent experiments performed in duplicate. *, P<0.05 versus the other three cell fractions.

View larger version (28K): [in this window] [in a new window]   Fig. 3. A CMV-endoglin transgene rescues hemangioblast activity in Eng-/- ES cells. (A) FACS analysis for endoglin expression in Eng+/-, Eng-/- and -/-:R5 (rescued clone) ES cells. Upper panels, undifferentiated cells (day 0); lower panels, day 3 of EB differentiation. Fluorescence intensity is indicated on the y axis. The x axis represents FITC, which provides a measure of autofluorescence. (B) Number of BL-CFCs on day 3 EBs for Eng+/-, Eng-/- and -/-:R5 ES cells. Error bars indicate standard deviations from two independent experiments performed in duplicate. *, P<0.01 versus -/-. (C) Representative blast colonies shown at similar magnification (200x).

View larger version (30K): [in this window] [in a new window]   Fig. 4. Defective erythropoiesis in the absence of endoglin. (A) Eng+/+, Eng+/- and Eng-/- ES cells were assayed for primitive erythroid development by plating cells from EBs differentiated for 3, 4 and 5 days (d3, d4 and d5, respectively) in methylcellulose (MCM) supplemented with IL-3, IL-6, SCF and EPO. (B) Relative levels of SCL, GATA2, RUNX1, GATA1, embryonic and adult globins, GPIIB (CD41), NFE2, and FMS from Eng+/+ and Eng-/- day 3, 4 and 7 EBs by real-time RT-PCR. Transcripts are normalized to GAPDH. (A,B) Error bars indicate standard deviations from three independent experiments performed in duplicate. *, P<0.05; **, P<0.005; ***, P<0.0001; versus controls (+/+ and -/-). (C) FACS analyses of day 6 Eng+/+ and Eng-/- EBs for GPIIB (CD41) and c-KIT. Fluorescence intensity for c-KIT is indicated on the y axis and for GPIIB (CD41) on the x axis.

View larger version (35K): [in this window] [in a new window]   Fig. 5. The effect of endoglin deletion on in vitro vascular sprout formation in differentiating EBs. (A) FACS analyses of day 6 Eng+/+ and Eng-/- EBs for VE-cadherin/FLK-1 (top) and VE-cadherin/TIE-2 (bottom). Fluorescence intensity for VE-cadherin is indicated on the y axis and for FLK-1 or TIE-2 on the x axis. (B) Percentage of EBs in each of the four classes of vascular sprout development in Eng+/+, Eng+/- and Eng-/- ES cells. Categories: I, no sprout formation; II, few sprouts; III, many sprouts but no network; and IV, many sprouts with network. Error bars indicate standard deviations from two independent experiments performed in duplicate. *, P<0.0001 versus controls (+/+ and +/-) on category IV. (C) Morphologic aspect of sprouting in Eng+/- (upper panels) and Eng-/- (lower panels) EBs.

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