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First published online July 21, 2003
doi: 10.1242/10.1242/dev.00589

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Tracking mesoderm induction and its specification to the hemangioblast during embryonic stem cell differentiation

¹ Department of Immunology, Medical Faculty/University Clinics Ulm, Germany
² Carl C. Icahn Center for Gene Therapy and Molecular Medicine, Mount Sinai School of Medicine, New York, NY 10029, USA
³ Department of Molecular Biology, UT Southwestern Medical Center, Dallas, TX 75390, USA

View larger version (17K): [in a new window]   Fig. 1. Brachyury expression in Flk1+ and Flk1– cells. (A) At day 2.75 of differentiation, wild-type EBs were harvested, dissociated and cells fractionated by cell sorting based on their level of Flk1 expression. (B) Blast-CFC potential was assayed in the unfractionated population (Presort), the Flk1+ and Flk1– fractions. Blast cell colonies (Blast) and secondary EBs (2nd EBs) were scored following 4 days of culture. Data are presented as the mean number of colonies from three dishes. Bars, where visible, represent s.d. of the mean. (C) Expression analysis of subpopulations positive and negative for Flk1. 3' cDNA from pools of sorted cells (1000 cells per lane) was prepared by RT-PCR and total cDNAs were separated on a 1.5% agarose gel, blotted and probed separately with 3' probes from the indicated genes. (D) Expression analysis of single cells. One-hundred individual cells from the Flk1+ sorted fraction were picked using a mouth pipette and deposited directly into the lysis buffer. 3' cDNA was prepared and analyzed as described above. A representative expression pattern for 40 such individual cells is shown here. Hybridization with a 3' probe from the L32 ribosomal protein gene was included to control for amounts of cDNA in each lane.

View larger version (28K): [in a new window]   Fig. 2. Generation of GFP-Bry ES cells. (A) Schematic structure of the mouse brachyury gene locus and the targeting vector. LoxP sites are indicated by black triangles on either side of the Neo gene. The hatched box indicates an exon derived from plasmid pBK-CMV, which contains the SV40 polyadenylation signal. TK: Herpes simplex thymidine kinase gene. H: HincII. (B) Blast-CFC potential: EBs from both ES cell lines were harvested on the indicated days of differentiation and cells assayed for BL-CFC. Colonies were scored after 4 days of culture and were referred to as 2nd EB (secondary embryoid bodies) and Blast (blast colonies). Data are presented as the mean number of colonies from three dishes. Bars, where visible, represent s.e. of the mean. (C) Fluorescence microscopy of Bry-GFP EBs at day 3 of differentiation. Wild-type EBs did not show detectable levels of fluorescence (not shown). Photos were taken with a Hamasura camera at 100x magnification. (D) EBs from the GFP-Bry ES cell line were harvested daily between day 1 and 6 of differentiation. Cells were used for expression analysis of brachyury by RT-PCR using gene specific primers and (E) flow cytometric analysis for detection of the GFP protein.

View larger version (36K): [in a new window]   Fig. 3. Analysis of GFP+ and GFP– EB-derived cells. (A) At day 3.5 of differentiation, GFP Bry EBs were harvested, dissociated and cells fractionated by cell sorting based on their GFP expression level. (B) RNA extraction was performed on the GFP–, GFP+ as well as the starting population (presort). Gene expression patterns were analyzed by RT-PCR using specific primers for each of the indicated genes. (C) Blast-CFC potential of the presort, GFP– and GFP+ populations. Colonies were scored after 4 days of culture. Data are presented as the mean number of colonies from three dishes. Bars, where visible, represent s.e. of the mean. The purity of sorted cells was 95%. (D) Expression analysis of day 6 reaggregated EBs generated from day 2.5 EB presort, GFP+ and GFP– cells. (E) Neurite potential of reaggregated EBs generated from GFP+ and GFP– populations. Data are presented as the % of EBs that form neurites. (F) Immunostaining demonstrating the expression of neuronal class III ß-tubulin (TuJ1) on neurites that develop from EBs derived from GFP– cells.

View larger version (39K): [in a new window]   Fig. 4. GFP-Bry expression relative to Flk1, CD31 and Kit. (A) At the indicated time points, GFP-Bry EBs were harvested and dissociated. The cells were stained with Flk1, c-Kit or CD31 mAb and analyzed by flow cytometry. (B) The level of Kit marker was assessed for each subpopulation delineated by Flk1 and GFP-Bry expression at day 3 of differentiation. The mean fluorescence intensity (MFI) is given for each graph. At day 0 of differentiation, the ES cells expressed Kit. The broken line in this graph represents the unstained control.

View larger version (22K): [in a new window]   Fig. 5. Blast potential and gene expression patterns of Flk1/GFP-Bry subpopulations. (A) At day 2.5 of differentiation, GFP-Bry EBs were harvested, dissociated and cells fractionated by cell sorting based on their respective GFP and Flk1 expression levels. (B) Blast-CFC potential of the GFP–Flk1–, GFP+Flk1– and GFP+Flk1+ populations. Colonies were scored following 4 days of culture. Data are presented as the mean number of colonies from three dishes. Bars, where visible, represent s.e. of the mean. The purity of sorted cells was 95%. (C) RNA extraction was performed on each fraction as well as the starting population (presort). Gene expression patterns were analyzed by RT-PCR using specific primers for each of the indicated genes. ß-actin expression was used to equilibrate the relative amount of each cDNA.

View larger version (38K): [in a new window]   Fig. 6. Relationship between the GFP–Flk1–, GFP+Flk1– and GFP+Flk1+ subpopulations. (A) At day 3 of differentiation, GFP-Bry EBs were harvested, dissociated and cells fractionated by cell sorting based on their respective GFP and Flk1 expression levels (pre-culture). Each subpopulation was allowed to reaggregate in culture for 20 hours. At this stage, EB-like structures were harvested, dissociated and stained for Flk1 expression (post-culture). (B) Blast-CFC potential of the GFP–Flk1–, GFP+Flk1– and GFP+Flk1+ populations immediately after cell sorting (pre-culture) or after the 20-hour culture step (post-culture). Colonies were scored after 4 days of culture. (C) Hematopoietic potential of the same fractions as above. Cells were replated in methylcellulose cultures containing cytokines appropriate for hematopoiesis differentiation. Colonies were scored after 6 to 8 days of culture and designated as follows: Mac, macrophages; Mix, multi-lineage; EryP, primitive erythroid. Data are presented as the mean number of colonies from three dishes. Bars, where visible, represent s.e. of the mean.

View larger version (29K): [in a new window]   Fig. 7. Model of ES cell differentiation to the BL-CFC.

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