Hematopoietic Gene Expression During Embryoid Body Development in Control and scl-Null ES Cells
| Gene . | Gene Expression During ES Cell Differentiation (days)* . | |||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| . | 0 . | 4 . | 6 . | 8 . | 12 . | Induction† . | Ratio‡ . | . | . | |||||
| . | c . | c . | null . | c . | null . | c . | null . | c . | null . | c . | null . | c:null . | . | . |
| Mesodermal markers | ||||||||||||||
| Brachyury | − | + | + | ++ | ++ | ± | ± | nd | nd | >500 | >500 | ∼1 | ||
| BMP R1A | + | ++ | ++ | ++ | ++ | +++ | +++ | nd | nd | ∼5 | ∼5 | ∼1 | ||
| BMP-4 | ± | ± | ± | + | + | ++ | ++ | nd | nd | ∼50 | ∼50 | ∼1 | ||
| Smad1 | + | + | + | ++ | ++ | ++ | ++ | ++ | ++ | >5 | >5 | ∼1 | ||
| Transcription factors | ||||||||||||||
| scl | − | ± | − | + | − | ++ | − | + | − | >500 | 0 | >500 | ||
| GATA-1 | ± | ± | ± | ± | ± | ++ | ± | ++ | ± | >5 | ∼1 | >5 | ||
| GATA-2 | − | + | + | ++ | ++ | ++ | ++ | ++ | ++ | >50 | >50 | ∼1 | ||
| EKLF | + | + | + | + | + | ++ | + | ++ | + | >5 | ∼1 | >5 | ||
| NF-E2 | ± | + | + | + | + | ++ | + | ++ | ++ | >5 | >5 | ∼1 | ||
| c-myb | + | ++ | ++ | + | + | + | ± | nd | nd | <5 | ∼1ρ | <5 | ||
| PU.1 | ± | ± | ± | ± | ± | + | ± | + | ± | >5 | ∼1 | >5 | ||
| Receptors | ||||||||||||||
| CD34 | ± | ± | ± | ± | ± | + | ± | ++ | ++ | >50 | >50 | ∼1 | ||
| flk-1 | − | + | + | +++ | +++ | +++ | +++ | nd | nd | >50 | >50 | ∼1 | ||
| flk-2 | ± | ± | ± | ± | ± | ± | ± | ± | ± | >5 | >5 | <5 | ||
| c-kit | ++ | ++ | ++ | ++ | ++ | ++ | ++ | nd | nd | ∼1 | ∼1 | ∼1 | ||
| Epo R | + | ++ | ++ | ++ | ++ | ++ | ++ | nd | nd | >5 | >5 | ∼1 | ||
| Erythroid and myeloid genes | ||||||||||||||
| ζ globin | − | − | − | ± | − | +++ | − | +++ | − | >500 | 0 | >500 | ||
| α globin | ± | ± | ± | ± | ± | +++ | ± | +++ | ± | >500 | ∼1 | >500 | ||
| βH1 globin | − | − | − | ± | − | +++ | − | +++ | − | >500 | 0 | >500 | ||
| ε2 globin | − | − | − | − | − | +++ | − | +++ | − | >500 | 0 | >500 | ||
| βmaj globin | − | − | − | − | − | +++ | − | +++ | − | >500 | 0 | >500 | ||
| MPO | − | ± | ± | ± | ± | + | ± | + | ± | >50 | <5 | >5 | ||
| Lysozyme | − | ± | ± | ± | ± | + | + | nd | nd | >50 | >50 | <5 | ||
| vav | + | + | + | + | + | + | ± | + | ± | ∼1 | 0ρ | >5 | ||
| LMO2 | − | + | + | ++ | ++ | ++ | ++ | nd | nd | >500 | >50 | <5 | ||
| Gene . | Gene Expression During ES Cell Differentiation (days)* . | |||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| . | 0 . | 4 . | 6 . | 8 . | 12 . | Induction† . | Ratio‡ . | . | . | |||||
| . | c . | c . | null . | c . | null . | c . | null . | c . | null . | c . | null . | c:null . | . | . |
| Mesodermal markers | ||||||||||||||
| Brachyury | − | + | + | ++ | ++ | ± | ± | nd | nd | >500 | >500 | ∼1 | ||
| BMP R1A | + | ++ | ++ | ++ | ++ | +++ | +++ | nd | nd | ∼5 | ∼5 | ∼1 | ||
| BMP-4 | ± | ± | ± | + | + | ++ | ++ | nd | nd | ∼50 | ∼50 | ∼1 | ||
| Smad1 | + | + | + | ++ | ++ | ++ | ++ | ++ | ++ | >5 | >5 | ∼1 | ||
| Transcription factors | ||||||||||||||
| scl | − | ± | − | + | − | ++ | − | + | − | >500 | 0 | >500 | ||
| GATA-1 | ± | ± | ± | ± | ± | ++ | ± | ++ | ± | >5 | ∼1 | >5 | ||
| GATA-2 | − | + | + | ++ | ++ | ++ | ++ | ++ | ++ | >50 | >50 | ∼1 | ||
| EKLF | + | + | + | + | + | ++ | + | ++ | + | >5 | ∼1 | >5 | ||
| NF-E2 | ± | + | + | + | + | ++ | + | ++ | ++ | >5 | >5 | ∼1 | ||
| c-myb | + | ++ | ++ | + | + | + | ± | nd | nd | <5 | ∼1ρ | <5 | ||
| PU.1 | ± | ± | ± | ± | ± | + | ± | + | ± | >5 | ∼1 | >5 | ||
| Receptors | ||||||||||||||
| CD34 | ± | ± | ± | ± | ± | + | ± | ++ | ++ | >50 | >50 | ∼1 | ||
| flk-1 | − | + | + | +++ | +++ | +++ | +++ | nd | nd | >50 | >50 | ∼1 | ||
| flk-2 | ± | ± | ± | ± | ± | ± | ± | ± | ± | >5 | >5 | <5 | ||
| c-kit | ++ | ++ | ++ | ++ | ++ | ++ | ++ | nd | nd | ∼1 | ∼1 | ∼1 | ||
| Epo R | + | ++ | ++ | ++ | ++ | ++ | ++ | nd | nd | >5 | >5 | ∼1 | ||
| Erythroid and myeloid genes | ||||||||||||||
| ζ globin | − | − | − | ± | − | +++ | − | +++ | − | >500 | 0 | >500 | ||
| α globin | ± | ± | ± | ± | ± | +++ | ± | +++ | ± | >500 | ∼1 | >500 | ||
| βH1 globin | − | − | − | ± | − | +++ | − | +++ | − | >500 | 0 | >500 | ||
| ε2 globin | − | − | − | − | − | +++ | − | +++ | − | >500 | 0 | >500 | ||
| βmaj globin | − | − | − | − | − | +++ | − | +++ | − | >500 | 0 | >500 | ||
| MPO | − | ± | ± | ± | ± | + | ± | + | ± | >50 | <5 | >5 | ||
| Lysozyme | − | ± | ± | ± | ± | + | + | nd | nd | >50 | >50 | <5 | ||
| vav | + | + | + | + | + | + | ± | + | ± | ∼1 | 0ρ | >5 | ||
| LMO2 | − | + | + | ++ | ++ | ++ | ++ | nd | nd | >500 | >50 | <5 | ||
Abbreviations: ES, embryonic stem; BMP, bone morphogenetic protein; Epo, erythropoietin; MPO, myeloperoxidase.
Gene expression profiles determined by RT-PCR after in vitro differentiation of ES cells. The mean level of expression is compared between two (day 12 samples) or three lines with at least one wild-type scl allele (c) and three lines with both scl alleles deleted (null). Samples were transferred to nylon membranes after electrophoresis, probed with 32P end-labeled specific internal oligonucleotides, and the signals quantitated on a phosphorimager. Scoring was as follows: −, no signal; ±, signal detected after hybridization only; +, ++, +++, signal of increasing intensity visible on ethidium bromide stained gel; nd, not determined.
Gene induction (fold) during differentiation compared with levels in undifferentiated ES cells.
The ratio of mean signal intensity in control versus null lines calculated at day 8 or 12 of differentiation.
ρ After initial induction, expression fell in null cell lines. See also Fig 2E and G.