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First published online 2 June 2004
doi: 10.1242/dev.01198

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Homotypic signalling regulates Gata1 activity in the erythroblastic island

Erasmus MC, Department of Cell Biology, PO Box 1738, 3000 DR Rotterdam, The Netherlands

View larger version (21K): [in a new window]   Fig. 1. (A) Summary of the different Gata1 mutants discussed and their outcome in terms of erythroid differentiation. FL, floxed; OX, overexpressing; KO, deleted Gata1 allele; WT, wild type. The progeny obtained in the crossing described in the present manuscript is indicated a-f, in accordance with Fig. 3A, Fig. 4C and Fig. 5A. (B) REDS signalling and erythroid differentiation. Heterotypic and homotypic cell-cell signalling mechanisms. 1 and 2 are different type of cells.

View larger version (32K): [in a new window]   Fig. 2. Recombination of the Gata1 floxed locus. (A) Maps of the floxed and the knockout Gata1 locus after recombination. N, NcoI; B, BamHI, X, XbaI; En2, murine engrailed 2 intronic sequence; SA, splice acceptor; GFP, green fluorescence protein. LoxP sites are indicated. (B) DNA samples of 13.5 dpc foetuses were digested with NcoI and blotted against the En2 probe for quantifying recombination of the Gata1 floxed locus. 1, XY wild-type male head DNA control; 2, XFLX female head DNA control; 3, XKOX female foetal liver DNA.

View larger version (47K): [in a new window]   Fig. 3. (A) Crossing strategy and phenotype of the different Gata1 mutant foetuses obtained at 13.5 dpc. A photograph of the foetus and foetal liver is included for each genotype. Mice a and d are wild type or Gata1-overexpressing, respectively, regardless of expression of the pEV-Cre transgene (in brackets). a-f are as in Fig. 1A, Fig. 4C and Fig. 5A. (B) Total cell number in foetal blood at 14.0 dpc. Average and s.d. are indicated. At least three foetuses were analysed per group.

View larger version (27K): [in a new window]   Fig. 4. (A) CASY analysis: the ratio of definitive versus primitive erythrocytes in foetal blood was compared at 13.5 and 14.0 dpc. The percentage of definitive cells in blood in each genotype at 13.5 dpc and 14.0 dpc is depicted below the graph. Average and s.d. are indicated. At least three foetuses were analysed per group. (B) CASY analysis: example of CASY graphs from wild-type male and compound female at 13.5 dpc. Peaks corresponding to enucleated and nucleated cells are indicated. The peak below 5 µm corresponds to cell debris. (C) Blood cytospins of each genotype at 13.5 dpc showing primitive nucleated erythrocyte (1), definitive enucleated erythrocyte (2), erythroid precursor (arrowhead). Pictures were taken at 100x magnification.

View larger version (67K): [in a new window]   Fig. 5. (A) Foetal liver cytospins of each genotype at 14.0 dpc. Consecutive differentiation stages are indicated in photograph c and f: large proerythroblast (1), basophilic erythroblast (2), polychromatic erythroblast (3) (which exhibit both basophilia and benzidine positivity), orthochromatic erythroblast (4) (which are strongly benzidine positive) and enucleated definitive erythrocyte (5). Pictures were taken at 100x magnification. (B,C) FACS analysis of (B)13.5 and (C) 14.0 dpc foetal liver cells at the day of collection. Percentage of alive erythroid (TER119+/7AAD–), alive non-erythroid (TER119–/7AAD–), dead erythroid (TER119+/7AAD+) and dead non-erythroid (TER119–/7AAD+) cells are depicted. Average and s.d. are indicated. At least three foetuses were analysed per group.

View larger version (20K): [in a new window]   Fig. 6. FACS analysis of foetal liver cells at 14.0 dpc after 2 days of hanging drop culture to induce differentiation. Differentiation is estimated as the percentage of TER119+/7AAD–/FSClow of the 50,000 events measured in total (light-grey bars) and as the percentage of TER119+/FSClow of the 7AAD– (alive) cells measured (dark-grey bars). Average and s.d. are indicated. At least three foetuses were analysed per group.

View larger version (38K): [in a new window]   Fig. 7. Gata1 levels decrease during terminal erythroid differentiation. (A) Whole cell extracts from wild-type foetal liver cells, after day 0, 1 and 2 of hanging drop culture were analysed by western blotting using antibodies against Gata1 with nucleophosmin as loading control. Two samples at day 0 are shown and cultures were done in triplicate. (B) Jo2 treatment of wild-type cells. Whole cell extracts at day 2 after standard (St) culture and culture with Jo2 1 (20 µg/ml) or Jo2 2 (40 µg/ml) were analysed by western blotting using antibodies against Gata1, and nucleophosmin as a loading control. (C) Jo2 treatment of erythroid cells from Gata1-overexpressing foetuses. Whole cell extracts at day 0 and 2 after standard culture and culture with Jo2 1 (20 µg/ml) were analysed by western blotting using antibodies against Gata1 with nucleophosmin as a loading control. Extracts from wild-type, XOXX female and XOXY male foetal liver cells are shown.