Figure 1. RasGEFE mutant cells are enriched in a genetic screen for modulators of nutritional bias.

(A) G− cells produce fewer spores than G+ cells in chimeric development. GFP-labelled Ax3 wild type cells were grown in either G+ or G− conditions and mixed 10:90 with wild type G+ cells. GFP spores were quantified by counting. Dotted line indicates the percentage GFP spores expected if there is no fate bias. Error bars represent SEM, p<0.0001. (B) G− growth biases cells towards pstO and pstB cell fates. Diagram shows organisation of different cell types along the anterior-posterior axis of the Dictyostelium slug. Patterning of GFP-labelled (*) G+ (i) and G− (ii) cells when mixed at 10:90 ratio with G+ cells. The reciprocal pattern was observed when GFP-labelled G+ (iii) and G− (iv) were mixed at 10:90 ratio with G− cells. (C) Schematic diagram of the genetic selection. REMI mutant cells were grown in G− and mixed 10:90 with wild type G+ GFP cells. Chimeric fruiting bodies were harvested and spores returned to growth medium after each developmental cycle. Wild type cells were removed with Blasticidin. (D) Generation of gefE⁻ mutants. REMI plasmid, pBSR1, inserted into 42 bp exon 1. RasGEF catalytic domain (blue) deleted by homologous recombination. (E) gefE⁻ mutant cells produce more spores than Ax3 wild type cells after G− growth. RFP-labelled wild type cells were grown in G+ medium and mixed at a 50:50 ratio with unlabelled wild type or gefE⁻ mutant cells grown in G−. Number of unlabelled spores was quantified by counting. Error bars represent SEM, p<0.04. (F) Comparison of the patterning of GFP-labelled (*) wild type (i) or gefE⁻ mutant (ii) cells grown in G− conditions when mixed at 10:90 ratio with unlabelled wild type G+ cells. AP axis in all slug images oriented from right-left with white bars showing regions of GFP enrichment.

DOI: http://dx.doi.org/10.7554/eLife.01067.003

Figure 1—figure supplement 1. Organisation of cell types in Dictyostelim slug and culminants.

Nomenclatures for parts of Dictyostelium slug (left) and fruiting body (right) are depicted. Yellow (prespore) cells eventually form sorus and red cells (pstA or AB) form stalk cells. Blue (pstO) and green (pstB) cells are the DIF dependent cell types which eventually form upper/lower cup and basal disc. ecmA promoter can be subdivided to pstA and O, and ecmB promoter to pstAB and pstB cell types.

DOI: http://dx.doi.org/10.7554/eLife.01067.004

Figure 1—figure supplement 2. Cell type specific effects of nutritional history in Dictyostelium slugs.

(A) and culminants (B) 10% labelled G+ cells (upper panels) or G− cells (lower panels) mixed with 90% unlabelled G+ or G− cells. Closed arrows indicate enrichment of label, open arrows an absence. In each panel, the top row shows the control mix and is comparable to the image directly beneath. General patterning differences between G+ and G− cells (A–D) are described in the main text and Figure 1. G+ cells labelled with ecmAO:lacZ occupy anterior-most pstA regions when mixed with G− cells (E and F). In the reciprocal mix, G− cells labelled with ecmAO:lacZ were absent from the tip and formed a collar in the pstO regions when mixed with unlabelled G+ cells (G and H). Expression of ecmB:lacZ was decreased in pstB regions of chimeras when labelled G+ cells were mixed with unlabelled G− mutant cells (I and J) and increased when labelled G− mutant cells were mixed with unlabelled G+ cells (K and L). Wild type cells labelled with pspA:lacZ occupy the prespore region when mixed with unlabelled G− cells (M and N). In the reciprocal mix, where G− cells labelled with pspA:lacZ were mixed with unlabelled G+ cells, expression was greatly reduced and restricted to the rear of the prespore compartment (O and P). The AP axis of all structures is aligned from right-left. Refer to Figure 1—figure supplement 1 to link marker expression to cell type localization within slug or culminant.

DOI: http://dx.doi.org/10.7554/eLife.01067.005

Figure 1—figure supplement 3. Enrichment of REMI mutants during screen correlates with change in cell fate preference during development.

(A) Patterning phenotype of REMI mutant pools in chimera with wild type cells. 10% wild type GFP cells were mixed with 90% REMI mutant cells from each pool at each round of selection. The dark regions in each slug show the location of the REMI mutant cells. In Rd0, REMI mutant cells localize in the pstO and pstB regions. By Rd7, wild GFP cells are almost entirely absent from prespore compartment, especially in pool 2. In pool 1 (top row), REMI mutant cells are enriched in the collar and back until Rd3. By Rd6 and 7, wild type GFP cells are enriched in the anterior prestalk region, suggesting the REMI mutant cells are in the prespore population. (B) Proportion of REMI mutants identified by iPCR that mapped to exon 1 of gefE locus in each round of selection. (C) Measurements of growth rate of wild type and gefE⁻ mutant cells in G+ or G− medium. (D) Measurement of spore hatching rate of wild type and gefE⁻ mutant cells. The percentage of hatched spores was scored at the indicated times after spores were placed in growth medium. The cell density after 24 hr of induction was also measured, revealing that the faster hatching rate of gefE⁻ mutant resulted in approximately twofold higher cell density after 24 hr. Error bars represent SEM.

DOI: http://dx.doi.org/10.7554/eLife.01067.006