The Role of DIF-1 Signaling in Dictyostelium Development Christopher R.L Thompson, Robert R Kay  Molecular Cell  Volume 6, Issue 6, Pages 1509-1514 (December 2000) DOI: 10.1016/S1097-2765(00)00147-7

Figure 1 The Des-Methyl-DIF-1 Methyltransferase Gene and Knockout Mutant (A) Developmental Northern blot. The dmtA mRNA is developmentally regulated and peaks at 12 hr, when tips are just forming. (B) Methyltransferase activity of the dmtA product, transcribed and translated in vitro. Methyltransferase activity was assayed by transfer of radioactive methyl groups to the substrates indicated at the top of the panel, with the products resolved by TLC. Enzyme activity was tested in a reticulocyte lysate programmed with dmtA mRNA, the lysate alone, a Dictyostelium lysate, or a no-protein control, as shown; dmtA mRNA produces an enzyme activity with the same specificity as that of the endogenous enzyme. (C) Expression of dmtA in growing cells results in their production of des-methyl-DIF-1 methyltransferase. Growing cells of an actin15-dmtA transformant were lysed, and des-methyl-DIF-1 methyltransferase activity was assayed. The TLC shows that these cells, but not their Ax2 parent, contain the activity. (D) The knockout vector, showing the site of insertion of the Bsr resistance cassette and relevant restriction sites. (E) Des-methyl-DIF-1 methyltransferase activity in high-speed supernatants of the knockout strains HM1030 and HM1031 and their parent Ax2. Substrate-independent bands on the TLC are due to methylation of endogenous substrates in the lysates. (F) Lack of detectable cell-associated DIF-1. Compounds, labeled by developing cells on 36Cl−, were extracted with chloroform/methanol, resolved by TLC, and detected by phosphorimaging. Ancymidol (a P450 inhibitor) was included in the agar, as indicated, to block DIF-1 metabolism after the formation of DIF-3. No DIF-1 or DIF-3 is detectable in the HM1030 mutant. LCCs are late-chlorinated compounds made by maturing stalk cells. Molecular Cell 2000 6, 1509-1514DOI: (10.1016/S1097-2765(00)00147-7)

Figure 2 Development of the dmtA Mutant and Rescue by DIF-1 Development of the HM1030 mutant strain was similar to wild-type Ax2 for aggregation (though there was sometimes a delay) and tip formation. First fingers were relatively long and thin; the 16 hr panel shows that the resulting slugs often broke up, and the 18 hr panel shows their stalked migration. By 24 hr the wild type had fruited, but few mutant structures had done so. The inclusion of 100 nM DIF-1 in the agar corrects the morphological defects of the mutant, and they go on to form normal fruiting bodies. Molecular Cell 2000 6, 1509-1514DOI: (10.1016/S1097-2765(00)00147-7)

Figure 3 Expression of Prestalk and Prespore Markers (A). Developmental time course of the expression of the ecmA and ecmB prestalk and the psA prespore marker mRNAs. Both prestalk markers are expressed by the HM1030 mutant. (B) Developmental time course of DIF-1 dechlorinase activity. This enzyme converts DIF-1 to DIF-3 and is one of the earliest prestalk markers; the mutant has substantially reduced levels. (C) The proportion of prestalk cells at the first finger stage. Prestalk cells are defined by lack of staining with an antibody against prespore vesicles. The mutant has a reduced complement of prestalk cells, but these can be restored by development on agar containing 100 nM DIF-1 (this amount is insufficient to affect wild-type proportioning). (D) Expression pattern in first fingers/early slugs of the intact ecmA promoter driving lacZ (ecmAO-gal) and of its pstA element (ecmA-gal). It is apparent that the HM1030 mutant has a reduced overall prestalk zone, but that the pstA zone remains. Molecular Cell 2000 6, 1509-1514DOI: (10.1016/S1097-2765(00)00147-7)

Figure 4 PstO Cells Are Abolished from the dmtA Mutant The HM1030 mutant essentially lacks pstO cells, as detected by expression of the ecmO-lacZ marker (C). PstO cells can be restored by mixed development with wild-type (F) or by development on 100 nM DIF-1 (D). Cells were developed on agar plates containing KK2 to the slug/first finger stage before fixation and staining. Cells in panels A, C, E, and G were developed without DIF-1, and panels B, D, F, and H were developed with 100 nM DIF-1 in the agar. Molecular Cell 2000 6, 1509-1514DOI: (10.1016/S1097-2765(00)00147-7)