Fig. 1

Experimental design for genome-wide RNAi screens to identify genes that interact with LKB1/par-4 or AMPK/aak(0). All the strains used for the three genome-wide screens contain a temperature-sensitive daf-2(e1370) mutation that allowed us to induce dauer by shifting synchronized L1 larvae to 25 °C. Importantly, all the strains carry a lag-2::GFP transgene that is expressed in the DTCs, marking the distal extremities of the germ line. This expression allowed us to indirectly evaluate the degree of germline stem cell hyperplasia [15]. a The screen was designed to identify genes that result in dauer germline hyperplasia when their function is compromised by RNAi. daf-2 mutants carrying the lag-2::GFP transgene were synchronized and L1 larvae were then put on plates containing IPTG to induce dsRNA expressed from each bacterial clone and were shifted to 25 °C to induce dauer formation. The dauer larvae were subsequently screened for increased DTC displacement; a proxy for germline hyperplasia [15]. The diagrams in b and c describe screens designed to identify suppressors of par-4- and aak(0)-induced dauer germline hyperplasia, respectively. For b, the screen was performed with a strain that contains a temperature-sensitive par-4 mutation, while in c an AMPK null mutant (aak(0)) that harboured deletions in both catalytic subunits was used. Upon dauer induction, animals were monitored for reduced displacement between the DTCs indicating suppression of the germline hyperplasia typical of both par-4 and aak(0) mutants. See Methods for more details