Fig. 6. Spt6 and Spt5 are required for an efficient heat shock response. (A-L) Lateral views of live embryos at 20 hpf (22-somite stage), anterior towards the left. Green fluorescence indicates production of GFP after a 1 hour heat shock and a 0.5 hour recovery period. Autofluorescence (yellow-green) is also visible in embryonic yolks. All photographs were taken with the same exposure time, and embryonic genotypes were confirmed after photography (see Materials and Methods). Genotypes are wild-type embryos (A,E,I); pan mutants (B,F,J); fog^s30 mutants (C,G,K); and fog^s30;pan double mutants (D,H,L). All embryos are heterozygous for the hsp70-egfp transgene (Halloran et al., 2000). Embryos maintained at normal temperature do not produce detectable GFP (A-D). After heat shock, pan and fog mutants produce less GFP than wild-type embryos, and double mutants do not produce any detectable GFP (E-H, with higher magnification views of the tail in I-L). Similar results were obtained when heat shock was administered from 15-16 hpf, before mutant phenotypes are morphologically apparent (data not shown). (M) Spt6 and Spt5 are required for efficient induction of hsp70 expression during a 1 hour heat shock. Graph of induction of endogenous hsp70 expression, detected by real-time RT-PCR. (See Materials and Methods for details of technique and data processing.) Wild-type embryos (black) induce significantly higher levels of hsp70 mRNA with more rapid kinetics than pan mutants (green) or fog^s30 mutants (blue). hsp70 induction is further inhibited in fog^s30;pan double mutants (red). Each data point represents the average degree of induction of hsp70 expression at a particular time point, relative to the low, but detectable, levels of hsp70 expression at time zero. Standard deviation from the mean is indicated by error bars. To account for small variances in RNA extraction and cDNA synthesis, levels of hsp70 expression were normalized relative to levels of stable ß-actin expression (see Materials and Methods).