Fig 4. Temporal regulation of Nacα-RNAi expression in the heart.

Using a temperature inducible driver specifically in the heart (HTT, Hand4.2-GAL4, tubulin-GAL80^ts; tubulin-GAL80^ts), Nacα-RNAi was expressed during specific stages of development by controlling ambient temperature to determine its contribution to cardiogenesis. Controls lacking RNAi are kept in similar temperature conditions to account for any developmental effects of temperature on the heart. A-H. Phalloidin staining to visualize cytoskeletal structural effects of Nacα knockdown (KD). A, As a test of GAL80 control of transcription, flies held at 18°C throughout development did not produce changes to heart structure indicating an inhibition of Nacα-RNAi transcription. B, Exposing flies to high temperatures (28°C) throughout development produced a no heart phenotype similar to the effects of driving Nacα-RNAi using Hand4.2-GAL4 alone, suggesting an induction of Nacα-RNAi transcription and subsequent Nacα KD with exposure to higher temperatures. * indicates absence of the heart. C, Exposing flies to high temperature during adulthood only for 1 week, D, pupae to eclosion, or E, mid-larvae to eclosion did not produce gross structural defects in the heart. F, Exposing embryos to high temperatures starting at egg-lay up until 24 hours resulted in the absence of the terminal chamber, indicated by white bar. Only thin alary muscles were present. G, Extending the high temperature exposure to 48 hours led to similar loss of the posterior heart, indicated by white bar. H, Only when the hearts were exposed to higher temperatures during embryonic stage (24 hours) and pupal stage until eclosion (~3 days), were we able to recapitulate the no heart phenotype produced by exposing the heart to constant high temperatures. I, Functional analysis of the adult heart following Nacα KD at various developmental stages. Maintaining flies at 18°C throughout development or exposure of adult flies to high temperature for 1 week led to no changes in diastolic diameter, systolic diameter, or fractional shortening. High temperature exposure from pupae to eclosion or from mid-larvae to eclosion led to subtle changes in diameters and fractional shortening. Exposure of embryos to high temperatures for 24hr led to constricted diastolic and systolic diameters. * p<0.05, ** p<0.01, *** p<0.001.