Figure 5. NO and PKG Are Utilized in a Hypoxic Block to S Phase.

(A and B) Hypoxia blocks S phase. Wild-type Sevelen embryos (stage 8) show BrdU incorporation into S phase cells during a 5 min labeling period (A). This incorporation is blocked when embryos are labeled from 5 to 10 min following imposition of hypoxia.

(C and D) The hypoxic block to S phase was mimicked by the ectopic expression of iNOS. Embryos (stage 10) carrying a hs-iNOS transgene were exposed to heat shock for 30 min at 37°C, allowed to recover, and then assayed for S phase using BrdU (D). Control embryos treated similarly (not shown) or nonheat-shocked transgenic embryos (C) had typical patterns of BrdU incorporation (Edgar and O’Farrell, 1990).

(E and F) The addition of the NO donor SNAP to stage 12 embryos resulted in a complete inhibition of BrdU incorporation. Embryos were exposed to 5 mM SNAP for 15 min prior to labeling with BrdU (F). Control embryos were left untreated (E).

(G and H) The addition of 8Br-cGMP to stage 14 embryos also resulted in a reduction of S phase as assayed by BrdU incorporation. Embryos were incubated in 5 mM 8Br-cGMP for 60 min prior to treatment with BrdU to detect S phase (H). Control embryos were similarly treated but incubated with 5 mM GMP (G).

(I–N) Inhibition of NO or PKG either genetically or pharmacologically resulted in a failure to block S phase during hypoxia. CNS (optic lobes shown) dissected from for^R larvae showed a block to S phase when exposed to hypoxia (I and K), whereas CNS dissected from for^s larvae continued to progress through S phase under hypoxia (J and L). Stage 11/12 embryos (360–480 min AED) that were incubated in 0.1 M D-NAME prior to hypoxia showed a block to S phase during hypoxia (M), whereas incubation of embryos in 0.1 M L-NAME allowed for continued BrdU incorporation during hypoxia (N). Scale bar is 50 μm. In (A–H) and (M and N), anterior is to the left and dorsal toward the top.