Cell cycle arrest associated with anoxia-induced quiescence, anoxic preconditioning, and embryonic diapause in embryos of the annual killifish Austrofundulus limnaeus

Abstract

Embryos of the annual killifish Austrofundulus limnaeus can enter into dormancy associated with diapause and anoxia-induced quiescence. Dormant embryos are composed primarily of cells arrested in the G(1)/G(0) phase of the cell cycle based on flow cytometry analysis of DNA content. In fact, most cells in developing embryos contain only a diploid complement of DNA, with very few cells found in the S, G(2), or M phases of the cell cycle. Diapause II embryos appear to be in a G(0)-like state with low levels of cyclin D1 and p53. However, the active form of pAKT is high during diapause II. Exposure to anoxia causes an increase in cyclin D1 and p53 expression in diapause II embryos, suggesting a possible re-entry into the cell cycle. Post-diapause II embryos exposed to anoxia or anoxic preconditioning have stable levels of cyclin D1 and stable or reduced levels of p53. The amount of pAKT is severely reduced in 12 dpd embryos exposed to anoxia or anoxic preconditioning. This study is the first to evaluate cell cycle control in embryos of A. limnaeus during embryonic diapause and in response to anoxia and builds a foundation for future research on the role of cell cycle arrest in supporting vertebrate dormancy.

Fig 1

Sampling regimen for embryos exposed to anoxia and subsequent recovery from anoxia for western blot analysis.

Fig 2

Flow cytometry analysis of DNA content (A) in cells isolated from A. limnaeus embryos during early development through diapause II (24 dpf) that were exposed to normoxia (green) or 24h of anoxia (red). Most cells have a diploid amount of DNA and thus are in the G₁/G₀ phase of the cell cycle during early development through diapause II. (B) There is an increase in the proportion of cells that are observed in G₁ (white) and a decrease in those in G₂ (gray) as embryos complete early development and enter diapause II. Bars are means ± range (n=2).

Fig 3

Flow cytometry analysis of DNA content (A) in cells isolated from post-diapause II embryos exposed to normoxia (N) or 24 h of anoxia (A). The age of the embryos is listed above the panels. Three independent replicates (n=3) are presented for each stage (green, red, blue). (B) The proportion of cells in G₁ (white) and G₂ (gray) is unaltered by exposure to anoxia (two-way ANOVA, p=0.15), but is significantly different in the different post-diapause II embryos incubated aerobically (Two-way ANOVA, F(4,10)=148.7, p<0.0001). Bars are means ± S.E.M. (n=3).

Fig 4

Survival of 12 dpd embryos of A. limnaeus exposed to anoxic preconditioning (48 h anoxia + 24 h recovery) is significantly enhanced (two-way ANOVA, F(7,56)=122.57, p<0.0001; Probit regression relative median potency analysis, p<0.05) in preconditioned (LT₅₀ = 10.2±0.8 d) compared to control embryos (LT₅₀ = 7.4±0.7 d). Symbols represent means±S.E.M. (n=5).

Fig 5

Protein expression of p53 in diapause II, 4 dpd and 12 dpd embryos exposed to normoxia, anoxia, or anoxic preconditioning (AP) as described in Fig 1. Diapause II, 4 dpd, and 12 dpd embryos were exposed to aerobic incubation (Normoxia), anoxia equal to 0.5*LT₅₀ (21/32 d or 72 h Anoxia), and 24 h of recovery from the anoxic exposure (24 h Rec.). Embryos at 12 dpd were also exposed to anoxic preconditioning (AP, 48 h anoxia + 24 h aerobic recovery) prior to exposure to anoxia for 0.5*LT₅₀ (72 h Anoxia) and subsquent aerobic recovery for 24 h (24 h Rec.). Bars represent means ± S.E.M. (n=3). A significant increase in p53 expression (asterisks) was observed in diapause II embryos exposed to anoxia (Dunnett’s post hoc, p<0.05). Diapause II embryos have signficantly less p53 under normoxic conditions compared to either post-diapause II stage (bars with different letters are statisically different, Tukey’s post hoc, p<0.05). Representative western blots are provided above the graph.

Fig 6

Cyclin D1 expression in diapause II, 4 dpd and 12 dpd embryos exposed to normoxia, anoxia, or anoxic preconditioning (AP) as described in Figure 1. Diapause II, 4 dpd, and 12 dpd embryos were exposed to aerobic incubation (Normoxia), anoxia equal to 0.5*LT₅₀ (21/32 d or 72 h Anoxia), and 24 h of recovery from the anoxic exposure (24 h Rec.). Embryos at 12 dpd were also exposed to anoxic preconditioning (AP, 48 h anoxia + 24 h aerobic recovery) prior to exposure to anoxia for 0.5*LT₅₀ (72 h Anoxia) and subsquent aerobic recovery for 24 h (24 h Rec.). Bars represent means ± S.E.M. (n=3). Anoxia causes a statistically significant increase in cyclin D1 levels (asterisks) in diapause II embryos (Dunnett’s post hoc, p<0.05). Normoxic 4 dpd embryos have significantly higher levels of cyclin D1 compared to embryos in diapause II and at 12 dpd (bars with different letters are statistically different, Tukey’s post hoc, p<0.05). Representative western blots are provided above the graph.

Fig 7

pAKT protein levels in normoxic embryos of A. limnaeus during embryonic development. Aside from the large spike observed in embryos 12 dpd, levels of pAKT are high in diapause II embryos and fall during post-diapause II development. Symbols represent mean ± S.E.M. (n=4). Representative western blots are provided above the graph.

Fig 8

Levels of pAKT plummet in 12 dpd embryos of A. limnaeus exposed to 48 h of anoxia and do not return to control levels after 24 h of aerobic recovery. Symbols represent mean ± S.E.M. (n=3). 1R = 48 h or anoxia + 1 h of aerobic recovery, 6R = 48 h of anoxia + 6 h of aerobic recovery, 24R = 48 h of anoxia + 24 h of aerobic recovery. Representative western blots are provided above the graph.