DEV017558 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Control experiments to confirm that GFP-PCNA is a single-cell marker of S phase in Fucus serratus zygotes. (A,B) Zygote co-loaded biolistically 2 hours after fertilization with both (A) GFP and (B) Texas Red dextran. These two markers co-localize, unlike GFP-PCNA and Texas Red dextran (compare with Fig. 1A), suggesting that the nuclear localization signal on the GFP-PCNA construct is functional. (C,D) Four-hour-old zygote microinjected 2 hours after fertilization with GFP-PCNA. Clear nuclear localization may be seen, which agrees with Fig. 1A. In addition, this image shows a separate area (D) of high intensity within the zygote nucleus. The identity of this area is not clear, but the most likely explanation is that it represents the sperm pronucleus immediately after pronuclear fusion. (E) Timecourse to show GFP-PCNA changes in a microinjected zygote. The zygote was microinjected 2 hours after fertilization (AF) and the nuclear GFP-PCNA signal can be seen to rise for 2 hours before falling. Microinjection is more demanding than biolistic loading and zygotes could not be injected less than 2 hours after fertilization.

• Supplemental Figure S2 -

  Fig. S2. Control experiments to confirm the timing of S phase in F. serratus zygotes. (A) Hoechst 33342 labelling reports increases in nuclear DNA content after fertilization. F. serratus zygotes were labelled with 100 µM Hoechst 33342 for 15 minutes at various times post-fertilization. Hoechst 33342 was excited with two-photon excitation as described in the Materials and methods. Images show different representative zygotes − it was not possible to follow a single individual throughout the full time course owing to relatively poor Hoechst loading and consequent high laser powers, leading to compromised cell integrity. (B) Timecourse to show the timing of S-phase onset. The nuclear signals from Hoechst 33342 images, such as those shown in A, were quantified in the presence and absence of 20 µM aphidicolin (applied from 1 hour after fertilization). Aphidicolin inhibits the increases in nuclear DNA content seen from 4 hours after fertilization, suggesting that S phase begins between 3 and 4 hours after fertilization. Data are given as mean±s.e.m. *P<0.05 (decreases relative to control.)