Su TT (2010), The effect of a DNA damaging agent on embryonic...

XB-ART-41815

PLoS One 2010 Jul 08;57:e11760. doi: 10.1371/journal.pone.0011760.

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The effect of a DNA damaging agent on embryonic cell cycles of the cnidarian Hydractinia echinata.

Su TT .

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The onset of gastrulation at the Mid-Blastula Transition can accompany profound changes in embryonic cell cycles including the introduction of gap phases and the transition from maternal to zygotic control. Studies in Xenopus and Drosophila embryos have also found that cell cycles respond to DNA damage differently before and after MBT (or its equivalent, MZT, in Drosophila). DNA checkpoints are absent in Xenopus cleavage cycles but are acquired during MBT. Drosophila cleavage nuclei enter an abortive mitosis in the presence of DNA damage whereas post-MZT cells delay the entry into mitosis. Despite attributes that render them workhorses of embryonic cell cycle studies, Xenopus and Drosophila are hardly representative of diverse animal forms that exist. To investigate developmental changes in DNA damage responses in a distant phylum, I studied the effect of an alkylating agent, Methyl Methanesulfonate (MMS), on embryos of Hydractinia echinata. Hydractinia embryos are found to differ from Xenopus embryos in the ability to respond to a DNA damaging agent in early cleavage but are similar to Xenopus and Drosophila embryos in acquiring stronger DNA damage responses and greater resistance to killing by MMS after the onset of gastrulation. This represents the first study of DNA damage responses in the phylum Cnidaria.

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	Figure 1. MMS inhibits mitosis in gastrula and cleavage stage embryos.(A, B) Gastrula stage embryos were incubated in filtered seawater (FSW) containing 0 (- MMS) or 0.1% (+ MMS) MMS for one hour before fixing and staining with antibodies to detect phospho-Histone H3 (red) and β-tubulin (green). The embryos are also stained with bisbenzamide to visualize DNA. (C) Mitotic index after 1-hour incubation in FSW containing various concentrations of MMS is quantified. The data are from 2690 cells in 11 embryos (0%), 3055 cells in 10 embryos (0.01%) and 2892 cells in 11 embryos (0.1%) in two different experiments. Error bars represent one standard deviation each. (D, E) Histograms show the percent of embryos that show nuclei number (n) per embryo as indicated when 2-cell (D) and 4-cell (E) stage embryos were incubated in 0 or 0.1% MMS in FSW for one hour, fixed and stained as in A and B. Mitotic figures from prophase to anaphase were counted as one nucleus each. Telophases (see Supplemental Figure S2) were counted as two nuclei each. Additional information on these data sets are in Table 1.
	Figure 2. Chromosome segregation failure in the presence of MMS and caffeine.4-cell stage embryos were incubated for one hour in FSW containing various drugs, fixed, and stained for PH3 (red) and β-tubulin (green). Anaphase figures are shown. Arrowheads indicate the metaphase plate and arrows indicate the leading edge of segregating chromosomes. (A, B) Control (A) and 0.1% MMS-treated (B) embryos show successful chromosome separation with little or no chromosome material remaining at the metaphase plate. PH3 signal is shown magnified in A' and B' respectively. (C, C') The presence of 5 mM caffeine in addition to MMS led to chromosome segregation failure. C' shows the PH3 signal after magnification. The extent of chromosome separation is similar to that in A' (compare leading edged), but most of the PH3 signal remains at the metaphase plate (arrowheads). (D, D') 5 mM caffeine alone allows successful chromosome separation. This figure is in later stage of anaphase then the preceding ones. Scale bar = 10 µM in A-D, 4 µM in A'-D'.
	Figure 3. Survival after MMS treatment of cleavage and gastrula stage embryos.(A, B, D, E) Embryos in the 4-cell stage were treated with 0% (−) or 0.1% (+) MMS for one hour. Embryos were transferred to drug-free FSW and examined after 24 hr. All of the control 4-cell stage embryos survived as indicated by their ability to reach the gastrula and planula stages (A, B). Most drug-treated 4-cell embryos did not survive as indicated by signs of disintegration (D, E). (C, F) Gastrula stage embryos at 14–16 hr after the first sign of spawning were treated with MMS and their survival examined as above. All of the control embryos and most of the MMS-treated embryos survived as indicated by their ability to reach the swimming planula stage. Development appears slower after incubation in MMS, but this issue remains to be studied rigorously. Animals in C and F were of similar size but oriented differently. Scale bar = 200 µM.

References [+] :

Conn, The DNA damage checkpoint in embryonic cell cycles is dependent on the DNA-to-cytoplasmic ratio. 2004, Pubmed, Xenbase