Developmentally arrested Austrofundulus limnaeus embryos have changes in post-translational modifications of histone H3

Abstract

Although vertebrate embryogenesis is typically a continuous and dynamic process, some embryos have evolved mechanisms to developmentally arrest. The embryos of Austrofundulus limnaeus, a killifish that resides in ephemeral ponds, routinely enter diapause II (DII), a reversible developmental arrest promoted by endogenous cues rather than environmental stress. DII, which starts at 24-26 days post-fertilization and can persist for months, is characterized by a significant decline in heart rate and an arrest of development and differentiation. Thus, A. limnaeus is a unique model to study epigenetic features associated with embryonic arrest. To investigate chromosome structures associated with mitosis or gene expression, we examined the post-translational modifications of histone H3 (phosphorylation of serine 10, mono-, di- and tri-methylation of lysine 4 or 27) in preDII, DII and postDII embryos. As seen by microscopy analysis, DII embryos have a significant decrease in the H3S10P marker for mitotic nuclei and an inner nuclear membrane localization of the H3K27me2 marker associated with silencing of gene expression. ELISA experiments reveal that the levels of methylation at H3K4 and H3K27 are significantly different between preDII, DII and postDII embryos, indicating that there are molecular differences between embryos of different chronological age and stage of development. Furthermore, in DII embryos relative to preDII embryos, there are differences in the level of H3K27me3 and H3K4me3, which may reflect critical chromatin remodeling that occurs prior to arrest of embryogenesis. This work helps lay a foundation for chromatin analysis of vertebrate embryo diapause, an intriguing yet greatly understudied phenomenon.

Keywords: Chromatin; Diapause; Killifish.

Fig. 1.

Representative images of pre-diapause II (preDII), DII and postDII embryos. The white arrow points to the anterior region of the preDII (18 days post-fertilization, dpf) embryo; the yolk and a large lipid droplet are indicated. Given that the embryo rotates within the chorion, forceps can be used to hold the embryo steady without damaging it. The black arrow in the 9 days postDII embryo points to developing vascular tissue. Scale bar, 500 µm.

Fig. 2.

The number of mitotic blastomeres decreases as embryos progress towards DII. Embryos were fixed and stained with anti-histone H3 serine 10 phosphate (H3S10P) to detect mitotic nuclei and Hoechst 33342 to stain DNA. (A) The anterior tail region is shown for all images: 18 dpf represents a preDII embryo, 24 dpf represents an embryo in early DII, and 32 dpf is a DII embryo. Scale bar, 50 µm. Representative embryos are shown (at least 18 embryos were analyzed; ≥6 embryos per trial, N=3, for each embryo stage). (B) The number of positive H3S10P cells was quantified in the tail region of embryos at various ages of preDII and DII. The data represent at least 18 embryos (analysis of ≥6 embryos per trial, N=3, for each embryo stage). The values are means±s.e.m. Identical letters indicate embryo groups with no significant difference; different letters indicate P<0.05. The P-values were determined using one-way ANOVA followed by a Holm–Sidak multiple comparison test.

Fig. 3.

PreDII and DII embryos display a similar distribution of H3K27me3. Embryos were stained with anti-H3K27me3 and Hoechst 33342. The anterior tail region is shown for all images. Scale bar, 50 µm. The data represent at least 18 embryos (analysis of ≥6 embryos per trial, N=3, for each embryo stage).

Fig. 4.

DII embryos stained with anti-H3K27me2 show an inner nuclear membrane localization pattern. Embryos were stained with anti-H3K27me2 and Hoechst 33342. The anterior tail region is shown for all images. Scale bar, 50 µm. The data represent at least 18 embryos (analysis of ≥6 embryos per trial, N=3, for each embryo stage).

Fig. 5.

Post-translational methylation of H3K27 or H3K4 relative to histone H3, determined in preDII, DII and postDII embryos. ELISA was used to quantify (A) H3K27me1, (B) H3K27me2, (C) H3K27me3, (D) H3K4me1, (E) H3K4me2 and (F) H3K4me3, in preDII (8 and 16 dpf), DII (24 and 32 dpf) and postDII embryos. Identical letters indicate embryo groups with no significant difference; different letters indicate P<0.05. The P-values were determined using one-way ANOVA followed by a Holm–Sidak multiple comparison test. The values are means±s.e.m. The data were obtained from 80–100 embryos per trial (N=3) for each embryo stage.

Fig. 6.

HSP70 and RNA Pol II S5P levels in preDII, DII and postDII embryos. ELISA was used to quantify the levels of (A) HSP70 and (B) RNA polymerase II C-terminal domain repeat YSPTSPS serine 5 phosphate (RNA Pol II S5P) from whole-embryo lysate, relative to total protein, in preDII (8 and 16 dpf), DII (24 and 32 dpf) and postDII embryos. Identical letters indicate embryo groups with no significant difference; different letters indicate P<0.05. The P-values were determined using one-way ANOVA followed by a Holm–Sidak multiple comparison test. The values are means±s.e.m. The data were obtained from 80–100 embryos per trial (N=3) for each embryo stage.