Journal: PLoS Biology

Article Title: A systemic cell cycle block impacts stage-specific histone modification profiles during Xenopus embryogenesis

doi: 10.1371/journal.pbio.3001377

Figure Lengend Snippet: Top part: a schematic representation of Xenopus laevis embryonic development. Developmental stages (NF) according to Nieuwkoop and Faber (1994). Stages used for mass spectrometry of histone modifications and embryonic analyses are characterized by the following features: NF13—late gastrula, germ layer specified; NF18—neurula, germ layer patterning and differentiation; NF25—tailbud stage, organogenesis; NF32—early tadpole, body plan established. Bottom part: We perform 2 types of experiments: Experiment type A (G1/S block)—embryos are split in 2 groups, which from NF10.5 on are continuously incubated in HUA solution or control solution (DMSO as carrier). Experiment type B (transient arrest)—embryos are split in 3 groups: continuous Mock, continuous HUA, and transient HUA. In the last group, HUA solution is replaced at NF13 with DMSO solution. DMSO, dimethyl sulfoxide; HUA, hydroxyurea and aphidicolin; HUAwo, HUA washout.

Article Snippet: Harris and Hartenstein used a combination of hydroxyurea and aphidicolin (HUA) to reveal that cell type determination and differentiation in embryonic central nervous system (CNS) are independent from cell division [ ].

Techniques: Mass Spectrometry, Blocking Assay, Incubation, Control

Journal: PLoS Biology

Article Title: A systemic cell cycle block impacts stage-specific histone modification profiles during Xenopus embryogenesis

doi: 10.1371/journal.pbio.3001377

Figure Lengend Snippet: (A) ICC for the mitotic histone mark H3S10Ph. Black dots represent mitotic cells. Elongated, older embryos are recorded as anterior halves, i.e., at the same magnification as younger stages, and in whole mount views as inserts. Scale bars: 1 mm. (B) Abundance of mitotic cells in Mock- and HUA-treated embryos. Box plots based on H3S10Ph-positive cells present on the recorded surface of embryos ( n = 3 biological replicates/condition; Student t test [unpaired, two-tailed]; *** p < 0.001). The individual quantitative observations can be found in . HUA, hydroxyurea and aphidicolin; ICC, immunocytochemical staining.

Article Snippet: Harris and Hartenstein used a combination of hydroxyurea and aphidicolin (HUA) to reveal that cell type determination and differentiation in embryonic central nervous system (CNS) are independent from cell division [ ].

Techniques: Two Tailed Test, Staining

Journal: PLoS Biology

Article Title: A systemic cell cycle block impacts stage-specific histone modification profiles during Xenopus embryogenesis

doi: 10.1371/journal.pbio.3001377

Figure Lengend Snippet: (A) During gastrulation, Mock and HUA embryos are indistinguishable. At NF18, the latter show a delay in neural tube closure. More severe malformations are detectable at stages NF25 and NF32, most notably reduced eye formation and absence of tail bud. Scale bars: 1 mm. (B) Cell size at the tailbud stage. Flattened Z-stack images show fields from embryonic skin at constant magnification (scale bars: 20 μm). Immunofluorescence detects cell borders (beta-catenin), nuclei (DAPI), and mitotic cells (H3S10Ph). GIFs from Z-stacks are presented in the Supporting information ( and Videos). (C) Whole mount RNA in situ hybridization for indicated marker genes. Images are representative for the majority of Mock- or HUA-treated embryos from 3 biological experiments. While mRNAs of α-tubulin and rx1 (skin, neuronal), α-globin, tnni3, actc1, and mhc-α (mesodermal) are detected in both conditions, xbra and foxd5a (tail blastema) are absent in HUA embryos. Numbers indicate embryos positive for the marker over the total number of analyzed embryos. Scale bars: 1 mm. HUA, hydroxyurea and aphidicolin.

Article Snippet: Harris and Hartenstein used a combination of hydroxyurea and aphidicolin (HUA) to reveal that cell type determination and differentiation in embryonic central nervous system (CNS) are independent from cell division [ ].

Techniques: Immunofluorescence, RNA In Situ Hybridization, Marker

Journal: PLoS Biology

Article Title: A systemic cell cycle block impacts stage-specific histone modification profiles during Xenopus embryogenesis

doi: 10.1371/journal.pbio.3001377

Figure Lengend Snippet: (A) Heatmap of the absolute histone modification states abundance, normalized to the corresponding R10 spiketides. Data in columns represent an average from 3 biological replicates/condition. Color key: row Z-score. The individual quantitative observations can be found in . (B) Ratio heatmap of relative histone modifications abundance for selected histone marks. In the first step, relative distributions of the indicated modification states were calculated in percentages within each tryptic peptide (see ). Then, HUA values were divided by Mock values to produce the relative change for each histone modification state between the 2 conditions. Color key is based on the Log10 scale. Numerical values in the cells indicate the fold-change. Values greater than 1.0 indicate an increase in HUA condition; values smaller than 1.0 indicate a higher abundance in Mock condition. NA, not applicable, when values in Mock were 0. Asterisks (*), adj. p < 0.1 (Benjamini–Hochberg procedure). “p,” propionylated (naturally unmodified); “un,” tryptic peptide, which has no modification state. The direct comparison of relative histone PTMs abundance is shown in , with p -values indicated in . HMP, histone modification profile; HUA, hydroxyurea and aphidicolin; PTM, posttranslational modification of histone.

Article Snippet: Harris and Hartenstein used a combination of hydroxyurea and aphidicolin (HUA) to reveal that cell type determination and differentiation in embryonic central nervous system (CNS) are independent from cell division [ ].

Techniques: Modification, Comparison

Journal: PLoS Biology

Article Title: A systemic cell cycle block impacts stage-specific histone modification profiles during Xenopus embryogenesis

doi: 10.1371/journal.pbio.3001377

Figure Lengend Snippet: Embryos, which were transiently incubated in HUA solution and thus mitotically arrested, were returned to Mock solution at NF13. (A) Abundance of mitotic cells in Mock, HUA-treated, and HUAwo embryos. Box plots based on H3S10Ph-positive cells present on the recorded surface of embryos, displayed in (n ≥ 3 biological replicates/condition; Student t test [unpaired, two-tailed]; *** p < 0.001; * p < 0.05; n.s., not significant). The individual quantitative observations can be found in . (B) Morphological and molecular features of embryos at the early tadpole stage. In contrast to continuous HUA-treated embryos, HUAwo embryos regain eye cups, fin, and tailbud structures. In addition, they express foxd5a and xbra mRNAs in the growth zone of the tailbud, comparable to Mock embryos. Numbers indicate embryos positive for the marker over the total number of analyzed embryos ( n = 3 biological replicates/condition). Scale bars: 1 mm. HUA, hydroxyurea and aphidicolin; HUAwo, HUA washout.

Article Snippet: Harris and Hartenstein used a combination of hydroxyurea and aphidicolin (HUA) to reveal that cell type determination and differentiation in embryonic central nervous system (CNS) are independent from cell division [ ].

Techniques: Incubation, Two Tailed Test, Marker

Journal: PLoS Biology

Article Title: A systemic cell cycle block impacts stage-specific histone modification profiles during Xenopus embryogenesis

doi: 10.1371/journal.pbio.3001377

Figure Lengend Snippet: (A) Heatmap of the relative histone modification states abundance under the indicated conditions. As before, the relative distributions of the indicated modification states are calculated in percentages within each tryptic peptide. Data in columns are collected from early tadpole stage embryos NF32 ( n = 1 biological replicate), independently from the data of experiment type A. Note: Relative differences between Mock and HUA samples of experiment type B are highly similar to the results of experiment type A . Color key, row Z-score. The individual quantitative observations can be found in . (B) Ratio heatmap of relative histone modification abundance for selected histone marks. First, the relative distributions of the indicated modification states are calculated as percentages within each tryptic peptide . Then, the relative values are presented as ratios between sample pairs indicated on top, showing the relative change for each histone modification state between the 2 conditions. Color key is based on the Log10 scale. Numerical values in the cells indicate the fold-change. “p,” propionylated (naturally unmodified). Values greater than 1.0 indicate an increase in HUA condition; values smaller than 1.0 indicate a higher abundance in Mock condition. NA, not applicable, when values in Mock were 0. The first 2 columns illustrate the similarity between experiment types A and B; the information in the third column shows the similarity between HUAwo and Mock samples. Asterisks (*), adj. p < 0.1 (Benjamini–Hochberg procedure) between indicated conditions. The direct comparison of relative histone PTMs abundance is shown in . HMP, histone modification profile; HUA, hydroxyurea and aphidicolin; HUAwo, HUA washout.

Article Snippet: Harris and Hartenstein used a combination of hydroxyurea and aphidicolin (HUA) to reveal that cell type determination and differentiation in embryonic central nervous system (CNS) are independent from cell division [ ].

Techniques: Modification, Comparison