The ascidian mouth opening is derived from the anterior neuropore: Reassessing the mouth/neural tube relationship in chordate evolution

AbstractThe relative positions of the brain and mouth are of central importance for models of chordate evolution. The dorsal hollow neural tube and the mouth have often been thought of as developmentally distinct structures that may have followed independent evolutionary paths. In most chordates however, including vertebrates and ascidians, the mouth primordia have been shown to fate to the anterior neural boundary. In ascidians such as Ciona there is a particularly intimate relationship between brain and mouth development, with a thin canal connecting the neural tube lumen to the mouth primordium at larval stages. This so-called neurohypophyseal canal was previously thought to be a secondary connection that formed relatively late, after the independent formation of the mouth primordium and the neural tube. Here we show that the Ciona neurohypophyseal canal is present from the end of neurulation and represents the anteriormost neural tube, and that the future mouth opening is actually derived from the anterior neuropore. The mouth thus forms at the anterior midline transition between neural tube and surface ectoderm. In the vertebrate Xenopus, we find that although the mouth primordium is not topologically continuous with the neural tube lumen, it nonetheless forms at this same transition point. This close association between the mouth primordium and the anterior neural tube in both ascidians and amphibians suggests that the evolution of these two structures may be more closely linked than previously appreciated

Epigenetic Regulation of Mammalian Cardiac Myocyte Cell Cycle

Cardiac myocyte (CM) proliferation is required for the heart regeneration seen in lower vertebrates and neonatal mammalian injury models. However, mammalian CMs stop proliferating soon after birth and subsequent heart growth comes from hypertrophy, limiting the adult heart’s regenerative potential after injury. The molecular events blocking CM proliferation in the adult heart remain poorly understood. We hypothesized repressive epigenetic mechanisms are responsible for the stable silencing of cell cycle genes in adult CMs (ACMs). Studies from our lab and others have suggested trimethylation of Lysine 9 of Histone H3 (H3K9me3) and H3K27me3, histone modifications associated with heterochromatin, are associated with permanent cell cycle exit. To test if depleting these repressive methylations in ACMs could relieve the silencing of cell cycle genes, we developed an adenoviral-gene-transduction model for combined H3K9me3- and H3K27me3-depletion in vitro. We tested this hypothesis in vivo using a transgenic mouse model where H3K9me3 is specifically removed by histone demethylase KDM4D in CMs. Loss of H3K9me3 in CMs disrupts ACM cell cycle gene silencing preferentially and results in increased CM cycling. Normalized heart mass was increased by postnatal day 14 (P14) and continued to increase until 9-weeks of age. ACM number, but not size, was significantly increased in BiTg hearts, suggesting CM hyperplasia accounts for the increased heart mass. Challenging H3K9me3-depleted hearts with a hypertrophic growth signal stimulated ACM mitotic activity. Thus, we demonstrated that H3K9me3 is required for cell cycle gene silencing in ACMs and depletion of H3K9me3 allows hyperplastic growth in vivo. To gain mechanistic understanding of the observed proliferation-competence we examined global chromatin structure and loci-specific DNA accessibility in H3K9me3-depleted and control ACMs. Combined with DNA methylation bisulfite sequencing (DNAme-Seq) and chromatin-immunoprecipitation (ChIP) studies, these data suggest a model where cell cycle genes have a unique chromatin signature, where the gene bodies are heterochromatinized and the gene promoters are regulated by canonical cell cycle transcription factor pathways that are modulated by H3K9me3

MOESM14 of Deletion of HP1γ in cardiac myocytes affects H4K20me3 levels but does not impact cardiac growth

Additional file 14: Fig. S10. Effect of HP1γ KO on ISO-induced heart growth. ISO (5 μg/g) was injected subcutaneously once a day for 6 days and hearts were harvested. Same volume of saline was injected as a vehicle control. Since there was no difference of HW normalized by BW at base line, all genotype of animals with vehicle injection are grouped as a vehicle control group. Upon ISO treatment, all genotype showed a significant increase in HW/BW compared to vehicle control; however, there was no difference between genotype, indicating that HP1γ KO doesn’t have a significant effect on ISO-induced heart growth. * p < 0.05 vs vehicle control

MOESM1 of Deletion of HP1γ in cardiac myocytes affects H4K20me3 levels but does not impact cardiac growth

Additional file 1: Fig. S1. HP1 localization with H3K9me3. Cardiac myocytes were isolated from 10 week adult mice and HP1s (green) were co-immunostained with H3K9me3 (red) using specific antibodies. Heterochromatin was visualized by Hoechst staining (Blue). Scale bar indicates 5 μ

MOESM14 of Deletion of HP1γ in cardiac myocytes affects H4K20me3 levels but does not impact cardiac growth

Additional file 14: Fig. S10. Effect of HP1γ KO on ISO-induced heart growth. ISO (5 μg/g) was injected subcutaneously once a day for 6 days and hearts were harvested. Same volume of saline was injected as a vehicle control. Since there was no difference of HW normalized by BW at base line, all genotype of animals with vehicle injection are grouped as a vehicle control group. Upon ISO treatment, all genotype showed a significant increase in HW/BW compared to vehicle control; however, there was no difference between genotype, indicating that HP1γ KO doesn’t have a significant effect on ISO-induced heart growth. * p < 0.05 vs vehicle control

MOESM7 of Deletion of HP1γ in cardiac myocytes affects H4K20me3 levels but does not impact cardiac growth

Additional file 7: Fig. S5. (A) No difference in DNA damage marker in HP1γ KO CMs. Nuclear extracts were prepared from purified CM at 8wk and WB was performed using specific antibody against γH2AX. As a positive control of γH2AX induction, C2C12 cells were treated with doxorubicin (1 μM) for 6 h. Representative pictures of 5 biologically independent experiments are shown here. (B and C) No induction of cell cycle inhibitors was seen. Gene expression of p21 (marker for DNA damage and senescence) and p16 (marker for senescence) were measured by qPCR. p21 gene expression is normalized by S26 expression. RNA from irradiated mouse embryonic fibroblasts (iMEF) was used for p16 positive control. p16 gene expression is shown with Ct value of qPCR. p16 was undetectable in both control and HP1γ KO CMs (n = 4–5)

MOESM6 of Deletion of HP1γ in cardiac myocytes affects H4K20me3 levels but does not impact cardiac growth

Additional file 6: Fig. S4. Localization of heterochromatic histone marks (A) and HP1s (B). 8-wk CMs from fl/fl control and KO (Cre;fl/fl) mice were isolated and stained with specific antibodies. Histone marks and HP1s are in red, cardiac troponin is in green and DNA is in blue. Scale bar indicates 10 μm

MOESM12 of Deletion of HP1γ in cardiac myocytes affects H4K20me3 levels but does not impact cardiac growth

Additional file 12: Fig. S8. No difference in cycling CM in HP1γ KO heart. TAC or Sham surgeries were performed at 10–12 weeks and hearts harvested 1 week after operations. Heart tissues were stained for Ki67 (white), Hoechst (red) and WGA (green). Scale bar indicates 100 μm

MOESM11 of Deletion of HP1Îł in cardiac myocytes affects H4K20me3 levels but does not impact cardiac growth

Additional file 11: Fig. S7. (A) Quantification of cardiac cross section area after TAC. More than 100 CM cross sections per animal were analyzed. Two-way ANOVA followed by multiple comparison was perform. TAC operation increased cross section area significantly; however, no interaction with genotype was detected. No significant difference was detected between genotype in Sham or TAC mice. (B) Quantification of cycling nuclear number in the heart. Heart sections were stained with Ki67 antibody and counted Ki67 positive nuclear number against total nuclear number. Since we could not find any cardiac nuclear positive for Ki67 in neither sham nor TAC condition, we estimated Ki67 positive nuclear number as cycling fibroblast number. Two-way ANOVA followed by multiple comparison was perform. TAC operation increased cycling fibroblast number significantly; however, no interaction with genotype was detected