Zebrafish mH2A expression pattern during early embryo development. Wild type zebrafish embryos were collected at different developmental stages. (A) Quantitative RT-PCR was performed for mH2A1, mH2A2 and two genes characteristic of undifferentiated stage (OCT4 and NANOG). Mean values (n = 3) ± SEM are plotted. Values indicate relative expression of the specific gene normalized to GAPDH/TUBULIN. (B) Whole-mount RNA in situ hybridization using mH2A1 and mH2A2 specific probes showing different temporal and spatial gene expression pattern (n = 50–60 embryos/probe with identical labeling).

Identification and characterization of mH2A promoter regions and fusion proteins during embryogenesis. (A) Schematic representation of mH2A constructs structure/generated. The origin of mH2A UTR variants were determined from genomic DNA sequence. Identified promoter region responsible for transcription of each mH2A isoform are shown (promoter locations are indicated relative to ORF starting nucleotide triplet, ATG). (B) For mH2A reporter constructs mH2A1.1 and mH2A2 GFP-fusion proteins and either ubiquitous promoter (EF1) (EF1:GFP-mH2A1.1) or endogenous promoter (mH2A1:GFP-mH2A1.1 or mH2A2:GFP-mH2A2) were cloned into pTOL vector (Tol2 sequences that flank each transgene include the terminal inverted repeats (red arrowheads). (C,D) Identified mH2A promoter regions match endogenous expression. Expression analysis was conducted by injecting reporter constructs into the 1–2 cell stage of wt AB zebrafish embryos and collecting them at different development stages. Quantitative RT-PCR was performed to detect GFP and endogenous mH2A1 (C) or mH2A2 (D) expression. Mean values (n = 3) ± SEM are plotted. Values indicate relative expression of the specific gene normalized to GAPDH/TUBULIN. (E) Transgenic embryos for both Tg(EF1:GFP-mH2A1.1) and Tg(mH2A1:GFP-mH2A1.1) at two stages (75% epiboly and 24 hpf) were collected to perform whole-mount RNA in situ hybridization using mH2A1, mH2A2, GFP and GSC specific probes. (n = 50–60 embryos/probe with uniform labeling).

GFP-mH2A1.1 and GFP-mH2A2 protein expression under endogenous promoter during zebrafish embryogenesis show distinct temporal and location pattern. In vivo confocal imaging of transgenic zf embryos expressing mH2A1:GFP-mH2A1.1 (A) or mH2A2:GFP-mH2A2 (B) reveals that mH2A1 is expressed in yolk syncytial layer (YSL) while mH2A2 is expressed within the embryo body becoming evident at 10 somites and 24 hpf stages. Figure shows a representative series of GFP and merged brightfield-GFP snapshots at different developmental stages during live image capturing. (Assay conducted in triplicate with n = 12 embryos/isoform).

mH2A isoforms differential colocalization with heterochromatin and mitotic marks in zebrafish embryos at epiboly stage. Transgenic zf embryos expressing mH2A1:GFP-mH2A1.1 (A–C,F) or mH2A2:GFP-mH2A2 (D,E,G,H) at late blastula (A) or 75% epiboly (B–H) stages were analysed using immunohistochemistry to detect the heterochromatin markers trimethyl Histone3 lysine K27 and K9 (H3K27me3 and H3K9me3) (A–E) or mitotic nuclei phosphohistone H3 (pHH3) (F–H). DAPI and Phalloidin-conjugate were used for nucleus and cytoskeleton labelling respectively. (A–C) Confocal microscope image projection of late blastula (A) and 75% epiboly stage (B) transgenic mH2A1:GFP-mH2A1.1 embryo shows YSL localization of GFP positive cells and lack of colocalization with H3K27me3. Apical pole and vegetal pole are pointed in the image. To the right of (A–C) there is a schematic illustration of the organization of the cortical cytoplasm of the yolk cell in relation to other cell types in zebrafish embryo during epiboly (draws at late blastula and 60% epiboly respectively). (C) Confocal microscope image section also shows absence H3K9m3 signal in the nuclei of YSL cells, where GFP-mH2A1.1 is expressed. (D,E) Confocal microscope image projection (D) and section (E) of 75% epiboly stage transgenic mH2A2:GFP-mH2A2 embryo stained with heterochromatin markers H3K27me3 and H3K9me3 shows heterochromatin labelling in the whole embryo while GFP (mH2A2) expression is still very weak. (F–H). Confocal microscope image projection of 75% epiboly stage transgenic mH2A1:GFP-mH2A1.1 (F) or mH2A2:GFP-mH2A2 (G,H) with labelled pHH3 mitotic nuclei. Figure show non-mitotic mH2A1 positive YSL nucleus and proliferative cells within the embryo body. (H) Higher magnification of images of mH2A2:GFP-mH2A2 transgenic fish clearly showing pHH3 positive cells while there is a low level GFP-mH2A2 expression (H). Each labelling assay was conducted in triplicate with n = 40–50 embryos/immunolabeling.

mH2A isoforms differential colocalization with heterochromatin and mitotic marks in zebrafish embryos at 24 hpf stage. Transgenic zf embryos expressing mH2A1:GFP-mH2A1.1 (A,B,E) or mH2A2:GFP-mH2A2 (C,D,F) at 24 hpf stage were analysed using immunohistochemistry to detect heterochromatin markers trimethyl Histone3 lysine K27 and K9 (H3K27me3 and H3K9me3) (A–D) or mitotic nuclei Phosphohistone H3 (pHH3) (E,F). DAPI was used for nucleus labelling. Confocal microscope image projection of 24 hpf transgenic fish. (A,B) mH2A1:GFP-mH2A1.1 embryo shows YSL localization of GFP positive cells and lack of colocalization with H3K27me3 (A,B) and H3K9m3 (C) throughout the embryo body. (C,D) mH2A2:GFP-mH2A2 embryo stained with heterochromatin markers H3K27me3 (D) and H3K9me3 (E) reveals areas of colocalization. (E,F) mH2A1:GFP-mH2A1.1 (E) or mH2A2:GFP-mH2A2 (F) with labelled pHH3 mitotic nuclei shows partial colocalization of GFP-mH2A2 isoform with mitotic nuclei. Each labelling assay was conducted in triplicate with n = 40–50 embryos/immunolabeling.

mH2A2 and mH2A1 are required for proper zebrafish embryo development. (A) An inhibitory morpholino (MO) was designed against the translational start site of zebrafish mH2A1 or mH2A2 mRNA (sequences at Supplementary Table S12). For the control mismatch morpholino we introduced a few base alterations. Total nuclei of zebrafish embryos were analysed by anti-mH2A1 or anti-mH2A2, and anti-histone H3 immunoblotting after specific morpholino injection. Full-length blots are presented in Supplementary Fig. S11. (B) Characterization of mH2A1 morphants and control embryos at the 18-somite stage. Lateral, apical and dorsal view of fixed zebrafish embryos show alterations in midbrain structure and organization (arrow). Somite number was correct, but their distribution along the embryo was shortened. Yolk sac extension and caudal fin was not properly formed after mH2A1 downregulation. In all experiments we co-injected with a morpholino against p53 (p53 MO) to mitigate the nonspecific dose-dependent neural toxicity widely reported⁵⁶. Rescues were generated by injection of embryos with mH2A1 morpholino in combination with mH2A1.1 mRNA. (C) Characterization of mH2A2 morphants and control embryos at the 10-somite stage. Lateral, apical and dorsal view of fixed zebrafish embryos show alterations in midbrain, optic primordium and somites and fin formation/structure after mH2A2 downregulation. In all experiments we co-injected with a morpholino against p53 (p53 MO) to mitigate the nonspecific dose-dependent neural toxicity widely reported⁵⁶. Rescues were generated by injection of embryos with mH2A2 morpholino in combination with mH2A2 mRNA. (D) Stacked bar graph representing the percentage of embryos which are normal, dead or showing described altered phenotype after injecting control and mH2A1 and mH2A2 morpholinos, and rescued with mH2A1.1 or mH2A2 mRNA at the developmental stages shown in figure (C), where n represents numbers of embryos injected.