The Polycomb group protein Ezh2 is conserved in zebrafish and ezh2 mRNA is maternally provided in zebrafish embryos.

(a) Schematic representation of Ezh2 orthologs in zebrafish, human, mouse, and Drosophila. Detailed alignments (Supplementary Fig. S1) show high conservation between the different species. This is 85% and 86% between zebrafish and human and mouse, respectively. Black boxes indicate the location of the SET domain. Grey boxes indicate the location of the WD domain. (b) In situ hybridization for ezh2 at 2 cells, 30% epiboly, 1, 2, and 3 dpf. ezh2 mRNA is maternally provided and at 2 and 3 dpf it is expressed in the pectoral fins, gut, tectum, eye, mid-hindbrain region, and the branchial arches (arrow heads). Scale bar is 200 µm.

Maternal zygotic ezh2 mutant embryos lack Ezh2 and H3K27me3, and show aberrant hox, pax, and shh gene expression.

(a) Schematic representation of germline transplantation at sphere stage to obtain germline mutant zebrafish. The progeny are maternal zygotic ezh2 mutant embryos (MZezh2^{hu5670/hu5670}). (b) In situ hybridization for ezh2 mRNA shows maternal contribution of ezh2 as well as zygotic expression in wildtype embryos. Maternal contribution of ezh2 is lost (3 hpf) in MZezh2^hu5670/+ and MZezh2^{hu5670/hu5670} embryos. Zygotic ezh2 expression (30% epiboly) is also lost in MZezh2^{hu5670/hu5670}. Scale bar is 200 µm. (c) Immunostaining for Ezh2 in wildtype and MZezh2^{hu5670/hu5670} embryos at 1 dpf. Ezh2 shows representative nuclear localization in the forebrain of wildtype embryos and is lost in MZezh2^{hu5670/hu5670} embryos. Scale bar is 10 µm. (d) Immunostaining for H3K27me3 in wildtype and MZezh2^{hu5670/hu5670} embryos at 1 dpf. H3K27me3 shows representative nuclear localization in the tail of wildtype embryos and is lost in MZezh2^{hu5670/hu5670} embryos. Scale bar is 10 µm. (e) In situ hybridization for hoxa9b, pax2, and shh mRNA in MZezh2^hu5670/+ and MZezh2^{hu5670/hu5670} embryos at 1 and 2 dpf. In MZezh2^hu5670/+ embryos a clear boundary of hoxa9b expression is visible (arrow head) as well as expression in the pectoral fin buds (arrows). Expression is shifted to anterior in MZezh2^{hu5670/hu5670} embryos (arrow head). The expression pattern of hoxa9b in MZezh2^hu5670/+ resembles that of wildtype embryos⁵⁴. Scale bar is 200 µm. In MZezh2^hu5670/+ embryos expression of pax2 is normal and amongst others restricted to the optic stalk, mid-hindbrain boundary, and the spinal cord neurons³². Expression in the optic stalk is spread throughout the eye in MZezh2^{hu5670/hu5670} embryos. Expression of shh is comparable to wildtype embryos in MZezh2^hu5670/+ embryos at 1 and 2 dpf³¹. In MZezh2^{hu5670/hu5670} embryos, expression of shh is outside the regular boundaries in the head region (arrow head) at 1 dpf and is still present at 2 dpf in the notochord, in contrast to MZezh2^hu5670/+ embryos (arrow head). Scale bar is 500 µm. The numbers indicate the number of embryos with the displayed phenotype compared to the total number of embryos analyzed.

Maternal zygotic ezh2 mutants form a normal body plan and display a pleiotropic phenotype at 2 dpf.

(a) MZezh2^{hu5670/hu5670} appear relatively normal at 1 dpf, although a clear mid-hindbrain boundary appears to be absent (arrow head). They display a pleiotropic phenotype at 2 dpf, having small eyes, a stringy heart, and blood accumulation (arrow heads). MZezh2^hu5670/+ show normal development. (b) The pleiotropic phenotypes of MZezh2^{hu5670/hu5670} can be rescued by injection of full-length ezh2 mRNA (300 pg). The numbers indicate the number of embryos with the displayed phenotype compared to the total number of embryos injected in two experiments. (c) Expression analysis of ezh1 and ezh2 in wildtype and MZezh2^{hu5670/hu5670} embryos at 0 hpf, 3.3 hpf, and 1 dpf. Expression of ezh1 is not detectable in MZezh2^{hu5670/hu5670} embryos and wildtype embryos at 0 hpf, 3.3 hpf, and 1 dpf. ezh1 is expressed in wildtype control embryos at 5 dpf. ezh2 is expressed in wildtype embryos at 0 hpf, 3.3 hpf, 1 dpf, and 5 dpf, showing a decrease in expression over time. ezh2 expression cannot be detected in MZezh2^{hu5670/hu5670} embryos. Relative expression was calculated based on expression of housekeeping genes β-actin and ef1α. Error bars represent standard deviation. n.d. is not done. (d) In situ hybridization for eng1 (muscle pioneer marker) and myoD (somite marker) at 1 dpf in MZezh2^{hu5670/hu5670} embryos and MZezh2^hu5670/+. Both eng1 and myoD are normally expressed in MZezh2^{hu5670/hu5670} and MZezh2^hu5670/+. Scale bar is 500 µm. (e) In situ hybridization for ntl at 1 dpf shows no difference in spatiotemporal expression between MZezh2^{hu5670/hu5670} embryos and the heterozygous siblings. At 2 dpf in situ hybridization for ntl showed expression in the notochord of MZezh2^{hu5670/hu5670} embryos, whereas this is not visible in MZezh2^hu5670/+ (arrow head). In situ hybridization for krox20 at 1 dpf showed normal expression in MZezh2^hu5670/+, but reduced expression in rhombomeres 3 and 5 in MZezh2^{hu5670/hu5670} embryos (arrow heads). Scale bar is 500 µm for lateral views and 250 µm for dorsal view of krox20 expression. The numbers indicate the number of embryos with the displayed phenotype compared to the total number of embryos analyzed.

Myocardial development is affected in MZezh2 embryos.

(a) In situ hybridization for different heart markers in MZezh2^hu5670/+ and MZezh2^{hu5670/hu5670} at various time points of development. hand2 is an early myocardial marker. myh6 is a marker for atrial cells. vmhc is a marker for ventricular cells. nppa is a late myocardial marker. nfat-c1 is an endocardial marker. All these markers are expressed in MZezh2^{hu5670/hu5670}, although vmhc, nppa, and nfat-c1 expression show a smaller number of positive cells. (b) In situ hybridization for nkx2.5 at different time points after fertilization in MZezh2^hu5670/+ and MZezh2^{hu5670/hu5670} embryos. Arrow heads point to cells of the pharyngeal arch artery progenitors. This is absent in MZezh2^{hu5670/hu5670}. (c) In situ hybridization for has2 and fgf24 at 2 dpf in MZezh2^{hu5670/hu5670} and their heterozygous siblings. In MZezh2^hu5670/+ expression is restricted to the heart (arrow heads), whereas in the MZezh2^{hu5670/hu5670} embryos expression is visible in the area surrounding the heart tube (encircled by dashed line). For fgf24 this is also shown from a lateral view (arrow heads). Scale bar is 200 µm. The numbers indicate the number of embryos with the displayed phenotype compared to the total number of embryos analyzed.

MZezh2 mutant embryos display impaired myocardial and gastrointestinal tissue maintenance.

(a) In situ hybridization for myl7 at different time points in MZezh2^hu5670/+ and MZezh2^{hu5670/hu5670}. At 1 dpf the heart of MZezh2^{hu5670/hu5670} is straight. In MZezh2^{hu5670/hu5670} embryos myl7 expressing cells are found outside the heart at 1.5 dpf (arrow heads). At 2 dpf the number of myl7 expressing cells is decreased in MZezh2^{hu5670/hu5670} compared to MZezh2^hu5670/+. (b) Stills of time lapse (Supplementary Movie S1, 2) imaging of Tg(myl7::GFP) MZezh2^hu5670/+ and MZezh2^{hu5670/hu5670} embryos from 1 to 2 dpf. In MZezh2^{hu5670/hu5670} embryos, GFP-positive cells are moving away from the heart (arrows). Arrow heads point at ventricle and atrium. (c) Immunostaining for GFP to visualize Tg(myl7::GFP) (brown precipitation, arrow heads) combined with in situ hybridization for nkx2.5 (purple staining, arrows) at 2 dpf. In MZezh2^hu5670/+ no nkx2.5 expressing cells are present. In MZezh2^{hu5670/hu5670} cells that are detached from the heart express nkx2.5. The right panel shows a zoom-in of the left panel (white square). Scale bar is 50 µm. (d) Immunostaining for GFP Tg(myl7::GFP) combined with in situ hybridization for nppa at 2 dpf in MZezh2^hu5670/+ and MZezh2^{hu5670/hu5670} embryos. Two embryos per genotype are shown. In MZezh2^{hu5670/hu5670} embryos nppa expression is absent in one and not ubiquitous in the other embryo (arrow heads). In MZezh2^hu5670/+ nppa is expressed in atrium and ventricle (arrow heads). Scale bar is 50 µm. (e) In situ hybridization for different gastrointestinal tract markers at 1 and 2 dpf in MZezh2^{hu5670/hu5670} and MZezh2^hu5670/+. Expression of gata6 is present in both MZezh2^{hu5670/hu5670} and MZezh2^hu5670/+ at 1 and 2 dpf. At 2 dpf the intestinal tube appears straight in the MZezh2^{hu5670/hu5670}, whereas structures like the liver and pancreas can be seen in MZezh2^hu5670/+ (arrow heads). MZezh2^{hu5670/hu5670} are able to form a gastrointestinal tract, observed by in situ hybridization for foxa3 at 2 dpf, although the organs are bilaterally formed (arrow heads). In MZezh2^{hu5670/hu5670} no expression of terminal differentiation markers for liver, fabp10, and exocrine pancreas, try, was observed. Scale bar is 100 µm. Numbers indicate the number of embryos with the displayed phenotype compared to the total number of embryos analyzed.