Fig 1.
Various facial deformities and perinatal lethality in Ezh2 null mice.
A) Ezh2 null mice displays abnormal craniofacial structures, which are visible at E15.5 and continues through P0 (Ezh2 null, n = 30/30). B) H&E staining shows absence of frontal face and ‘pigeon’s chest’ (marked in asterisk) (Ezh2 null, n = 4/12). C) Kaplan-Meier survival curve demonstrates perinatal lethality in Ezh2 null mice within first 40 minutes after birth (n = 35, p = 0.0194, Fisher’s exact test). D) Absence of outer ear in Ezh2 null mice marked in white box. E) Absence of the outer ear in an H&E-stained transverse section of the head of a Ezh2 null P0 mouse, and higher magnification of an H&E-stained transverse section of the head revealing aberrant development of the middle ear in the Ezh2 null P0 mice. Wnt1-Cre-Red arrow: Developing outer ear (pinna and external ear canal), Black arrow: Developing middle ear (ossicles), Red asterisks: Developing inner ear (left: cochlea; right: cochlea + semicircular canals). Ezh2flox/flox;Wnt1-CreT/+- Red asterisks: Developing inner ear (cochlea + semicircular canals). There was no evidence of proper middle ear and outer ear development in any of the transverse sections of the head of the mutant mice. F) The dissected eyes of P0 pups have a 2/3 reduction in size in Ezh2 null mutants (n = 5/10) compared to the wild-type littermates. G) Circle: Shows primordium of oral cavity. Long arrow: Points to the space corresponding to the nasal chamber. Short arrow: points to a space that appears to correspond to the nasopharynx (junction of nasal cavity and pharynx). H) Insufficient closure of the eyelid can be seen in Ezh2 null P0 pups (marked arrowheads) (n = 30/30). H&E staining shows complete absence of cornea and conjunctiva. I) Toluidine blue staining of P0 pups showed staining only in the eyelid region of Ezh2 mutants (n = 4/4). J) Cleft lip (or bifid nose) visible in region of the upper lips of Ezh2 null mice. High magnification of an H&E-stained transverse section of the head of an Ezh2 mutant P0 mice. The arrow in red points to the nasal pits, and the surrounding cartilage plates are marked with (c) in red. The oral cavity in the center is rudimentary.
Fig 2.
Hypoganglionosis in Ezh2 null mice.
Whole-mount AChE staining in wild type and Ezh2 null E18.5 mice. AChE stained proximal and distal colon of wild-type littermate (top panel) and two Ezh2 mutants (bottom panel). The two Ezh2 null mice are representative of mild (middle panel, Ezh2 null n = 4/7, p<0.0001) hypogangliosis and severe (bottom panel, Ezh2 null n = 3/7, p<0.0001) aganglionosis cases. The bar graph is the quantification of the AChE staining in the proximal colon. (scale bar = 0.25 mm).
Fig 3.
NCC developmental gene expression in Ezh2 null mice guts.
NCC developmental gene expression levels were compared between wild-type (grey, Ezh2flox/+Wnt1-CreT/+, Ezh2flox/+ Wnt1-Cre+/+, Ezh2+/+ Wnt1-CreT/+, were all combined as wild type control), and Ezh2 mutant (red) gut tissues with qRT-PCR. The relative gene expression fold difference between Ezh2 null and wild type littermates are presented. Each bar graph represents a biological replicate. Among the eleven genes tested, four genes displayed significant expression changes in the Ezh2 null. Phox2b (n = 6, p = 0.0035) and Sox10 (n = 6, p = 0.00239) were down-regulated, and Zic1 (n = 6, p = 0.003) and Pax3 (n = 6, p = 0.0019) were up-regulated in the Ezh2 null mice.
Fig 4.
NCC gene expression analysis and H3K27me3 ChIP assays in myenteric plexus.
A) Dissection and isolation of myenteric plexus from P0 wild-type and Ezh2 null mice for qRT-PCR and ChIP assays. B) qRT-PCR of Pax3, Zic1 and Sox10 in myenteric plexus. In the Ezh2 null myenteric plexus, Pax3 was upregulated 20–40 fold (n = 3, p = 0.0010), Sox10 was upregulated 5–30 fold (n = 3, p = 0.0263), and Zic1 was upregulated 40–100 fold (n = 3, p = 0.0071) compared to its wild type littermates. C) H3K27me3 ChIP-qPCR in Ezh2 mutant (in orange) and wild-type littermate (in grey) P0 gut tissues. The amount of precipitated DNA in promoter regions of NCC developmental genes and HoxA9 are presented as a relative value (%) to that of the input DNA normalized over a negative locus (beta actin) (y-axis). Student’s t-test (two-tailed) value shows significant difference in H3K27me3 enrichment between Ezh2 null (n = 6) and wild-type (n = 8) littermates (p<0.01).
Fig 5.
Immunohistochemistry of Hu and GFAP in E16 Ezh2 null mice myenteric plexus.
The distribution of Hu expression (green) and GFAP expression (red) in wild type (top, n = 4) and Ezh2 null (bottom, n = 4) E16 myenteric plexus (scale bar = 100 μm).
Fig 6.
Double immunofluorescence of Sox10 and Hu in myenteric plexus of P0 neonates.
Immunohistochemical analysis of Sox10 (red) in P0 neonates show non-overlapping expression of Sox10 (red) and Hu (green) in wild-type littermates (top, n = 4), while Ezh2 null mutants (n = 4) display aberrant overexpression of Sox10 proteins that partially overlap with Hu expression (bottom) (overlap of Sox10 and Hu marked with white arrow) (p<0.0001, scale bar = 100 μm).
Fig 7.
Double immunofluorescence of Sox10 and GFAP in myenteric plexus of P0 neonates.
Co-expression analysis of Sox10 (red) and GFAP (green) in wild type (top panel, n = 4) and Ezh2 null (bottom panel, n = 4) myenteric plexus (p<0.001, scale bar = 100 μm).