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. 2018 Nov 15;443(2):173-187.
doi: 10.1016/j.ydbio.2018.09.014. Epub 2018 Sep 14.

Stage-specific Roles of Ezh2 and Retinoic Acid Signaling Ensure Calvarial Bone Lineage Commitment

Free PMC article

Stage-specific Roles of Ezh2 and Retinoic Acid Signaling Ensure Calvarial Bone Lineage Commitment

James W Ferguson et al. Dev Biol. .
Free PMC article


Development of the skull bones requires the coordination of two stem progenitor populations, the cranial neural crest cells (CNCC) and head paraxial mesoderm (PM), to ensure cell fate selection and morphogenesis. The epigenetic methyltransferase, Ezh2, plays a role in skull bone formation, but the spatiotemporal function of Ezh2 between the CNCC- and PM-derived bone formation in vivo remains undefined. Here, using a temporally-inducible conditional deletion of Ezh2 in both the CNCC- and PM- derived cranial mesenchyme between E8.5 and E9.5, we find a reduction of the CNCC-derived calvarial bones and a near complete loss of the PM-derived calvarial bones due to an arrest in calvarial bone fate commitment. In contrast, deletion of Ezh2 after E9.5 permits PM-derived skull bone development, suggesting that Ezh2 is required early to guide calvarial bone progenitor commitment. Furthermore, exposure to all-trans Retinoic acid at E10.0 can mimic the Ezh2 mutant calvarial phenotype, and administration of the pan retinoic acid receptor (RAR) antagonist, BMS-453, to Ezh2 mutants partially restores the commitment to the calvarial bone lineage and PM-derived bone development in vivo. Exogenous RA signaling activation in the Ezh2 mutants leads to synergistic activation of the anti-osteogenic factors in the cranial mesenchyme in vivo. Thus, RA signaling and EZH2 can function in parallel to guide calvarial bone progenitor commitment by balancing the suppression of anti-osteogenic factors.

Keywords: BMS453; Cell fate selection; Incoherent feedforward loop; Mesenchyme stem cells; Skull.

Conflict of interest statement

Conflict of interest: The authors declare that they have no competing interests.


Figure 1:
Figure 1:. Inducible and conditional deletion of Ezh2 at E8.5 in both the CNCC-derived and PM- derived CM.
(A) Mating strategy and gavage regimen for conditional Ezh2 deletion for E8.5-CMEzh2 and E9.5-CMEzh2 mutants. Tamoxifen was administered by oral gavage starting at E8.5 or E9.5 (purple shaded) at a concentration of 25µg/g mouse body weight. (B) Anatomy of mouse embryo between E8.5 and E9.5. PdgfrαCreER is active in the CM, frontonasal prominence, maxillary process, and BA1. Plane I corresponds to the future frontal bone, and plane II corresponds to the future parietal bone. (C) PdgfrαCreER/+;Ezh2fl/fl Rosa 26 Reporter lineage-marked CM in coronal sections. E8.5+E9.5 gavages is sufficient to induce Cre-ER recombination in cranial mesenchyme in frontal bone and parietal bone primordia in plane I and plane II, respectively (scale bar = 200µm). (D) Schematic representing manual enrichment of the cranial mesenchyme (CM). The ectoderm was manually removed and all the CM above the eye was collected. (E) RT-qPCR for Ezh2 in the manually enriched CM at E13.5. (F) Western blot for EZH2 in the manually enriched cranial mesenchyme. Band intensities were quantified using ImageJ/Fiji. (G) Western blot for H3K27me3 in manually enriched CM. (H) Whole mount skeletal staining of controls and E8.5-CMEzh2 and E9.5-CMEzh2 mutants at E17.5. All embryos were imaged at the same magnification. White arrows mark the coronal suture and yellow arrow marks the lambdoid suture. F = frontal bone; P = parietal bone; T= temporal; IP = interparietal bone; O = occipital bone; Fa = facial bones; M = mandible, Tympanic ring = TR; TN = Trigeminal neurons.
Figure 2:
Figure 2:. E8.5-CMEzh2 leads to truncation of CNCC-derived bones and a severe reduction in PM-derived bones.
(A) Schematic and key representing the primary bones examined in E8.5-CMEzh2 embryos. (B) Psuedo-colored 3D images from microCT at E17.5 of E8.5-CMEzh2 embryos. * indicates ear bones which are lost in the mutants. Arrows point to the reduced/lost PM-derived bones. Numbers represent landmarks used for morphometric measurements (scale bar = 2mm). (C) Quantification of changes in combined bone volume of the calvaria, mandible, maxilla, premaxilla, and nasal bones. (D) Quantification of changes in bone volume in the bones of the calvaria. (E) Morphometric analysis of the frontal and interparietal bone. Both the left and right frontal bones were measured and plotted. Pairs of colored dots correspond with each left-right pair. Definition of landmarks: (1,5,6) Most posterior-superior point of the frontal bone (2) Most anterior-superior point of the frontal bone (3) Most posterior-inferior point of the frontal bone (4) Most posterior-lateral intersection of the frontal and parietal bone (7,8) Lateral points of the parietal bone.
Figure 3:
Figure 3:. Arrest during bone differentiation in PM-derived parietal bone in E8.5-CMEzh2 mutants.
(A) Schematic of E13.5 mouse embryo and coronal sections. Plane I refers to future frontal bone and plane II refers to future parietal bone. (B) Immunofluorescence of bone precursor marker MSX1/2 in E11.5 coronal sections. Quantification of total number of cells positive for MSX1/2 in plane I and plane II. (C) Immunofluorescence for bone progenitor marker RUNX2 in E13.5 coronal sections. Arrows indicate expanded domains. Quantification of total number of cells positive for RUNX2 in plane I and plane II. (D) Immunofluorescence for bone progenitor marker OSX in E13.5 coronal sections. Arrows indicate lost expression in plane II. Quantification for total number of cells positive for OSX in plane I. Quantification in plane II was not performed due to lost expression in the mutant. TN: Trigeminal nerve. Scale bars = 200µm.
Figure 4:
Figure 4:. Activation of retinoic acid signaling inhibits skull bone formation and positively regulates Ezh2.
(A) Gavage regimen for at-RA (yellow shaded) in cre- control embryos and schematic for obtaining manually enriched E13.5 CM. At-RA was administered at E10.0. (B) Up regulation of known RA-signaling target, HoxA1, in manually enriched E13.5 CM. (C) Expression levels of PRC2 components in manually enriched CM. (D) Quantification of total area of the frontal and parietal bones in E17.5 skeletal staining. (E) Immunofluorescence for RARγ in the frontal bone primordia of E10.5 at-RA treated cre- control embryos. Embryos were administered at-RA at E9.5. (F) Immunofluorescence for OSX in the frontal and parietal bone primordia of at-RA treated embryos. At-RA was administered at E9.5. White arrows mark OSX+ domain. (G) Summary of the regulation of skull bone formation by Ezh2 and at-RA by which Ezh2 maintain a balance of activation and suppression of anti-osteogenic genes. Following administration of at-RA, the balance is shifted towards activation of anti-osteogenic genes.
Figure 5:
Figure 5:. Pharmacological inhibition of RA signaling partially rescues the E8.5-CMEzh2 mutant phenotype and restores the PM-derived bones.
(A) Gavage regimen for tamoxifen and RA-antagonist BMS-453 in E8.5-CMEzh2 mutants and schematic of obtaining manually enriched CM without the ectoderm. (B) RT-qPCR for Ezh2 in Cre- control and BMS-453 treated embryos from E13.5 manually enriched CM. (C) Skeletal staining of E17.5 3.5µg/gm BMS-453 treated Ezh2 mutants. C' and C'' represent two different litters. Alcian blue marks cartilage and alizarin red marks bone (scale bar = 2mm). (D) Quantification of the Alizarin Red stained area outlined in (C) using ImageJ/Fiji. (E) Immunofluorescence of OSX in the parietal bone at E13.5. Arrows indicate partial restoration of OSX (scale bar = 200µm). (F) Schematic with proposed model by which, in the absence of Ezh2, inhibition of RA-signaling prevents the activation of the anti-osteogenic genes restoring OSX expression and skull bone formation.
Figure 6:
Figure 6:. Ezh2 and RA-signaling maintain a balance of anti-osteogenic genes in the CM.
(A) Relative mRNA quantity for HoxA1, HoxC8 and Hand2 expression levels in E13.5 manually enriched CM in Cre- control, E8.5-CMEzh2 mutant, 100µg/gm body weight at-RA exposure at E10.0 in Cre- control, and E8.5-CMEzh2 mutants treated with 100µg/gm at-RA. (B) Integrated Genome Viewer representation of H3K27me3 ChiP-seqencing of E13.5 cranial mesenchyme control. (C) Protein expression of HOXC8 in in the parietal bone primordia of E13.5 control, E8.5-CMEzh2, and 3.5ug/gm body weight treated BMS-453 at E8.5, 9.5, and 11.5. embryo. Scale bars: 200µm. (D) Hypothetical model explaining that EZH2 and RA signaling balance the inhibition of anti-osteogenic factors to ensure calvarial bone commitment and development.

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