Circadian BMAL1 regulates mandibular condyle development by hedgehog pathway

Homozygous BMAL1‐deficiency (Bmal1^‐/‐, B6.129‐Arntl^tm1Bra/J mice) mice18 were produced by breeding heterozygous BMAL1‐deficienct mating pairs (Bmal1^+/–) which were obtained originally from the Jackson Laboratory and kindly provided by Dr Ying Xu (Soochow University, China). Bmal1^fl/fl mice were also obtained from Dr Ying Xu, and Twist2‐Cre mice were purchased from the Jackson Laboratories (No. 008712). All experimental mice were maintained on a C57BL/6J genetic background. All procedures were performed according to the ethical guidelines and approved by the Institutional Animal Care and Use Committee of Tongji Medical College (IACUC number: S739).

The mandibles of experimental mice were harvested and fixed. Scanning was performed at a voxel size of 9 μm by Micro‐CT (SkyScan 1176, Broker). The images were used to reconstruct the tomography scans and quantify. Bone mass was evaluated according to bone mineral density (BMD), bone volume/total volume (BV/TV), trabecular thickness (Tb.Th), bone surface area/bone volume (BS/BV), trabecular number (Tb.N) and trabecular separation (Tb.Sp).

Total RNA was extracted from distally mandibular condyle tissues of Bmal1^‐/‐ mice and wild‐type mice (each 3 pairs of 4‐, 6‐, 8‐ and 10‐week‐old). The samples were sent to BGI Tech. for quantification, RNA‐Seq library construction, sequencing and bioinformatics analysis (Appendix S1).

Alcian blue and alizarin red staining, Edu staining assay, etc, were performed. Please see the Appendixfor more details. S1

Unless otherwise stated, all data were shown as mean ± standard deviation. The GraphPad Prism 7.0 software was used for statistical analysis. Statistically significant differences were determined by Student's t test or ANOVA. A P‐value <.05 was considered with statistical significance.

To determine the functions of circadian clock in the growth of MCC, we measured the expression patterns and levels of core clock genes in MCC from various week‐old mice. We revealed that the phases of circadian rhythms were consistent across different developmental stages (Figure 1A). Meanwhile, we detected that the expression levels of circadian regulator BMAL1 decreased gradually in MCC, rather than other circadian proteins, including circadian locomotor output cycles kaput (CLOCK), period homologue drosophila 1/2 (PER1/2), cryptochrome 1/2 (CRY1/2), reverse‐erythroblastosis virus alpha (REV‐ERBα) and RAR‐related orphan receptor alpha (RORα) in MCC tissues of (1‐8)‐week‐old mice (Figure 1B, 1 and Figure S1). Traditionally, the thicknesses of proliferative layer zone (PZ) and hypertrophic layer zone (HZ) in MCC is gradually decreased in wild‐type mice (Figure 2E‐G). These results suggested that circadian regulator BMAL1 is closely correlated with the development of MCC.

To further confirm the role of BMAL1 in development and growth of MCC, we constructed global BMAL1‐deficiency (Bmal1^‐/‐) mice (Figure S2A). Here, we observed that the chondrogenesis of Bmal1^‐/‐ mice was slower and lesser compared with wild‐type mice at E14.5, E16.5 and E18.5 stages (Figure S2B, C). Consistently, the mandibular condyles of Bmal1^‐/‐ mice were smaller and shorter in height than those of the age‐matched wild‐type mice (Figure 2A). Micro‐CT analysis showed that the bone mineral density (BMD), bone volume/total volume (BV/TV), trabecular thickness (Tb.Th) and trabecular number (Tb.N) of Bmal1^‐/‐ mice significantly decreased and trabecular separation (Tb.Sp) increased relative to those wild‐type mice in the mandibular condylar regions (Figure 2B‐D). Morphological evaluation of MCC further verified that the height of PZ and HZ was diminished obviously, and the number of chondrocytes per column in PZ was decreased significantly compared to the age‐matched wild‐type mice (Figure 2E‐G). The basic components of cartilage matrix type II collagen (COL2α1), aggrecan (ACAN) and type X collagen (COL10α1) were dramatically less in Bmal1^‐/‐ mice at prenatal and postnatal development stage (Figure 2H and Figure S2D). Immunohistochemical staining for Ki67 revealed that chondrocyte proliferation was significantly attenuated in PZ (Figure 2H and Figure S2E). Moreover, TUNEL staining showed that cell apoptosis of chondrocyte was increased in MCC of Bmal1^‐/‐ mice compared with the age‐matched wild‐type mice (Figure 2I and Figure S2F).

To test whether BMAL1 has intrinsic functionality in chondrocyte proliferation and cartilage matrix productions, we constructed conditional Bmal1 knockout mice in mesenchymal stem cells (MSCs) (Bmal1^fl/fl; Twist2‐Cre) (Figure 3A). The results showed that the chondrogenesis and endochondral ossification of the conditional Bmal1 knockout mice were significantly reduced, suggesting that BMAL1 in MSCs was closely associated with chondrogenesis and endochondral ossification (Figure 3B). To further verify the function of BMAL1 in chondrocytes, we extracted primary chondrocytes from mandibular condylar tissues of wild‐type rat and knocked down Bmal1 using CRISPR/CAS9 system (Figure 3C). We found that BMAL1 deficiency obviously decreased cell proliferation and increased cell apoptosis in chondrocytes (Figure 3D‐F). The critical components of cartilage matrix COL2α1, ACAN and COL10α1, and the chondrocyte proliferation markers ex‐determining region Y (SRY)‐box 9 (SOX9) and CyclinD1, were all significantly downregulated in BMAL1‐deficient chondrocytes (Figure 3K, M). Consistently, BMAL1 overexpression yielded the opposite results in chondrocytes (Figure 3G‐J, L and M). Collectively, these findings verified that BMAL1 deficiency inhibits chondrogenesis and endochondral ossification in mandibular condyle by reducing sequential chondrocyte differentiation.

BMAL1 is a widely acknowledged transcriptional activator.19 To determine the transcriptional effects of BMAL1 on the development of MCC, we performed a genome‐wide RNA sequencing to acquire the transcriptional profile in mandibular condyles from 4‐, 6‐, 8‐ and 10‐week‐old Bmal1^‐/‐ mice and the age‐matched wild‐type mice (Figure 4A). Similarity of transcriptional profiles among the three replicates in the same group was determined by a Pearson correlation coefficient (PCC) analysis. The three replicates showed high correlation with each other (Figure S3). Importantly, we found that the number of differentially expressed genes (DEGs) in the group of 4 weeks of age was the largest, with 206 differential genes, and the 10 weeks of age was the least, with 58 differential genes (Figure 4B). GO analysis revealed that many DEGs were enriched in development process, differentiation and cell death. Remarkably, the number of DEGs in development and differentiation during prepuberty and puberty (4 and 6 weeks of age) was almost twice as many as in young adulthood (8 and 10 weeks of age; Figure 4C). Then, we focused on the candidate gene set that related to chondrogenesis and endochondral ossification, including Gli1, Col2α1 and Col10α1. We found that these genes had the similar spatio‐temporal expression patterns to Bmal1, with a higher expression level during prepuberty and puberty (Figure 4D). Moreover, the expression changes of these genes after knocking down Bmal1 were greater during prepuberty and puberty than young adulthood (Figure 4D). These results verified that the transcriptional effects of BMAL1 in MCC are gradually decreased which is along with the development of mandibular condyle.

Due to the higher expression levels of Bmal1 and the genes related to chondrogenesis and endochondral ossification in prepuberty and puberty, the data of RNA sequencing from 4‐week‐old mice were analysed and further verified by quantitative PCR and immumohistochemical staining. We found that Ihh, Ptch1 and Gli1 were significantly downregulated along with the genes related to chondrogenesis and endochondral ossification (Figure 4D‐F), suggesting that hedgehog (Hh) pathway was under the strict control of BMAL1 in mandibular condyles. Hedgehog pathway plays critical roles in sequential chondrocyte differentiation.20 To verify the effects of Hh signalling in chondrocytes from mandibular condyle, we used Hh signalling activator smoothened agonist (SAG; 0.1, 0.5 or 1.0 μmol/L) or inhibitor vismodegib (GDC‐0449; 0.1, 0.5 or 1.0 μmol/L). We observed increased proliferation and decreased apoptosis after SAG supplement (Figure S4A‐D). In contrast, GDC‐0449 resulted in significant decrease of cell proliferation and increase of cell apoptosis (Figure S4A‐D). Western blot assay showed that the expression levels of indian hedgehog (IHH), patched homologue 1 (PTCH1), GLI‐Kruppel family member (GLI1), COL2α1, ACAN, COL10α1, matrix metallopeptidase 13 (MMP13), cell apoptosis regulators apoptosis regulator Bcl‐2 (BCL2) and bcl‐2‐like protein 1 (BCL‐XL), chondrocyte proliferation markers SOX9 and CyclinD1 increased with SAG supplement (Figure S4E, F), while the expression levels of all above genes decreased with GDC‐0449 treatment (Figure S4G, H). These results suggested that the loss of BMAL1 may decrease chondrocyte proliferation and cartilage matrix production by downregulating Hh signalling.

As a classical transcription activator, BMAL1 plays its roles by combining the E‐box site in the promoter of downstream target genes and activating their transcription.11 To further clarify the underlying mechanisms in which BMAL1 controls Hh signalling, the ChIP assays were employed to examine BMAL1‐binding site within Ptch1 and Ihh promoter regions. Our results indicated that BMAL1 selectively bound to the promoter (−1800/‐1542, 259 bp) of Ptch1 (Figure 5A). We also found that BMAL1 could bind to Ihh promoter region which was identical with the similar report 21 (Figure 5B). Dual‐luciferase assay further verified that BMAL1 improved the activity of Ptch1 and Ihh promoters, but not those of the mutants with BMAL1‐binding site being either deleted or mutated. In addition, we found that BMAL1 overexpression could further improve the activity of both Ptch1 and Ihh promoters (Figure 5C, 5).

To verify the necessary function of PTCH1 in BMAL1‐regulated chondrogenesis and endochondral ossification, we explored whether PTCH1 overexpression or Hh signalling activator SAG could rescue the phenotype caused by BMAL1 deficiency in chondrocytes. The results indicated that PTCH1 overexpression or SAG reversed the reduction of chondrocyte proliferation and cartilage matrix production and the increase of chondrocyte apoptosis induced by BMAL1 knockdown (Figure 5E‐N and Figure S5). Taken together, these findings indicated that the loss of BMAL1 inhibits sequential chondrocyte differentiation through directly activating Ptch1 and Ihh transcription.

To determine whether activating Hh signalling can rescue the short malformation phenotypes of mandible caused by BMAL1 deficiency, SAG was injected intraperitoneally into Bmal1^‐/‐ mice during the prepuberty and early puberty periods (2 weeks of age) and young adulthood (8 weeks of age), respectively (20μg/g, three times a week, 4 weeks). Immunohistochemical staining for GLI1 demonstrated significant activation of the Hh pathway as defined by increased expression of GLI1 after the application of SAG (Figure 6A). Micro‐CT results showed that skeletal development in the puberty‐SAG‐injected Bmal1^‐/‐ mice was substantially ameliorative (Figure 6B‐E). H&E staining showed a significant increase in the number of PZ chondrocytes (Figure 6F and Figure S6A). Furthermore, there were more Ki67‐positive chondrocytes in PZ and less TUNEL‐positive chondrocytes in MCC from the Bmal1^‐/‐ mice at puberty‐SAG‐injected (Figure 6G, H). Consistently, the basic components of cartilage matrix COL2α1 were significantly increased after injecting SAG (Figure S6C). However, young adulthood‐SAG‐injected Bmal1^‐/‐ mice did not response effectively (Figure 6B‐E and Figure S6B). Taken together, our results revealed that Hh signalling activator plays potential roles in improving bone mass reduction caused by BMAL1 deficiency, which was effective in prepuberty and early puberty periods intervention, rather than young adulthood treatment. These findings provide a new strategy for the clinical treatment of facial dysmorphism.