Pth1r Signal in Gli1+ Cells Maintains Postnatal Cranial Base Synchondrosis

Abstract

Cranial base synchondroses are the endochondral ossification centers for cranial base growth and thus indispensable for proper skull, brain, and midfacial development. The synchondroses are composed of mirror-image growth plates that are continuously maintained from the embryonic to postnatal stage through chondrocyte differentiation. Several factors, including Pth1r signaling, are known to control fetal synchondrosis development. However, there are currently no reports regarding any role for Pth1r signaling in postnatal cranial base and synchondrosis development. Also, the mesenchymal cells that source Pth1r signaling for synchondroses are not known. Here, we employed an inducible mouse model, a hedgehog-responsive Gli1-CreERT2 driver, focusing on the postnatal study. We performed 2 inducible protocols using Gli1-CreERT2;Tomatofl/+ mice that uncovered distinct patterning of Gli1-positive and Gli1-negative chondrocytes in the synchondrosis cartilage. Moreover, we generated Gli1-CreERT2;Pth1rfl/fl;Tomatofl/+ mice to assess their functions in postnatal synchondrosis and found that the mutants had survived postnatally. The mutant skulls morphologically presented unambiguous phenotypes where we noticed the shortened cranial base and premature synchondrosis closure. Histologically, gradual disorganization in mutant synchondroses caused an uncommon remaining central zone between hypertrophic zones on both sides while the successive differentiation of round, flat, and hypertrophic chondrocytes was observed in control sections. These mutant synchondroses disappeared and were finally replaced by bone. Of note, the mutant fusing synchondroses lost their characteristic patterning of Gli1-positive and Gli1-negative chondrocytes, suggesting that loss of Pth1r signaling alters the distribution of hedgehog-responsive chondrocytes. Moreover, we performed laser microdissection and RNA sequencing to characterize the flat proliferative and round resting chondrocytes where we found flat chondrocytes have a characteristic feature of both chondrocyte proliferation and maturation. Taken together, these data demonstrate that Pth1r signaling in Gli1-positive cells is essential for postnatal development and maintenance in cranial base synchondroses. Our findings will elucidate previously unknown aspects of Pth1r functions in cranial biology and development.

Pth1r in Neural Crest Cells Regulates Nasal Cartilage Differentiation

“…14). To reinforce our findings in vivo, we employed a hedgehog responsive Gli1-Cre ERT2 driver and have recently established Gli1-Cre ERT2 ;Pth1r fl/fl ; Tomato fl/+ mice, an inducible model in which Pth1r is conditionally deleted in Gli1-positive cells (Amano et al 2023). Tamoxifen was interperitoneally injected at P3, and the samples were harvested at P10 (Fig.…”

Mesenchymal stem cells in craniofacial reconstruction: a comprehensive review

“…Guo et al employed cell flow cytometry to demonstrate that craniofacial Gli1+ cells express high levels of markers specific to mesenchymal stem cells (MSCs), such as Sca1 and CD44, and do not express CD34, suggesting that these cells are predominantly MSCs ( Guo et al, 2018 ). K Amano et al distinguished Gli1+ and Gli1-chondrocytes in the cranial base using Gli1-CreERT2; Tomatofl/+ and Gli1-CreERT2; Pth1rfl/fl; Tomatofl/+ mice and validated the crucial role of Gli1+ cells in early cranial development and adulthood through the Pth1r signaling pathway ( Amano et al, 2023 ). Zhang et al also studied the distribution of Gli1+ cells in the mandible using Gli1-CreERT2 mice and found that Gli1+ cells are present in specific regions of the periosteum and condyle, with stronger proliferative capacity in the condyle region ( Zhang N. et al, 2023 ).…”