FGF8 Signaling Alters the Osteogenic Cell Fate in the Hard Palate

Abstract

Fibroblast growth factor (FGF) signaling has been implicated in the regulation of osteogenesis in both intramembranous and endochondral ossifications. In the developing palate, the anterior bony palate forms by direct differentiation of cranial neural crest (CNC)-derived mesenchymal cells, but the signals that regulate the osteogenic cell fate in the developing palate remain unclear. In the present study, we investigated the potential role of FGF signaling in osteogenic fate determination of the palatal mesenchymal cells. We showed that locally activated FGF8 signaling in the anterior palate using a Shox2 knock-in allele and an R26R allele leads to a unique palatal defect: a complete loss of the palatine process of the maxilla as well as formation of ectopic cartilaginous tissues in the anterior palate. This aberrant developmental process was accompanied by a significantly elevated level of cell proliferation, which contributes to an abnormally thickened palatal tissue, where the palatine process of the maxilla would normally form, and by a complete inhibition of Osterix expression, which accounts for the lack of bone formation. The coexpression of Runx2 initially with Sox9 and subsequently with Col II in the ectopic cartilaginous tissues indicates a conversion of osteogenic fate to a chondrogenic one. Consistent with the unique palatal phenotype, RNA-Sequencing analysis revealed that the augmented FGF8 signaling downregulated genes involved in ossification, biomineral tissue development, and bone mineralization but upregulated genes involved in cell proliferation, cartilage development, and cell fate commitment, which was further supported by quantitative real-time reverse transcription polymerase chain reaction validation of selected genes. Our results demonstrate that FGF8 signaling functions as a negative regulator of osteogenic fate and is sufficient to convert a subset of CNC cell-derived mesenchymal cells into cartilage in the anterior hard palate, which will have implications in future directed differentiation of CNC-derived precursor cells for clinical application.

Exogenous FGF8 signaling in osteocytes leads to mandibular hypoplasia in mice

“…Therefore, micrognathia is an isolated phenotype and the lethality is possibly associated with impaired swallowing problem attributing to micrognathia. Similar to our previous observations that enhanced FGF8 signaling in CNC‐derived mesenchymal cells (Shao et al, ; Xu et al, ) inhibits osteogenesis, we found bone loss in the maxillomandibular region including the maxillary region (data not shown). Since the maxillary bone hypoplasia does not contribute significantly to micrognathia, we focused on our analyses on mandibular phenotype.…”

“…As presented in our previous studies, enhanced FGF8 signaling promotes cell proliferation in CNC‐derived mesenchymal cells in vitro and in Shox2 + linage cells in the developing palate in vivo (Shao et al, ; Xu et al, ). Similarly, we indeed found a significant difference in the levels of cell proliferation rate of the mesenchymal cells in the region of forming mandibular bone (except Meckel's cartilage) between the control and Dmp1 Cre ; R26R Fgf8 mandible (Figure ).…”

“…We found that the defective osteogenesis appeared discernible at E15.5 and become much severe at the birth. Given that our recent work showing that FGF8 signaling alters osteogenic cell fate of Shox2 + palatal mesenchymal cells and induces ectopic chondrogenic differentiation (Xu et al, ), we also examined whether cartilage‐like tissues or chondrocytes were present in the maxillomandibular region of Dmp1 Cre ; R26R Fgf8 mice, but we did not find such tissues or cells (data not shown). Thus, the augmented FGF8 signaling in osteocytes functions to inhibit osteogenesis but cannot convert osteogenic cells to chondrocytes.…”

“…In our previous study, we found that augmented FGF8 signaling in cranial neural crest cells results in early embryonic lethal as well as dramatic disruption of maxillomandibular ossification (Shao et al, ). In addition, ectopic FGF8 signaling in Shox2 + linage cells (anterior palatal mesenchymal cells) also leads to the deficiency of palatal bone the alteration of osteogenic fate in the anterior bony palate (Xu et al, ). These results demonstrate crucial importance of finely tuned level of FGF8 signaling in craniofacial bone formation.…”

Extracellular Matrix Composition and Remodeling: Current Perspectives on Secondary Palate Formation, Cleft Lip/Palate, and Palatal Reconstruction

“…Once palatal fusion is complete, the anterior two-thirds mineralize by intramembranous ossification (hard palate) ( Figure 1E), and the posterior third will give rise to a fibromuscular tissue (soft palate) under the signaling by numerous factors, particularly bone morphogenetic proteins (BMPs), fibroblast growth factors (FGFs), hedgehog (HH), vascular endothelial growth factor (VEGF), and Wnt/β-catenin signaling, which drive the palatal mesenchyme to undergo osteoblast differentiation for mineralization (Wu et al, 2008;Baek et al, 2011;Nelson et al, 2011;Pan et al, 2013;Smith et al, 2013;Hill et al, 2014Hill et al, , 2015Nassif et al, 2014;Iyyanar and Nazarali, 2017;Zhang et al, 2017;Xu J. et al, 2018;Thompson et al, 2019).…”