Abstract
Sperm flagellar protein 2 (SPEF2) is essential for motile cilia, and lack of SPEF2 function causes male infertility and primary ciliary dyskinesia. Cilia are pointing out from the cell surface and are involved in signal transduction from extracellular matrix, fluid flow and motility. It has been shown that cilia and cilia-related genes play essential role in commitment and differentiation of chondrocytes and osteoblasts during bone formation. Here we show that SPEF2 is expressed in bone and cartilage. The analysis of a Spef2 knockout (KO) mouse model revealed hydrocephalus, growth retardation and death prior to five weeks of age. To further elucidate the causes of growth retardation we analyzed the bone structure and possible effects of SPEF2 depletion on bone formation. In Spef2 KO mice, long bones (tibia and femur) were shorter compared to wild type, and X-ray analysis revealed reduced bone mineral content. Furthermore, we showed that the in vitro differentiation of osteoblasts isolated from Spef2 KO animals was compromised. In conclusion, this study reveals a novel function for SPEF2 in bone formation through regulation of osteoblast differentiation and bone growth.
Highlights
Skeletogenesis occurs through endochondral and intramembranous ossification
We report the results from the conventional KO mouse model, in which the Spef[2] gene was inactivated in all tissues examined
The genotype of pups was confirmed by PCR using primers flanking the LoxP site after exon 5 to amplify 478 bp product from mutated allele and 379 bp product from wild type (WT) allele (Supplemental Fig. S1A)
Summary
Skeletogenesis occurs through endochondral and intramembranous ossification. During intramembranous ossification, mesenchymal stem cells (MSC) directly differentiate into osteoblasts. MSCs first differentiate to chondrocytes forming the cartilage, which is subsequently replaced by bone. It has been shown that commitment and differentiation to chondrocytes and osteoblasts are dependent on cilia and cilia-related genes[23] and e.g. blocking of the primary cilia formation using IFT88- siRNA in the MSCs caused the loss of cell adhesion and cell type specific differentiation[23]. Depletion of the IFT complex proteins, IFT20 and IFT80 in osteoblasts caused decreased bone mass and impaired osteoblast differentiation[24,25], indicating an important role for functional IFT in osteoblast differentiation Both osteoblasts and cartilage forming chondrocytes are derived from MSC, which have been shown to express primary cilia on their surface[23,26]. We generated a mouse model with a stop codon located after exon 2 of Spef[2] gene to further investigate the role of SPEF2 in ciliated tissues in mice
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