The Role Of Foxp2 In Regulating Craniofacial Shaping And Long Bone Remodeling

Fundamental human traits,such as language and bipedalism,are associated with anatomical adaptations in craniofacial shaping and skeletal remodeling.However,it is unclear how such morphological features arose during human evolution.Clinical observations have reported a rare speech and language disorder of developmental verbal dyspraxia?DVD?,which is typically characterized by the symptom of defects at expressing language and mild mental retard.Genetic analysis has positioned this symptom in several genetic loci including FOXP2,FOXP1 etc.A KE family of DVD symptom has been revealed to carry a FOXP2 R553H point mutation.Meanwhile,FOXP1,a paralog of FOXP2 protein,is also predisposed to the symptom of language impairment and autism.Through the genetic analysis in mouse or bird,Foxp1 or Foxp2 has been revealed to exert their roles in language development by their action in neuronal circuits.Foxp1and Foxp2 usually form a heterodimers to redundantly regulate the development of long bones or lung organ.Interestingly,FOXP2 protein is highly conserved across mammalian species from mouse to apes.Only a two-amino-acid conversion?T303N,N325S?is detected in human version of FOXP2 since the split of human from primates.Genetic analysis indicates that the humanized conversion attributes to the emergence of language and cognition during human evolution.Given the importance of adaption in skull shaping for a language-ready brain,it is plausible to know whether Foxp2 has more extensive and fundamental roles in anatomical substances involved in speech and vocalization.It would also be interesting to know whether Foxp1/2 cooperatively controls the skull shaping and long bone remodeling,as well as skeletal characteristics involved in bipedal locomotion.Here we first examined the expression of Foxp2 in chondrocytes at the synchondrosis of cranial base and mesenchymal stem cells?MSC?from long bone marrow by immunohistochemical analysis.We found that Foxp2was relatively enriched in skeletal progenitor cells in synchondrosis,and its expression progressively inclined with osteogenic differentiation.To investigate the role of Foxp2 in skull shaping,we generated conditional knockout mice with single or compound deletion of Foxp1/2 alleles in chondrocytes by using Col2-Cre.The Foxp2 deficiency mice appeared shorter nasal bone,attenuated mineralization of cranial base elements including basioccipital bone?Bo?,the basisphenoid bone?Bs?,and deformities in presphenoid and a shortened basisphenoid.Dysmorphogenesis of larynx and impaired maintenance of intervertebral disc were also observed in those Foxp2 knockout mice.Immunohistochemistry showed that the chondrocyte hypertrophy and ossification within the mutant spheno-occipital synchondrosis were delayed,which may account for the defective cranial base development.Similar deformities in nasal bone and presphenoid were observed in Foxp1^?/?_col2 mice.Of note,shortened nasal bone and defective sphenoid were more pronounced in Foxp1/2^?/?_Col2 mice as compared to each single mutant.Acoustic analysis showed that both USVs of Foxp1^?/?_Col2 and Foxp2_Col2^?/?mice at P10 were perturbed.Similar defects of prespenoid and larynx were observed in Foxp2^R552H/+mice,which mimiced human DVD symptom in KE family.In addition,these deformities in cranialfacial bones were more profound in homozygous Foxp2^R552H/R552H mice.Next,we generated conditional knockout mice with single Foxp2deficiency or compound Foxp1/2 deficiency strictly in mesenchymal progenitor cells by employing with Prx1-Cre.The resulting Foxp2^?/?_Prx1mice showed shortened long bone,impaired maintenance in cartilage of growth plate and joint,delayed fusion of interperietal bone.Foxp2deficiency also increased bone mass of long bone,perturbed osteogenic differentiation,and augmented MSC expansion capacity.Three points bending tests indicated a higher load but a lower stiffness of bone at loss of Foxp2.The delayed fusion of lambdoid suture in skull,and impaired osteogenic differentiation of MSCs were more pronounced in mice with double Foxp1/2 knockout mice.And more,we generated conditional knockout mice with Foxp2 deletion in osteoclasts using Ctsk-Cre.The Foxp2^?/?_Ctsk mutant mice showed increased bone mass with impaired osteoclast formation,indicating a positive role of Foxp2 in sustaining osteoclast differentiation.Molecularly,we identified that Foxp2 can inhibit the transactivation of Notch signaling by forming a complex with Foxp1 and RBPj?protein,thus,it promoted osteogenic differentiaion whereas repressed MSC proli-feration.Taken together,our findings indicate that Foxp2 can regulate skull shaping by coordinating the development of cranial base bones,interperietal bone and laryngeal cartilage,which are fundamental substrates for vocalization.And more,Foxp2 could form skeletal features adaptive for upright walking by maintaining the integrity of growth plate,articular cartilage in joint and interveribral disc,and forges strong legs by accelerating the pace of bone remodeling.In light of the known roles of Foxp2 in brain circuitry that are important for motor-skills and spoken language,we suggest that Foxp2 may have been well-placed to contribute to parallel coevolution of neural and anatomical adaptations in forming a language-ready brain and bipedal locomotion.