Foxp2 regulates anatomical features that may be relevant for vocal behaviors and bipedal locomotion Shuqin Xua, Pei Liua, Yuanxing Chena, Yi Chenb, Wei Zhanga, Haixia Zhaoa, Yiwei Caoa, Fuhua Wanga, Nana Jianga, Shifeng Lina, Baojie Lia, Zhenlin Zhangc, Zhanying Weic, Ying Fanc, Yunyun Jind, Lin Hea, Rujiang Zhoua, Joseph D. Dekkere, Haley O. Tuckere, Simon E. Fisherf,g, Zhengju Yaoa, Quansheng Liub,1, Xuechun Xiaa,1, and Xizhi Guoa,1 aBio-X-Renji Hospital Research Center, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, 200240 Shanghai, China; bGuangdong Key Laboratory of Animal Conservation and Resource Utilization, Guangdong Public Laboratory of Wild Animal Conservation and Utilization, Guangdong Institute of Applied Biological Resources, 510260 Guangzhou, China; cThe Sixth People’s Hospital of Shanghai, School of Medicine, Shanghai Jiao Tong University, 200240 Shanghai, China; dInstitute of Biomedical Sciences and School of Life Sciences, East China Normal University, 200241 Shanghai, China; eInstitute for Cellular and Molecular Biology, University of Texas at Austin, Austin, TX 78712; fLanguage and Genetics Department, Max Planck Institute for Psycholinguistics, Nijmegen 6525 XD, The Netherlands; and gDonders Institute for Brain, Cognition and Behaviour, Radboud University, Nijmegen 6500 HE, The Netherlands Edited by Joseph S. Takahashi, Howard Hughes Medical Institute and University of Texas Southwestern Medical Center, Dallas, TX, and approved July 12, 2018 (received for review December 18, 2017) Fundamental human traits, such as language and bipedalism, are on the lineage that led to modern humans (24, 25). Two amino associated with a range of anatomical adaptations in craniofacial acid substitutions occurred in human FOXP2 after splitting from shaping and skeletal remodeling. However, it is unclear how such our common ancestor with the chimpanzee. Investigations of morphological features arose during hominin evolution. FOXP2 is these substitutions in partially humanized mice suggest they af- a brain-expressed transcription factor implicated in a rare disorder fect connectivity and plasticity of cortico-basal ganglia circuits, involving speech apraxia and language impairments. Analysis of impacting learning mechanisms (26, 27). its evolutionary history suggests that this gene may have contrib- Morphological correlation or covariation, a concept going as uted to the emergence of proficient spoken language. In the far back as Darwin’s On the Origin of Species, is an essential present study, through analyses of skeleton-specific knockout driving force for evolution. The emergence of human speech Foxp2 mice, we identified roles of in skull shaping and bone involved not only neural changes, but also modifications in an- Foxp2 remodeling. Selective ablation of in cartilage disrupted atomical features of the vocal tract, including configuration of pup vocalizations in a similar way to that of global Foxp2 mutants, superficial vocal folds, trachea, and oral cavities. For instance, which may be due to pleiotropic effects on craniofacial morpho- the importance of a relatively descended larynx for human genesis. Our findings also indicate that Foxp2 helps to regulate strength and length of hind limbs and maintenance of joint carti- speech has been a topic of much discussion (28). While multiple lage and intervertebral discs, which are all anatomical features studies of Foxp2 have focused on neuronal functions, none have that are susceptible to adaptations for bipedal locomotion. In light tested its potential contributions to vocal anatomical geometry. Of of the known roles of Foxp2 in brain circuits that are important for note, a comparison of transcriptional regulation by human and motor skills and spoken language, we suggest that this gene may chimpanzee versions of FOXP2 reported enrichment of differential have been well placed to contribute to coevolution of neural and targets involved in craniofacial formation and cartilage development anatomical adaptations related to speech and bipedal locomotion. (29). Moreover, in a previous study, we demonstrated cooperative Foxp2 | vocalization | bipedalism | cranial base | bone remodeling Significance BIOLOGY DEVELOPMENTAL poken language and bipedalism are two behavioral traits that Speech and bipedalism are key aspects of behavior that Sdistinguish humans from other living apes, each with a emerged during human evolution. FOXP2, a gene implicated in complex evolutionary history. The emergence of such derived a human speech and language disorder, has been suggested to traits was accompanied by various changes in skeletal anatomy. contribute to language evolution. Here, through knockout For example, as well as long-term increases in overall cranial studies of mouse Foxp2, we show that this gene is not only capacity over the course of primate evolution, more recent al- important for neural circuits involved in vocal behaviors, it also terations in skull shape occurred in our ancestors, changes that helps regulate relevant anatomical substrates. We additionally some hypothesize as important for language evolution (1, 2). demonstrate that Foxp2 influences skeletal features that may Advances in genomics are uncovering genes of relevance for be relevant for bipedal locomotion. Our findings raise the distinct human traits like language (3). In particular, disruptions possibility that FOXP2 might be important for anatomical fea- of the FOXP2 transcription factor are implicated in a monogenic tures contributing to derived human traits, including speech disorder involving childhood apraxia of speech (CAS) and ex- and bipedalism. pressive–receptive language impairments (4–7). The first etio- logical FOXP2 mutation was identified in a family (KE) in which Author contributions: X.G. designed research; S.X., P.L., Yuanxing Chen, Yi Chen, W.Z., H.Z., Yiwei Cao, F.W., N.J., S.L., Y.J., R.Z., Z.Y., Q.L., and X.X. performed research; B.L., Z.Z., all affected members carried an R553H substitution within the Z.W., Y.F., L.H., J.D.D., H.O.T., S.E.F., and Q.L. contributed new reagents/analytic tools; Forkhead-box DNA-binding domain. In addition, mutations Q.L., X.X., and X.G. analyzed data; and X.G. wrote the paper. of FOXP1, the closest paralogue of FOXP2, cause a neuro- The authors declare no conflict of interest. developmental syndrome including speech and language im- This article is a PNAS Direct Submission. – pairments (8 11), partially overlapping with deficits associated Published under the PNAS license. – with FOXP2 variants in multiple different cases (12 14). The 1To whom correspondence may be addressed. Email: [email protected], xuechun.xia@sjtu. functions of Foxp2 in vocal behaviors have been assessed edu.cn, or [email protected]. through analysis of ultrasonic vocalizations (USVs) in mouse This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. models (15–20), or learned song in songbirds (21–23). Foxp2 is 1073/pnas.1721820115/-/DCSupplemental. highly conserved across species, but underwent positive selection Published online August 13, 2018. www.pnas.org/cgi/doi/10.1073/pnas.1721820115 PNAS | August 28, 2018 | vol. 115 | no. 35 | 8799–8804 Downloaded by guest on September 27, 2021 Δ/Δ fl/fl functions of Foxp1/2 in regulating endochondral ossification during Foxp2Col2 mice were smaller in size than Foxp2 littermates at embryonic bone development (30). embryonic stage 15.5 days (E15.5) and P13 (Fig. 1C). Endo- Building on our demonstration of a Foxp2 role in embryonic chondral ossification of the Bo, the basisphenoid bone (Bs) and Δ/Δ bone development, and in light of prior hypothesized involve- the nasal bone, were attenuated in Foxp2Col2 mice at E15.5, as ment of this gene in human evolution, we here used skeleton- evidenced by diminished Alizarin red staining (arrows in Fig. 1C). specific loss-of-function analyses in mice to investigate how it In particular, broader funnel-shaped presphenoids were observed Δ/Δ might help regulate anatomy. Unexpectedly, skeletal Foxp2 loss in Foxp2Col2 mice at P13 (arrows in Fig. 1 D and F). Minor led to disruption of pup vocalizations, similar to phenotypes alterations in presphenoid morphology were also detected at P7 in R552H/+ previously reported for global mutant or knockout lines (17, 31). Foxp2 mutant mice, which carry a point mutation matching Most interestingly, loss of Foxp2 in skeletal tissue also led to that found in affected members of the KE family (Fig. 1 E and G pleiotropic deficits in skull shaping and bone strengthening. Our and SI Appendix,Fig.S1A). Altered presphenoid morphology was R552H/R552H findings reveal regulatory roles of Foxp2 in helping build ana- much more evident in homozygous Foxp2 mice at P0 tomic substrates that are important for vocal behaviors, and (arrows in Fig. 1H and SI Appendix, Fig. S1B). At the histological Δ/Δ suggest that it might also be considered a candidate for skeletal level, Foxp2Col2 mice showed delayed chondrocyte hypertrophy adaptations relevant to bipedal locomotion. and ossification within sphenooccipital synchondroses, as revealed by Safranin O staining and immunohistochemistry (IHC) and Results immunofluorescence (IF) examination using Col X, Osterix (Osx), Cartilage-Specific Deletion of Foxp2 Impairs Cranial Base Development. and Foxp2 antibodies (SI Appendix, Figs. S1C and S2A). Collec- Cranial base morphogenesis is a major determinant of skull tively, these data indicate that Foxp2 is important for sphenoid shaping (32). Basicranial