Inactivation of Cntnap4 in Cranial Neural Crest Cells Results in Craniofacial Bone Deformities and Hydrocephalus
Pin Ha, M.D., D.D.S, M.S., Xiangyou Luo, Ph.D., D.D.S., Chenshuang Li, Ph.D., D.D.S., Jean Woo Mok, B.S., Samantha S. Lee, B.S., Nicolette K. Golnazarian, B.S., Eric Chen, D.D.S., Zhong Zheng, Ph.D., Kang Ting, D.M.D., D.Med.Sc, Xinli Zhang, M.D., Ph.D., Chia Soo, M.D., FASC.
UCLA, Los Angeles, CA, USA.
PURPOSE: Contactin-associated protein-like 4 (Cntnap4) is a member of the neurexin superfamily of transmembrane molecules that have critical functions in neuronal cell communication. For example, global Cntnap4 knockout mice exhibit autism spectrum disorder (ASD)-like behavioral phenotypes. Interestingly, Cntnap4 was recently identified as a specific cell surface receptor of the osteogenic protein, Nell-1, and has a critical role in Nell-1-mediated osteogenesis. By using cranial neural crest cells (CNCCs) specific Cntnap4 knockout mice, this study provides direct evidence that Cntnap4 is a pivotal factor in the development and growth of the craniofacial tissues, thereby introducing a new target for future clinical interventions related to craniofacial anomalies. METHODS: Cntnap4flox/flox; Wnt1-Cre (Cntnap4Wnt1KO) mice were generated with specific inactivation of Cntnap4 in Wnt1 expressing CNCCs for comprehensive analysis. Cre negative littermates of the same age and/or gender were used as wild type (WT) control. Mice at neonatal and early postnatal stages were used for this study. Gross examination was performed with particular attention to craniofacial phenotypes. The calvarial vault and base were evaluated using multiple approaches including neonatal skeletal staining, high resolution Micro-CT, histology with cell lineage validation of Cntnap4 inactivation, and immunohistochemistry. Regional calvarial cells from either the frontonasal or parietal bones were used for in vitro osteogenic differentiation experiments. RESULTS: Forty-seven out of 97 (48%) Cntnap4Wnt1KO mice were born alive and matured into adulthood. In comparison to WT controls, neonatal KO revealed severe frontonasal bone defects with mild parietal bone defects in terms of bone density and bone volume at 100% penetrance. The cranial base appeared indistinguishable between KO and WT mice in the bones and synchondroses regardless of being born dead or alive at birth. Among the surviving Cntnap4Wnt1KO mice, 32% died of hydrocephalus with premature ossification of the intrasphenoidal synchondrosis and widened frontal, sagittal, and coronal sutures several weeks after birth as revealed by micro-CT and histological analyses. At week 3, both the frontal and parietal bones of KO mice showed a 17% lower bone volume (BV) compared to WT mice. This percentage remained almost the same for the frontal bone at week 7, while a smaller difference (8.5% lower) was observed for the parietal bone in KO mice. Similarly, the bone mineral density (BMD) at week 7 was found to be 7% and 8.8% lower in the frontal and parietal bones of KO mice, respectively. Moreover, the osteogenic capacity of calvarial cells from CNCCs-derived frontonasal bones of KO mice was significantly impaired, which is in agreement with the reduced OCN expression in the tissue sections of KO craniofacial bones.
CONCLUSION: Collectively, Cntnap4 was validated for the first time as a novel regulator of cranial neural crest cells that is essential for the normal development and growth of the cranial vault and base. Moreover, Cntnap4 inactivation is associated with the pathogenesis of postnatal hydrocephalus.
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