Abstract
Upregulation of Nell-1 has been associated with craniosynostosis (CS) in humans, and validated in a mouse transgenic Nell-1 overexpression model. Global Nell-1 inactivation in mice by N-ethyl-N-nitrosourea (ENU) mutagenesis results in neonatal lethality with skeletal abnormalities including cleidocranial dysplasia (CCD)-like calvarial bone defects. This study further defines the role of Nell-1 in craniofacial skeletogenesis by investigating specific inactivation of Nell-1 in Wnt1 expressing cell lineages due to the importance of cranial neural crest cells (CNCCs) in craniofacial tissue development. Nell-1flox/flox; Wnt1-Cre (Nell-1Wnt1 KO) mice were generated for comprehensive analysis, while the relevant reporter mice were created for CNCC lineage tracing. Nell-1Wnt1 KO mice were born alive, but revealed significant frontonasal and mandibular bone defects with complete penetrance. Immunostaining demonstrated that the affected craniofacial bones exhibited decreased osteogenic and Wnt/β-catenin markers (Osteocalcin and active-β-catenin). Nell-1-deficient CNCCs demonstrated a significant reduction in cell proliferation and osteogenic differentiation. Active-β-catenin levels were significantly low in Nell-1-deficient CNCCs, but were rescued along with osteogenic capacity to a level close to that of wild-type (WT) cells via exogenous Nell-1 protein. Surprisingly, 5.4% of young adult Nell-1Wnt1 KO mice developed hydrocephalus with premature ossification of the intrasphenoidal synchondrosis and widened frontal, sagittal, and coronal sutures. Furthermore, the epithelial cells of the choroid plexus and ependymal cells exhibited degenerative changes with misplaced expression of their respective markers, transthyretin and vimentin, as well as dysregulated Pit-2 expression in hydrocephalic Nell-1Wnt1 KO mice. Nell-1Wnt1 KO embryos at E9.5, 14.5, 17.5, and newborn mice did not exhibit hydrocephalic phenotypes grossly and/or histologically. Collectively, Nell-1 is a pivotal modulator of CNCCs that is essential for normal development and growth of the cranial vault and base, and mandibles partially via activating the Wnt/β-catenin pathway. Nell-1 may also be critically involved in regulating cerebrospinal fluid homeostasis and in the pathogenesis of postnatal hydrocephalus.
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23 March 2020
A Correction to this paper has been published: https://doi.org/10.1038/s41418-020-0529-9
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Acknowledgements
We would like to thank Drs Dan Pan and Zhong Zheng at the University of California, Los Angeles for their excellent technical assistance and scientific input. This study was supported by the NIH-NIAMS (grants R01AR066782, R01AR068835, R01AR061399), NIH-NIDCR (grant K08DE026805), UCLA/NIH CTSI (grant UL1TR000124), the National Aeronautical and Space Administration (“NASA”, GA- 2014–154), the National Natural Science Foundation of China [81670972, 81400538], and the Zhejiang Provincial Natural Science Foundation of China [LY19H140002].
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XZ, KT, CS, and HW conceived and designed the study, interpreted data, and approved the manuscript; XC, HW, MY, and XZ executed the study, collected and analyzed the data, and wrote the manuscript; CK, HQ, PH, WJ, XL, and EC collected and analyzed the data and edited the manuscript; RBN, CY, LB, JS, and JHK provided research materials and collected data.
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XZ, KT, and CS are inventors of Nell-1 related patents. XZ, KT, and CS are founders and/or previous board members of Bone Biologics Inc./Bone Biologics Corp., which sublicenses Nell-1 patents from the UC Regents, which also hold equity in the company. XZ, KT, and CS = Xinli Zhang, Kang Ting, Chia Soo
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Chen, X., Wang, H., Yu, M. et al. Cumulative inactivation of Nell-1 in Wnt1 expressing cell lineages results in craniofacial skeletal hypoplasia and postnatal hydrocephalus. Cell Death Differ 27, 1415–1430 (2020). https://doi.org/10.1038/s41418-019-0427-1
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DOI: https://doi.org/10.1038/s41418-019-0427-1
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