Deficiency of lrp4 in zebrafish and human LRP4 mutation induce aberrant activation of Jagged–Notch signaling in fin and limb development
Low-density lipoprotein receptor-related protein 4 (LRP4) is a multi-functional protein implicated in bone, kidney and neurological diseases including Cenani-Lenz syndactyly (CLS), sclerosteosis, osteoporosis, congenital myasthenic syndrome and myasthenia gravis. Why different LRP4 mutation alleles cause distinct and even contrasting disease phenotypes remain unclear. Herein, we utilized the zebrafish model to search for pathways affected by a deficiency of LRP4. The lrp4 knockdown in zebrafish embryos exhibits cyst formations at fin structures and the caudal vein plexus, malformed pectoral fins, defective bone formation and compromised kidney morphogenesis; which partially phenocopied the human LRP4 mutations and were reminiscent of phenotypes resulting form a perturbed Notch signaling pathway. We discovered that the Lrp4-deficient zebrafish manifested increased Notch outputs in addition to enhanced Wnt signaling, with the expression of Notch ligand jagged1b being significantly elevated at the fin structures. To examine conservatism of signaling mechanisms, the effect of LRP4 missense mutations and siRNA knockdowns, including a novel missense mutation c.1117C > T (p.R373W) of LRP4, were tested in mammalian kidney and osteoblast cells. The results showed that LRP4 suppressed both Wnt/β-Catenin and Notch signaling pathways, and these activities were perturbed either by LRP4 missense mutations or by a knockdown of LRP4. Our finding underscore that LRP4 is required for limiting Jagged–Notch signaling throughout the fin/limb and kidney development, whose perturbation representing a novel mechanism for LRP4-related diseases. Moreover, our study reveals an evolutionarily conserved relationship between LRP4 and Jagged–Notch signaling, which may shed light on how the Notch signaling is fine-tuned during fin/limb development.
KeywordsSkeletogenesis Bone disorders Pronephros Morphant Phenocopy EGF-like domain wt1b HES1
We are indebted to the family for kindly partaking in this study. We are grateful to Prof. Bernd Wollnik, Dr. Thomas J. Carney and Prof. David Virshup for the kind provision of plasmids. We also thank Prof. Baojie Li for the kind gift of MC3T3-E1 cell line; Prof. Christoph Englert for the kind gift of the Tg(wt1b:GFP)(line 1) zebrafish strain; Dr. Xingang Wang and Ms. Pang Zhan for assistance on gene cloning; Mr. Kuan-Chieh Wang for statistical help; Ms. Wei-Ru (Lydia) Hsiao and Chia-Yu Chang for aquarium care; the Taiwan Zebrafish Core Facility at NHRI (TZCF@NHRI), the Taiwan Zebrafish Core Facility at Academia Sinica (TZCAS) and Northwest University Zebrafish Core Facility for assistance with fish culture.
JT and Y-WL conceived the study, designed the experiments and prepared the manuscript. JS, CL, H-YH, C-WC, GL, YK, Y-HC, M-JC, ZL, W-LC, Y-FC and Y-HS prepared the samples, performed the experiments and analyzed the data. MS, ME-K, and OQS diagnosed the patient.
This work was supported by Natural Science Foundation of Shaanxi Province, China (2016JM3018); Opening Foundation of State Key Laboratory of Freshwater Ecology and Biotechnology, China (2018FB10); Ministry of Science and Technology, Taiwan (MOST) 106-2313-B-029-002-MY3, 105-2313-B-029-002 and 102-2628-B-029-002-MY3.
Compliance with ethical standards
Conflict of interest
The authors declare no competing financial interests.
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