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Deficiency of lrp4 in zebrafish and human LRP4 mutation induce aberrant activation of Jagged–Notch signaling in fin and limb development

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Abstract

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.

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References

  1. Nakamura T, Gehrke AR, Lemberg J, Zymaszek JS, Shubin NH (2016) Digits and fin rays share common developmental histories. Nature 537:225

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Grandel H, Schulte-Merker S (1998) The development of the paired fins in the zebrafish (Danio rerio). Mech Dev 79:99–120

    Article  CAS  PubMed  Google Scholar 

  3. Tian J, Ling L, Shboul M et al (2010) Loss of CHSY1, a secreted FRINGE enzyme, causes syndromic brachydactyly in humans via increased NOTCH Signaling. Am J Hum Genet 87:768–778

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Carney TJ, Feitosa NM, Sonntag C et al (2010) Genetic analysis of fin development in zebrafish identifies furin and hemicentin 1 as potential novel fraser syndrome disease genes. PLoS Genet 6:e1000907

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Lieschke GJ, Currie PD (2007) Animal models of human disease: zebrafish swim into view. Nat Rev Genet 8:353–367

    Article  CAS  PubMed  Google Scholar 

  6. Gerlach GF, Wingert RA (2013) Kidney organogenesis in the zebrafish: insights into vertebrate nephrogenesis and regeneration. Wiley Interdiscip Rev Dev Biol 2:559–585

    Article  CAS  PubMed  Google Scholar 

  7. Cenani A, Lenz W (1967) Total syndactylia and total radioulnar synostosis in 2 brothers. A contribution on the genetics of syndactylia. Z Kinderheilkd 101:181–190

    Article  CAS  PubMed  Google Scholar 

  8. Leupin O, Piters E, Halleux C et al (2011) Bone overgrowth-associated mutations in the LRP4 gene impair sclerostin facilitator function. J Biol Chem 286:19489–19500

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Ohkawara B, Cabrera-Serrano M, Nakata T et al (2014) LRP4 third beta-propeller domain mutations cause novel congenital myasthenia by compromising agrin-mediated MuSK signaling in a position-specific manner. Hum Mol Genet 23:1856–1868

    Article  CAS  PubMed  Google Scholar 

  10. Rivadeneira F, Styrkarsdottir U, Estrada K et al (2009) Twenty bone-mineral-density loci identified by large-scale meta-analysis of genome-wide association studies. Nat Genet 41:1199-U1158

    Google Scholar 

  11. Pevzner A, Schoser B, Peters K et al (2012) Anti-LRP4 autoantibodies in AChR- and MuSK-antibody-negative myasthenia gravis. J Neurol 259:427–435

    Article  CAS  Google Scholar 

  12. Johnson EB, Hammer RE, Herz J (2005) Abnormal development of the apical ectodermal ridge and polysyndactyly in Megf7-deficient mice. Hum Mol Genet 14:3523–3538

    Article  CAS  PubMed  Google Scholar 

  13. Simon-Chazottes D, Tutois S, Kuehn M et al (2006) Mutations in the gene encoding the low-density lipoprotein receptor LRP4 cause abnormal limb development in the mouse. Genomics 87:673–677

    Article  CAS  PubMed  Google Scholar 

  14. Weatherbee SD, Anderson KV, Niswander LA (2006) LDL-receptor-related protein 4 is crucial for formation of the neuromuscular junction. Development 133:4993–5000

    Article  CAS  PubMed  Google Scholar 

  15. Tanahashi H, Tian QB, Hara Y, Sakagami H, Endo S, Suzuki T (2016) Polyhydramnios in Lrp4 knockout mice with bilateral kidney agenesis: defects in the pathways of amniotic fluid clearance. Sci Rep 6:20241

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Li Y, Pawlik B, Elcioglu N et al (2010) LRP4 mutations alter Wnt/beta-catenin signaling and cause limb and kidney malformations in Cenani-Lenz syndrome. Am J Hum Genet 86:696–706

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Khan TN, Klar J, Ali Z, Khan F, Baig SM, Dahl N (2013) Cenani-Lenz syndrome restricted to limb and kidney anomalies associated with a novel LRP4 missense mutation. Eur J Med Genet 56:371–374

    Article  PubMed  Google Scholar 

  18. Tamai K, Semenov M, Kato Y et al (2000) LDL-receptor-related proteins in Wnt signal transduction. Nature 407:530–535

    Article  CAS  PubMed  Google Scholar 

  19. Pinson KI, Brennan J, Monkley S, Avery BJ, Skarnes WC (2000) An LDL-receptor-related protein mediates Wnt signalling in mice. Nature 407:535–538

    Article  CAS  PubMed  Google Scholar 

  20. Kim N, Stiegler AL, Cameron TO et al (2008) Lrp4 is a receptor for Agrin and forms a complex with MuSK. Cell 135:334–342

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Zhang B, Luo SW, Wang Q, Suzuki T, Xiong WC, Mei L (2008) LRP4 serves as a coreceptor of agrin. Neuron 60:285–297

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Choi HY, Dieckmann M, Herz J, Niemeier A (2009) Lrp4, a novel receptor for Dickkopf 1 and Sclerostin, Is expressed by osteoblasts and regulates bone growth and turnover In vivo. PLoS One 4:e7930

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Ahn Y, Sims C, Logue JM, Weatherbee SD, Krumlauf R (2013) Lrp4 and Wise interplay controls the formation and patterning of mammary and other skin appendage placodes by modulating Wnt signaling. Development 140:583–593

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Karner CM, Dietrich MF, Johnson EB et al (2010) Lrp4 regulates initiation of ureteric budding and is crucial for kidney formation—a mouse model for Cenani-Lenz syndrome. PLoS One 5:e10418

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Choi HY, Liu Y, Tennert C et al (2013) APP interacts with LRP4 and agrin to coordinate the development of the neuromuscular junction in mice. Elife 2:e00220

    Article  PubMed  PubMed Central  Google Scholar 

  26. Ohazama A, Johnson EB, Ota MS et al (2008) Lrp4 modulates extracellular integration of cell signaling pathways in development. PLoS One 3:e4092

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Ahn Y, Sims C, Murray MJ et al (2017) Multiple modes of Lrp4 function in modulation of Wnt/beta-catenin signaling during tooth development. Development 144:2824–2836

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Lu Y, Tian QB, Endo S, Suzuki T (2007) A role for LRP4 in neuronal cell viability is related to apoE-binding. Brain Res 1177:19–28

    Article  CAS  PubMed  Google Scholar 

  29. Westerfield M (2000) The zebrafish book: guide for the laboratory use of zebrafish (Danio rerio). Univ. of Oregon Press, Eugene

    Google Scholar 

  30. Kimmel CB, Ballard WW, Kimmel SR, Ullmann B, Schilling TF (1995) Stages of embryonic development of the zebrafish. Dev Dynam 203:253–310

    Article  CAS  Google Scholar 

  31. Chou CW, Zhuo YL, Jiang ZY, Liu YW (2014) The hemodynamically-regulated vascular microenvironment promotes migration of the steroidogenic tissue during its interaction with chromaffin cells in the zebrafish embryo. PLoS One 9:e107997

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Tian J, Yam C, Balasundaram G, Wang H, Gore A, Sampath K (2003) A temperature-sensitive mutation in the nodal-related gene cyclops reveals that the floor plate is induced during gastrulation in zebrafish. Development 130:3331–3342

    Article  CAS  PubMed  Google Scholar 

  33. Chou CW, Lin J, Hou HY, Liu YW (2016) Visualizing the interrenal steroidogenic tissue and its vascular microenvironment in zebrafish. J Vis Exp 118:e54820

    Google Scholar 

  34. Tucker B, Lardelli M (2007) A rapid apoptosis assay measuring relative acridine orange fluorescence in zebrafish embryos. Zebrafish 4:113–116

    Article  PubMed  Google Scholar 

  35. Louw JJ, Bastos RN, Chen XW et al (2018) Compound heterozygous loss-of-function mutations in KIF20A are associated with a novel lethal congenital cardiomyopathy in two siblings. PLoS Genet 14:e1007138

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Liu YW, Guo L (2006) Endothelium is required for the promotion of interrenal morphogenetic movement during early zebrafish development. Dev Biol 297:44–58

    Article  CAS  PubMed  Google Scholar 

  37. Chou CW, Lin J, Jiang YJ, Liu YW (2017) Aberrant global and Jagged-mediated Notch signaling disrupts segregation between wt1-expressing and steroidogenic tissues in zebrafish. Endocrinology 158:4206–4217

    Article  PubMed  Google Scholar 

  38. Pan YH, Liu ZY, Shen J, Kopan R (2005) Notch 1 and 2 cooperate in limb ectoderm to receive an early Jagged2 signal regulating interdigital apoptosis. Dev Biol 286:472–482

    Article  CAS  PubMed  Google Scholar 

  39. Sidow A, Bulotsky MS, Kerrebrock AW et al (1997) Serrate2 is disrupted in the mouse limb-development mutant syndactylism. Nature 389:722–725

    Article  CAS  PubMed  Google Scholar 

  40. Hilton MJ, Tu XL, Wu XM et al (2008) Notch signaling maintains bone marrow mesenchymal progenitors by suppressing osteoblast differentiation. Nat Med 14:306–314

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Kariminejad A, Stollfuss B, Li Y et al (2013) Severe Cenani-Lenz syndrome caused by loss of LRP4 function. Am J Med Genet A 161A:1475–1479

    Article  CAS  PubMed  Google Scholar 

  42. Lindy AS, Bupp CP, McGee SJ et al (2014) Truncating mutations in LRP4 lead to a prenatal lethal form of Cenani-Lenz syndrome. Am J Med Genet A 164:2391–2397

    Article  CAS  Google Scholar 

  43. Watanabe N, Tezuka Y, Matsuno K et al (2003) Suppression of differentiation and proliferation of early chondrogenic cells by Notch. J Bone Miner Metab 21:344–352

    Article  CAS  PubMed  Google Scholar 

  44. Tao JN, Chen S, Yang T et al (2010) Osteosclerosis Owing to Notch Gain of Function Is Solely Rbpj-Dependent. J Bone Miner Res 25:2175–2183

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Simpson MA, Irving MD, Asilmaz E et al (2011) Mutations in NOTCH2 cause Hajdu-Cheney syndrome, a disorder of severe and progressive bone loss. Nat Genet 43:303–305

    Article  CAS  PubMed  Google Scholar 

  46. Kung AWC, Xiao SM, Cherny S et al (2010) Association of JAG1 with bone mineral density and osteoporotic fractures: a genome-wide association study and follow-up replication studies. Am J Hum Genet 86:229–239

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Murea M, Park JK, Sharma S et al (2010) Expression of Notch pathway proteins correlates with albuminuria, glomerulosclerosis, and renal function. Kidney Int 78:514–522

    Article  CAS  PubMed  Google Scholar 

  48. Niranjan T, Bielesz B, Gruenwald A et al (2008) The Notch pathway in podocytes plays a role in the development of glomerular disease. Nat Med 14:290–298

    Article  CAS  PubMed  Google Scholar 

  49. Ma M, Jiang Y-J (2007) Jagged2a-notch signaling mediates cell fate choice in the zebrafish pronephric duct. PLoS Genet 3:e18

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Liu Y, Pathak N, Kramer-Zucker A, Drummond IA (2007) Notch signaling controls the differentiation of transporting epithelia and multiciliated cells in the zebrafish pronephros. Development 134:1111–1122

    Article  CAS  PubMed  Google Scholar 

  51. McGregor L, Makela V, Darling SM et al (2003) Fraser syndrome and mouse blebbed phenotype caused by mutations in FRAS1/Fras1 encoding a putative extracellular matrix protein. Nat Genet 34:203–208

    Article  CAS  PubMed  Google Scholar 

  52. Slavotinek AM, Tifft CJ (2002) Fraser syndrome and cryptophthalmos: review of the diagnostic criteria and evidence for phenotypic modules in complex malformation syndromes. J Med Genet 39:623–633

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

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.

Funding

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.

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Authors and Affiliations

  1. The College of Life Sciences, Northwest University, #229 Taibai North Road, Xi’an, 710069, China

    Jing Tian, Jinhui Shao, Cong Liu, Guo-Qing Li, Yao Kou & Zhaojie Lyu

  2. Department of Life Science, Tunghai University, No. 1727, Sec. 4, Taiwan Boulevard, Xitun District, Taichung, 40704, Taiwan

    Hsin-Yu Hou, Chih-Wei Chou, Yu-Hsuan Chen, Mei-Jen Chen, Wei-Leng Chen, Yu-Fu Chen & Yi-Wen Liu

  3. Department of Medical Laboratory Sciences, Jordan University of Science and Technology, Irbid, Jordan

    Mohammad Shboul

  4. National Center for Diabetes, Endocrinology and Genetics, Amman, Jordan

    Mohammad El-Khateeb

  5. Orthopedic Division, Special Surgery Department, School of Medicine, The University of Jordan, Amman, Jordan

    Omar Q. Samarah

  6. State Key Laboratory of Freshwater Ecology and Biotechnology, Wuhan, China

    Jing Tian & Yong-Hua Sun

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  1. Jing Tian
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  2. Jinhui Shao
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  3. Cong Liu
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  4. Hsin-Yu Hou
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  5. Chih-Wei Chou
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  17. Yi-Wen Liu
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Contributions

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.

Corresponding authors

Correspondence to Jing Tian or Yi-Wen Liu.

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The authors declare no competing financial interests.

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Figure S1.

Alignment of human, mouse and zebrafish LRP4 amino acid sequences. The zebrafish sequence lacks 39 amino acid residues at the C-terminal region. (PDF 982 kb)

Figure S2.

Homology modeling of human, mouse and zebrafish LRP4 proteins. Template-based structure prediction of LRP4 protein structures was performed by SWISS-MODEL, and the predicted 3D structures from three species were superimposed by PyMOL. The templates used for modeling each region are: Region I, Low-density lipoprotein receptor (1n7d.1.A); Region II and III, Low-density lipoprotein receptor-related protein 6 chain A (5gje.1.A). AA, amino acids. (PDF 551 kb)

Figure S3.

The effect of lrp4-sdMO on the RNA splicing of lrp4. (A) RT-PCR analysis was used to examine whether the lrp4-sdMO affects the splicing of intron 15 of lrp4 pre-mRNA. The primer set was designed to amplify the cDNA sequence spanning through the junction between exons 15 and 16. The exon–intron organization was predicted by BLASTing the lrp4 cDNA sequence to GRCz10 (Ensembl). Retention of partial intron 15 of the lrp4 pre-mRNA sequence (#2 band of RT-PCR products) was detected in the lrp4 morphant at 10 hpf, which is predicted to cause an in-frame early stop codon (highlighted in light blue). (B) Increasing doses of lrp4-sdMO led to reductions of lrp4 mRNA expression as analyzed by semi-quantitative RT-PCR. The gel picture shown is a representative of triplicate experiments. The intensity of #1 band from the gel picture was subject to densitometric analysis (lower panel) for semi-quantitative measurements of wild-type lrp4 mRNA expressions. The average of three separate experiments shows a significant dose-dependent decrease of #1 band product from 0.2 to 1.2 pmol of lrp4-sdMO injections. Apart from the #2 band of mis-spliced product, one more mis-spliced product was present in trace amounts (indicated by yellow star). (C) Quantitative analysis of mis-spliced products by qRT-PCR using the intron-15 specific primer. A representative gel picture of the mis-spliced product and its housekeeping control (eelf1a) from qRT-PCR is shown in the lower panel. The results of semi-quantitative RT-PCR in (B) and qRT-PCR in (C) were the average of triplicate experiments using 10 embryos in each treatment group for one RT reaction. *P<0.05. (Student’s t test) (PDF 579 kb)

Figure S4.

The posterior trunk and tail phenotypes of lrp4-sd morphants. (A) Injections of lrp4-sd MO (0.4 pmol/embryo), tp53MO (0.5 pmol/embryo) and lrp4-sdMO/tp53MO led to mild dysmorphogenesis in the posterior trunk and tail regions at 24 hpf. (B) Abnormal accumulation of blood cells (red arrows) at the caudal vein plexus in the class I to III of lrp4-sd morphants was captured by live imaging. (C) Acridine orange staining revealed apoptotic cells (orange arrows) in the tail region of the control embryo, which were apparently reduced in the tp53 morphant; whereas no apoptotic cells were detected in either lrp4-sd morphants or lrp4-sd/tp53 double morphants. Scale bar: 100 μm. (PDF 373 kb)

Figure S5.

The effect of lrp4-atgMO on the median fin fold phenotype and the Notch pathway. (A) lrp4-sdMO led to blistering phenotype in the caudal vein plexus and the median fin fold. Phenotypic classification is according to what is described in Fig. 2H. (B) The expression of genes which are components of Notch pathway was analyzed by qRT-PCR for lrp4-atg morphants and STD-MO injected control embryos at 2 dpf. The results were the average of triplicate experiments using 10 embryos in each treatment group for one RT reaction. ***P<0.0001 (Student’s t test). (PDF 212 kb)

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Tian, J., Shao, J., Liu, C. et al. Deficiency of lrp4 in zebrafish and human LRP4 mutation induce aberrant activation of Jagged–Notch signaling in fin and limb development. Cell. Mol. Life Sci. 76, 163–178 (2019). https://doi.org/10.1007/s00018-018-2928-3

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