Humans carrying homozygous loss-of-function mutations in the Wnt co-receptor, low-density lipoprotein receptor–related protein 5 (LRP5), develop osteoporosis and a defective retinal vasculature known as familial exudative vitreoretinopathy (FEVR) due to disruption of the Wnt signaling pathway. The purpose of this study was to use CRISPR-Cas9–mediated gene editing to create strains of Lrp5-deficient rats and to determine whether knockout of Lrp5 resulted in phenotypes that model the bone and retina pathology in LRP5-deficient humans. Knockout of Lrp5 in rats produced low bone mass, decreased bone mineral density, and decreased bone size. The superficial retinal vasculature of Lrp5-deficient rats was sparse and disorganized, with extensive exudates and decreases in vascularized area, vessel length, and branch point density. This study showed that Lrp5 could be predictably knocked out in rats using CRISPR-Cas9, causing the expression of bone and retinal phenotypes that will be useful for studying the role of Wnt signaling in bone and retina development and for research on the treatment of osteoporosis and FEVR.
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References
1.
JoinerDM, KeJ, ZhongZ, et al.LRP5 and LRP6 in development and disease. Trends Endocrin Met. 2013; 24:31–39. DOI: 10.1016/j.tem.2012.10.003.
GongY, SleeRB, FukaiN, et al.LDL receptor-related protein 5 (LRP5) affects bone accrual and eye development. Cell. 2001; 107:513–523. DOI: 10.1016/s0092-8674(01)00571-2.
4.
MalteseP, ZiccardiL, IarossiG, et al.Osteoporosis-pseudoglioma syndrome: report of two cases and a manifesting carrier. Ophthalmic Genet. 2017; 38:473–479. DOI: 10.1080/13816810.2016.1253107.
5.
HolmenS, GiambernardiT, ZylstraC, et al.Decreased BMD and limb deformities in mice carrying mutations in both Lrp5 and Lrp6. J Bone Miner Res. 2004; 19:2033–2040. DOI: 10.1359/JBMR.040907.
6.
CuiY, NiziolekPJ, MacDonaldBT, et al.Lrp5 functions in bone to regulate bone mass. Nat Med. 2011; 17:684-U673. DOI: 10.1038/nm.2388.
7.
JoengKS, SchumacherCA, Zylstra-DiegelCR, et al.Lrp5 and Lrp6 redundantly control skeletal development in the mouse embryo. Dev Biol. 2011; 359:222–229. DOI: 10.1016/j.ydbio.2011.08.020.
8.
MaupinKA, DroschaCJ, WilliamsBO. A comprehensive overview of skeletal phenotypes associated with alterations in Wnt/beta-catenin signaling in humans and mice. Bone Res. 2013; 1. DOI: 10.4248/BR201301004.
9.
RiddleRC, DiegelCR, LeslieJM, et al.Lrp5 and Lrp6 exert overlapping functions in osteoblasts during postnatal bone acquisition. PLoS One. 2013; 8. DOI: 10.1371/journal.pone.0063323.
10.
WilliamsBO. Insights into the mechanisms of sclerostin action in regulating bone mass accrual. J Bone Miner Res. 2014; 29:24–28. DOI: 10.1002/jbmr.2154.
11.
AiM, HeegerS, BartelsCF, et al.Clinical and molecular findings in osteoporosis-pseudoglioma syndrome. Am J Hum Genet. 2005; 77:741–753. DOI: 10.1086/497706.
12.
ToomesC, BottomleyHM, JacksonRM, et al.Mutations in LRP5 or FZD4 underlie the common familial exudative vitreoretinopathy locus on chromosome 11q. Am J Hum Genet. 2004; 74:721–730. DOI: 10.1086/383202.
13.
GilmourDF. Familial exudative vitreoretinopathy and related retinopathies. Eye (Lond). 2015; 29:1–14. DOI: 10.1038/eye.2014.70.
14.
WardenSM, AndreoliCM, MukaiS. The Wnt signaling pathway in familial exudative vitreoretinopathy and Norrie disease. Semin Ophthalmol. 2007; 22:211–217. DOI: 10.1080/08820530701745124.
15.
HuangXY, ZhuangH, WuJH, et al.Targeted next-generation sequencing analysis identifies novel mutations in families with severe familial exudative vitreoretinopathy. Mol Vis. 2017; 23:605–613.
16.
LiuCC, PearsonC, BuG. Cooperative folding and ligand-binding properties of LRP6 beta-propeller domains. J Biol Chem. 2009; 284:15299–15307. DOI: 10.1074/jbc.M807285200.
17.
AhnVE, ChuML, ChoiHJ, et al.Structural basis of Wnt signaling inhibition by Dickkopf binding to LRP5/6. Dev Cell. 2011; 21:862–873. DOI: 10.1016/j.devcel.2011.09.003.
18.
MacDonaldBT, HeX. Frizzled and LRP5/6 receptors for Wnt/β-catenin signaling. Cold Spring Harb Perspect Biol. 2012; 4. DOI: 10.1101/cshperspect.a007880.
19.
ChengZ, BiecheleT, WeiZ, et al.Crystal structures of the extracellular domain of LRP6 and its complex with DKK1. Nat Struct Mol Biol. 2011; 18:1204–1210. DOI: 10.1038/nsmb.2139.
20.
BourhisE, TamC, FrankeY, et al.Reconstitution of a frizzled8.Wnt3a.LRP6 signaling complex reveals multiple Wnt and Dkk1 binding sites on LRP6. J Biol Chem. 2010; 285:9172–9179. DOI: 10.1074/jbc.M109.092130.
21.
BourhisE, WangW, TamC, et al.Wnt antagonists bind through a short peptide to the first β-propeller domain of LRP5/6. Structure. 2011; 19:1433–1442. DOI: 10.1016/j.str.2011.07.005.
22.
ChenS, BubeckD, MacDonaldBT, et al.Structural and functional studies of LRP6 ectodomain reveal a platform for Wnt signaling. Dev Cell. 2011; 21:848–861. DOI: 10.1016/j.devcel.2011.09.007.
23.
EttenbergSA, CharlatO, DaleyMP, et al.Inhibition of tumorigenesis driven by different Wnt proteins requires blockade of distinct ligand-binding regions by LRP6 antibodies. Proc Natl Acad Sci U S A. 2010; 107:15473–15478. DOI: 10.1073/pnas.1007428107.
24.
GongY, BourhisE, ChiuC, et al.Wnt isoform-specific interactions with coreceptor specify inhibition or potentiation of signaling by LRP6 antibodies. PLoS One. 2010; 5:e12682. DOI: 10.1371/journal.pone.0012682.
25.
WangZ, LiuCH, SunY, et al.Pharmacologic activation of wnt signaling by lithium normalizes retinal vasculature in a murine model of familial exudative vitreoretinopathy. Am J Pathol. 2016; 186:2588–2600. DOI: 10.1016/j.ajpath.2016.06.015.
26.
WangZ, LiuCH, HuangS, et al.Wnt Signaling in vascular eye diseases. Prog Retin Eye Res. 2019; 70:110–133. DOI: 10.1016/j.preteyeres.2018.11.008.
27.
ChenJ, StahlA, KrahNM, et al.Retinal expression of Wnt-pathway mediated genes in low-density lipoprotein receptor-related protein 5 (Lrp5) knockout mice. PLoS One. 2012; 7:e30203. DOI: 10.1371/journal.pone.0030203.
28.
HuangW, LiQ, Amiry-MoghaddamM, et al.Critical endothelial regulation by Lrp5 during retinal vascular development. PLoS One. 2016; 11:e0152833. DOI: 10.1371/journal.pone.0152833.
29.
HombergJR, WöhrM, AleninaN. Comeback of the rat in biomedical research. ACS Chem Neurosci. 2017; 8:900–903. DOI: 10.1021/acschemneuro.6b00415.
BäckS, NecarsulmerJ, WhitakerLR, et al.Neuron-specific genome modification in the adult rat brain using CRISPR-Cas9 transgenic rats. Neuron. 2019; 102:105–119.e108. DOI: 10.1016/j.neuron.2019.01.035.
33.
MaY, ShenB, ZhangX, et al.Heritable multiplex genetic engineering in rats using CRISPR/Cas9. PLoS One. 2014; 9:e89413. DOI: 10.1371/journal.pone.0089413.
34.
ChengX, WaghuldeH, MellB, et al.Positional cloning of quantitative trait nucleotides for blood pressure and cardiac QT-interval by targeted CRISPR/Cas9 editing of a novel long non-coding RNA. PLoS Genet. 2017; 13:e1006961. DOI: 10.1371/journal.pgen.1006961.
35.
WaghuldeH, ChengX, GallaS, et al.Attenuation of microbiotal dysbiosis and hypertension in a CRISPR/Cas9 gene ablation rat model of GPER1. Hypertension. 2018; 72:1125–1132. DOI: 10.1161/hypertensionaha.118.11175.
ChallaAK, BoitetER, TurnerAN, et al.Novel hypomorphic alleles of the mouse tyrosinase gene induced by CRISPR-Cas9 nucleases cause non-albino pigmentation phenotypes. PLoS One. 2016; 11:e0155812. DOI: 10.1371/journal.pone.0155812.
38.
HolmenS, SalicA, ZylstraC, et al.A novel set of Wnt-frizzled fusion proteins identifies receptor components that activate beta-catenin-dependent signaling. J Biol Chem. 2002; 277:34727–34735. DOI: 10.1074/jbc.M204989200.
39.
XuQ, WangY, DabdoubA, et al.Vascular development in the retina and inner ear: control by Norrin and Frizzled-4, a high-affinity ligand-receptor pair. Cell. 2004; 116:883–895. DOI: 10.1016/s0092-8674(04)00216-8.
40.
WilliamsB, BarishG, KlymkowskyM, et al.A comparative evaluation of beta-catenin and plakoglobin signaling activity. Oncogene. 2000; 19:5720–5728. DOI: 10.1038/sj.onc.1203921.
41.
ZudaireE, GambardellaL, KurczC, et al.A computational tool for quantitative analysis of vascular networks. PLoS One. 2011; 6:e27385. DOI: 10.1371/journal.pone.0027385.
42.
KashaniAH, BrownKT, ChangE, et al.Diversity of retinal vascular anomalies in patients with familial exudative vitreoretinopathy. Ophthalmology. 2014; 121:2220–2227. DOI: 10.1016/j.ophtha.2014.05.029.
43.
YonekawaY, ThomasBJ, DrenserKA, et al.Familial exudative vitreoretinopathy: spectral-domain optical coherence tomography of the vitreoretinal interface, retina, and choroid. Ophthalmology. 2015; 122:2270–2277. DOI: 10.1016/j.ophtha.2015.07.024.
44.
XiaCH, LiuH, CheungD, et al.A model for familial exudative vitreoretinopathy caused by LPR5 mutations. Hum Mol Genet. 2008; 17:1605–1612. DOI: 10.1093/hmg/ddn047.
45.
Díaz-CoránguezM, RamosC, AntonettiDA. The inner blood-retinal barrier: cellular basis and development. Vision Res. 2017; 139:123–137. DOI: 10.1016/j.visres.2017.05.009.
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