title: Functional Investigation of a Non-coding Variant Associated with Adolescent Idiopathic Scoliosis in Zebrafish: Elevated Expression of the Ladybird Homeobox Gene Causes Body Axis Deformation.
abstract: Previously, we identified an adolescent idiopathic scoliosis susceptibility locus near human ladybird homeobox 1 (LBX1) and FLJ41350 by a genome-wide association study. Here, we characterized the associated non-coding variant and investigated the function of these genes. A chromosome conformation capture assay revealed that the genome region with the most significantly associated single nucleotide polymorphism (rs11190870) physically interacted with the promoter region of LBX1-FLJ41350. The promoter in the direction of LBX1, combined with a 590-bp region including rs11190870, had higher transcriptional activity with the risk allele than that with the non-risk allele in HEK 293T cells. The ubiquitous overexpression of human LBX1 or either of the zebrafish lbx genes (lbx1a, lbx1b, and lbx2), but not FLJ41350, in zebrafish embryos caused body curvature followed by death prior to vertebral column formation. Such body axis deformation was not observed in transcription activator-like effector nucleases mediated knockout zebrafish of lbx1b or lbx2. Mosaic expression of lbx1b driven by the GATA2 minimal promoter and the lbx1b enhancer in zebrafish significantly alleviated the embryonic lethal phenotype to allow observation of the later onset of the spinal curvature with or without vertebral malformation. Deformation of the embryonic body axis by lbx1b overexpression was associated with defects in convergent extension, which is a component of the main axis-elongation machinery in gastrulating embryos. In embryos overexpressing lbx1b, wnt5b, a ligand of the non-canonical Wnt/planar cell polarity (PCP) pathway, was significantly downregulated. Injection of mRNA for wnt5b or RhoA, a key downstream effector of Wnt/PCP signaling, rescued the defective convergent extension phenotype and attenuated the lbx1b-induced curvature of the body axis. Thus, our study presents a novel pathological feature of LBX1 and its zebrafish homologs in body axis deformation at various stages of embryonic and subsequent growth in zebrafish.
authors.affiliation: Department of Cellular Differentiation, Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan.
authors.forename: Long
authors.initials: L
authors.lastname: Guo
authors.affiliation: Department of Molecular Biology and Biochemistry, Division of Basic Life Sciences, Institute of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Yamashita
authors.affiliation: Laboratory for Bone and Joint Diseases, RIKEN Center for Integrative Medical Sciences, Tokyo, Japan.
authors.forename: Ikuyo
authors.initials: I
authors.lastname: Kou
authors.affiliation: Department of Cellular Differentiation, Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan.
authors.forename: Aki
authors.initials: A
authors.lastname: Takimoto
authors.affiliation: Division of Functional Genomics, Advanced Science Research Center, Kanazawa University, Kanazawa, Japan.
authors.forename: Makiko
authors.initials: M
authors.lastname: Meguro-Horike
authors.affiliation: Division of Functional Genomics, Advanced Science Research Center, Kanazawa University, Kanazawa, Japan.
authors.forename: Shin-ichi
authors.initials: S
authors.lastname: Horike
authors.affiliation: Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, Higashi-Hiroshima, Hiroshima, Japan.
authors.forename: Tetsushi
authors.initials: T
authors.lastname: Sakuma
authors.affiliation: Department of Cellular Differentiation, Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan.
authors.forename: Shigenori
authors.initials: S
authors.lastname: Miura
authors.affiliation: Department of Biomechanics, Research Center for Nano Medical Engineering, Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan.
authors.forename: Taiji
authors.initials: T
authors.lastname: Adachi
authors.affiliation: Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, Higashi-Hiroshima, Hiroshima, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Yamamoto
authors.affiliation: Laboratory for Bone and Joint Diseases, RIKEN Center for Integrative Medical Sciences, Tokyo, Japan.
authors.forename: Shiro
authors.initials: S
authors.lastname: Ikegawa
authors.affiliation: Department of Cellular Differentiation, Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan.
authors.forename: Yuji
authors.initials: Y
authors.lastname: Hiraki
authors.affiliation: Department of Cellular Differentiation, Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan.|Department of Molecular Biology and Biochemistry, Division of Basic Life Sciences, Institute of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan.
authors.forename: Chisa
authors.initials: C
authors.lastname: Shukunami
mesh_terms: D000293:Adolescent; D000818:Animals; D016764:Cell Polarity / Q000235:genetics; D018507:Gene Expression Regulation, Developmental; D057809:HEK293 Cells; D018398:Homeodomain Proteins / Q000096:biosynthesis / Q000235:genetics*; D006801:Humans; D009024:Morphogenesis / Q000235:genetics*; D020641:Polymorphism, Single Nucleotide; D012600:Scoliosis / Q000235:genetics* / Q000473:pathology; D014157:Transcription Factors / Q000096:biosynthesis / Q000235:genetics*; D051153:Wnt Proteins / Q000096:biosynthesis / Q000235:genetics*; D060449:Wnt Signaling Pathway / Q000235:genetics; D000071818:Wnt-5a Protein; D015027:Zebrafish; D029961:Zebrafish Proteins / Q000096:biosynthesis / Q000235:genetics*
chemical_list: D018398:Homeodomain Proteins; C542809:LBX1 protein, human; D014157:Transcription Factors; D051153:Wnt Proteins; D000071818:Wnt-5a Protein; C480282:Wnt5a protein, zebrafish; D029961:Zebrafish Proteins
references.citation: Spine (Phila Pa 1976). 2008 Dec 15;33(26):2898-908
references.pmid: 19092622
references.citation: Nat Genet. 2005 Jan;37(1):31-40
references.pmid: 15608638
references.citation: Curr Top Dev Biol. 2012;101:263-95
references.pmid: 23140633
references.citation: Am J Hum Genet. 2015 Aug 6;97(2):337-42
references.pmid: 26211971
references.citation: Eur Spine J. 2013 Mar;22 Suppl 2:S81-95
references.pmid: 23099524
references.citation: Nat Genet. 2011 Dec;43(12):1237-40
references.pmid: 22019779
references.citation: BMC Biotechnol. 2008;8:81
references.pmid: 18954456
references.citation: Dev Dyn. 2014 Dec;243(12):1646-57
references.pmid: 25283277
references.citation: Nat Neurosci. 2005 Nov;8(11):1510-5
references.pmid: 16234809
references.citation: Proc Natl Acad Sci U S A. 2008 Jan 29;105(4):1255-60
references.pmid: 18202183
references.citation: Genetics. 2006 Oct;174(2):639-49
references.pmid: 16959904
references.citation: Med Hypotheses. 2005;65(3):501-8
references.pmid: 15913901
references.citation: Dev Dyn. 2009 Sep;238(9):2409-17
references.pmid: 19653328
references.citation: Dev Biol. 2014 Feb 1;386(1):72-85
references.pmid: 24333517
references.citation: Neuron. 2002 May 16;34(4):535-49
references.pmid: 12062038
references.citation: Dev Dyn. 1995 Jul;203(3):253-310
references.pmid: 8589427
references.citation: Hum Genet. 2005 Apr;116(5):416-9
references.pmid: 15717203
references.citation: Spine (Phila Pa 1976). 1999 Dec 15;24(24):2592-600
references.pmid: 10635522
references.citation: Development. 2000 Jan;127(2):437-45
references.pmid: 10603359
references.citation: Cell Signal. 2006 Mar;18(3):359-72
references.pmid: 16019189
references.citation: Am J Med Genet A. 2010 May;152A(5):1178-88
references.pmid: 20425822
references.citation: Dev Cell. 2002 Jun;2(6):695-706
references.pmid: 12062082
references.citation: Sports Health. 2012 Sep;4(5):394-403
references.pmid: 23016112
references.citation: J Pineal Res. 2004 Mar;36(2):132-9
references.pmid: 14962065
references.citation: J Pediatr Orthop. 2007 Jan-Feb;27(1):106-16
references.pmid: 17195809
references.citation: Development. 2000 Jan;127(2):413-24
references.pmid: 10603357
references.citation: Nucleic Acids Res. 1991 May 11;19(9):2499
references.pmid: 2041787
references.citation: Clin Orthop Relat Res. 2002 Aug;(401):27-31
references.pmid: 12151879
references.citation: Gene. 1999 Nov 15;240(1):239-44
references.pmid: 10564832
references.citation: Development. 2005 Jun;132(11):2503-12
references.pmid: 15890825
references.citation: Mech Dev. 2003 Apr;120(4):467-76
references.pmid: 12676324
references.citation: Spine (Phila Pa 1976). 2007 Apr 1;32(7):735-41
references.pmid: 17414906
references.citation: Development. 2006 Jan;133(2):195-208
references.pmid: 16339190
references.citation: Nat Genet. 2013 Jun;45(6):676-9
references.pmid: 23666238
references.citation: Isr Med Assoc J. 2008 Mar;10(3):186-8
references.pmid: 18494229
references.citation: Mech Dev. 2001 Jul;105(1-2):157-62
references.pmid: 11429291
references.citation: Trends Genet. 2003 Feb;19(2):97-106
references.pmid: 12547519
references.citation: Eur Spine J. 2013 Feb;22(2):282-6
references.pmid: 23096252
references.citation: Stem Cells Int. 2011;2011:130970
references.pmid: 21941564
references.citation: Dev Cell. 2004 Jul;7(1):133-44
references.pmid: 15239961
references.citation: Orthop Traumatol Surg Res. 2013 Feb;99(1 Suppl):S124-39
references.pmid: 23337438
references.citation: J Med Genet. 2014 Jun;51(6):401-6
references.pmid: 24721834
references.citation: Mech Dev. 1995 Nov;53(3):345-56
references.pmid: 8645601
references.citation: J Cell Biol. 2003 Sep 1;162(5):889-98
references.pmid: 12952939
references.citation: Nat Commun. 2014;5:4777
references.pmid: 25182715
references.citation: Genes Dev. 2012 Jan 1;26(1):11-24
references.pmid: 22215806
references.citation: Sci Rep. 2013;3:3379
references.pmid: 24287550
references.citation: Nat Commun. 2015;6:6452
references.pmid: 25784220
references.citation: Genesis. 2011 Oct;49(10):803-10
references.pmid: 21956886
references.citation: J Hum Genet. 2012 Apr;57(4):244-6
references.pmid: 22301463
references.citation: Genome. 2011 Dec;54(12):973-85
references.pmid: 22077099
references.citation: Dev Biol. 2014 Jun 15;390(2):231-46
references.pmid: 24662046
references.citation: Spine (Phila Pa 1976). 1993 Sep 15;18(12):1609-15
references.pmid: 8235839
references.citation: Development. 2013 Apr;140(8):1807-18
references.pmid: 23533179
references.citation: PLoS One. 2013;8(1):e53234
references.pmid: 23308168
references.citation: Cell. 2011 May 27;145(5):650-63
references.pmid: 21620133
references.citation: Dev Dyn. 2007 Nov;236(11):3088-99
references.pmid: 17937395
references.citation: Curr Opin Genet Dev. 2002 Oct;12(5):548-57
references.pmid: 12200160
references.citation: Circ Res. 2003 Jan 10;92(1):73-80
references.pmid: 12522123
references.citation: J Child Orthop. 2013 Feb;7(1):3-9
references.pmid: 24432052
references.citation: Nature. 2000 May 4;405(6782):76-81
references.pmid: 10811221
references.citation: EMBO J. 2001 Dec 17;20(24):7174-83
references.pmid: 11742994