Unravelling The Molecular Mechanism Of VRTN Causative Mutations For Thoracic Vertebral Number In Pigs

Vertebral number is an economically important trait in the pig industry because of its effect on carcass（body）length,meat production and teat number.In previous studies,we and other researchers mapped a major quantitative trait locus（QTL）for the number of thoracic vertebrae（TVN）on pig chromosome（SSC）7,at which one allele increases approximately 0.5 vertebra.Vertnin（VRTN）has been considered as a strong candidate gene for this locus and two candidate quantitative trait nucleotides（QTNs）underlying the QTL effect have been identified in the VRTN gene.The candidate QTNs are likely of Chinese origin and have been historically introgressed from Chinese pigs into European pigs.We herein performed an in-depth investigation to verify the causality of VRTN in relation to TVN,and to identify the molecular features of VRTN and its role in controlling the formation of thoracic somites.We first conducted genome-wide association analyses（GWAS）for TVN in an European hybrid pig poulation derived from a three-way cross:Duroc×（Landrace×Large White）（thereafter referred to as DLY,N=609）using Illumina Porcine 60K chip.The most significantly associated SNP was MARC0038565(P=1.87×10^-51)at 103.5 Mb on SSC7.The 95%confidence interval for the major QTL was defined within a 400 kb segment containing the VRTN gene.To refine the location of the QTL,we increased the SNP density from 2 to 11 in the 400 kb region and conducted an association analysis in the DLY population.Four variants in the VRTN gene（g.8063G>A,g.13066C>T,g.19034A>C and g.20311₂0312ins291）showed the strongest association signal(P=5.79×10^-62)and explained 68.7%of phenotypic variance in this DLY population.When the effect of the VRTN variants was removed from the analysis,no association signal was observed for TVN across the genome.The findings support the causality of VRTN in relation to TVN.Further,we show that VRTN is highly expressed from 7.5 dpc to 9.5 dpc in mice,and thoracic somites are formed during this embryonic stage.The temporal expression pattern of VRTN is consistent with the critical role of this gene in regulating TVN.To identify causative mutations in the VRTN gene,we constructed the luciferase reporter recombinant plasmids of both allelic for the four candidate QTNs,and then performed a dual luciferase reporter assay in HEK293T cells.The results demonstrated that two candidate QTNs（g.19034A>C and g.20311₂0312ins291）additively affect the expression of target genes.Next,we generated VRTN QTN-LacZ transgenic mice and examined whether the QTNs up-regulated the expression of VRTN at the embryonic stage of the formation of thoracic somites.To mimic the additive effect of the two candidate QTNs,we cloned two copies of a 291 bp fragment corresponding to the mutant allele at the QTN site（g.20311₂0312ins291）into an upstream region of a LacZ reporter gene.The reporter constructs were linearized and injected into fertilized mouse oocytes.The transgenic and wild-type embryos were collected at 9.5 dpc and then stained to visualize LacZ reporter gene activity.We found that the two QTNs act as an enhancer to up-regulate the expression of target gene in structures along the anterior-posterior（AP）axis for the formation of somites.Moreover,QQ（mutatant）pig embryos at 17.5 dpc（corresponding to9.5 dpc in mice）showed higher mRNA and protein levels of VRTN than qq（wild-type）individuals at the two QTN sites.These observations suggest that g.19034A>C and g.20311₂0312ins291 are most likely causative mutations.We also illustrate that VRTN is a novel DNA-binding transcription factor as it localizes exclusively in the nucleus,binds to DNA on a genome-wide scale and regulates the transcription of a set of genes that harbor VRTN binding motifs.To investigate the function of VRTN during the thoracic vertebral development,we generated Vrtn knockout mice using TAL-effector endonuclease（TALEN）technology.Vrtn-null embryos have irregular and fewer（less than 13）somites at 9.5 dpc and 10.5 dpc,and the development ceased after E10.5.In addition,more than half adult Vrtn^+/-animals have significantly fewer thoracic vertebra and ribs than wild-type animals.These findings provide the first biological evidence that Vrtn is indispensable for the formation of thoracic somites and,hence,for the determination of TVN.To understand the molecular mechanism for the regulation of TVN by VRTN,we conducted an RNA-seq experiment using three homozygous（QQ）and three wild-type（qq）pig embryos at day 17.5 of pregnancy.We identified 42 differential expressed genes（DEGs）,of which NOTCH2 and HSPG2 have been implicated in abnormal vertebral segmentation in human and animal models.These DEGs were enriched in developmental processes,and the top 20 KEGG-enriched pathways included the Notch signaling pathway and Dorso-Ventral axis formation.Intriguingly,NOTCH2 has a similar expression pattern with VRTN.NOTCH2 belongs to the Notch pathway associated with coupling of Notch and Delta.The Notch signaling pathway is a major component of the clock and wavefront model.We confirmed the RNA-seq result of NOTCH2 by RT-qPCR.Next,we constructed two luciferase reporter plasmids with fragments derived from the promoters of the human and pig HES1 genes,which is a downstream gene of Notch signaling pathway.We then performed a dual luciferase reporter assay in PK-15 cells.The overexpression of VRTN resulted in significantly higher（P<0.05）reporter activities compared with the controls.These results indicate that VRTN mediates the expression of NOTCH2 and regulates its downstream genes in the Notch signaling pathway.It is known that a faster segmentation clock gives rise to a reduced periodicity of somitogenesis and more but shorter segments in mice.In pigs,compared with wild-type homozygotes（qq）,the number of thoracic vertebrae was increased by one in homozygous mutant（QQ）animals,and the total length of the thoracic vertebrae in homozygous mutant DLY pigs was increased by 1.56 cm（P<0.001）,which is nearly half（1.56/3.23）the size of one thoracic vertebra.Therefore,we expect that the increased NOTCH2 levels driven by the VRTN causative variants may accelerate the synchronized oscillations of cells in PSM,which in turn accelerate the segmentation clock via the Notch signaling pathway,and consequently affect the number of thoracic somites and ribs as well.In conclusion,we identified the VRTN gene as a causative gene,and g.19034A>C and g.20311₂0312ins291 in this gene as causative mutationsfor TVN on SSC7 using a battery of assays.We also show that VRTN causative mutations（QTN）possibly affect TVN through the Notch pathway.The VRTN QTNs provide a robust molecular breeding tool to improve pork production by increasing TVN.Moreover,our results provide novel insight to clinical investigation of human supernumerary vertebrae.