VRTN is Required for the Development of Thoracic Vertebrae in Mammals

Abstract

Vertnin (VRTN) variants are associated with thoracic vertebral number (TVN) in pigs. However, the biological function of VRTN remains poorly understood. Here we first conducted a range of experiments to demonstrate that VRTN is a responsible gene for TVN and two causative variants in the regulatory region of VRTN additively regulate TVN. Then, we show 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. Next, we illustrate that VRTN is essential for the development of thoracic vertebrae. Vrtn-null embryos display somitogenesis defect with the failure of axial rotation and fewer somites at the thoracic somite stage. Half of Vrtn heterozygous mice show abnormal spinal development with fewer thoracic vertebrae and ribs than their wild-type littermates. Lastly, we reveal that VRTN could modulate somite segmentation via the Notch signaling pathway. The findings advance our understanding of the mechanisms underlying the development of thoracic vertebrate in mammals, and VRTN causative variants provide a robust tool to improve pork production by selecting the alleles increasing the number of thoracic vertebrae and ribs.

Keywords: VRTN; somitogenesis; thoracic vertebrae; transcription factor.

Figure 1

Genome-wide association study (GWAS) mapping supports VRTN as one of the genes responsible for the number of thoracic vertebrae. (A) Manhattan plots of a GWAS for the number of thoracic vertebrae in 609 European hybrid pigs. Negative log₁₀ P-values of the qualified SNPs were plotted against their genomic positions. Different colors indicate distinct chromosomes. The red dots represent the nine variants including four VRTN variants within a region of 400 kb that were not included in Illumina Porcine 60K Beadchips, and the top SNP (MARC0038565) identified in the GWAS using the Beadchip is indicated. (B) When the results were conditional on the effect of the VRTN candidate QTN, no other SNPs on chromosome 7 showed an association signal. The solid and dashed lines indicate the 5% genome-wide and chromosome-wide Bonferroni-corrected thresholds, respectively. (C) Regional association plot for the number of thoracic vertebrae within the 95% confidence interval of the QTL on chromosome 7. The vertical dashed lines delineate the boundary region of the top GWAS SNPs, which are four VRTN variants. The two flanking markers (MARC0038565 and 103407089A>G) within an interval of 88 kb have a much lower association strength (LOD less than 14) values than the VRTN variants. The genes encoded on this part of the chromosome are indicated. (D) A haplotype view of all polymorphisms shown in panel C. The top four significant SNPs including g.8063G>A, g.13066C>T, g.19034A>C and g.20311_20312ins291 in the VRTN gene define a single haplotype block (r² > 0.98).

Figure 2

Two candidate causative variants additively affect the expression of VRTN. (A) Luciferase activity driven by four pig promoter sequences in human HEK293T cells. The schematic diagram (left panel) illustrates the sequence of each construct, and the right panel shows the relative luciferase activity of each construct. Ins indicates the ins allele at the g.20311_20312ins291 mutation site. C or A represents the C or A allele at the g.19034A>C mutation site. The relative luciferase activity (firefly luciferase light units/Renilla luciferase light units) is represented as the mean ± standard error (S.E) of triplicate experiments. (B) The VRTN candidate causative mutations direct the expression of LacZ reporter in transgenic mouse embryos at E9.5. Two copies of 291 bp fragment corresponding to the Q allelic form at the candidate QTN site (g.20311_20312ins291) were cloned into LacZ reporter plasmids (HSP68-lacZ), which were then used to generate Q mice. Lateral views of representative X-gal-stained transgenic (Q) and control (q) embryos at 9.5 dpc are shown with sections of stained tissues. One enlarged areas (Q') correspond to the region indicated by dashed boxes in the left panel. Three independent transgenic lines displayed virtually identical expression patterns (Fig. S6). (C) mRNA expression levels of VRTN in mutant (QQ, n = 3), heterozygous (Qq, n = 18), and wild-type (qq, n = 7) pig embryos at E17.5. Values were determined via RT-qPCR and are expressed as the mean ± S.E. of the triplicate experiments after normalization to 18S mRNA levels. The expression level of the qq samples was set as 1. Significance of difference between each paired group was determined by student's t-test. A P value of smaller than 0.05 was considered statistically significant. (D) VRTN expression in whole pig embryos at E17.5. Whole mount immunohistochemistry was performed with an anti-VRTN antibody. VRTN expression is shown in green and nuclei in blue. The full names of the abbreviations used by the arrowheads are as follows: HE, head; HT, heart; T, tail; TS, thoracic somites. Embryos were all imaged via confocal microscopy at the same magnification. Scale bar: 100 µm. (E) Quantitative analysis of relative VRTN immunostaining intensities in the head (HE), thoracic somite (TS), heart (HT) and tail (T, presomitic mesoderm) regions of the mutant (QQ) and heterozygous (Qq) pig embryos at E17.5. The data represent the fold increase (mean ± S.E. of triplicate experiments) relative to the staining intensity in the tails of Qq embryos.*, P < 0.05 as determined by student's t-test.

Figure 3

VRTN is a novel transcription factor. (A) VRTN localizes to the nucleus. PK-15 cells were transfected with pEGFP-C1-VRTN plasmids. Subcellular localization of VRTN was determined via GFP fluorescence at 24 hours post-transfection. Cell nuclei were counterstained with DAPI. Scale bar: 10 µm. (B) VRTN binds to DNA. PK-15 cells expressing GFP or the GFP-VRTN fusion protein were imaged before and during recovery after briefly photobleaching the fluorescence in a small strip spanning the nucleus. The recovery rate of fluorescence was recorded over time. The GFP-VRTN showed a slower recovery of fluorescence compared with GFP alone. (C) Genomic distribution of VRTN binding peaks identified via ChIP sequencing. The schematic diagram (lower panel) shows the definitions of the intergenic, upstream, promoter, exon and intron regions. TSS, transcription start site. (D) VRTN binding motif revealed by an enrichment analysis of ChIP sequencing. (E) VRTN regulates the expression of target genes. VRTN peak proximity scores for each of 514 differentially expressed genes (DEGs) were calculated and plotted together with the log₂ values of fold changes in DEGs. Blue dots indicate negative and the red dots positive significant fold changes. The horizontal dashed line at the peak proximity score of 10 represents the chosen cut-off for defining a gene likely to be a direct target of VRTN.

Figure 4

Vrtn is essential for the development of thoracic veterbrae in mice. (A) At 9.5 and 10.5 dpc, Vrtn-null embryos (Vrtn^-/-, right) cannot complete axial rotation and have 8-12 somites. Embryos were all imaged at the same magnification (4×). Scale bar: 100 µm. (B) Compared with the Vrtn wild-type (Vrtn^+/+, left) individual, Vrtn^-/- mouse embryos at 9.5 dpc (middle and right) show thoracic somite defects (abnormal and fewer (less than 13) somite segments) and delays in axial turning and neural tube closure. (C) Compared with Vrtn^+/+ mice (left), some Vrtn^+/- animals had one less thoracic vertebrae (middle) or were missing a rib from the last thoracic element on one side (right). The positions of the first (R1) and last (R12 or R13) rib are labeled. (D) Comparison of the number of thoracic vertebrae and ribs between Vrtn^+/+ and Vrtn^+/- mice (n = 26). NTV, number of thoracic vertebrae. Data are expressed as means ± standard deviations. Student t-test was used to evaluate phenotypic difference between the two groups. A P value of smaller than 0.05 was considered statistically significant. (E) Phenotypic variation in the number of thoracic vertebrae and ribs in Vrtn^+/- mice (n = 26). 12/12, 12 thoracic vertebrae with 12 pairs of ribs; 13/12, 13 thoracic vertebrae with a rib missing from the last thoracic element on one side; 13/13, 13 thoracic vertebrae with 13 pairs of ribs.

Figure 5

VRTN regulates the Notch pathway. (A) NOTCH2 mRNA levels in VRTN mutant (QQ, n = 3), heterozygous (Qq, n = 18) and wild-type (qq, n = 7) pig embryos at E17.5. Values were determined via RT-qPCR and are expressed as the mean ± standard error (S.E) of triplicate experiments after normalization to BACT mRNA levels. The expression level of the qq samples was set as 1. (B) VRTN up-regulates the expression of NOTCH2. VRTN expression and control vectors were transfected into pig PK-15 cells. Values were determined via RT-qPCR and are expressed as the mean ± S.E. of triplicate experiments after normalization to BACT mRNA levels. (C) Transcriptional activation of HES1 by VRTN. PK-15 cells were co-transfected with luciferase reporter plasmids driven by a pig (pGL4.20-pHES1) / human (pGL4.20-hHES1) promoter and VRTN expression plasmids (red) or control vectors (blue). The ratio of firefly luciferase to Renilla activity was defined as the relative luciferase activity. The data represent fold increases (mean ± S.E. of triplicate experiments) relative to empty vector-transfected values. Student t-test was performed to determine the significance of difference between each paired group. A P value of smaller than 0.05 was considered statistically significant.