Multi-stage transcriptomic analysis of mouse embryonic lethality induced by homozygous knockout of the Vrtn gene
Received date: 2025-07-04
Revised date: 2025-10-27
Online published: 2025-11-21
Supported by
National Natural Science Foundation of China(32060139);Natural Science Foundation of Guangxi Zhuang Autonomous Region(2025JJA130063);Natural Science Foundation of Guangxi Zhuang Autonomous Region(2020GXNSFBA297026);Graduate Research Program of Guilin Medical University(GYYK2025024);Guangxi Zhuang Autonomous Region Innovation and Entrepreneurship Training Program for College Students(S202510601101)
The Vrtn gene, a recently identified regulator of embryonic development and stem cell pluripotency, is essential for embryonic survival, as its homozygous knockout (Vrtn-/-) leads to lethality in mice at approximately embryonic day 12.5 (E12.5). To elucidate the underlying lethal mechanism, an integrated approach combining morphological observation, multi-stage transcriptomic analysis, and functional validation experiments was employed to systematically investigate the developmental disorders caused by Vrtn deficiency. Morphological observation showed that Vrtn-/- embryos exhibited significant abnormalities at developmental stages E9.0, E9.5, E10.0, E10.5, and E11.0, including shortened body axis, defective neural tube closure, aberrant somite differentiation, and cardiovascular malformations, accompanied by overall developmental delay. At the molecular level, through RNA sequencing and qRT-PCR validation revealed that Vrtn deficiency not only suppressed the expression of genes critical for somitogenesis (Hoxa2, Hes5), neurodevelopment (Nefm, Nefl), and the hematopoietic system (Hbb-bh1, Klf1), but also aberrantly activated genes associated with apoptosis (Crabp2, Fam162a) and lipid metabolism (Apom, Apoe). TUNEL staining showed that the level of apoptosis was significantly increased in Vrtn-/- embryos. Meanwhile, immunofluorescence detection of Hif-1α indicated that the hypoxic stress response was aberrantly activated. Furthermore, the widespread dysregulation of genes involved in thyroid hormone transport (Ttr), DNA damage stress (Ddit4), and lipid metabolism (Apoa4, Apoa1) collectively exacerbated the developmental imbalance, ultimately leading to embryonic death. A cross-species analysis demonstrated that VRTN knockdown in human embryonic stem cells (hESCs) significantly suppressed the expression of core angiogenic genes (VEGFA, COL1A1 and HES1), a finding consistent with public database analyses indicating a strong association between VRTN and the hypoxic response. In conclusion, this study elucidates that Vrtn functions as a regulatory gene that maintains embryonic homeostasis by orchestrating multiple key developmental processes, including somitogenesis, neural differentiation, angiogenesis, and the hypoxic stress response. This discovery not only deepens the understanding of the role of Vrtn in embryonic development but also provides a new perspective for deciphering the pathogenesis of related hereditary diseases.
Key words： Vrtn gene; gene knockout; transcriptome; embryonic lethality
Yaxin Deng, Baojun Ding, Hongchun Li, Song Chen, Ying Zhang, Bo Zhou, Zhen Zhang . Multi-stage transcriptomic analysis of mouse embryonic lethality induced by homozygous knockout of the Vrtn gene[J]. Hereditas(Beijing), 2026 , 48(3) : 313 -330 . DOI: 10.16288/j.yczz.25-190
| [1] | Lewis J, Schuh M, Hanna JH, Zernicka-Goetz M, Srivastava M, Tan T, Behjati S, Liu Z, Petridou NI, Mendjan S. Developmental and stem cell biology's bright future. Cell, 2024, 187(13): 3224-3228. |
| [2] | White JK, Gerdin AK, Karp NA, Ryder E, Buljan M, Bussell JN, Salisbury J, Clare S, Ingham NJ, Podrini C, Houghton R, Estabel J, Bottomley JR, Melvin DG, Sunter D, Adams NC, Sanger Institute Mouse Genetics Project, Tannahill D, Logan DW, Macarthur DG, Flint J, Mahajan VB, Tsang SH, Smyth I, Watt FM, Skarnes WC, Dougan G, Adams DJ, Ramirez-Solis R, Bradley A, Steel KP. Genome-wide generation and systematic phenotyping of knockout mice reveals new roles for many genes. Cell, 2013, 154(2): 452-464. |
| [3] | Wu Y, Zhang J, Peng BY, Tian D, Zhang D, Li Y, Feng XY, Liu JH, Li J, Zhang T, Liu XY, Lu J, Chen BA, Wang SL. Generating viable mice with heritable embryonically lethal mutations using the CRISPR-Cas9 system in two-cell embryos. Nat Commun, 2019, 10(1): 2883. |
| [4] | Mikawa S, Sato S, Nii M, Morozumi T, Yoshioka G, Imaeda N, Yamaguchi T, Hayashi T, Awata T. Identification of a second gene associated with variation in vertebral number in domestic pigs. BMC Genet, 2011, 12: 5. |
| [5] | Li CY, Li M, Li XY, Ni W, Xu YR, Yao R, Wei B, Zhang MD, Li HX, Zhao Y, Liu L, Ullah Y, Jiang Y, Hu SW. Whole-genome resequencing reveals loci associated with thoracic vertebrae number in sheep. Front Genet, 2019, 10: 674. |
| [6] | Jiang NJ, Liu CX, Lan TX, Zhang Q, Cao Y, Pu G, Niu PP, Zhang ZP, Li Q, Zhou J, Li XK, Hou LM, Huang RH, Li PH. Polymorphism of VRTN gene g.20311_20312ins291 was associated with the number of ribs, carcass diagonal length and cannon bone circumference in Suhuai pigs. Animals (Basel), 2020, 10(3): 484. |
| [7] | Shao M, Wang M, Liu YY, Ge YW, Zhang YJ, Shi DL. Vegetally localised Vrtn functions as a novel repressor to modulate bmp2b transcription during dorsoventral patterning in zebrafish. Development, 2017, 144(18): 3361-3374. |
| [8] | Lemmens M, Perner J, Potgeter L, Zogg M, Thiruchelvam S, Müller M, Doll T, Werner A, Gilbart Y, Couttet P, Martus HJ, Libertini S. Identification of marker genes to monitor residual iPSCs in iPSC-derived products. Cytotherapy, 2023, 25(1): 59-67. |
| [9] | Yilmaz A, Peretz M, Aharony A, Sagi I, Benvenisty N. Defining essential genes for human pluripotent stem cells by CRISPR-Cas9 screening in haploid cells. Nat Cell Biol, 2018, 20(5): 610-619. |
| [10] | Duan YY, Zhang H, Zhang Z, Gao J, Yang J, Wu ZP, Fan Y, Xing YY, Li L, Xiao SJ, Hou Y, Ren J, Huang LS. VRTN is required for the development of thoracic vertebrae in mammals. Int J Biol Sci, 2018, 14(6): 667-681. |
| [11] | Rushlow CA, Shvartsman SY. Temporal dynamics, spatial range, and transcriptional interpretation of the Dorsal morphogen gradient. Curr Opin Genet Dev, 2012, 22(6): 542-546. |
| [12] | Zhao YL, Zheng DY, Cvekl A. A comprehensive spatial- temporal transcriptomic analysis of differentiating nascent mouse lens epithelial and fiber cells. Exp Eye Res, 2018, 175: 56-72. |
| [13] | Hadas R, Rubinstein H, Mittnenzweig M, Mayshar Y, Ben-Yair R, Cheng SF, Aguilera-Castrejon A, Reines N, Orenbuch AH, Lifshitz A, Chen DY, Elowitz MB, Zernicka- Goetz M, Hanna JH, Tanay A, Stelzer Y. Temporal BMP4 effects on mouse embryonic and extraembryonic development. Nature, 2024, 634(8034): 652-661. |
| [14] | He QL, Yang XY, Su XT, Yao LF, Zhang Y, Tang JH, Zhang Z. Structural and tissue expression of Vrtn protein and its functional analysis in mouse embryonic development. Heilongjiang Anim Sci Vet Med, 2023, (9): 13-23+127-129. |
| 何秋玲, 杨筱韵, 苏晓田, 姚陆丰, 张勇, 唐建红, 张震. 小鼠Vrtn蛋白结构和组织表达及Vrtn基因在胚胎发育中的功能分析. 黑龙江畜牧兽医, 2023, (9): 13-23+127- 129. | |
| [15] | Ray JB, Arab S, Deng YP, Liu P, Penn L, Courtman DW, Ward ME. Oxygen regulation of arterial smooth muscle cell proliferation and survival. Am J Physiol Heart Circ Physiol, 2008, 294(2): H839-H852. |
| [16] | Mense SM, Sengupta A, Zhou M, Lan C, Bentsman G, Volsky DJ, Zhang L. Gene expression profiling reveals the profound upregulation of hypoxia-responsive genes in primary human astrocytes. Physiol Genomics, 2006, 25(3): 435-449. |
| [17] | Rivera-Pérez JA, Hadjantonakis AK. The dynamics of morphogenesis in the early mouse embryo. Cold Spring Harb Perspect Biol, 2014, 7(11): a015867. |
| [18] | Kalli AC, Reithmeier RAF. Organization and dynamics of the red blood cell Band 3 anion exchanger SLC4A1: insights from molecular dynamics simulations. Front Physiol, 2022, 13: 817945. |
| [19] | Han A, Yermalovich A, Najia MAT, Pearson DS, Fujiwara Y, Bolgov M, Kubaczka C, North TE, Lundin V, Orkin S, Daley GQ. RNA editors sculpt the transcriptome during terminal erythropoiesis. Res Sq, 2025, rs.3.rs-6355281. |
| [20] | Ogawa Y, Kakumoto K, Yoshida T, Kuwako KI, Miyazaki T, Yamaguchi J, Konno A, Hata J, Uchiyama Y, Hirai H, Watanabe M, Darnell RB, Okano H, Okano HJ. Elavl3 is essential for the maintenance of Purkinje neuron axons. Sci Rep, 2018, 8(1): 2722. |
| [21] | Sohrabi N, Habibi L, Akrami SM. Regulatory role of insulin on endogenous L 1 ORF1 and NEFM gene expression through PI3K signaling pathway specifically in neuroblastoma cell line. Iran J Public Health, 2023, 52(3): 633-645. |
| [22] | Cordes R, Schuster-Gossler K, Serth K, Gossler A. Specification of vertebral identity is coupled to Notch signalling and the segmentation clock. Development, 2004, 131(6): 1221-1233. |
| [23] | Scala M, Accogli A, De Grandis E, Allegri A, Bagowski CP, Shoukier M, Maghnie M, Capra V. A novel pathogenic MYH3 mutation in a child with Sheldon-Hall syndrome and vertebral fusions. Am J Med Genet A, 2018, 176(3): 663-667. |
| [24] | Matonóg A, Drosdzol-Cop A. Alpha-fetoprotein level in fetuses, infants, and children with ovarian masses: a literature review. Front Endocrinol (Lausanne), 2024, 15: 1307619. |
| [25] | Carmeliet P, Ferreira V, Breier G, Pollefeyt S, Kieckens L, Gertsenstein M, Fahrig M, Vandenhoeck A, Harpal K, Eberhardt C, Declercq C, Pawling J, Moons L, Collen D, Risau W, Nagy A. Abnormal blood vessel development and lethality in embryos lacking a single VEGF allele. Nature, 1996, 380(6573): 435-439. |
| [26] | Dong XY, Sun BC, Zhao XL, Liu ZY, Gu Q, Zhang DF, Zhao N, Wang JJ, Chi JD. Expression of relative-protein of hypoxia-inducible factor-1α in vasculogenesis of mouse embryo. J Biol Res (Thessalon), 2014, 21(1): 4. |
| [27] | Shimizu R, Yamamoto M. GATA-related hematologic disorders. Exp Hematol, 2016, 44(8): 696-705. |
| [28] | Gerber HP, Mcmurtrey A, Kowalski J, Yan M, Keyt BA, Dixit V, Ferrara N. Vascular endothelial growth factor regulates endothelial cell survival through the phosphatidylinositol 3′-kinase/Akt signal transduction pathway. Requirement for Flk-1/KDR activation. J Biol Chem, 1998, 273(46): 30336-30343. |
| [29] | Iyer NV, Kotch LE, Agani F, Leung SW, Laughner E, Wenger RH, Gassmann M, Gearhart JD, Lawler AM, Yu AY, Semenza GL. Cellular and developmental control of O2 homeostasis by hypoxia-inducible factor 1 alpha. Genes Dev, 1998, 12(2): 149-162. |
| [30] | Fritsch M, Günther SD, Schwarzer R, Albert MC, Schorn F, Werthenbach JP, Schiffmann LM, Stair N, Stocks H, Seeger JM, Lamkanfi M, Krönke M, Pasparakis M, Kashkar H. Caspase-8 is the molecular switch for apoptosis, necroptosis and pyroptosis. Nature, 2019, 575(7784): 683-687. |
| [31] | Fiore R, Rahim B, Christoffels VM, Moorman AFM, Püschel AW. Inactivation of the Sema5a gene results in embryonic lethality and defective remodeling of the cranial vascular system. Mol Cell Biol, 2005, 25(6): 2310-2319. |
| [32] | Huang LS, Voyiaziakis E, Markenson DF, Sokol KA, Hayek T, Breslow JL. apo B gene knockout in mice results in embryonic lethality in homozygotes and neural tube defects, male infertility, and reduced HDL cholesterol ester and apo A-I transport rates in heterozygotes. J Clin Invest, 1995, 96(5): 2152-2161. |
| [33] | Zhang N, Gridley T. Defects in somite formation in lunatic fringe-deficient mice. Nature, 1998, 394(6691): 374-377. |
| [34] | Deprez PML, Nichane MG, Lengelé BG, Rezsöhazy R, Nyssen-Behets C. Molecular study of a Hoxa2 gain-of- function in chondrogenesis: a model of idiopathic proportionate short stature. Int J Mol Sci, 2013, 14(10): 20386-20398. |
| [35] | Della Marina A, Hentschel A, Czech A, Schara-Schmidt U, Preusse C, Laner A, Abicht A, Ruck T, Weis J, Choueiri C, Lochmüller H, Kölbel H, Roos A. Novel genetic and biochemical insights into the spectrum of NEFL- associated phenotypes. J Neuromuscul Dis, 2024, 11(3): 625-645. |
| [36] | Kulczynska-Figurny K, Bieker JJ, Siatecka M. Severe anemia caused by dominant mutations in Krüppel-like factor 1 (KLF1). Mutat Res Rev Mutat Res, 2020, 786: 108336. |
| [37] | Liang XS, Song C, Lin JR, Li SF, Li LP, Dai GK, Zhang RH, Zou OM, Yao HY, Zhou LB, Zou Y. Transthyretin, a novel prognostic marker of POCD revealed by time-series RNA-sequencing analysis. Mol Psychiatry, 2025, 30(7): 3103-3119. |
| [38] | Monk JA, Sims NA, Dziegielewska KM, Weiss RE, Ramsay RG, Richardson SJ. Delayed development of specific thyroid hormone-regulated events in transthyretin null mice. Am J Physiol Endocrinol Metab, 2013, 304(1): E23-E31. |
| [39] | Mesa-Ciller C, Turiel G, Guajardo-Grence A, Lopez- Rodriguez AB, Egea J, De Bock K, Aragonés J, Urrutia AA. Unique expression of the atypical mitochondrial subunit NDUFA4L 2 in cerebral pericytes fine tunes HIF activity in response to hypoxia. J Cereb Blood Flow Metab, 2023, 43(1): 44-58. |
| [40] | Ding FD, Gao F, Zhang S, Lv XT, Chen YT, Liu QC. A review of the mechanism of DDIT 4 serve as a mitochondrial related protein in tumor regulation. Sci Prog, 2021, 104(1): 36850421997273. |
| [41] | Sun YQ, Zhang J, Wang YF, Wang L, Song MY, Khan A, Zhang L, Niu B, Zhao H, Li MN, Luo TE, He QW, Xie XH, Liu ZZ, Xie J. miR-222-3p is involved in neural tube closure by directly targeting Ddit4 in RA induced NTDs mouse model. Cell Cycle, 2021, 20(22): 2372-2386. |
| [42] | Blanchard JW, Akay LA, Davila-Velderrain J, Von Maydell D, Mathys H, Davidson SM, Effenberger A, Chen CY, Maner-Smith K, Hajjar I, Ortlund EA, Bula M, Agbas E, Ng A, Jiang XQ, Kahn M, Blanco-Duque C, Lavoie N, Liu LW, Reyes R, Lin YT, Ko T, R'bibo L, Ralvenius WT, Bennett DA, Cam HP, Kellis M, Tsai LH. APOE4 impairs myelination via cholesterol dysregulation in oligodendrocytes. Nature, 2022, 611(7937): 769-779. |
| [43] | Chen G, Long J, Zhang YG, Zhou XH, Gao BT, Qin ZJ, Zhu YH, Song BY, Cui ZW, Liu ZZ, Xu M, Yu Z, Song BQ, Zhang ZA. Fascia-derived stem cells enhance fat graft retention by promoting vascularization through the HMOX1-HIF-1α pathway. Stem Cell Res Ther, 2025, 16(1): 92. |
| [44] | Xie XS, Yuan Y, Huang YL, Hong X, Hong SC, Chen G, Chen YH, Lin Y, Lu WF, Fu WG, Wang LX. Effects of COL1A1 and SYTL2 on inflammatory cell infiltration and poor extracellular matrix remodeling of the vascular wall in thoracic aortic aneurysm. Chin Med J (Engl), 2024, 137(9): 1105-1114. |
| [45] | Liu J, Wang W, Wang L, Chen S, Tian B, Huang K, Corrigan CJ, Ying S, Wang W, Wang C. IL-33 initiates vascular remodelling in hypoxic pulmonary hypertension by up-regulating HIF-1α and VEGF expression in vascular endothelial cells. EBioMedicine, 2018, 33: 196-210. |
| [46] | Chen YB, Lan YW, Hung TH, Chen LG, Choo KB, Cheng WTK, Lee HS, Chong KY. Mesenchymal stem cell-based HSP70 promoter-driven VEGFA induction by resveratrol promotes angiogenesis in a mouse model. Cell Stress Chaperones, 2015, 20(4): 643-652. |
| [47] | Sakai D, Murakami Y, Shigeta D, Tomosugi M, Sakata- Haga H, Hatta T, Shoji H. Glycolytic activity is required for the onset of neural plate folding during neural tube closure in mouse embryos. Front Cell Dev Biol, 2023, 11: 1212375. |
| [48] | Baloglu E. Hypoxic stress-dependent regulation of Na, K-ATPase in ischemic heart disease. Int J Mol Sci, 2023, 24(9): 7855. |
| [49] | Pan MM. The study of Vrtn functions in cardiomyocyte proliferation[Dissertation]. Gannan Medical University, 2023. |
| 潘蒙蒙. Vrtn对心肌细胞增殖的影响及机制研究[学位论文]. 赣南医学院, 2023. |
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