Expression of Shh and Ihh during the formation of intervertebral discs in mouse embryo

doi:10.3969/j.issn.2095-4344.2855

Abstract

Abstract:

BACKGROUND: Studies have shown that Sonic Hedgehog (Shh) is expressed in mouse Notochordal cells, and India Hedgehog (Ihh) is mainly expressed in chondrocyte-like cells and end plates of mouse embryonic vertebral bodies. However, the expression of Shh and Ihh during the formation of intervertebral discs in mouse embryo is unclear.

OBJECTIVE: To investigate the expression of Shh and Ihh during the formation of intervertebral discs in mouse embryo.

METHODS: Male Shh-CreER^T2, R26-mTmG/+ mice mated with female R26-mTmG/+ mice to obtain pregnant rats at different periods of pregnancy (E8.5, E11.5, E12.5, E14.5, E16.5, E18.5), followed by injection of 10 g/L tamoxifen at a dose of 10 μL/g. Intervertebral disc tissues were isolated from the mouse embryo at P0, followed by genotype identification. At the same time, male C57BL/6 mice mated with female C57BL/6 mice to obtain pregnant rats at different periods of pregnancy (E11.5, E12.5, E14.5, E16.5, E18.5), and the intervertebral disc tissues of mouse embryo were taken and analyzed. The expression of Shh and Ihh during the formation of intervertebral discs in mouse embryo was detected using immunofluorescence and immunohistochemistry, respectively. Implementation of animal experiments was approved by the Animal Ethics Committee of Soochow University.

RESULTS AND CONCLUSION: Shh-CreER^T2; R26-mTmG/+ mouse embryos were identified by PCR amplification. The results of immunofluorescent staining showed a gradual decrease in the expression of Shh in nucleus pulposus cells during the formation of intervertebral discs in mouse embryo. The results of immunohistochemical staining showed a gradual increase in expression of Ihh in nucleus pulposus cells during the formation of intervertebral discs in mouse embryo. In summary, Shh and Ihh are dynamically expressed during the formation of intervertebral discs in mouse embryo, and provide a basis for further research on the molecular mechanism of intervertebral disc degeneration.

Key words: intervertebral disc generation, notochord sheath, nucleus pulposus, Shh, Ihh, Shh-CreERT2, R26-mTmG/+

CLC Number:

Gong Tingting, Chen Jianquan. Expression of Shh and Ihh during the formation of intervertebral discs in mouse embryo[J]. Chinese Journal of Tissue Engineering Research, 2020, 24(32): 5097-5101.

Figures/Tables 4

References

[1] WEI W, TANG HY, LI YZ, et al. Effectiveness of extracorporeal shock wave for low back pain: A protocol of systematic review. Medicine (Baltimore).2019; 98(7): e14511.

[2] ANDERSSON GBJ. Epidemiological features of chronic low-back pain. The Lancet.1999;354(9178):581-585.

[3] HOY D, MARCH L, BROOKS P, et al. The global burden of low back pain: estimates from the Global Burden of Disease 2010 study. Ann Rheum Dis.2014; 73(6): 968-974.

[4] ZHENG CJ, CHEN J. Disc degeneration implies low back pain. Theor Biol Med Model.2015;12(24).

[5] 余城墙,张宇,谢程欣,等. 椎间盘退变分子生物学机制及再生治疗的优势与未来[J].中国组织工程研究, 2019,23(30):4889-4893.

[6] ZHENG Y, FU X, LIU Q, et al. Characterization of Cre recombinase mouse lines enabling cell type-specific targeting of postnatal intervertebral discs.J Cell Physiol. 2019. doi: 10.1002/jcp.28166.

[7] DOWDELL J, ERWIN M, CHOMA T, et al. Intervertebral Disk Degeneration and Repair. Neurosurgery.2017; 80(3S): S46-S54.

[8] 海宝,祝斌,刘晓光. 椎间盘退变动物模型的研究进展[J].中国实验动物学报,2019(3):279-281.

[9] HUANG YC, URBAN JP, LUK KD. Intervertebral disc regeneration: do nutrients lead the way?. Nat Rev Rheumatol.2014; 10(9): 561-566.

[10] LAWSON L, HARFE BD. Notochord to Nucleus Pulposus Transition. Current Osteoporosis Reports.2015;13(5): 336-341.

[11] KYUNG-SUK CHOI BDH. Hedgehog signaling is required for formation of the notochord sheath and patterning of nuclei pulposi within the intervertebral discs. Proc Natl Acad Sci U S A. 2011;108(23): 9484-9489.

[12] LEE RT, ZHAO Z, INGHAM PW. Hedgehog signalling. Development. 2016;143(3): 3673-3672.

[13] CHOI KS, LEE C, HARFE BD. Sonic hedgehog in the notochord is sufficient for patterning of the intervertebral discs. Mech Dev.2012; 129(9-12): 255-262.

[14] RAJESH D, DAHIA CL. Role of Sonic Hedgehog Signaling Pathway in Intervertebral Disc Formation and Maintenance. Curr Mol Biol Rep. 2018;4(4):173-179.

[15] DIPAOLA CP, FARMER JC, MANOVA K,et al. Molecular signaling in intervertebral disk development.J Orthop Res. 2005;23(5):1112-1119.

[16] MCLELLAN MA, ROSENTHAL NA, PINTO AR. Cre-loxP-Mediated Recombination: General Principles and Experimental Considerations. Curr Protoc Mouse Biol.2017;7(1):1-12.

[17] KIM H, KIM M, IM SK, et al. Mouse Cre-LoxP system: general principles to determine tissue-specific roles of target genes. Lab Anim Res.2018;34(4):147-159.

[18] CHEN M, LI S, XIE W, et al. Col2CreER(T2), a mouse model for a chondrocyte-specific and inducible gene deletion. Eur Cell Mater. 2014;28:236-245.

[19] KENNON JC, AWAD ME, CHUTKAN N, et al. Current insights on use of growth factors as therapy for Intervertebral Disc Degeneration. Biomol Concepts.2018; 9(1): 43-52.

[20] MOHANTY S, DAHIA CL. Defects in intervertebral disc and spine during development, degeneration, and pain: New research directions for disc regeneration and therapy. Wiley Interdiscip Rev Dev Biol.2019; 8(4): e343.

[21] 陶晖.干细胞治疗椎间盘退变性疾病的研究进展[J].中国脊柱脊髓杂志, 2013,6(23):565-568.

[22] CHITRA LEKHA D, MAHONEY EJ, DURRANI AA, et al. Intercellular signaling pathways active during intervertebral disc growth, differentiation, and aging. Spine.2009;34(5): 456-462.

[23] COLOMBIER P, CLOUET J, HAMEL O, et al. The lumbar intervertebral disc: from embryonic development to degeneration. Joint Bone Spine. 2014; 81(2): 125-129.

[24] 俞佳斌,王宸,马雪倩,等. 椎间盘退行性变机制的研究进展[J].脊柱外科杂志,2017,15(6):374-379.

[25] LUDWINSKI FE, KANNA G, RICHARDSON SM, et al. Understanding the native nucleus pulposus cell phenotype has important implications for intervertebral disc regeneration strategies. Regen Med. 2013;8(1): 75-87.

[26] YANG J, ANDRE P, YE L, et al. The Hedgehog signalling pathway in bone formation. Int J Oral Sci.2015;7(2):73-79.

[27] CHEN H, WANG Y, GE R, et al. Leydig cell stem cells: Identification, proliferation and differentiation. Mol Cell Endocrinol.2017; 44(5)65-73.

[28] ZOU SS, ZHENG L, HU HL. Desert hedgehog regulates the proliferation and differentiation of Leydig cells: an update. Zhonghua Nan Ke Xue. 2012;18(2):172-175.