Characterization of Cre recombinase  mouse lines enabling cell type‐specific targeting of postnatal intervertebral discs

Abstract

Cre/loxP technology is an important tool for studying cell type‐specific gene functions. Cre recombinase mouse lines, including Agc1‐CreERT2, Col2a1‐Cre; Col2a1‐CreERT2, Shh‐Cre, Shh‐CreERT2, and Osx‐Cre, have been proven to be valuable tools to elucidate the biology of long bones, yet the information for their activity in postnatal intervertebral disc (IVD) tissues was very limited. In this study, we used R26‐mTmG fluorescent reporter to systematically analyze cell specificity and targeting efficiency of these six mouse lines in IVD tissues at postnatal growing and adult stages. We found that Agc1‐CreERT2 is effective to direct recombination in all components of IVDs, including annulus fibrosus (AF), nucleus pulposus (NP), and cartilaginous endplate (CEP), upon tamoxifen induction at either 2 weeks or 2 months of ages. Moreover, Col2a1‐Cre targets most of the cells in IVDs, except for some cells in the outer AF (OAF) and NP. In contrast, the activity of Col2a1‐CreERT2 is mainly limited to the IAF of IVD tissues at either stage of tamoxifen injection. Similarly, Shh‐Cre directs recombination specifically in all NP cells, whereas Shh‐CreERT2 is active only in a few NP cells when tamoxifen is administered at either stage. Finally, Osx‐Cre targets cells in the CEP, but not in the NP or AF of IVDs tissues at these two stages. Thus, our data demonstrated that all these Cre lines can direct recombination in IVD tissues at postnatal stages with different cell type specificity and/or targeting efficiency, and can, therefore, serve as valuable tools to dissect cell type‐specific gene functions in IVD development and homeostasis.

LRP5-deficiency in OsxCreERT2 mice models intervertebral disc degeneration by aging and compression

“…The OsxCreERT2 mouse is commonly used to target bone cells in the osteoblastic lineage (Nakashima et al, 2002) and, while osterix is not expressed in growing mice (Zheng et al, 2019), we note that this inducible-Cre targets adult IVD cells in the nucleus pulposus and outer annulus fibrosus (Table 2). The OsxCreERT2 did not targeting cells in the inner annulus fibrosus as demonstrated by lack of expression of osterix by immunoflourescence and immunohistochemistry and by expression of WNT signaling in the inner annulus fibrosus following conditional deletion of LRP5 in osterix expressing cells.…”

Serial blockface SEM suggests that stem cells may participate in adult notochord growth in an invertebrate chordate, the Bahamas lancelet

“…On the one hand, tracing of labeled proliferating cells indicates that augmentation of cell number in the nucleus pulposus depends on import of chondrocytes from more peripheral tissues [ 78 – 80 ]. On the other hand, cell type-specific targeting in Cre-Lox mice indicates that endogenous cell proliferation occurs within the adult nucleus pulposus itself [ 81 , 82 ].…”

Characterization of Krt19CreERT allele for targeting the nucleus pulposus cells in the postnatal mouse intervertebral disc

“…An inducible Shh CreERT2 (Harfe et al, 2004) allele is also available. However, very few NP cells were targeted following tamoxifen administration to Shh CreERT2 allele in the postnatal mouse IVDs (Zheng et al, 2019). In addition to node or notochord specific alleles, the use of tamoxifen-inducible alleles under the extracellular matrix genes including aggrecan using Agc1 CreERT2 (Henry et al, 2009; Henry et al, 2012), and collagen type 2a1 using Col2a1 CreERT2 (Chen et al, 2007) are used to target NP cells in the postnatal stages (Alkhatib et al, 2018; Alvarez-Garcia et al, 2018; Chen et al, 2007; Henry et al, 2012; Liao et al, 2019; Novais et al, 2019; Zheng et al, 2018).…”