Spatial distribution of type II collagen gene expression in the mouse intervertebral disc

Abstract

Genetic tools such as the Cre-Lox reporter system are powerful aids for tissue-specific cell tracking. For example, it would be useful in examining intervertebral disc (IVD) cell populations in normal and diseased states. A Cre recombinase and its recognition site, loxP have been adapted from the bacteriophage for use in genetic manipulation. The reporter mice used here express the red fluorescent protein, tdTomato with flanking LoxP sites (Rosa26 TdTomato mice). We compared two different Collagen type II (Col2) promoter constructs that drive Cre-recombinase expression in mice: (a) Col2-Cre, which allows constitutive Cre-recombinase expression under the control of the Col2 promoter/enhancer and (b) Col2-CreER, which contains a shorter promoter/enhancer region than Col2-Cre, but has human estrogen binding elements that bind tamoxifen, resulting in Cre-recombinase expression. The goal of the study is to characterize Cre-recombinase distribution pattern in Col2-Cre and Col2-CreER mice using tdTomato as reporter in the spine. The expression patterns of these two mice were further compared with Col2 gene expression in the native mouse NP and AF tissues by real-time PCR. We crossed Col2-Cre mice or Col2-CreER mice with the tdTomato reporter mice, and compared the tdTomato expression patterns. Col2-CreER/tdTomato mice were injected with tamoxifen at postnatal day 7 to activate the Cre-recombinase. TdTomato in the constitutively active Col2-Cre mice was detected in the nucleus pulposus (NP), the entire annulus fibrosus (AF), and in cartilaginous endplate and growth plate cells in the lower lumbar and coccygeal spine. In contrast, when Col2-CreER activity was induced by tamoxifen at P7, tdTomato was limited to the inner AF, and was absent from the NP. We have described the differences in Col2 reporter gene expression, in Col2-Cre/tdTomato and Col2-Cre-ER/tdTomato mouse IVD. The information provided here will help to guide future investigations of IVD biology.

Keywords: Cre‐Lox reporter system; gene regulation; intervertebral disc; mouse model; type II collagen (Col2).

Figure 1

Col2‐Cre and Col2‐CreER gene construct. Col2‐Cre gene includes a 3 kb Col2a1 promoter fragment, Col2a1 exon 1, and a 3.02 kb Col2a1 intron 1. Col2‐CreER gene includes 1 kb Col2a1 promoter, estrogen receptor that binds to tamoxifen (ERTam), and 650 bp Col2a1 intron 1. Both constructs included the Cre Recombinase gene (Cre) and a Simian Virus 40 polyadenylation signal (SV40 pA). Diagram adapted from Ovchinnikov et al and Nakamura et al

Figure 2

Sagittal section of Col2‐Cre/tdTomato or Col2‐CreER/tdTomato 6‐week old mouse lower lumbar or coccygeal intervertebral disc (IVD). Left panel, confocal images of mouse lumbar, and coccygeal IVDs, with the red fluorescent protein (Tomato) under the control of Col2‐Cre or Col2‐CreER promoters. Red: type II collagen expressing cells; blue: cell nuclei stained with DAPI. A,B, Lumbar spine IVD. C,D, Coccygeal IVD. AF, annulus fibrosus. Scale bar: 250 μm. Right panel, percent (%) of AF cells positive for Tomato in lumbar IVDs of Col2‐CreER mice. Error bar: SE of the mean. *P < .05

Figure 3

Col2‐Cre/tdTomato 6‐week old mouse lower lumbar and coccygeal spine intervertebral disc (IVD). A‐C, Lumbar spine IVD. A′‐C′, Nucleus pulposus (NP) of the lumbar spine IVD. A″‐C″, Posterior annulus fibrosus (AF) of the lumbar spine. D‐F, Coccygeal IVD. Scale bars: 250 μm for the low magnification images and 25 μm for the high magnification images

Figure 4

Col2‐CreER/tdTomato 6‐week old mouse lower lumbar and coocygeal spine intervertebral disc (IVD). A‐C, Lumbar spine IVD; A′‐C′, Nucleus pulposus (NP) of the lumbar spine IVD. A″‐C″, posterior annulus fibrosus (AF) of the lumbar spine IVD. D‐F, Coccygeal IVD. Scale bars: 250 μm for the low magnification images and 25 μm for the high magnification images

Figure 5

F‐actin aligns along the cell membrane in the mouse lumbar spine intervertebral disc (IVD). A,B, Low magnification images of lumbar spine IVD. C,D, High magnification images of the nucleus pulposus (NP). Scale bars: 250 μm for the low magnification images and 25 μm for the high magnification images

Figure 6

Type II collagen (Col2a1) and type I collagen (Col1a1) gene expression in the 8‐week old murine nucleus pulposus (NP) and annulus fibrosus (AF). A, Col2a1 to b2m gene expression ratio. B, Col1a1 to b2m gene expression ratio in mice. **P ≤ .01