Generation and characterization of col10a1‐mcherry reporter mice

Abstract

SummaryWe report here on the generation of a new fluorescent protein reporter transgenic mouse line, Col10a1-mCherry, which can be used as a tool to study chondrocyte biology and pathology. Collagen, Type X, alpha 1 (Col10a1) is highly expressed in hypertrophic chondrocytes and commonly used as a gene marker for this cell population. The Col10a1-mCherry reporter line was generated using a bacterial recombination strategy with the mouse BAC clone RP23-192A7. To aid in the characterization of this animal model, we intercrossed Col10a1-mCherry mice with Collagen, Type II, alpha 1 (Col2a1) enhanced cyan fluorescent protein (ECFP) reporter mice and characterized the expression of both chondrocyte reporters during embryonic skeletal development from days E10.5 to E17.5. Additionally, at postnatal day 0, Col10a1-mCherry reporter expression was compared to endogenous Col10a1 mRNA expression in long bones and revealed that mCherry fluorescence extended past the Col10a1 expression domain. However, in situ hybridization for mCherry was consistent with the zone of Col10a1 mRNA expression, indicating that the persistent detection of mCherry fluorescence was a result of the long protein half life of mCherry in conjunction with a very rapid phase of skeletal growth and not due to aberrant transcriptional regulation. Taking advantage of the continued fluorescence of hypertrophic chondrocytes at the chondro-osseus junction, we intercrossed Col10a1-mCherry mice with two different Collagen, Type 1, alpha 1, (Col1a1) osteoblast reporter mice, pOBCol3.6-Topaz and pOBCol2.3-Emerald to investigate the possibility that hypertrophic chondrocytes transdifferentiate into osteoblasts. Evaluation of long bones at birth suggests that residual hypertrophic chondrocytes and osteoblasts in the trabecular zone exist as two completely distinct cell populations. genesis 49:410-418, 2011.

Evolution of the vertebrate bone matrix: An expression analysis of the network forming collagen paralogues in amphibian osteoblasts

“…Below, we discuss how the expression of each of the network forming collagen paralogues evolved following speciation and duplication events. First, our results show that the osteoblastic expression of Col10a1 is shared between amphibians and actinopterygians and supports the concept that this feature represents a synapomorphy of osteichthyans which was secondarily lost in the amniote lineage (Kim et al, ; Avaron et al, ; Laue et al, ; Li et al, ; Albertson et al, ; Maye et al, ; Eames et al, ). Second, Col8a1 represents another case of expression divergence between tetrapods.…”

“…The expression of Col10a1 represents an interesting case of expression pattern evolution between skeletal cell types. This is because independent studies have shown that Col10a1 is strongly expressed in osteoblasts in a variety of actinopterygian species, but not in mammals (Warman et al, ; Kwan et al, ; Kim et al, ; Avaron et al, ; Laue et al, ; Li et al, ; Albertson et al, ; Maye et al, ; Eames et al, ). However, the complete lack of data from amphibians limits our ability to polarize evolutionary change for this gene.…”

Drug-induced modulation of gp130 signalling prevents articular cartilage degeneration and promotes repair

“…To identify compounds that could inhibit hypertrophy, we isolated total limb cells from Col10a1 -mCherry mice27 and cultured them in the presence of bone morphogenetic protein (BMP)-4, a driver of developmental chondrocyte hypertrophy;28 170 000 compounds were assayed for their ability to decrease the mCherry signal (figure 1A). Seventy-five were chosen for follow-up (figure 1B), and additional vetting based on reproducibility and magnitude of Col10a1 inhibition led to the selection of regulator of cartilage growth and differentiation (RCGD) 423 for continued characterisation (figure 1C).…”

Antagonistic interaction between Wnt and Notch activity modulates the regenerative capacity of a zebrafish fibrotic liver model

“…10). Further analysis of Notch activity by comparing the expression dynamics of VenusPEST and mCherry, of which the half‐life is comparable to that of GFP, revealed two distinct populations of mCherry‐positive cells at 0 hpa: bright‐ and dim‐red (Supporting Fig. 11A,A′,G).…”