Tgfbr2 is required in Acan‐expressing cells for maintenance of the intervertebral and sternocostal joints

Abstract

Background Members of the transforming growth factor beta (TGF‐β) family are secreted proteins that regulate skeletal development. TGF‐β signaling is critical in embryonic development of the annulus fibrosus (AF) of the intervertebral disc (IVD). To address the question of the role of TGF‐β signaling in postnatal development and maintenance of the skeleton, we generated mice in which Tgfbr2 was deleted at 2‐weeks of age in Aggrecan (Acan)‐expressing cells using inducible Cre/LoxP recombination. Methods Localization of Cre recombination was visualized by crossing Acan tm1(cre/ERT2)Crm mice to fluorescent mTmG reporter mice. Acan tm1(cre/ERT2)Crm mice were mated to Tgfbr2 LoxP/LoxP mice and Cre recombinase was activated by tamoxifen injection at 2‐weeks postnatally. Following tamoxifen injection, mice were aged to 3, 6, and 12‐months and control mice were compared to the experimental (cKO) group. Mice were initially analyzed using X‐ray and skeletal preparations. Sternocostal joints and IVD tissues were further analyzed histologically by hematoxylin and eosin (H&E), Safranin O, and Picrosirius Red staining as well as Col10 immunostaining. Results Cre recombination was observed in the IVD and sternocostal joints. X‐ray analysis revealed osteophyte formation within the disc space of 12‐month‐old cKO mice. Skeletal preparations confirmed calcification within the IVD and the sternocostal joints in cKO mice. H&E staining of cKO IVD revealed disorganized growth plates, delay in the formation of the bony endplate, and Col10 staining in the AF indicative of ectopic endochondral bone formation. Furthermore, proteoglycan loss was observed and collagen bundles within the inner AF were thinner and less organized. Alterations in the IVD were apparent beginning at 3 months and were progressively more visible at 6 and 12 months. Similarly, histological analysis of cKO sternocostal joints revealed joint calcification, proteoglycan loss, and disorganization of the collagen architecture at 12 months of age. Conclusions TGF‐β signaling is important for postnatal development and maintenance of fibrocartilaginous IVD and sternocostal joints.

Scaffoldless tissue‐engineered cartilage for studying transforming growth factor beta‐mediated cartilage formation

“…22 The toluidine blue protocol is described as follows. Samples were processed and then embedded in paraffin.…”

“…H&E and picrosirius red protocols were described previously. 22 The toluidine blue protocol is described as follows. Sections were deparaffinized and rehydrated in two washes of xylene for 5 min each, two washes of 100% ethanol for 2 min each, and one wash of 90% ethanol for 1 min.…”

Protocol for parallel proteomic and metabolomic analysis of mouse intervertebral disc tissues

“…We have explored the effect of mechanical loading on the IVD in mice [42][43][44] and used transgenic mice to study IVD development, 45 disc degeneration, 16,46 and diffuse idiopathic skeletal hyperostosis (DISH). [47][48][49][50] Important insights have likewise been provided by others using mouse models to study disc development, [51][52][53] inflammation, 54 IVD degeneration, 10,13,[55][56][57][58] calcification, 59,60 and scoliosis. [61][62][63] The multitude of available mouse models of spine pathologies allows for global molecular comparisons to uncover novel biological insights.…”

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

“…The currently available driver lines to target conditional alleles in the NP cells are the non-inducible Noto Cre (McCann et al, 2012), Shh Cre (Harfe et al, 2004), and Foxa2 Cre (Uetzmann et al, 2008) alleles, or tamoxifen-inducible Agc1 CreERT2 (Henry et al, 2009), Col2a1 CreERT2 (Chen et al, 2007), and Col2a1 CreER (Nakamura et al, 2006). And the Noto Cre (Bedore et al, 2013; McCann et al, 2012), Shh Cre (Winkler et al, 2014; Wu et al, 2013), Foxa2 Cre (Merceron et al, 2014; Uetzmann et al, 2008), Agc1 CreERT2 (Alkhatib et al, 2018; Alvarez-Garcia et al, 2018; Henry et al, 2009; Liao et al, 2019; Novais et al, 2019) and Col2a1 CreERT2 (Chen et al, 2007; Zheng et al, 2018) alleles have been employed to understand the development, regulation and aging of the mouse NP cells in the IVD. However, none of these driver lines are appropriate to target NP cells during the postnatal stages due to the constraint of spatial and temporal regulation of their action, and specificity to NP cells.…”

“…Hence, this study aims to characterize the Krt19 CreERT allele for tamoxifen-induced recombination in the NP cells using conditional fluorescent reporter alleles in postnatal mice at an early neonatal stage, at skeletal maturity, middle age and by two years of age. The postnatal stages analyzed for characterization of the Krt19 CreERT are based on the current literature on mouse model system to understand the role of NP cells in the postnatal IVD (Alkhatib et al, 2018; Alvarez-Garcia et al, 2018; Bonavita et al, 2018; Dahia et al, 2012; Vincent et al, 2019; Wu et al, 2013).…”