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Mesenchymal Stem Cell-Specific and Preosteoblast-Specific Ablation of TSC1 in Mice Lead to Severe and Slight Spinal Dysplasia, Respectively

Background TSC1-related signaling plays a pivotal role in intramembranous and endochondral ossification processes during skeletogenesis. This study was aimed at determining the significance of the TSC1 gene at different stages of spinal development. Materials and Methods. TSC1-floxed mice (TSC1flox/flox) were crossed with Prrx1-Cre or BGLAP-Cre transgenic mice or mesenchymal stem cell- and osteoblast-specific TSC1-deficient mice, respectively. Somatic and vertebral differences between WT and Prrx1-TSC1 null mice were examined at 4 weeks after birth. Results No apparent body size abnormalities were apparent in newborn and 4-week- to 2-month-old mice with BGLAP-Cre driver-depleted TSC1. Vertebral and intervertebral discs displayed strong dysplasia in Prrx1-TSC1 null mice. In contrast, vertebrae were only slightly affected, and intervertebral discs from skeletal preparations displayed no apparent changes in BGLAP-TSC1 null mice. Conclusion Our data suggest that the TSC1 gene is crucial for endochondral ossification during postnatal spine development but plays discriminative roles at different stages. Mesenchymal stem cell-specific ablation of TSC1 led to severe spinal dysplasia at early stages of endochondral ossification while osteoblast-specific deletion of TSC1 affected vertebrae slightly and had no detectable effects on intervertebral discs.


Introduction
Endochondral and intramembranous ossifications are two types of skeletal ossification processes [1]. Endochondral ossification is the dominant pathway of limb and vertebral growth after embryonic development [2,3]. During this process, mesenchymal stem cells (MSC) in the cartilaginous template differentiate into chondrocytes, following which the chondrification centers are eventually replaced with bone-containing osteoblasts and osteoclasts [4]. Classically, disruption of spinal endochondral ossification can result in spinal dysplasia and consequent development of congenital spinal deformities [5][6][7]. However, the mechanisms underlying spinal endochondral ossification are yet to be clarified.
In the present study, Cre/LoxP technology was applied to delete tuberous sclerosis 1 (TSC1, a major upstream inhibitory regulator of mTORC1) in Prrx-1-positive mesenchymal stem cells and osteocalcin (BGLAP)-positive preosteoblasts in mice and postnatal spinal development examined, with a view to determining the functions of TSC1 in MSCs and preosteoblasts involved in spinal endochondral ossification.

Skeletal Preparations.
Whole-mount skeletal preparations were prepared by removing the skin and internal organs of mice before immersion in 95% ethanol overnight. Specimens were stained with 0.015% Alcian Blue 8GX (Sigma-Aldrich, St Louis, MO, USA) in 80% ethanol/20% acetic acid and 0.005% Alizarin Red S (Sigma-Aldrich) in 1% KOH after digestion with 2% KOH overnight. Next, specimens were cleared with 1% KOH/20% glycerol solution and stored in a 1 : 1 mixture of glycerol and 95% ethanol.

Appearance and Body Length Are Altered in Prrx-1-TSC1
Null but Not BGLAP-TSC1 Null Mice during Development.
The body lengths of TSC1 +/+ -Prrx1-Cre (WT) and Prrx1-TSC1 null mice were measured on days 1, 7, 28, and 60 after birth. As shown in Figure 1, no appearance alterations in BGLAP-TSC1 and Prrx1-TSC1 null mice relative to their WT counterparts were observed on days 1 and 7 (Figures 1(a), 1(b), and 1(i)). However, the body lengths of Prrx1-TSC1 null mice appeared significantly decreased, compared with WT mice, on days 28 and 60 after birth (Figures 1(c), 1(d), and 1(i)). In contrast, no skeletal abnormalities and body length changes were apparent in BGLAP-TSC1 null mice from the newborn stage to day 60, compared with WT mice (Figures 1(e)-1(h), and 1(j)). In contrast, the vertebral heights of BGLAP-TSC1 null mice were similar to that of WT mice. In this group, the structures of growth plates and intervertebral discs were well arranged, and the cartilage endplate, fiber ring, and nucleus pulposus showed no differences from those of WT mice (Figures 4(C1 Taken together, our results suggest that knockout of the TSC1 gene in mesenchymal stem cells leads to severe spinal dysplasia while ablation in preosteoblasts exerts relatively minor effects.

Discussion
As a vital regulator of cell metabolism and proliferation, mTORC1 is ubiquitously expressed in all mammalian cell types [20]. A key role of mTORC1 in bone formation was originally identified in almost all skeleton-related cell lineages. However, conflicting findings regarding the precise mechanisms and functional effects of mTORC1 have been reported. Recent studies suggest that rapamycin inhibits the early stages of osteoblast differentiation in BMSCs and preosteoblasts [21]. Other experiments have demonstrated  (n = 6, " * " represents p < 0:05; " * * " represents p < 0:01; " * * * " represents p < 0:001).

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BioMed Research International overactivation of mTORc1 in both BMSCs and preosteoblastmaintained adult bone mass in vivo and in vitro [15,22].
Here, we hypothesized that mTORC1 is highly expressed in BMSc during the first phase of osteoblastogenesis and weakly downregulated in the last phase of mature osteoblast formation. Upon deletion of TSC1, mTORC1 activity was elevated significantly in BMSCs but only slightly in mature osteoblasts. As hyperactive mTORC1 may impair osteoblastic differentiation [18], spinal dysplasia was more marked in Prrx1-TSC1 null than in BGLAP-TSC1 null mice. Indeed, research has shown that the earlier modulation of mTOR-C1expression in osteoblasts, once differentiated, is associated with more significant of spinal development [18]. Experiments in the current study focused on illustrating the potential significance of the TSC1 gene at different stages of spinal development.
Previously, we reported that specific ablation of TSC1 in chondrocytes leads to retardation of chondrocyte hypertrophic and terminal differentiation [19]. Moreover, congenital spinal deformities of flat chest and thoracolumbar kyphosis were observed in chondrocyte-specific TSC1 null mice [23]. This group of mice further developed chronic wasting and seizures associated with premature death, potentially attributable to leakage of Cre in noncartilaginous tissues [24].

BioMed Research International
Similar to chondrocyte-specific TSC1 null mice, premature death at postnatal days 60-90 was recorded in both Prrx1-TSC1 null and BGLAP-TSC1 null mice. However, the specific causes of death remain to be established.
During mammalian spine development, the chondrification center appears at the embryonic stage and is transformed into chondrified vertebrae continuously at birth [25,26]. Next, endochondral ossification manipulates spinal development gradually after birth and contributes to spinal growth until skeletal maturation [27]. In our experiments, although both Prrx1-TSC1 null and BGLAP-TSC1 null mice presented normal spine at birth, spinal dysplasia developed at postnatal week 4. In keeping with previous studies, our data suggest that overactivation of mTORC1 in chondrocytes leads to embryonic chondral dysplasia that finally results in congenital spinal deformity while overactivation at the preosteoblastic lineage disrupts postnatal endochondral ossification, triggering spinal dysplasia [23]. These findings provide indications of the specific roles of TSC1 in spinal development, both timely and spatial.
Spinal scoliosis is the most common congenital spinal deformity in the clinic followed by kyphosis [28,29]. In our experiments, despite shortened vertebrae and spine, Prrx1-TSC1 null and BGLAP-TSC1 null mice exhibited neither scoliosis nor kyphosis. While this may be derived from premature death, compared with the congenital spinal kyphosis of Col2-TSC1 null mice reported previously by our group, this phenomenon suggests that overactivation of mTORC1 in MSCs or preosteoblasts may not contribute to spinal deformity.
In summary, we have focused on the potential significance of TSC1 gene in osteoblastic lineage cells and spinal development in this study. Deletion of the TSC1 gene in both Prrx1-positive and BGLAP-positive cell osteoblastic lineages, which target osteoblast precursors, resulted in spinal dysplasia. Interestingly, the extent of spinal dysplasia was severe in Prrx1-TSC1 null mice but relatively slight in BGLAP-TSC1 null mice, suggesting that TSC1 gene plays different roles in diversiform osteoblast precursors for mammalian endochondral ossification. Further studies are required for comprehensive understanding of the mechanisms underlying this phenomenon.

Data Availability
All data generated or analysed during this study are included in this published article.