A comparative analysis of TonEBP conditional knockout mouse models reveals inter-dependency between compartments of the intervertebral disc.

Interactions between notochord and sclerotome are required for normal embryonic spine patterning, but whether the postnatal derivatives of these tissues also require interactions for postnatal intervertebral disc (IVD) growth and maintenance is less established. We report here the comparative analysis of four conditional knockout mice deficient for TonEBP, a transcription factor known to allow cells to adapt to changes in extracellular osmotic pressure, in specific compartments of the IVD. We show that TonEBP deletion in nucleus pulposus (NP) cells does not affect their survival or aggrecan expression, but promoted cell proliferation in the NP and in adjacent vertebral growth plates (GPs).

Sites of Cre-recombinase activity in mouse lines targeting skeletal cells.

The Cre/Lox system is a powerful tool in the biologist's toolbox, allowing loss-of-function and gain-of-function studies, as well as lineage tracing, through gene recombination in a tissue-specific and inducible manner. Evidence indicates, however, that Cre transgenic lines have a far more nuanced and broader pattern of Cre activity than initially thought, exhibiting "off-target" activity in tissues/cells other than the ones they were originally designed to target. With the goal of facilitating the comparison and selection of optimal Cre lines to be used for the study of gene function, we have summarized in a single manuscript the major sites and timing of Cre activity of the main Cre lines available to target bone mesenchymal stem cells, chondrocytes, osteoblasts, osteocytes, tenocytes, and osteoclasts, along with their reported sites of "off-target" Cre activity.

Advantages and Limitations of Cre Mouse Lines Used in Skeletal Research.

The Cre-LoxP technology permits gene ablation in specific cell lineages, at chosen differentiation stages of this lineage and in an inducible manner. It has allowed tremendous advances in our understanding of skeleton biology and related pathophysiological mechanisms, through the generation of loss/gain of function or cell tracing experiments based on the creation of an expanding toolbox of transgenic mice expressing the Cre recombinase in skeletal stem cells, chondrocytes, osteoblasts, or osteoclasts. In this chapter, we provide an overview of the different Cre-LoxP systems and Cre mouse lines used in the bone field, we discuss their advantages, limitations, and we outline best practices to interpret results obtained from the use of Cre mice.

A step-by-step protocol for isolation of murine nucleus pulposus cells.

The intervertebral disc (IVD) is composed of three separate tissues with distinct origins and properties. Elucidating changes occurring in these tissues in response to injury or age is paramount to identify new therapies to better manage disc and spine degenerative conditions, including low back pain. Despite their small size and different mechanical load pattern compared to higher species, the use of mouse models represents a cost-effective and powerful approach to better understand the formation, maintenance, and degeneration of the IVD.

Promoter Cre-Specific Genotyping Assays for Authentication of Cre-Driver Mouse Lines.

The Cre-LoxP system gene knockout (KO) technology provides cell- and time-specificity of gene ablation to investigate cell-autonomous gene function , and is paramount for understanding the function of genes involved in bone development, remodeling, and repair. This approach permits gene ablation in a cell- or tissue-specific, differentiation stage-specific, and inducible manner, thanks to the use of well-chosen promoters that drive expression of the Cre recombinase in selected cells/tissues. The generation of these powerful tools has led to the expansion of Cre mouse lines available to the research community, which are often shared within and between laboratories.

PiT1/Slc20a1 Is Required for Endoplasmic Reticulum Homeostasis, Chondrocyte Survival, and Skeletal Development.

During skeletal mineralization, the sodium-phosphate co-transporter PiT1Slc20a1 is assumed to meet the phosphate requirements of bone-forming cells, although evidence is missing. Here, we used a conditional gene deletion approach to determine the role of PiT1 in growth plate chondrocytes. We show that PiT1 ablation shortly after birth generates a rapid and massive cell death in the center of the growth plate, together with an uncompensated endoplasmic reticulum (ER) stress, characterized by morphological changes and increased Chop, Atf4, and Bip expression.

Phosphate (P)-regulated heterodimerization of the high-affinity sodium-dependent P transporters PiT1/Slc20a1 and PiT2/Slc20a2 underlies extracellular P sensing independently of P uptake.

Extracellular phosphate (P) can act as a signaling molecule that directly alters gene expression and cellular physiology. The ability of cells or organisms to detect changes in extracellular P levels implies the existence of a P-sensing mechanism that signals to the body or individual cell. However, unlike in prokaryotes, yeasts, and plants, the molecular players involved in P sensing in mammals remain unknown.

Expression of phosphate transporters in optimized cell culture models for dental cells biomineralization.

Phosphate is a key component of dental mineral composition. The physiological role of membrane proteins of dental cells is suspected to be crucial for mineralization mechanisms. Contrary to published data related to calcium, data on regulation of phosphate flux through membrane of mineralizing cells are scarce.