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Osteoclasts are bone-resorbing cells that play an essential role in homeostatic bone remodeling and pathological bone erosion. Macrophage colony stimulating factor (M-CSF) is abundant in rheumatoid arthritis (RA). However, the role of M-CSF in arthritic bone erosion is not completely understood. Here, we show that M-CSF can promote osteoclastogenesis by triggering the proteolysis of c-FMS, a receptor for M-CSF, leading to the generation of FMS intracellular domain (FICD) fragments. Increased levels of FICD fragments positively regulated osteoclastogenesis but had no effect on inflammatory responses. Moreover, myeloid cell-specific FICD expression in mice resulted in significantly increased osteoclast-mediated bone resorption in an inflammatory arthritis model. The FICD formed a complex with DAP5, and the FICD/DAP5 axis promoted osteoclast differentiation by activating the MNK1/2/EIF4E pathway and enhancing NFATc1 protein expression. Moreover, targeting the MNK1/2 pathway diminished arthritic bone erosion. These results identified a novel role of c-FMS proteolysis in osteoclastogenesis and the pathogenesis of arthritic bone erosion.

Intervertebral disc degeneration and associated back pain are relatively common but sparsely understood conditions, affecting over 70% of the population during some point of life. Disc degeneration is often associated with a loss of nucleus pulposus (NP) cells. Genetic mouse models offer convenient avenues to understand the cellular and molecular regulation of the disc during its formation, growth, maintenance, and aging. However, due to the lack of inducible driver lines to precisely target NP cells in the postnatal mouse disc, progress in this area of research has been moderate. NP cells are known to express cytokeratin 19 (Krt19), and tamoxifen (Tam)‐inducible Krt19 CreERT allele is available. The current study describes the characterization of Krt19 CreERT allele to specifically and efficiently target NP cells in neonatal, skeletally mature, middle‐aged, and aged mice using two independent fluorescent reporter lines. The efficiency of recombination at all ages was validated by immunostaining for KRT19. Results show that following Tam induction, Krt19 CreERT specifically drives recombination of NP cells in the spine of neonatal and aged mice, while no recombination was detected in the surrounding tissues. Knee joints from skeletally mature Tam‐treated Krt19 CreERT/+; R26 tdTOM mouse show the absence of recombination in all tissues and cells of the knee joint. Thus, this study provides evidence for the use of Krt19 CreERT allele for genetic characterization of NP cells at different stages of the mouse life., An inducible driver line to precisely target nucleus pulposus (NP) cells during the postnatal is required to understand the role of these cells during development and aging. The current study describes the characterization of Krt19CreERT allele to efficiently target NP cells in neonatal, skeletally mature, middle‐aged, and aged mice using two independent fluorescent reporter lines. Moreover, this allele did not target the surrounding cells in the spine or any cell in the knee joint, validating its specificity to target NP cells in the postnatal mouse.

This perspective summarizes the genesis, development, and potential future directions of the multispeciesJOR Spinehistopathology series.

Mice have been increasingly used as preclinical model to elucidate mechanisms and test therapeutics for treating intervertebral disc degeneration (IDD). Several intervertebral disc (IVD) histological scoring systems have been proposed, but none exists that reliably quantitate mouse disc pathologies. Here, we report a new robust quantitative mouse IVD histopathological scoring system developed by building consensus from the spine community analyses of previous scoring systems and features noted on different mouse models of IDD. The new scoring system analyzes 14 key histopathological features from nucleus pulposus (NP), annulus fibrosus (AF), endplate (EP), and AF/NP/EP interface regions. Each feature is categorized and scored; hence, the weight for quantifying the disc histopathology is equally distributed and not driven by only a few features. We tested the new histopathological scoring criteria using images of lumbar and coccygeal discs from different IDD models of both sexes, including genetic, needle‐punctured, static compressive models, and natural aging mice spanning neonatal to old age stages. Moreover, disc sections from common histological preparation techniques and stains including H&E, SafraninO/Fast green, and FAST were analyzed to enable better cross‐study comparisons. Fleiss's multi‐rater agreement test shows significant agreement by both experienced and novice multiple raters for all 14 features on several mouse models and sections prepared using various histological techniques. The sensitivity and specificity of the new scoring system was validated using artificial intelligence and supervised and unsupervised machine learning algorithms, including artificial neural networks, k‐means clustering, and principal component analysis. Finally, we applied the new scoring system on established disc degeneration models and demonstrated high sensitivity and specificity of histopathological scoring changes. Overall, the new histopathological scoring system offers the ability to quantify histological changes in mouse models of disc degeneration and regeneration with high sensitivity and specificity., We have developed a new Mouse intErveRtebral disC histopathologY (MERCY) system using a step‐wise approach that included building consensus in the spine community, testing reliability using various mouse disc degeneration models for agreement by multiple raters, validating for high sensitivity and specificity using AI and machine learning algorithms, and applied on established models of murine disc degeneration. Hence, this new system can be broadly applied to quantify mouse IVD histopathology in disc degeneration and regeneration models.

The fifth biennial ORS PSRS International Spine Research Symposium took place from November 3 to 7, 2019, at Skytop Lodge in northeastern Pennsylvania. Organized jointly by the Orthopaedic Research Society and the Philadelphia Spine Research Society, the symposium attracted more than 180 participants from 10 different countries to share the latest advances in basic and preclinical spine research. Following the symposium, participants were invited to submit full‐length manuscripts to this special issue of JOR Spine.

Aging is a major risk factor for numerous painful, inflammatory, and degenerative diseases including disc degeneration. A better understanding of how the somatosensory nervous system adapts to the changing physiology of the aging body will be of great significance for our expanding aging population. Previously, we reported that chronological aging of mouse lumbar discs is pathological and associated with behavioral changes related to pain. It is established that with age and degeneration the lumbar discs become inflammatory and innervated. Here we analyze the aging lumbar dorsal root ganglia (DRGs) and spinal cord dorsal horn (SCDH) in mice between 3 and 24 months of age for age‐related somatosensory adaptations. We observe that as mice age there are signs of peripheral sensitization, and response to inflammation at the molecular and cellular level in the DRGs. From 12 months onwards the mRNA expression of vasodilator and neurotransmitter, Calca (CGRP); stress (and survival) marker, Atf3; and neurotrophic factor, Bdnf, increases linearly with age in the DRGs. Further, while the mRNA expression of neuropeptide, Tac1, precursor of Substance P, did not change at the transcriptional level, TAC1 protein expression increased in 24‐month‐old DRGs. Additionally, elevated expression of NFκB subunits, Nfkb1 and Rela, but not inflammatory mediators, Tnf, Il6, Il1b, or Cox2, in the DRGs suggest peripheral nerves are responding to inflammation, but do not increase the expression of inflammatory mediators at the transcriptional level. These results identify a progressive, age‐related shift in the molecular profile of the mouse somatosensory nervous system and implicates nociceptive sensitization and inflammatory response., Age‐related degeneration including disc degeneration induces Atf3 expression in the DRGs, possibly through the Npy, Bdnf and TRPA1 pathways. Intact small‐diameter nerves respond by inducing Calca expression in DRGs, its transport to the SCDH, and increased Calca expression by the SCDH neurons, leading to central sensitization. Expression of TNFalpha in aging degenerative tissues or the circulatory system induces NFκB pathway, causing increased expression of nociceptive sodium channels, NaV1.8 and NaV1.9, which further stimulates SCDH neuron.

Intervertebral disc degeneration is the most significant, and least understood, cause of chronic back pain, affecting almost one in seven individuals at some point of time. Each intervertebral disc has three components; central nucleus pulposus (NP), concentric layers of annulus fibrosus (AF), and a pair of end plate (EP) that connects the disc to the vertebral bodies. Understanding the molecular and cellular basis of intervertebral disc growth, health, and aging will generate significant information for developing therapeutic approaches. Rapid and efficient preparations of homogeneous and pure cells are crucial for meaningful and rigorous downstream analysis at the cellular, molecular, and biochemical level. Cross‐sample contamination may influence the interpretation of the results. In addition to altering gene expression, slow or delayed isolation procedures will also cause the degradation of cells and biomolecules that create a bias in the outcomes of the study. The mouse model system is extensively used to understand the intervertebral disc biology. Here we describe two protocols: (a) for efficient isolation of pure NP, AF, and EP cells from mouse lumbar intervertebral disc. We validated the purity of the NP and AF cells using ShhCre/+; R26mT/mG/+ dual‐fluorescent reporter mice where all NP cells are GPF+ve, and by the sensitive approach of qPCR analysis using TaqMan probes for Shh, and Brachyury as NP‐specific markers, Tenomodulin as AF‐specific marker, and Osteocalcin as bone‐specific marker. (b) For isolation of high‐quality intact RNA with RIN of 9.3 to 10 from disc cells. These methods will be useful for the rigorous analysis of NP and AF cells, and improve our understanding of intervertebral disc biology.

A) PURPOSE OF REVIEW: The intervertebral discs (IVD) are an essential component of the spine. Degeneration of the discs, commonly due to age or injury, is a leading cause of chronic lower back pain. Despite its high prevalence, there is no effective treatment for disc disease due to limited understanding of disc at the cellular and molecular level. B) RECENT FINDINGS: Recent research has demonstrated the importance of the intracellular developmental pathway sonic hedgehog (Shh) during the formation and postnatal maintenance of the IVD. Recent studies corroborate that the down-regulation of SHH expression is associated with pathological changes in the IVDs and demonstrate the reactivation of the hedgehog pathway as a promising avenue for rescuing health disc structure and function. C) SUMMARY: Understanding the role of developmental signaling pathways that regulate disc formation and maintenance may help develop strategies to recapitulate the same mechanism for disc treatment and hence improve the quality and longevity of patient lives.

Aging is a major risk factor of intervertebral disc degeneration and a leading cause of back pain. Pathological changes associated with disc degeneration include the absence of large, vacuolated and reticular‐shaped nucleus pulposus cells, and appearance of smaller cells nested in lacunae. These small nested cells are conventionally described as chondrocyte‐like cells; however, their origin in the intervertebral disc is unknown. Here, using a genetic mouse model and a fate mapping strategy, we have found that the chondrocyte‐like cells in degenerating intervertebral discs are, in fact, nucleus pulposus cells. With aging, the nucleus pulposus cells fuse their cell membranes to form the nested lacunae. Next, we characterized the expression of sonic hedgehog (SHH), crucial for the maintenance of nucleus pulposus cells, and found that as intervertebral discs age and degenerate, expression of SHH and its target Brachyury is gradually lost. The results indicate that the chondrocyte‐like phenotype represents a terminal stage of differentiation preceding loss of nucleus pulposus cells and disc collapse.

Intervertebral discs are cartilaginous joints present between vertebrae. The centers of the intervertebral discs consist of a gelatinous nucleus pulposus derived from the embryonic notochord. With age or injury, intervertebral discs may degenerate, causing neurological symptoms including back pain, which affects millions of people worldwide. Back pain is a multifactorial disorder, and disc degeneration is one of the primary contributing factors. Recent studies in mice have identified the key molecules involved in the formation of intervertebral discs. Several of these key molecules including sonic hedgehog and Brachyury are not only expressed by notochord during development, but are also expressed by neonatal mouse nucleus pulposus cells, and are crucial for postnatal disc maintenance. These findings suggest that intrinsic signals in each disc may maintain the nucleus pulposus microenvironment. However, since expression of these developmental signals declines with age and degeneration, disc degeneration may be related to the loss of these intrinsic signals. In addition, findings from mouse and other mammalian models have identified similarities between the patterning capabilities of the embryonic notochord and young nucleus pulposus cells, suggesting that mouse is a suitable model system to understand disc development and aging. Future research aimed at understanding the upstream regulators of these developmental signals and the modes by which they regulate disc growth and maintenance will likely provide mechanistic insights into disc growth and aging. Further, such findings will likely provide insights relevant to the development of effective therapies for treatment of back pain and reversing the disc degenerative process. This article is categorized under: Birth Defects > Organ Anomalies Vertebrate Organogenesis > Musculoskeletal and Vascular Adult Stem Cells, Tissue Renewal, and Regeneration > Regeneration Adult Stem Cells, Tissue Renewal, and Regeneration > Stem Cells and Aging.

With the increased burden of low back pain (LBP) in our globally aging population there is a need to develop preclinical models of LBP that capture clinically relevant features of physiological aging, degeneration, and disability. Here we assess the validity of using a mouse model system for age-related LBP by characterizing aging mice for features of intervertebral disc (IVD) degeneration, molecular markers of peripheral sensitization, and behavioral signs of pain. Compared to three-month-old and one-year-old mice, two-year-old mice show features typical of IVD degeneration including loss of disc height, bulging, innervation and vascularization in the caudal lumbar IVDs. Aging is also associated with the loss of whole-body bone mineral density in both male and female mice, but not associated with percent lean mass or body fat. Additionally, two-year-old mice have an accumulation of TRPA1 channels and sodium channels Na(v)1.8 and Na(v)1.9 in the L4 and L5 lumbar dorsal root ganglia consistent with changes in nociceptive signaling. Lastly, the effect of age, sex, and weight on mobility, axial stretching and radiating pain measures was assessed in male and female mice ranging from two months to two years in a general linear model. The model revealed that regardless of sex or weight, increased age was a predictor of greater reluctance to perform axial stretching and sensitivity to cold, but not heat in mice.

This paper is a concise review aiming to assemble the most relevant topics presented by the authors at ORS-Philadelphia Spine Research Society Fourth International Spine Research Symposium. It centers on the latest advances in disc development, its main structural entities, and the populating cells, with emphasis on the advances in pivotal molecular pathways responsible for forming the intervertebral discs (IVD). The objective of finding and emphasizing pathways and mechanisms that function to control tissue formation is to identify and to explore modifications occurring during normal aging, disease, and tissue repair. Thus, to comprehend that the cellular and molecular basis of tissue degeneration are crucial in the study of the dynamic interplay that includes cell-cell communication, gene regulation, and growth factors required to form a healthy and functional tissue during normal development.

In humans, the sacrum forms an important component of the pelvic arch, and it transfers the weight of the body to the lower limbs. The sacrum is formed by collapse of the intervertebral discs (IVDs) between the five sacral vertebrae during childhood, and their fusion to form a single bone. We show that collapse of the sacral discs in the mouse is associated with the down-regulation of sonic hedgehog (SHH) signaling in the nucleus pulposus (NP) of the disc, and many aspects of this phenotype can be reversed by experimental postnatal activation of hedgehog (HH) signaling. We have previously shown that SHH signaling is essential for the normal postnatal growth and differentiation of intervertebral discs elsewhere in the spine, and that loss of SHH signaling leads to pathological disc degeneration, a very common disorder of aging. Thus, loss of SHH is pathological in one region of the spine but part of normal development in another., Summary: Loss of SHH signaling is associated with the collapse of the sacral discs and formation of the sacrum. Conditional reactivation of SHH re-awakens the sacral disc, suggesting its potential for disc regeneration.

The structure and composition of the native enthesis is not recapitulated following tendon-to-bone repair. Indian Hedgehog (IHH) signaling has recently been shown to be important in enthesis development in a mouse model but no studies have evaluated IHH signaling in a healing model. Fourteen adult male rats underwent ACL reconstruction using a flexor tendon graft. Rats were assigned to two groups based on whether or not they received 0N or 10N of pre-tension of the graft. Specimens were evaluated at 3 and 6 weeks post-operatively using immunohistochemistry for three different protein markers of IHH signaling. Quantitative analysis of staining area and intensity using custom software demonstrated that IHH signaling was active in interface tissue formed at the healing tendon-bone interface. We also found increased staining area and intensity of IHH signaling proteins at 3 weeks in animals that received a pre-tensioned tendon graft. No significant differences were seen between the 3-week and 6-week time points. Our data suggests that the IHH signaling pathway is active during the tendon-bone healing process and appears to be mechanosensitive, as pre-tensioning of the graft at the time of surgery resulted in increased IHH signaling at three weeks.

The effect of Follicle Stimulating Hormone (FSH) deprivation, or hyperthyroidism, on the expression of selected genes in Sertoli cells of neonatal rats was compared, and significant differences were revealed. While hyperthyroidism resulted in both arrest of proliferation and expression of differentiation markers, FSH deprivation only resulted in arrest of proliferation, as assessed by a decrease in such proliferation markers as connective tissue growth factor (CTGF) and collagen 12A1 and the expression of differentiation markers, such as androgen binding protein, transferrin and clusterin. In Sertoli cells isolated from hyperthyroid rats, a decrease in the expression of FSH receptor was also observed. Microarray analysis of RNA from FSH-deprived or hyperthyroid neonatal rat Sertoli cells suggests that molecular events associated with arrest of proliferation by hyperthyroidism appear to be different from arrest of proliferation by FSH deprivation.

STUDY DESIGN Vertebral growth plates at different postnatal ages were assessed for active intercellular signaling pathways. OBJECTIVE To generate a spatial and temporal map of the major signaling pathways active in the postnatal mouse lumbar vertebral growth plate. SUMMARY OF BACKGROUND DATA The growth of all long bones is known to occur by cartilaginous growth plates. The growth plate is composed of layers of chondrocyets that actively proliferate, differentiate, die and, are replaced by bone. The role of major cell signaling pathways has been suggested for regulation of the fetal long bones. But not much is known about the molecular or cellular signals that control the postnatal vertebral growth plate and hence postnatal vertebral bone growth. Understanding such molecular mechanisms will help design therapeutic treatments for vertebral growth disorders such as scoliosis. METHODS Antibodies against activated downstream intermediates were used to identify cells in the growth plate responding to BMP, TGFβ, and FGF in cryosections of lumbar vertebrae from different postnatal age mice to identify the zones that were responding to these signals. Reporter mice were used to identify the chondrocytes responding to hedgehog (Ihh), and Wnt signaling. RESULTS We present a spatial/temporal map of these signaling pathways during growth, and differentiation of the mouse lumbar vertebral growth plate. CONCLUSION During growth and differentiation of the vertebral growth plate, its different components respond at different times to different intercellular signaling ligands. Response to most of these signals is dramatically downregulated at the end of vertebral growth.

Sertoli cells under the influence of Follicle Stimulating Hormone are known to provide a microenvironment for the growth and differentiation of germ cells. Follicle Stimulating Hormone regulates proliferation of Sertoli cells only during the early neonatal period and during adult period it regulates functional parameters like Transferrin, Androgen binding protein and other factors needed for germ cell differentiation and how this is achieved is not known. In an attempt to understand the differential action of Follicle Stimulating Hormone on the Sertoli cells, we employed the approach of neutralization of endogenous Follicle Stimulating Hormone in neonatal rats for a very limited period and study the effect on the regulation of gene expression by Differential Display RT-PCR analysis. One of the transcripts was found to be down regulated following neutralization of endogenous Follicle Stimulating Hormone in the neonatal rats and it shared identity with Intracisternal A particle element, an endogenous retrovirus. Often integration of Intracisternal A particle into host genes and its regulation result in growth factor independence suggesting a role in proliferation. Interestingly, the expression of Intracisternal A particle transcript is not regulated by Follicle Stimulating Hormone in the adult rat Sertoli cells suggesting a possible reason for stimulation of proliferation of Sertoli cells by Follicle Stimulating Hormone only in the neonatal rats.

FSH receptor has been shown to be specifically expressed only in the Sertoli cells in males. In one of our studies that consisted of deprival of endogenous FSH in immature rats and adult bonnet monkeys, atrophy of the epididymis was observed, cauda region being the most affected. Although epididymis is an androgen-dependent tissue, the changes in histology of the cauda region were observed without any associated change in the levels of testosterone in FSH-deprived animals. Considering this, it was of interest to evaluate the possibility of epididymis being a direct target for FSH action. In the present study, we have examined the expression of FSH receptor in the epididymis of rat and monkey. In the cauda region of rat epididymis, FSH receptor expression was demonstrated by RT-PCR and Northern and Western blot analyses. FSH receptor was found to be functional as observed by its ability to bind 125IoFSH, by an increase in cAMP production, and by BrdU incorporation following addition of FSH under in vitro conditions. These results suggest the possibility of a role for FSH in regulating the growth of the epididymis.

Sertoli cells support the development of germ cells by providing a microenvironment in the seminiferous tubules. FSH stimulates Sertoli cell proliferation only during neonatal period till day 18 in the immature rat whereas FSH regulates only functional parameters in the adult rat Sertoli cells. This suggests that FSH exerts differential action in immature and adult Sertoli cells. In an attempt to elucidate the mechanism by which FSH exerts the differential effects, we have carried out both in vivo and in vitro studies using Sertoli cells isolated from immature (7-10 days old) and adult (90 days old) rats. The differential role of FSH was studied at the receptor as well as at the signaling level. Monitoring the level of expression of FSH receptor by RTPCR and northern blot analysis revealed that the expression was more in immature Sertoli cells. Furthermore, it was found that FSH up (1.8-fold) regulates its receptor level only in the immature Sertoli cells and not in the adult. Results also revealed that PKIbeta and calcium, which are the downstream signaling molecules, are involved in FSH regulated Sertoli cells proliferation. It was also observed that FSH up (1.4-fold) regulates the levels of expression of IL-6 mRNA only in the immature rat Sertoli cells suggesting the possibility of its involvement in FSH regulated Sertoli cell proliferation.

Study design This study follows postnatal intervertebral disc (IVD) growth and differentiation in the mouse. OBJECTIVE.: To initiate use of the mouse as a model system for postnatal IVD differentiation and growth, and to serve as a basis for assaying changes caused by disease or genetic or experimental perturbation. Summary of background data Lower back pain caused by disc degeneration is one of the most common clinical conditions worldwide. There is currently no completely effective treatment, largely because of a lack of basic knowledge of the molecular and cellular controls of disc growth, differentiation, and maintenance after birth. Methods Conventional histology of decalcified IVDs, differential interference contrast, polarizing optics, immunocytochemistry, laser capture microscopy followed by molecular analysis of the dissected cells by reverse transcriptase polymerase chain reaction. Results There is a single postnatal growth spurt in the mouse IVD, between birth and 9 weeks of age. Cell proliferation was found in the nucleus pulposus (NP) and anulus fibrosus (AF) only until 3 weeks of age. Most of the postnatal growth of the IVD is due to accumulating extracellular matrix. NP cell numbers decline steadily after 2 weeks of age, because of apoptosis. Laser capture microscopy was used to dissect NP cells from the disc, and showed that these cells express markers of the embryonic notochord. The postnatal AF appears initially as a continuous structure surrounding the NP. This structure differentiates, during the first 2 postnatal weeks, to form the mineralized, but nonossified endplate over the surfaces of the vertebral growth plates, and the mature fibrous AF (fAF) passing between adjacent vertebrae. The fact that the mature fAF and the endplate form from an originally continuous layer of cells explains the anatomic relationship between these 2 structures, in which the fAF inserts into the vertebral endplate. Conclusion Growth of the IVD takes place during the first 9 postnatal weeks, although cell proliferation ceases after 3 weeks. After birth, the early postnatal IVD differentiates into 3 tissue types, the NP, the fAF between the vertebrae, and the mineralized endplates over the surfaces of the vertebrae.

STUDY DESIGN Intervertebral discs at different postnatal ages were assessed for active intercellular signaling pathways. OBJECTIVE To generate a spatial and temporal map of the signaling pathways active in the postnatal intervertebral disc (IVD). SUMMARY OF BACKGROUND DATA The postnatal IVD is a complex structure, consisting of 3 histologically distinct components, the nucleus pulposus, fibrous anulus fibrosus, and endplate. These differentiate and grow during the first 9 weeks of age in the mouse. Identification of the major signaling pathways active during and after the growth and differentiation period will allow functional analysis using mouse genetics and identify targets for therapy for individual components of the disc. METHODS Antibodies specific for individual cell signaling pathways were used on cryostat sections of IVD at different postnatal ages to identify which components of the IVD were responding to major classes of intercellular signal, including sonic hedgehog, Wnt, TGFbeta, FGF, and BMPs. RESULTS We present a spatial/temporal map of these signaling pathways during growth, differentiation, and aging of the disc. CONCLUSION During growth and differentiation of the disc, its different components respond at different times to different intercellular signaling ligands. Most of these are dramatically downregulated at the end of disc growth.

A full list of authors and affiliations appears on page 5 of this article., The extent to which low-frequency (minor allele frequency (MAF) between 1-5%) and rare (MAF ≤ 1%) variants contribute to complex traits and disease in the general population is mainly unknown. Bone mineral density (BMD) is highly heritable, a major predictor of osteoporotic fractures, and has been previously associated with common genetic variants, as well as rare, population-specific, coding variants. Here we identify novel non-coding genetic variants with large effects on BMD (ntotal = 53,236) and fracture (ntotal = 508,253) in individuals of European ancestry from the general population. Associations for BMD were derived from whole-genome sequencing (n = 2,882 from UK10K (ref. 10); a population-based genome sequencing consortium), whole-exome sequencing (n = 3,549), deep imputation of genotyped samples using a combined UK10K/1000 Genomes reference panel (n = 26,534), and de novo replication genotyping (n = 20,271). We identified a low-frequency non-coding variant near a novel locus, EN1, with an effect size fourfold larger than the mean of previously reported common variants for lumbar spine BMD (rs11692564(T), MAF = 1.6%, replication effect size = +0.20 s.d., Pmeta = 2 × 10-14), which was also associated with a decreased risk of fracture (odds ratio = 0.85; P = 2 × 10-11; ncases = 98,742 and ncontrols = 409,511). Using an En1cre/flox mouse model, we observed that conditional loss of En1 results in low bone mass, probably as a consequence of high bone turnover. We also identified a novel low-frequency non-coding variant with large effects on BMD near WNT16 (rs148771817(T), MAF = 1.2%, replication effect size = +0.41 s.d., Pmeta = 1 × 10-11). In general, there was an excess of association signals arising from deleterious coding and conserved non-coding variants. These findings provide evidence that low-frequency non-coding variants have large effects on BMD and fracture, thereby providing rationale for whole-genome sequencing and improved imputation reference panels to study the genetic architecture of complex traits and disease in the general population., peer-reviewed

Introduction: Degenerative disc disease is a major cause of lower back pain. However the molecular signals that control normal growth and differentiation of the disc are not well defined. We hypothesize that nucleus pulposus (NP) cells, which originate from the embryonic notochord, provide signals that control growth and differentiation of the postnatal disc. Methods: Postnatal day 4 mouse IVDs were cultured in serum-free DMEM Ham/F12 medium at 37 °C in 5% CO2 for 2–5 days on type IV collagen-coated cell culture inserts. Cyclopamine (250 μM) was used as a Shh antagonist, and its specificity was tested by add-back experiments using 100 nM recombinant Shh (rShh). Cell proliferation was assayed using pulselabelling with BrdU. 6 μm thick cryosections were immunostained (IHC) using antibodies specific to IVD differentiation markers, and Cy5 conjugated secondary antibodies. Imaging was carried out using confocal microscope. To test the role of Shh in vivo, triple transgenic mice [(tetO)7CMV-cre:R26 rtTA:Shhflx/flx] were injected with 2 μg of doxycycline on postnatal day 4 to conditionally delete Shh, and the lumbar spine was collected 5 days later. Results were compared to vehicle treated controls. Results: Both cyclopamine treatment in vitro and conditional Shh knockout in vivo caused rounding up and aggregation of the NP cells, and loss of orientation of the layers of the annulus fibrosus (AF). There was also a loss of expression of both NP and AF differentiation markers. BrdU incorporation studies showed loss of proliferation of NP cells. IHC for activated caspase-3 showed increased levels of apoptosis of NP cells rShh replacement and reversed the cyclopamine phenotype in vitro. Conclusions: These results show that Shh is essential for postnatal growth and differentiation of both the NP and AF. Significance: Identification of signals controlling IVD growth and differentiation offers the opportunity for the development of biological repair as an alternative to surgery.