Project description:Recapitulation of developmental signals represents a promising strategy for treating intervertebral disc degeneration. During development, embryonic notochord-derived cells (NDCs) are the direct progenitors of cells that populate the adult nucleus pulposus (NP) and are an important source of secreted signaling molecules. The objective of this study was to define global gene expression profiles of NDCs at key stages of embryonic disc formation. NDCs were isolated from Shh-cre;ROSA:YFP mice at embryonic day 12.5 and postnatal day 0, representing opposite ends of the notochord to NP transformation. Differences in global mRNA abundance across this developmental window were established using RNA-Seq. Protein expression of selected molecules was confirmed using immunohistochemistry. Principal component analysis revealed clustering of gene expression at each developmental stage with more than 5000 genes significantly differentially expressed between E12.5 and P0. There was significantly lower mRNA abundance of sonic hedgehog pathway elements at P0 vs E12.5, while abundance of elements of the transforming growth factor-beta and insulin-like growth factors pathways, and extracellular matrix components including collagen 6 and aggrecan, were significantly higher at P0. This study represents the first transcriptome-wide analysis of embryonic NDCs. Results suggest signaling and biosynthesis of NDCs change dramatically as a function of developmental stage.
Project description:The adult nucleus pulposus originates from the embryonic notochord, but loss of notochordal cells with skeletal maturity in humans is thought to contribute to the onset of intervertebral disc degeneration. Thus, defining the phenotype of human embryonic/fetal notochordal cells is essential for understanding their roles and for development of novel therapies. However, a detailed transcriptomic profiling of human notochordal cells has never been achieved. In this study, the notochord-specific marker CD24 was used to specifically label and isolate (using FACS) notochordal cells from human embryonic and fetal spines (7.5-14 weeks post-conception). Microarray analysis and qPCR validation identified CD24, STMN2, RTN1, PRPH, CXCL12, IGF1, MAP1B, ISL1, CLDN1 and THBS2 as notochord-specific markers. Expression of these markers was confirmed in nucleus pulposus cells from aged and degenerate discs. Ingenuity pathway analysis revealed molecules involved in inhibition of vascularisation (WISP2, Noggin and EDN2) and inflammation (IL1-RN) to be master regulators of notochordal genes. Importantly, this study has, for the first time, defined the human notochordal cell transcriptome and suggests inhibition of inflammation and vascularisation may be key roles for notochordal cells during intervertebral disc development. The molecules and pathways identified in this study have potential for use in developing strategies to retard/prevent disc degeneration, or regenerate tissue.
Project description:Dysregulation of ribosome production can lead to a number of developmental disorders called ribosomopathies. Despite the ubiquitous requirement for these cellular machines used in protein synthesis, ribosomopathies manifest in a tissue-specific manner, with many affecting the development of the face. Here we reveal yet another connection between craniofacial development and making ribosomes through the protein Paired Box 9 (PAX9). PAX9 functions as an RNA Polymerase II transcription factor to regulate the expression of proteins required for craniofacial and tooth development in humans. We now expand this function of PAX9 by demonstrating that PAX9 acts outside of the cell nucleolus to regulate the levels of proteins critical for building the small subunit of the ribosome. This function of PAX9 is conserved to the organism Xenopus tropicalis, an established model for human ribosomopathies. Depletion of pax9 leads to craniofacial defects due to abnormalities in neural crest development, a result consistent with that found for depletion of other ribosome biogenesis factors. This work highlights an unexpected layer of how the making of ribosomes is regulated in human cells and during embryonic development.
Project description:Intervertebral disc degeneration describes the vicious cycle of the deterioration of intervertebral discs and can eventually result in degenerative disc disease (DDD), which is accompanied by low-back pain, the musculoskeletal disorder with the largest socioeconomic impact world-wide. In more severe stages, intervertebral disc degeneration is accompanied by loss of joint space, subchondral sclerosis, and osteophytes, similar to osteoarthritis (OA) in the articular joint. Inspired by this resemblance, we investigated the analogy between human intervertebral discs and articular joints. Although embryonic origin and anatomy suggest substantial differences between the two types of joint, some features of cell physiology and extracellular matrix in the nucleus pulposus and articular cartilage share numerous parallels. Moreover, there are great similarities in the response to mechanical loading and the matrix-degrading factors involved in the cascade of degeneration in both tissues. This suggests that the local environment of the cell is more important to its behavior than embryonic origin. Nevertheless, OA is widely regarded as a true disease, while intervertebral disc degeneration is often regarded as a radiological finding and DDD is undervalued as a cause of chronic low-back pain by clinicians, patients and society. Emphasizing the similarities rather than the differences between the two diseases may create more awareness in the clinic, improve diagnostics in DDD, and provide cross-fertilization of clinicians and scientists involved in both intervertebral disc degeneration and OA.
Project description:Previously, we showed that deletion of the TGF-beta type II receptor (Tgfbr2) in Type II Collagen (Col2a) expressing cells results in defects in the development of the axial skeleton. Defects included a reduction in size and alterations in the shape of specific vertebral elements. Anterior lateral and dorsal elements of the vertebrae were missing or irregularly shaped. Vertebral bodies were only mildly affected, but the intervertebral disc (IVD) was reduced or missing. In this manuscript, we show that alterations in the initiation or proliferation of cartilage are not detected in the axial skeleton. However, the expression domain of Fibromodulin (Fmod), a marker of the IVD, was reduced and the area of the future IVD contained peanut agglutinin (PNA) staining cartilage. Next, we show that the expression domains of Pax1 and Pax9, which are preferentially expressed in the caudal sclerotome, are expanded over the entire rostral to caudal length of the sclerotome segment. Dorsal-ventral patterning was not affected in these mice as accessed by expression of Pax1, Pax9, and Msx1. Proliferation was modestly reduced in the loose cells of the sclerotome. The results suggest that signaling through Tgfbr2 regulates the maintenance of boundaries in the sclerotome and developing axial skeleton.
Project description:Pax1 and Pax9 play redundant, synergistic functions in the patterning and differentiation of the sclerotomal cells that give rise to the vertebral bodies and intervertebral discs (IVD) of the axial skeleton. They are conserved in mice and humans, whereby mutation/deficiency of human PAX1/PAX9 has been associated with kyphoscoliosis. By combining cell-type-specific transcriptome and ChIP-sequencing data, we identified the roles of Pax1/Pax9 in cell proliferation, cartilage development and collagen fibrillogenesis, which are vital in early IVD morphogenesis. Pax1 is up-regulated in the absence of Pax9, while Pax9 is unaffected by the loss of Pax1/Pax9 We identified the targets compensated by a single- or double-copy of Pax9 They positively regulate many of the cartilage genes known to be regulated by Sox5/Sox6/Sox9 and are connected to Sox5/Sox6 by a negative feedback loop. Pax1/Pax9 are intertwined with BMP and TGF-B pathways and we propose they initiate expression of chondrogenic genes during early IVD differentiation and subsequently become restricted to the outer annulus by the negative feedback mechanism. Our findings highlight how early IVD development is regulated spatio-temporally and have implications for understanding kyphoscoliosis.
Project description:Purpose of review:Intervertebral discs (IVD) are derived from embryonic notochord and sclerotome. The nucleus pulposus is derived from notochord while other connective tissues of the spine are derived from sclerotome. This manuscript will review the past 5 years of research into IVD development. Recent findings:Over the past several years, advances in understanding the step-wise process that govern development of the nucleus pulposus and the annulus fibrosus have been made. Generation of tissues from induced or embryonic stem cells into nucleus pulposus and paraxial mesoderm derived tissues has been accomplished in vitro using pathways identified in normal development. A balance between BMP and TGF-? signaling as well as transcription factors including Pax1/Pax9, Mkx and Nkx3.2 appear to be very important for cell fate decisions generating tissues of the IVD. Summary:Understanding how the IVD develops will provide the foundation for future repair, regeneration, and tissue engineering strategies for IVD disease.
Project description:Recapitulation of developmental signals represents a promising strategy for treating intervertebral disc degeneration. During development, embryonic notochord-derived cells (NDCs) are the direct progenitors of cells that populate the adult nucleus pulposus (NP) and are an important source of secreted signaling molecules. The objective of this study was to define global gene expression profiles of NDCs at key stages of embryonic disc formation. NDCs were isolated from Shh-cre;ROSA:YFP mice at embryonic day 12.5 and postnatal day 0, representing opposite ends of the notochord to NP transformation. Differences in global mRNA abundance across this developmental window were established using RNA-Seq. Principal component analysis revealed clustering of gene expression at each developmental stage with more than 5000 genes significantly differentially expressed between E12.5 and P0. This study represents the first transcriptome-wide analysis of embryonic NDCs. Results suggest signaling and biosynthesis of NDCs change dramatically as a function of developmental stage. Overall design: For these studies, we used the Shh-cre;ROSA:YFP mouse model, previously established and shown to express YFP in SHH-expressing notochord cells and their progeny at any developmental stage. Animals were euthanized at two developmental stages, embryonic day 12.5 (E12.5) and postnatal day 0 (P0), representing opposite ends of the notochord to NP transformation. Biological replicates (n=4 at both time points) each consisted of pooled embryos or pups (~6) from a single litter.
Project description:The intervertebral disc contributes to motion, weight bearing, and flexibility of the spine, but is susceptible to damage and morphological changes that contribute to pathology with age and injury. Engineering strategies that rely upon synthetic materials or composite implants that do not interface with the biological components of the disc have not met with widespread use or desirable outcomes in the treatment of intervertebral disc pathology. Here we review bioengineering advances to treat disc disorders, using cell-supplemented materials, or acellular, biologically based materials, that provide opportunity for cell-material interactions and remodeling in the treatment of intervertebral disc disorders. While a field still in early development, bioengineering-based strategies employing novel biomaterials are emerging as promising alternatives for clinical treatment of intervertebral disc disorders.
Project description:Background/objective:Intervertebral disc degeneration (IDD) remains to be an intractable clinical challenge. Although IDD is characterised by loss of notochordal cells (NCs) and dysfunction of nucleus pulposus (NP) cells, little is known about the origin, heterogeneity, fate and maintenance of NCs and NP cells, which further stunts the therapeutic development. Thus, effective tools to spatially and temporally trace specific cell lineage and clarify cell functions in intervertebral disc (IVD) development and homoeostasis are urgently required. Methods:In this study, NP specimens were obtained from 20 patients with degenerative disc disease or scoliosis. LepR-Cre mice was crossed with R26R-Tdtomato mice to generate LepR-Cre; R26R-Tdtomato mice, which enabled fate-mapping of NPs from embryo stage to late adult. LMNA G609G/G609G mice was used to determine the effect of premature-aging induced IDD on LepR NPs. X-ray imaging was used to measure lumber disc height of mice. Results:Here, we provide the first evidence that the leptin receptor (LepR) is preferentially expressed in NCs at embryonic stages and notochord-derived cells in the postnatal IVD. By using R26R-Tdtomato fluorescent reporter mice, we systematically analysed the specificity of activity and targeting efficiency of leptin receptor-Cre (LepR-Cre) in IVD tissues from the embryonic stage E15.5 to 6-month-old LepR-Cre; Rosa26-Tdtomato (R26R-Tdtomato) mice. Specifically, LepR-Cre targets a distinct subpopulation of notochord-derived cells closely associated with disc homoeostasis. The percentage of LepR-expressing NP cells markedly decreases in the postnatal mouse IVD and, more importantly, in the human IVD with the progression of IDD. Moreover, both spine instability-induced and premature ageing-induced IDD mouse models display the phenotype of IDD with decreased percentage of LepR-expressing NP cells. These findings uncover a potential role of LepR-expressing notochord-derived cells in disc homoeostasis and open the gate for therapeutically targeting the NP cell subpopulation. Conclusion:In conclusion, our data prove LepR-Cre mice useful for mapping the fate of specific subpopulations of IVD cells and uncovering the underlying mechanisms of IDD. The translational potential of this article:The translation potential of article is that we first identified LepR as a candidate marker of subpopulation of nucleus pulposus (NP) cells and provided LepR as a potential target for the treatment of intervertebral disc degeneration (IDD), which have certain profound significance.