Genes In Skeletal Development Research Articles

DLX Genes in the Development and Maintenance of the Vertebrate Skeleton: Implications for Human Pathologies.

Skeletal shape and mechanical properties define, to a large extent, vertebrate morphology and physical capacities. During development, skeletal morphogenesis results from dynamic communications between chondrocytes, osteoblasts, osteoclasts, and other cellular components of the skeleton. Later in life, skeletal integrity depends on the regulatory cascades that assure the equilibrium between bone formation and resorption. Finally, during aging, skeletal catabolism prevails over anabolism resulting in progressive skeletal degradation. These cellular processes depend on the transcriptional cascades that control cell division and differentiation in each cell type. Most Distal-less (Dlx) homeobox transcription factors are directly involved in determining the proliferation and differentiation of chondrocytes and osteoblasts and, indirectly, of osteoclasts. While the involvement of Dlx genes in the regulation of skeletal formation has been well-analyzed thanks to several mutant mouse models, the role of these genes in the maintenance of bone integrity has been only partially studied. The importance of Dlx genes for adult bone tissues is evidenced by their central role in the regulatory pathways involving Osx/Sp7 and Runx2, the two major master genes of osteogenesis. Dlx genes appear to be involved in several bone pathologies including, for example, osteoporosis. Indeed, at least five large-scale GWAS studies which aimed to detect loci associated with human bone mineral density (BMD) have identified a known DLX5/6 regulatory region within chromosome 7q21.3 in proximity of SEM1/FLJ42280/DSS1 coding sequences, suggesting that DLX5/6 expression is critical in determining healthy BMD. This review aims to summarize the major findings concerning the involvement of Dlx genes in skeletal development and homeostasis and their involvement in skeletal aging and pathology.

Lipolysis of bone marrow adipocytes is required to fuel bone and the marrow niche during energy deficits.

To investigate roles for bone marrow adipocyte (BMAd) lipolysis in bone homeostasis, we created a BMAd-specific Cre mouse model in which we knocked out adipose triglyceride lipase (ATGL, Pnpla2 gene). BMAd-Pnpla2-/- mice have impaired BMAd lipolysis, and increased size and number of BMAds at baseline. Although energy from BMAd lipid stores is largely dispensable when mice are fed ad libitum, BMAd lipolysis is necessary to maintain myelopoiesis and bone mass under caloric restriction. BMAd-specific Pnpla2 deficiency compounds the effects of caloric restriction on loss of trabecular bone in male mice, likely due to impaired osteoblast expression of collagen genes and reduced osteoid synthesis. RNA sequencing analysis of bone marrow adipose tissue reveals that caloric restriction induces dramatic elevations in extracellular matrix organization and skeletal development genes, and energy from BMAd is required for these adaptations. BMAd-derived energy supply is also required for bone regeneration upon injury, and maintenance of bone mass with cold exposure.

Probiotics Enhance Bone Growth and Rescue BMP Inhibition: New Transgenic Zebrafish Lines to Study Bone Health.

Zebrafish larvae, especially gene-specific mutants and transgenic lines, are increasingly used to study vertebrate skeletal development and human pathologies such as osteoporosis, osteopetrosis and osteoarthritis. Probiotics have been recognized in recent years as a prophylactic treatment for various bone health issues in humans. Here, we present two new zebrafish transgenic lines containing the coding sequences for fluorescent proteins inserted into the endogenous genes for sp7 and col10a1a with larvae displaying fluorescence in developing osteoblasts and the bone extracellular matrix (mineralized or non-mineralized), respectively. Furthermore, we use these transgenic lines to show that exposure to two different probiotics, Bacillus subtilis and Lactococcus lactis, leads to an increase in osteoblast formation and bone matrix growth and mineralization. Gene expression analysis revealed the effect of the probiotics, particularly Bacillus subtilis, in modulating several skeletal development genes, such as runx2, sp7, spp1 and col10a1a, further supporting their ability to improve bone health. Bacillus subtilis was the more potent probiotic able to significantly reverse the inhibition of bone matrix formation when larvae were exposed to a BMP inhibitor (LDN212854).

Genetic variant of WIF1 gene is functionally associated with developmental dysplasia of the hip in Han Chinese population

Developmental dysplasia of the hip (DDH) is a common skeletal disorder. Studies have demonstrated a significant role of WIF1 gene in skeletal development. The present study was conducted to reveal the association between DDH and gene WIF1. A two-stage case-control candidate gene association study was conducted in total 1573 samples (586 DDH patients and 987 healthy controls) in this study. Polymorphism rs3782499 was genotyped in all samples. Difference of WIF1 expression in hip joint tissue was compared between the patients and the controls. WIF1 expression was compared among different genotypes in DDH patients. The SNP rs3782499 was found significantly associated with DDH in the two-stage study with 585 patients and 987 controls. There was a significant difference in allele frequency (p = 4.37 * 10−5) and genotype distribution in a recessive model (AG + GG vs. AA). DDH patients were found to have significantly higher WIF1 expression than controls. Moreover, Patients with rs3782499 genotype AA have a significantly increased expression of WIF1 than those with GG. To conclude, polymorphism rs3782499 of WIF1 gene is a functional variant regulating the expression of WIF1 in DDH in Chinese Han population, which might be a potential biomarker for the early diagnosis of DDH.

Dlx2 overexpression enhanced accumulation of type II collagen and aggrecan by inhibiting MMP13 expression in mice chondrocytes

Genetic studies revealed a crucial role of Distal-homebox (Dlx) genes in skeletal development, and our previous study demonstrated overexpressing Dlx2 in neural crest cells led to abnormal cartilage structure, including ectopic cartilage in the maxillary region and nasal bone in mice. The aim of this study was to investigate how Dlx2 overexpression affects chondrogenesis in mouse chondroblast cell line TMC23 and the underlying mechanism. We first demonstrated that Dlx2 expression was upregulated during chondrogenesis in TMC23 cells. Moreover, forced overexpression of Dlx2 in TMC23 cells led to increased accumulation of aggrecan and type II collagen, markers of early chondrocyte differentiation, but had little effect on mRNA and protein levels of Aggrecan and Col2α1, type II collagen gene. Importantly, Dlx2 overexpression decreased mRNA and protein levels of MMP13, a major collagenase degrading aggrecan and type II collagen during late stages of chondrogenesis. Luciferase-reporter and Chromatin-immunoprecipitation analysis demonstrated that MMP13 promoter contained two Dlx2-response elements, and Dlx2 inhibited MMP13 expression by directly binding to these two elements. Based on these observations, we propose that forced overexpression of Dlx2 enhances early chondrocyte differentiation by increasing accumulation of type II collagen and aggrecan, but interferes later stages of chondrocyte differentiation through inhibiting MMP13 expression.

Deletion of the Fanconi Anemia C Gene in Mice Leads to Skeletal Anomalies and Defective Bone Mineralization and Microarchitecture.

Fanconi anemia (FA) is a rare genetic disorder associated with a progressive decline in hematopoietic stem cells leading to bone marrow failure. FA is also characterized by a variety of developmental defects including short stature and skeletal malformations. More than half of children affected with FA have radial-ray abnormalities, and many patients have early onset osteopenia/osteoporosis. Although many Fanconi anemia genes have been identified and a molecular pathway defined, the underlying mechanism leading to bone defects remains elusive. To understand the role of FA genes in skeletal development and bone microarchitecture, we evaluated bone physiology during embryogenesis and in adult FancA- and FancC-deficient mice. We found that both FancA-/- and FancC-/- embryos have abnormal skeletal development shown by skeletal malformations, growth delay, and reduced bone mineralization. FancC-/- adult mice present altered bone morphology and microarchitecture with a significant decrease in cortical bone mineral density in a sex-specific manner. Mechanical testing revealed that male but not female FancC-/- mice show reduced bone strength compared with their wild-type littermates. Ex vivo cultures showed that FancA-/- and FancC-/- bone marrow-derived mesenchymal stem cells (BM MSC) have impaired differentiation capabilities together with altered gene expression profiles. Our results suggest that defective bone physiology in FA occurs in utero and possibly results from altered BM MSC function. These results provide valuable insights into the mechanism involved in FA skeletal defects. © 2018 American Society for Bone and Mineral Research.

Filamin-interacting proteins, Cfm1 and Cfm2, are essential for the formation of cartilaginous skeletal elements

Mutations of Filamin genes, which encode actin-binding proteins, cause a wide range of congenital developmental malformations in humans, mainly skeletal abnormalities. However, the molecular mechanisms underlying Filamin functions in skeletal system formation remain elusive. In our screen to identify skeletal development molecules, we found that Cfm (Fam101) genes, Cfm1 (Fam101b) and Cfm2 (Fam101a), are predominantly co-expressed in developing cartilage and intervertebral discs (IVDs). To investigate the functional role of Cfm genes in skeletal development, we generated single knockout mice for Cfm1 and Cfm2, as well as Cfm1/Cfm2 double-knockout (Cfm DKO) mice, by targeted gene disruption. Mice with loss of a single Cfm gene displayed no overt phenotype, whereas Cfm DKO mice showed skeletal malformations including spinal curvatures, vertebral fusions and impairment of bone growth, showing that the phenotypes of Cfm DKO mice resemble those of Filamin B (Flnb)-deficient mice. The number of cartilaginous cells in IVDs is remarkably reduced, and chondrocytes are moderately reduced in Cfm DKO mice. We observed increased apoptosis and decreased proliferation in Cfm DKO cartilaginous cells. In addition to direct interaction between Cfm and Filamin proteins in developing chondrocytes, we showed that Cfm is required for the interaction between Flnb and Smad3, which was reported to regulate Runx2 expression. Furthermore, we found that Cfm DKO primary chondrocytes showed decreased cellular size and fewer actin bundles compared with those of wild-type chondrocytes. These results suggest that Cfms are essential partner molecules of Flnb in regulating differentiation and proliferation of chondryocytes and actin dynamics.

Roles of natriuretic peptide receptor 2 and a novel gene in skeletal development, ovulation, and spermatogenesis

Roles of natriuretic peptide receptor 2 and a novel gene in skeletal development, ovulation, and spermatogenesis

Evidence for Positive Selection on the Osteogenin (BMP3) Gene in Human Populations

BackgroundHuman skeletal system has evolved rapidly since the dispersal of modern humans from Africa, potentially driven by selection and adaptation. Osteogenin (BMP3) plays an important role in skeletal development and bone osteogenesis as an antagonist of the osteogenic bone morphogenetic proteins, and negatively regulates bone mineral density.Methodology/Principal FindingsHere, we resequenced the BMP3 gene from individuals in four geographically separated modern human populations. Features supportive of positive selection in the BMP3 gene were found including the presence of an excess of nonsynonymous mutations in modern humans, and a significantly lower genetic diversity that deviates from neutrality. The prevalent haplotypes of the first exon region in Europeans demonstrated features of long-range haplotype homogeneity. In contrast with findings in European, the derived allele SNP Arg192Gln shows higher extended haplotype homozygosity in East Asian. The worldwide allele frequency distribution of SNP shows not only a high-derived allele frequency in Asians, but also in Americans, which is suggestive of functional adaptation.Conclusions/SignificanceIn conclusion, we provide evidence for recent positive selection operating upon a crucial gene in skeletal development, which may provide new insight into the evolution of the skeletal system and bone development.

MEF2C Transcription Factor Controls Chondrocyte Hypertrophy and Bone Development

Chondrocyte hypertrophy is essential for endochondral bone development. Unexpectedly, we discovered that MEF2C, a transcription factor that regulates muscle and cardiovascular development, controls bone development by activating the gene program for chondrocyte hypertrophy. Genetic deletion of Mef2c or expression of a dominant-negative MEF2C mutant in endochondral cartilage impairs hypertrophy, cartilage angiogenesis, ossification, and longitudinal bone growth in mice. Conversely, a superactivating form of MEF2C causes precocious chondrocyte hypertrophy, ossification of growth plates, and dwarfism. Endochondral bone formation is exquisitely sensitive to the balance between MEF2C and the corepressor histone deacetylase 4 (HDAC4), such that bone deficiency of Mef2c mutant mice can be rescued by an Hdac4 mutation, and ectopic ossification in Hdac4 null mice can be diminished by a heterozygous Mef2c mutation. These findings reveal unexpected commonalities in the mechanisms governing muscle, cardiovascular, and bone development with respect to their regulation by MEF2 and class II HDACs.

Functional Study of Gene using Inducible Cre System

Gene manipulation by disrupting important genes using homologous recombination in mammals has provided important insights into their function and development with regard to disease. However, many questions related to the genetic pathways that regulate cellular differentiation and function remain to be clarified. In particular, analysis of genetic pathways that control embryonic skeletal development is often hindered by the disruption of critical genes that function in early embryogenesis, thereby leading to embryonic or perinatal death and thus preventing study of the role of these genes in skeletal development and physiology postnatally. To overcome this problem gene-targeting methods, using site- and time-specific recombination based methods with the Cre/loxP system, have been used to delete particular genes in specific tissues and stages during development. Thus, the generation and characterization of transgenic mice expressing Cre recombinase, under the control of a tissue-specific and stage-specific promoter, has become prerequisite for study of the physiology and homeostasis of specific tissues during a specific time frame. In this report, we introduce the principles and methods of site-specific and time-specific recombination using the Cre/loxP and inducible Cre system, and discuss the potential applications for applying this system to the study of the development and physiology of the skeletal system. (J Kor Endocrinol Soc 21:364~369, 2006)

Reactivation of Hox gene expression during bone regeneration

Previous studies have explored the link between bone regeneration and skeletogenesis. Although a great deal is known regarding tissue and cell based events, especially those involving ossification and chondrogenesis, much remains unknown about the molecular similarity of repair and development. Since the functional significance of Homeobox (Hox) genes in embryonic skeletogenesis has been well documented through knockout and deficiency studies, we chose to investigate whether members of this family are reactivated during fracture repair. Specifically, we examined the temporal and spatial expression of Msx-1, Msx-2, rHox, Hoxa-2 and Hoxd-9, because of their involvement in limb development. Utilizing quantitative reverse transcriptase RT-PCR (qPCR), mRNA levels from all five genes were shown to be upregulated during fracture repair at all times tested (post-fracture day 3–21), as compared to intact bone. Further, using in situ hybridization and immunohistochemistry, spatial expression of these genes was localized to osteoblasts, chondrocytes and periosteal osteoprogenitor cells found within the fracture callus, the foremost cells responsible for the reparative phase of the healing process. Given the contribution of Hox genes in skeletal development, our results suggest that these genes are involved in either the patterning or formation of the fracture callus, further supporting the notion that bone regeneration recapitulates skeletal development.

Roles of FGF receptors in mammalian development and congenital diseases.

Four fibroblast growth factor receptors (FGFR1-4) constitute a family of transmembrane tyrosine kinases that serve as high affinity receptors for at least 22 FGF ligands. Gene targeting in mice has yielded valuable insights into the functions of this important gene family in multiple biological processes. These include mesoderm induction and patterning; cell growth, migration, and differentiation; organ formation and maintenance; neuronal differentiation and survival; wound healing; and malignant transformation. Furthermore, discoveries that mutations in three of the four receptors result in more than a dozen human congenital diseases highlight the importance of these genes in skeletal development. In this review, we will discuss recent progress on the roles of FGF receptors in mammalian development and congenital diseases, with an emphasis on signal transduction pathways.

Role of Runx Genes in Chondrocyte Differentiation

Runx2/Cbfa1 plays a central role in skeletal development as demonstrated by the absence of osteoblasts/bone in mice with inactivated Runx2/Cbfa1 alleles. To further investigate the role of Runx2 in cartilage differentiation and to assess the potential of Runx2 to induce bone formation, we cloned chicken Runx2 and overexpressed it in chick embryos using a retroviral system. Infected chick wings showed multiple phenotypes consisting of (1) joint fusions, (2) expansion of carpal elements, and (3) shortening of skeletal elements. In contrast, bone formation was not affected. To investigate the function of Runx2/Cbfa1 during cartilage development, we have generated transgenic mice that express a dominant negative form of Runx2 in cartilage. The selective inactivation of Runx2 in chondrocytes results in a severe shortening of the limbs due to a disturbance in chondrocyte differentiation, vascular invasion, osteoclast differentiation, and periosteal bone formation. Analysis of the growth plates in transgenic mice and in chick limbs shows that Runx2 is a positive regulator of chondrocyte differentiation and vascular invasion. The results further indicate that Runx2 promotes chondrogenesis either by maintaining or by initiating early chondrocyte differentiation. Furthermore, Runx2 is essential but not sufficient to induce osteoblast differentiation. To analyze the role of runx genes in skeletal development, we performed in situ hybridization with Runx2- and Runx3-specific probes. Both genes were coexpressed in cartilaginous condensations, indicating a cooperative role in the regulation of early chondrocyte differentiation and thus explaining the expansion/maintenance of cartilage in the carpus and joints of infected chick limbs.

Function of homeobox genes in skeletal development.

Function of homeobox genes in skeletal development.

The roles of collagen genes in skeletal development and morphogenesis.

Molecular genetic analyses of osteochondrodysplasias in mice and humans have recently led to the identification of mutations in genes encoding structural proteins growth factor receptors and sulphate transporters. Further analyses of such inherited disorders, using rapid techniques for gene mapping, positional cloning and mutation detection, will undoubtedly uncover other genes that are important for skeletal development. Together with studies of transgenic mice, in which specific genes that are expressed in the skeleton are mutated, these analyses will provide insight into genes that are essential for skeletal morphogenesis.

The Sry-related gene Sox9 is expressed during chondrogenesis in mouse embryos.

Mutations in the human SRY-related gene, SOX9, located on chromosome 17, have recently been associated with the sex reversal and skeletal dysmorphology syndrome, campomelic dysplasia. In order to clarify the role of this gene in skeletal development, we have studied the expression of mouse Sox9 during embryogenesis. Sox9 is expressed predominantly in mesenchymal condensations throughout the embryo before and during the deposition of cartilage, consistent with a primary role in skeletal formation. Interspecific backcross mapping has localized mouse Sox9 to distal chromosome 11. The expression pattern and chromosomal location of Sox9 suggest that it may be the gene defective in the mouse skeletal mutant Tail-short, a potential animal model for campomelic dysplasia.