In vivo evidence for GDP-fucose transport in the absence of transporter SLC35C1 and putative transporter SLC35C2.

Slc35c1 encodes an antiporter that transports GDP-fucose into the Golgi and returns GMP to the cytoplasm. The closely related gene Slc35c2 encodes a putative GDP-fucose transporter and promotes Notch fucosylation and Notch signaling in cultured cells. Here, we show that HEK293T cells lacking SLC35C1 transferred reduced amounts of O-fucose to secreted epidermal growth factor-like repeats from NOTCH1 or secreted thrombospondin type I repeats from thrombospondin 1. However, cells lacking SLC35C2 did not exhibit reduced fucosylation of these epidermal growth factor-like repeats or thrombospondin type I repeats. To investigate SLC35C2 functions in vivo, WW6 embryonic stem cells were targeted for Slc35c2. Slc35c2[-/-] mice were viable and fertile and exhibited no evidence of defective Notch signaling during skeletal or T cell development. By contrast, mice with inactivated Slc35c1 exhibited perinatal lethality and marked skeletal defects in late embryogenesis, typical of defective Notch signaling. Compound Slc35c1[-/-]Slc35c2[-/-] mutants were indistinguishable in skeletal phenotype from Slc35c1[-/-] embryos and neonates. Double mutants did not exhibit the exacerbated skeletal defects predicted if SLC35C2 was functionally important for Notch signaling in vivo. In addition, NOTCH1 immunoprecipitated from Slc35c1[-/-]Slc35c2[-/-] neonatal lung carried fucose detected by binding of Aleuria aurantia lectin. Given that the absence of both SLC35C1, a known GDP-fucose transporter, and SLC35C2, a putative GDP-fucose transporter, did not lead to afucosylated NOTCH1 nor to the severe Notch signaling defects and embryonic lethality expected if all GDP-fucose transport were abrogated, at least one more mechanism of GDP-fucose transport into the secretory pathway must exist in mammals.

SCUBE3 loss-of-function causes a recognizable recessive developmental disorder due to defective bone morphogenetic protein signaling.

Signal peptide-CUB-EGF domain-containing protein 3 (SCUBE3) is a member of a small family of multifunctional cell surface-anchored glycoproteins functioning as co-receptors for a variety of growth factors. Here we report that bi-allelic inactivating variants in SCUBE3 have pleiotropic consequences on development and cause a previously unrecognized syndromic disorder. Eighteen affected individuals from nine unrelated families showed a consistent phenotype characterized by reduced growth, skeletal features, distinctive craniofacial appearance, and dental anomalies. In vitro functional validation studies demonstrated a variable impact of disease-causing variants on transcript processing, protein secretion and function, and their dysregulating effect on bone morphogenetic protein (BMP) signaling. We show that SCUBE3 acts as a BMP2/BMP4 co-receptor, recruits the BMP receptor complexes into raft microdomains, and positively modulates signaling possibly by augmenting the specific interactions between BMPs and BMP type I receptors. Scube3-/- mice showed craniofacial and dental defects, reduced body size, and defective endochondral bone growth due to impaired BMP-mediated chondrogenesis and osteogenesis, recapitulating the human disorder. Our findings identify a human disease caused by defective function of a member of the SCUBE family, and link SCUBE3 to processes controlling growth, morphogenesis, and bone and teeth development through modulation of BMP signaling.

Age assessment in unaccompanied minors: assessing uniformity of protocols across Europe.

Age assessment of migrants is crucial, particularly for unaccompanied foreign minors, a population facing legal, social, and humanitarian challenges. Despite existing guidelines, there is no unified protocol in Europe for age assessment.The Forensic Anthropology Society of Europe (FASE) conducted a comprehensive questionnaire to understand age estimation practices in Europe. The questionnaire had sections focusing on the professional background of respondents, annual assessment numbers, requesting parties and reasons, types of examinations conducted (e.g., physical, radiological), followed protocols, age estimation methods, and questions on how age estimates are reported.The questionnaire's findings reveal extensive engagement of the forensic community in age assessment in the living, emphasizing multidisciplinary approaches. However, there seems to be an incomplete appreciation of AGFAD guidelines. Commonalities exist in examination methodologies and imaging tests. However, discrepancies emerged among respondents regarding sexual maturity assessment and reporting assessment results. Given the increasing importance of age assessment, especially for migrant child protection, the study stresses the need for a unified protocol across European countries. This can only be achieved if EU Member States wholeheartedly embrace the fundamental principles outlined in EU Directives and conduct medical age assessments aligned with recognized standards such as the AGFAD guidelines.

[Role of Fibroblast Growth Factor 7 in Craniomaxillofacial Development]. / æˆçº¤ç»´ç»†èƒžç”Ÿé•¿å› å­7åœ¨é¢ é¢Œé¢å‘è‚²ä¸­çš„ä½œç”¨.

Craniomaxillofacial development involves a series of highly ordered temporal-spatial cellular differentiation processes in which a variety of cell signaling factors, such as fibroblast growth factors, play important regulatory roles. As a classic fibroblast growth factor, fibroblast growth factor 7 (FGF7) serves a wide range of regulatory functions. Previous studies have demonstrated that FGF7 regulates the proliferation and migration of epithelial cells, protects them, and promotes their repair. Furthermore, recent findings indicate that epithelial cells are not the only ones subjected to the broad and powerful regulatory capacity of FGF7. It has potential effects on skeletal system development as well. In addition, FGF7 plays an important role in the development of craniomaxillofacial organs, such as the palate, the eyes, and the teeth. Nonetheless, the role of FGF7 in oral craniomaxillofacial development needs to be further elucidated. In this paper, we summarized the published research on the role of FGF7 in oral craniomaxillofacial development to demonstrate the overall understanding of FGF7 and its potential functions in oral craniomaxillofacial development.

The visible skeleton 2.0: phenotyping of cartilage and bone in fixed vertebrate embryos and foetuses based on X-ray microCT.

For decades, clearing and staining with Alcian Blue and Alizarin Red has been the gold standard to image vertebrate skeletal development. Here, we present an alternate approach to visualise bone and cartilage based on X-ray microCT imaging, which allows the collection of genuine 3D data of the entire developing skeleton at micron resolution. Our novel protocol is based on ethanol fixation and staining with Ruthenium Red, and efficiently contrasts cartilage matrix, as demonstrated in whole E16.5 mouse foetuses and limbs of E14 chicken embryos. Bone mineral is well preserved during staining, thus the entire embryonic skeleton can be imaged at high contrast. Differences in X-ray attenuation of ruthenium and calcium enable the spectral separation of cartilage matrix and bone by dual energy microCT (microDECT). Clearing of specimens is not required. The protocol is simple and reproducible. We demonstrate that cartilage contrast in E16.5 mouse foetuses is adequate for fast visual phenotyping. Morphometric skeletal parameters are easily extracted. We consider the presented workflow to be a powerful and versatile extension to the toolkit currently available for qualitative and quantitative phenotyping of vertebrate skeletal development.

Embryonic and skeletal development of the albino African clawed frog (Xenopus laevis).

The normal stages of embryonic development for wild-type Xenopus laevis were established by Nieuwkoop and Faber in 1956, a milestone in the history of understanding embryonic development. However, this work lacked photographic images and staining for skeleton structures from the corresponding stages. Here, we provide high-quality images of embryonic morphology and skeleton development to facilitate studies on amphibian development. On the basis of the classical work, we selected the albino mutant of X. laevis as the observation material to restudy embryonic development in this species. The lower level of pigmentation makes it easier to interpret histochemical experiments. At 23°C, albino embryos develop at the same rate as wild-type embryos, which can be divided into 66 stages as they develop into adults in about 58 days. We described the complete embryonic development system for X. laevis, supplemented with pictures of limb and skeleton development that are missing from previous studies, and summarized the characteristics and laws of limb and skeleton development. Our study should aid research into the development of X. laevis and the evolution of amphibians.

Building a Co-ordinated Musculoskeletal System: The Plasticity of the Developing Skeleton in Response to Muscle Contractions.

The skeletal musculature and the cartilage, bone and other connective tissues of the skeleton are intimately co-ordinated. The shape, size and structure of each bone in the body is sculpted through dynamic physical stimuli generated by muscle contraction, from early development, with onset of the first embryo movements, and through repair and remodelling in later life. The importance of muscle movement during development is shown by congenital abnormalities where infants that experience reduced movement in the uterus present a sequence of skeletal issues including temporary brittle bones and joint dysplasia. A variety of animal models, utilising different immobilisation scenarios, have demonstrated the precise timing and events that are dependent on mechanical stimulation from movement. This chapter lays out the evidence for skeletal system dependence on muscle movement, gleaned largely from mouse and chick immobilised embryos, showing the many aspects of skeletal development affected. Effects are seen in joint development, ossification, the size and shape of skeletal rudiments and tendons, including compromised mechanical function. The enormous plasticity of the skeletal system in response to muscle contraction is a key factor in building a responsive, functional system. Insights from this work have implications for our understanding of morphological evolution, particularly the challenging concept of emergence of new structures. It is also providing insight for the potential of physical therapy for infants suffering the effects of reduced uterine movement and is enhancing our understanding of the cellular and molecular mechanisms involved in skeletal tissue differentiation, with potential for informing regenerative therapies.

Runt-related Transcription Factors and Gene Regulatory Mechanisms in Skeletal Development and Diseases.

PURPOSE OF REVIEW: Runt-related transcription factors (RUNX) play critical roles in skeletal development, metabolism, and diseases. In mammals, three RUNX members, namely RUNX1, RUNX2, and RUNX3, play distinct and redundant roles, although RUNX2 is a dominant factor in skeletal development and several skeletal diseases. This review is to provide an overview of the current understanding of RUNX-mediated transcriptional regulation in different skeletal cell types. RECENT FINDINGS: Advances in chromatin immunoprecipitation and next-generation sequencing (ChIP-seq) have revealed genome-wide RUNX-mediated gene regulatory mechanisms, including their association with cis-regulatory elements and putative target genes. Further studies with genome-wide analysis and biochemical assays have shed light on RUNX-mediated pioneering action and involvements of RUNX2 in lipid-lipid phase separation. Emerging multi-layered mechanisms of RUNX-mediated gene regulations help us better understanding of skeletal development and diseases, which also provides clues to think how genome-wide studies can help develop therapeutic strategies for skeletal diseases.

Role of the Peripheral Nervous System in Skeletal Development and Regeneration: Controversies and Clinical Implications.

PURPOSE OF REVIEW: This review examines the diverse functional relationships that exist between the peripheral nervous system (PNS) and bone, including key advances over the past century that inform our efforts to translate these discoveries for skeletal repair. RECENT FINDINGS: The innervation of the bone during development, homeostasis, and regeneration is highly patterned. Consistent with this, there have been nearly 100 studies over the past century that have used denervation approaches to isolate the effects of the different branches of the PNS on the bone. Overall, a common theme of balance emerges whereby an orchestration of both local and systemic neural functions must align to promote optimal skeletal repair while limiting negative consequences such as pain. An improved understanding of the functional bidirectional pathways linking the PNS and bone has important implications for skeletal development and regeneration. Clinical advances over the next century will necessitate a rigorous identification of the mechanisms underlying these effects that is cautious not to oversimplify the in vivo condition in diverse states of health and disease.

Intergenerational toxic effects of parental exposure to [Cn mim]NO3 (n = 2,4,6) on nervous and skeletal development in zebrafish offspring.

Ionic liquids (ILs) are thought to have negative effects on human health. Researchers have explored the effects of ILs on zebrafish development during the early stages, but the intergenerational toxicity of ILs on zebrafish development has rarely been reported. Herein, parental zebrafish were exposed to different concentrations (0, 12.5, 25, and 50 mg/L) of [Cn mim]NO3 (n = 2, 4, 6) for 1 week. Subsequently, the F1 offspring were cultured in clean water for 96 h. [Cn mim]NO3 (n = 2, 4, 6) exposure inhibited spermatogenesis and oogenesis in F0 adults, even causing obvious lacunae in the testis and atretic follicle oocytes in ovary. After parental exposure to [Cn mim]NO3 (n = 2, 4, 6), the body length and locomotor behavior were measured in F1 larvae at 96 hours post-fertilization (hpf). The results showed that the higher the concentration of [Cn mim]NO3 (n = 2, 4, 6), the shorter the body length and swimming distance, and the longer the immobility time. Besides, a longer alkyl chain length of [Cn mim]NO3 had a more negative effect on body length and locomotor behavior. RNA-seq analysis revealed several downregulated differentially expressed genes (DEGs)-grin1b, prss1, gria3a, and gria4a-enriched in neurodevelopment-related pathways, particularly the pathway for neuroactive ligand-receptor interaction. Moreover, several upregulated DEGs, namely col1a1a, col1a1b, and acta2, were mainly associated with skeletal development. Expression of DEGs was tested by RT-qPCR, and the outcomes were consistent with those obtained from RNA-Seq. We provide evidence showing the effects of parental exposure to ILs on the regulation of nervous and skeletal development in F1 offspring, demonstrating intergenerational effects.

Emerging RUNX2-Mediated Gene Regulatory Mechanisms Consisting of Multi-Layered Regulatory Networks in Skeletal Development.

Skeletal development is tightly coordinated by chondrocytes and osteoblasts, which are derived from skeletal progenitors, and distinct cell-type gene regulatory programs underlie the specification and differentiation of cells. Runt-related transcription factor 2 (Runx2) is essential to chondrocyte hypertrophy and osteoblast differentiation. Genetic studies have revealed the biological functions of Runx2 and its involvement in skeletal genetic diseases. Meanwhile, molecular biology has provided a framework for our understanding of RUNX2-mediated transactivation at a limited number of cis-regulatory elements. Furthermore, studies using next-generation sequencing (NGS) have provided information on RUNX2-mediated gene regulation at the genome level and novel insights into the multiple layers of gene regulatory mechanisms, including the modes of action of RUNX2, chromatin accessibility, the concept of pioneer factors and phase separation, and three-dimensional chromatin organization. In this review, I summarize the emerging RUNX2-mediated regulatory mechanism from a multi-layer perspective and discuss future perspectives for applications in the treatment of skeletal diseases.

Effects of joint loading on the development of capital femoral epiphysis morphology.

INTRODUCTION: The deleterious influence of increased mechanical forces on capital femoral epiphysis development is well established; however, the growth of the physis in the absence of such forces remains unclear. The hips of non-ambulatory cerebral palsy (CP) patients provide a weight-restricted (partial weightbearing) model which can elucidate the influence of decreased mechanical forces on the development of physis morphology, including features related to development of slipped capital femoral epiphysis (SCFE). Here we used 3D image analysis to compare the physis morphology of children with non-ambulatory CP, as a model for abnormal hip loading, with age-matched native hips. MATERIALS AND METHODS: CT images of 98 non-ambulatory CP hips (8-15 years) and 80 age-matched native control hips were used to measure height, width, and length of the tubercle, depth, width, and length of the metaphyseal fossa, and cupping height across different epiphyseal regions. The impact of age on morphology was assessed using Pearson correlations. Mixed linear model was used to compare the quantified morphological features between partial weightbearing hips and full weightbearing controls. RESULTS: In partial weightbearing hips, tubercle height and length along with fossa depth and length significantly decreased with age, while peripheral cupping height increased with age (r > 0.2, P < 0.04). Compared to normally loaded (full weightbearing) hips and across all age groups, partially weightbearing hips' epiphyseal tubercle height and length were smaller (P < .05), metaphyseal fossa depth was larger (P < .01), and posterior, inferior, and anterior peripheral cupping heights were smaller (P < .01). CONCLUSIONS: Smaller epiphyseal tubercle and peripheral cupping with greater metaphyseal fossa size in partial weightbearing hips suggests that the growing capital femoral epiphysis requires mechanical stimulus to adequately develop epiphyseal stabilizers. Deposit low prevalence and relevance of SCFE in CP, these findings highlight both the role of normal joint loading in proper physis development and how chronic abnormal loading may contribute to various pathomorphological changes of the proximal femur (i.e., capital femoral epiphysis).

Deletion of ATAD3A inhibits osteogenesis by impairing mitochondria structure and function in pre-osteoblast.

BACKGROUND: ATPase family AAA-domain containing protein 3A (ATAD3A) is a nuclear encoded mitochondrial membrane protein that spans inner and outer membrane, and it has been shown to regulate mitochondrial dynamics and cholesterol metabolism. Since the mitochondrial functions have been implicated for osteogenic differentiation, a role of ATAD3A in skeletal development has been investigated. RESULTS: Mesenchyme-specific ATAD3 knockout mice displayed severe defects in skeletal development. Additionally, osteoblast-specific deletion of ATAD3 in mice caused significant reduction in bone mass, while cartilage-specific ATAD3 knockout mice did not show any significant phenotypes. Consistent with these in vivo findings, ATAD3A knockdown impaired mitochondrial morphology and function in calvarial pre-osteoblast cultures, which, in turn, suppressed osteogenic differentiation in vitro. CONCLUSIONS: The current findings suggest that ATAD3A plays a crucial role in mitochondria homeostasis, which is required for osteogenic differentiation during skeletal development.

Growth orientations, rather than heterogeneous growth rates, dominate jaw joint morphogenesis in the larval zebrafish.

In early limb embryogenesis, synovial joints acquire specific shapes which determine joint motion and function. The process by which the opposing cartilaginous joint surfaces are moulded into reciprocal and interlocking shapes, called joint morphogenesis, is one of the least understood aspects of joint formation and the cell-level dynamics underlying it are yet to be unravelled. In this research, we quantified key cellular dynamics involved in growth and morphogenesis of the zebrafish jaw joint and synthesised them in a predictive computational simulation of joint development. Cells in larval zebrafish jaw joints labelled with cartilage markers were tracked over a 48-h time window using confocal imaging. Changes in distance and angle between adjacent cell centroids resulting from cell rearrangement, volume expansion and extracellular matrix (ECM) deposition were measured and used to calculate the rate and direction of local tissue deformations. We observed spatially and temporally heterogeneous growth patterns with marked anisotropy over the developmental period assessed. There was notably elevated growth at the level of the retroarticular process of the Meckel's cartilage, a feature known to undergo pronounced shape changes during zebrafish development. Analysis of cell dynamics indicated a dominant role for cell volume expansion in growth, with minor influences from ECM volume increases and cell intercalation. Cell proliferation in the joint was minimal over the timeframe of interest. Synthesising the dynamic cell data into a finite element model of jaw joint development resulted in accurate shape predictions. Our biofidelic computational simulation demonstrated that zebrafish jaw joint growth can be reasonably approximated based on cell positional information over time, where cell positional information derives mainly from cell orientation and cell volume expansion. By modifying the input parameters of the simulation, we were able to assess the relative contributions of heterogeneous growth rates and of growth orientation. The use of uniform rather than heterogeneous growth rates only minorly impacted the shape predictions, whereas isotropic growth fields resulted in altered shape predictions. The simulation results suggest that growth anisotropy is the dominant influence on joint growth and morphogenesis. This study addresses the gap of the cellular processes underlying joint morphogenesis, with implications for understanding the aetiology of developmental joint disorders such as developmental dysplasia of the hip and arthrogryposis.

Patellar shape is associated with femoral trochlear morphology in individuals with mature skeletal development.

BACKGROUND: As several studies have detected correlations between patellar and femoral trochlear development, this raises the question of whether patellar shape is associated with trochlear developmental outcomes. METHODS: Patellar shape and femoral trochlear morphology were retrospectively analyzed in 183 subjects, of whom 61 each were classified as having Wiberg type I, II, and III patellae (groups A, B, and C, respectively). The sulcus angle (SA), lateral trochlea inclination angle (LTA), medial trochlear inclination angle (MTA), lateral facet length (LFL), medial facet length (MFL), lateral trochlear height (LTH), medial trochlear height (MTH), trochlea sulcus height (TH), and lateral-medial trochlear facet distance (TD) were analyzed as a means of evaluating trochlear morphology. Trochlear depth, trochlear condyle asymmetry, and trochlear facet asymmetry were additionally calculated, and differences in trochlear morphology and correlations between trochlear morphology and patellar shape were evaluated. RESULTS: The femoral trochlear parameters of patients in group A differed significantly from those of patients in groups B and C. No significant differences between groups B and C were evident. Patellar shape was positively correlated with LTA, MTA, MFL, trochlear condyle asymmetry, and trochlear facet asymmetry, and was negatively correlated with SA. CONCLUSIONS: These data indicated that patellar shape and trochlear morphology are related to one another,which suggest normalized patella morphology surgery and trochlear surgery are better choices for patients with patella instability. TRIAL REGISTRATION: Retrospectively registered.

Fibroblast Growth Factors and Cellular Communication Network Factors: Intimate Interplay by the Founding Members in Cartilage.

Fibroblast growth factors (FGFs) constitute a large family of signaling molecules that act in an autocrine/paracrine, endocrine, or intracrine manner, whereas the cellular communication network factors (CCN) family is composed of six members that manipulate extracellular signaling networks. FGFs and CCNs are structurally and functionally distinct, except for the common characteristics as matricellular proteins. Both play significant roles in the development of a variety of tissues and organs, including the skeletal system. In vertebrates, most of the skeletal parts are formed and grow through a process designated endochondral ossification, in which chondrocytes play the central role. The growth plate cartilage is the place where endochondral ossification occurs, and articular cartilage is left to support the locomotive function of joints. Several FGFs, including FGF-2, one of the founding members of this family, and all of the CCNs represented by CCN2, which is required for proper skeletal development, can be found therein. Research over a decade has revealed direct binding of CCN2 to FGFs and FGF receptors (FGFRs), which occasionally affect the biological outcome via FGF signaling. Moreover, a recent study uncovered an integrated regulation of FGF and CCN genes by FGF signaling. In this review, after a brief introduction of these two families, molecular and genetic interactions between CCN and FGF family members in cartilage, and their biological effects, are summarized. The molecular interplay represents the mutual involvement of the other in their molecular functions, leading to collaboration between CCN2 and FGFs during skeletal development.

Making and shaping endochondral and intramembranous bones.

Skeletal elements have a diverse range of shapes and sizes specialized to their various roles including protecting internal organs, locomotion, feeding, hearing, and vocalization. The precise positioning, size, and shape of skeletal elements is therefore critical for their function. During embryonic development, bone forms by endochondral or intramembranous ossification and can arise from the paraxial and lateral plate mesoderm or neural crest. This review describes inductive mechanisms to position and pattern bones within the developing embryo, compares and contrasts the intrinsic vs extrinsic mechanisms of endochondral and intramembranous skeletal development, and details known cellular processes that precisely determine skeletal shape and size. Key cellular mechanisms are employed at distinct stages of ossification, many of which occur in response to mechanical cues (eg, joint formation) or preempting future load-bearing requirements. Rapid shape changes occur during cellular condensation and template establishment. Specialized cellular behaviors, such as chondrocyte hypertrophy in endochondral bone and secondary cartilage on intramembranous bones, also dramatically change template shape. Once ossification is complete, bone shape undergoes functional adaptation through (re)modeling. We also highlight how alterations in these cellular processes contribute to evolutionary change and how differences in the embryonic origin of bones can influence postnatal bone repair.

How faithfully does intramembranous bone regeneration recapitulate embryonic skeletal development?

Postnatal intramembranous bone regeneration plays an important role during a wide variety of musculoskeletal regeneration processes such as fracture healing, joint replacement and dental implant surgery, distraction osteogenesis, stress fracture healing, and repair of skeletal defects caused by trauma or resection of tumors. The molecular basis of intramembranous bone regeneration has been interrogated using rodent models of most of these conditions. These studies reveal that signaling pathways such as Wnt, TGFß/BMP, FGF, VEGF, and Notch are invoked, reminiscent of embryonic development of membranous bone. Discoveries of several skeletal stem cell/progenitor populations using mouse genetic models also reveal the potential sources of postnatal intramembranous bone regeneration. The purpose of this review is to compare the underlying molecular signals and progenitor cells that characterize embryonic development of membranous bone and postnatal intramembranous bone regeneration.

The tissues and regulatory pattern of limb chondrogenesis.

Initial limb chondrogenesis offers the first differentiated tissues that resemble the mature skeletal anatomy. It is a developmental progression of three tissues. The limb begins with undifferentiated mesenchyme-1, some of which differentiates into condensations-2, and this tissue then transforms into cartilage-3. Each tissue is identified by physical characteristics of cell density, shape, and extracellular matrix composition. Tissue specific regimes of gene regulation underlie the diagnostic physical and chemical properties of these three tissues. These three tissue based regimes co-exist amid a background of other gene regulatory regimes within the same tissues and time-frame of limb development. The bio-molecular indicators of gene regulation reveal six identifiable patterns. Three of these patterns describe the unique bio-molecular indicators of each of the three tissues. A fourth pattern shares bio-molecular indicators between condensation and cartilage. Finally, a fifth pattern is composed of bio-molecular indicators that are found in undifferentiated mesenchyme prior to any condensation differentiation, then these bio-molecular indicators are upregulated in condensations and downregulated in undifferentiated mesenchyme. The undifferentiated mesenchyme that remains in between the condensations and cartilage, the interdigit, contains a unique set of bio-molecular indicators that exhibit dynamic behaviour during chondrogenesis and therefore argue for its own inclusion as a tissue in its own right and for more study into this process of differentiation.

The MAPK Erk5 is necessary for proper skeletogenesis involving a Smurf-Smad-Sox9 molecular axis.

Erk5 belongs to the mitogen-activated protein kinase (MAPK) family. Following its phosphorylation by Mek5, Erk5 modulates several signaling pathways in a number of cell types. In this study, we demonstrated that Erk5 inactivation in mesenchymal cells causes abnormalities in skeletal development by inducing Sox9, an important transcription factor of skeletogenesis. We further demonstrate that Erk5 directly phosphorylates and activates Smurf2 (a ubiquitin E3 ligase) at Thr249, which promotes the proteasomal degradation of Smad proteins and phosphorylates Smad1 at Ser206 in the linker region known to trigger its proteasomal degradation by Smurf1. Smads transcriptionally activated the expression of Sox9 in mesenchymal cells. Accordingly, removal of one Sox9 allele in mesenchymal cells from Erk5-deficient mice rescued some abnormalities of skeletogenesis. These findings highlight the importance of the Mek5-Erk5-Smurf-Smad-Sox9 axis in mammalian skeletogenesis.