The proteasome subunit psmb1 is essential for craniofacial cartilage maturation and morphogenesis.

Craniofacial dysmorphisms are among the most common birth defects. Proteasome mutations frequently result in craniofacial dysmorphisms, including lower jaw malformations; however, the underlying mechanisms are unknown. Here, we used a zebrafish proteasome subunit ß 1 (psmb1) mutant to define the cellular mechanisms underlying proteasome mutation-induced craniofacial dysmorphisms. psmb1 mutants exhibited a flattened ceratohyal and smaller Meckel's and palatoquadrate cartilages. Ceratohyal flattening was a result of failed chondrocyte convergent extension, accompanied by reduced numbers of chondrocytes in the lower jaw due to defects in chondrocyte differentiation. Morphogenesis of craniofacial muscles and tendons was similarly perturbed. psmb1 mutants lacked the hyohyal muscles, and craniofacial tendons were shortened and disorganized. We additionally identified a critical period for proteasome function in craniofacial development, specifically during chondrocyte and muscle differentiation. psmb1 overexpression in sox10+ cells of mutant embryos rescued both cartilage and tendon phenotypes but induced only a partial rescue of the muscle phenotype, indicating that psmb1 was required in both tissue-autonomous and nonautonomous fashions during craniofacial development. Overall, our work demonstrates that psmb1 is required for craniofacial cartilage, tendon, and muscle differentiation and morphogenesis.

FOXC1 and FOXC2 regulate growth plate chondrocyte maturation towards hypertrophy in the embryonic mouse limb skeleton.

The Forkhead box transcription factors FOXC1 and FOXC2 are expressed in condensing mesenchyme cells at the onset of endochondral ossification. We used the Prx1-cre mouse to ablate Foxc1 and Foxc2 in limb skeletal progenitor cells. Prx1-cre;Foxc1Δ/Δ;Foxc2Δ/Δ limbs were shorter than controls, with worsening phenotypes in distal structures. Cartilage formation and mineralization was severely disrupted in the paws. The radius and tibia were malformed, whereas the fibula and ulna remained unmineralized. Chondrocyte maturation was delayed, with fewer Indian hedgehog-expressing, prehypertrophic chondrocytes forming and a smaller hypertrophic chondrocyte zone. Later, progression out of chondrocyte hypertrophy was slowed, leading to an accumulation of COLX-expressing hypertrophic chondrocytes and formation of a smaller primary ossification center with fewer osteoblast progenitor cells populating this region. Targeting Foxc1 and Foxc2 in hypertrophic chondrocytes with Col10a1-cre also resulted in an expanded hypertrophic chondrocyte zone and smaller primary ossification center. Our findings suggest that FOXC1 and FOXC2 direct chondrocyte maturation towards hypertrophic chondrocyte formation. At later stages, FOXC1 and FOXC2 regulate function in hypertrophic chondrocyte remodeling to allow primary ossification center formation and osteoblast recruitment.

3D atlas of the human fetal chondrocranium in the middle trimester.

The chondrocranium provides the key initial support for the fetal brain, jaws and cranial sensory organs in all vertebrates. The patterns of shaping and growth of the chondrocranium set up species-specific development of the entire craniofacial complex. The 3D development of chondrocranium have been studied primarily in animal model organisms, such as mice or zebrafish. In comparison, very little is known about the full 3D human chondrocranium, except from drawings made by anatomists many decades ago. The knowledge of human-specific aspects of chondrocranial development are essential for understanding congenital craniofacial defects and human evolution. Here advanced microCT scanning was used that includes contrast enhancement to generate the first 3D atlas of the human fetal chondrocranium during the middle trimester (13 to 19 weeks). In addition, since cartilage and bone are both visible with the techniques used, the  endochondral ossification of cranial base was mapped since this region is so critical for brain and jaw growth. The human 3D models are published as a scientific resource for human development.

Hox-A2 protein expression in avian jaws cartilages and muscle primordia development.

OBJECTIVE: To elucidate the branchial origin of the articular and the square (homology of the malleus and the incus of mammals), we used immunohistochemistry to analyse the expression of the Hox-A2 protein during cephalogenesis in chickens. MATERIALS AND METHODS: Immunohistochemistry on paraffin sections of embryos from stage HH16 to HH40. RESULTS: In addition to the columella (equivalent to the mammalian stapes), the joint between the articular and the quadrate bones, and the retro-articular process of the articular (homologous to the short process of the malleus) express Hox-A2, suggesting an intervention of the 2nd arch in their formation. However, we fortuitously observed very intense expression within the early muscle plate of the second arch, which then generalized to all cephalic muscles, and extended to the trunk's myotomes. In the cartilage, the presence of the protein disappeared at stage 35. DISCUSSION AND CONCLUSION: The present results, while confirming the contribution of the second arch to the development of avian equivalents of the mammalian ear ossicles, strongly suggest that the Hox-A2 gene plays a role in muscle development, which remains to be elucidated by more sophisticated techniques.

Temporospatial regulation of intraflagellar transport is required for the endochondral ossification in mice.

Ciliogenic components, such as the family of intraflagellar transport (IFT) proteins, are recognized to play key roles in endochondral ossification, a critical process to form most bones. However, the unique functions and roles of each IFT during endochondral ossification remain unclear. Here, we show that IFT20 is required for endochondral ossification in mice. Utilizing osteo-chondrocyte lineage-specific Cre mice (Prx1-Cre and Col2-Cre), we deleted Ift20 to examine its function. Although chondrocyte-specific Ift20 deletion with Col2-Cre mice did not cause any overt skeletal defects, mesoderm-specific Ift20 deletion using Prx1-Cre (Ift20:Prx1-Cre) mice resulted in shortened limb outgrowth. Primary cilia were absent on chondrocytes of Ift20:Prx1-Cre mice, and ciliary-mediated Hedgehog signaling was attenuated in Ift20:Prx1-Cre mice. Interestingly, loss of Ift20 also increased Fgf18 expression in the perichondrium that sustained Sox9 expression, thus preventing endochondral ossification. Inhibition of enhanced phospho-ERK1/2 activation partially rescued defective chondrogenesis in Ift20 mutant cells, supporting an important role for FGF signaling. Our findings demonstrate that IFT20 is a critical regulator of temporospatial FGF signaling that is required for endochondral ossification.

A comprehensive series of Irx cluster mutants reveals diverse roles in facial cartilage development.

Proper function of the vertebrate skeleton requires the development of distinct articulating embryonic cartilages. Irx transcription factors are arranged in co-regulated clusters that are expressed in the developing skeletons of the face and appendages. IrxB cluster genes are required for the separation of toes in mice and formation of the hyoid joint in zebrafish, yet whether Irx genes have broader roles in skeletal development remains unclear. Here, we perform a comprehensive loss-of-function analysis of all 11 Irx genes in zebrafish. We uncover conserved requirements for IrxB genes in formation of the fish and mouse scapula. In the face, we find a requirement for IrxAb genes and irx7 in formation of anterior neural crest precursors of the jaw, and for IrxBa genes in formation of endodermal pouches and gill cartilages. We also observe extensive joint loss and cartilage fusions in animals with combinatorial losses of Irx clusters, with in vivo imaging revealing that at least some of these fusions arise through inappropriate chondrogenesis. Our analysis reveals diverse roles for Irx genes in the formation and later segmentation of the facial skeleton.

Intrinsic and growth-mediated cell and matrix specialization during murine meniscus tissue assembly.

The incredible mechanical strength and durability of mature fibrous tissues and their extremely limited turnover and regenerative capacity underscores the importance of proper matrix assembly during early postnatal growth. In tissues with composite extracellular matrix (ECM) structures, such as the adult knee meniscus, fibrous (Collagen-I rich), and cartilaginous (Collagen-II, proteoglycan-rich) matrix components are regionally segregated to the outer and inner portions of the tissue, respectively. While this spatial variation in composition is appreciated to be functionally important for resisting complex mechanical loads associated with gait, the establishment of these specialized zones is poorly understood. To address this issue, the following study tracked the growth of the murine meniscus from its embryonic formation through its first month of growth, encompassing the critical time-window during which animals begin to ambulate and weight bear. Using histological analysis, region specific high-throughput qPCR, and Col-1, and Col-2 fluorescent reporter mice, we found that matrix and cellular features defining specific tissue zones were already present at birth, before continuous weight-bearing had occurred. These differences in meniscus zones were further refined with postnatal growth and maturation, resulting in specialization of mature tissue regions. Taken together, this work establishes a detailed timeline of the concurrent spatiotemporal changes that occur at both the cellular and matrix level throughout meniscus maturation. The findings of this study provide a framework for investigating the reciprocal feedback between cells and their evolving microenvironments during assembly of a mechanically robust fibrocartilage tissue, thus providing insight into mechanisms of tissue degeneration and effective regenerative strategies.

Development of the cartilaginous connecting apparatuses in the fetal sphenoid, with a focus on the alar process.

The human fetal sphenoid is reported to have a cartilaginous connecting apparatus known as the alar process (AP), which connects the ala temporalis (AT) (angle of the greater wing of the sphenoid) to the basisphenoid (anlage of the sphenoid body). However, how the AP develops in humans is unclear. In addition, although the AP is a common structure of the mammalian chondrocranium, little is known about whether it is really a fundamental feature in mammals. This study examined the histological sections of 20 human embryos and fetuses from 6 to 14 weeks of development, of 20 mouse embryos from embryonic days 12-18, and of 4 rats embryos form embryonic days 17 and 20. In addition, we reconsidered the definition of the AP by comparing humans and rats with mice. In humans, the AP was continuous with the basisphenoid but was separated from the AT by a thick perichondrium. Then, the AP-AT connection had a key-and-keyhole structure. Unlike a joint, no cavitation developed in this connection. In mice, there was no boundary between the AT and the basisphenoid, indicating the absence of the AP in the mouse chondrocranium. In rats, the AP was, however, separated from the AT by a thick perichondrium. Therefore, the AP can be defined as follows: the AP is temporally separated from the AT by a thick perichondrium or a key-and-keyhole structure during the fetal period. This is the first study that confirms the absence of the alar process in the mice skull, and its presence in other mammals skull should be further investigated.

The Mandibular and Hyoid Arches-From Molecular Patterning to Shaping Bone and Cartilage.

The mandibular and hyoid arches collectively make up the facial skeleton, also known as the viscerocranium. Although all three germ layers come together to assemble the pharyngeal arches, the majority of tissue within viscerocranial skeletal components differentiates from the neural crest. Since nearly one third of all birth defects in humans affect the craniofacial region, it is important to understand how signalling pathways and transcription factors govern the embryogenesis and skeletogenesis of the viscerocranium. This review focuses on mouse and zebrafish models of craniofacial development. We highlight gene regulatory networks directing the patterning and osteochondrogenesis of the mandibular and hyoid arches that are actually conserved among all gnathostomes. The first part of this review describes the anatomy and development of mandibular and hyoid arches in both species. The second part analyses cell signalling and transcription factors that ensure the specificity of individual structures along the anatomical axes. The third part discusses the genes and molecules that control the formation of bone and cartilage within mandibular and hyoid arches and how dysregulation of molecular signalling influences the development of skeletal components of the viscerocranium. In conclusion, we notice that mandibular malformations in humans and mice often co-occur with hyoid malformations and pinpoint the similar molecular machinery controlling the development of mandibular and hyoid arches.

Foxc1 establishes enhancer accessibility for craniofacial cartilage differentiation.

The specification of cartilage requires Sox9, a transcription factor with broad roles for organogenesis outside the skeletal system. How Sox9 and other factors gain access to cartilage-specific cis-regulatory regions during skeletal development was unknown. By analyzing chromatin accessibility during the differentiation of neural crest cells into chondrocytes of the zebrafish head, we find that cartilage-associated chromatin accessibility is dynamically established. Cartilage-associated regions that become accessible after neural crest migration are co-enriched for Sox9 and Fox transcription factor binding motifs. In zebrafish lacking Foxc1 paralogs, we find a global decrease in chromatin accessibility in chondrocytes, consistent with a later loss of dorsal facial cartilages. Zebrafish transgenesis assays confirm that many of these Foxc1-dependent elements function as enhancers with region- and stage-specific activity in facial cartilages. These results show that Foxc1 promotes chondrogenesis in the face by establishing chromatin accessibility at a number of cartilage-associated gene enhancers.

Animals with backbones (or vertebrates) have body shape determined, in part, by their skeletons. These emerge in the embryo in the form of cartilage structures that will then get replaced by bone during development. The neural crest is a group of embryonic cells that can become different tissues. In the head, it forms the cartilage scaffold for some of the facial bones and the base of the skull. During this process, a protein called Sox9 is required for neural crest cells to morph into cartilage. This transcription factor binds to regulatory sequences in the genome to turn cartilage genes on. But Sox9 is also required to form non-cartilage tissues in organs such as the liver, lung, and kidneys. How, then, does Sox9 only turn on the genes required for cartilage formation in the embryonic face? This specificity can be controlled by which regulatory sequences Sox9 can physically access in a cell: controlling which regulatory sequences Sox9 can access determines which genes it can activate, and which type of tissue a cell will become. Xu, Yu et al. wanted to understand exactly how Sox9 switches on the genes that turn neural crest cells into facial cartilage. They studied the genomes of zebrafish embryos, which have a cartilaginous skeleton similar to other vertebrates, and found out which areas were accessible to transcription factors in the neural crest cells that became facial cartilage. Analyzing these regions suggested that sites where Sox9 could bind were often close to binding sites for another protein, called Foxc1. When zebrafish embryos were genetically modified to inactivate Foxc1 proteins, many of the regulatory sequences in cartilage failed to become accessible, and the cartilaginous skeleton did not form properly. These results support a model in which Foxc1 opens up the genomic regions that Sox9 needs to bind for cartilage to form, as opposed to the regions that Sox9 would bind to make different organ cell types. The findings of Xu, Yu et al. uncover the stepwise process by which cartilage cells are made during development. Further research based on these results could allow scientists to develop new ways of replacing cartilage in degenerative conditions such as arthritis.

Zebrafish model for spondylo-megaepiphyseal-metaphyseal dysplasia reveals post-embryonic roles of Nkx3.2 in the skeleton.

The regulated expansion of chondrocytes within growth plates and joints ensures proper skeletal development through adulthood. Mutations in the transcription factor NKX3.2 underlie spondylo-megaepiphyseal-metaphyseal dysplasia (SMMD), which is characterized by skeletal defects including scoliosis, large epiphyses, wide growth plates and supernumerary distal limb joints. Whereas nkx3.2 knockdown zebrafish and mouse Nkx3.2 mutants display embryonic lethal jaw joint fusions and skeletal reductions, respectively, they lack the skeletal overgrowth seen in SMMD patients. Here, we report adult viable nkx3.2 mutant zebrafish displaying cartilage overgrowth in place of a missing jaw joint, as well as severe dysmorphologies of the facial skeleton, skullcap and spine. In contrast, cartilage overgrowth and scoliosis are absent in rare viable nkx3.2 knockdown animals that lack jaw joints, supporting post-embryonic roles for Nkx3.2. Single-cell RNA-sequencing and in vivo validation reveal increased proliferation and upregulation of stress-induced pathways, including prostaglandin synthases, in mutant chondrocytes. By generating a zebrafish model for the skeletal overgrowth defects of SMMD, we reveal post-embryonic roles for Nkx3.2 in dampening proliferation and buffering the stress response in joint-associated chondrocytes.

Development pattern of tracheal cartilage in human embryos.

INTRODUCTION: Congenital tracheal anomalies are associated with high morbidity and mortality. The etiology of congenital tracheal anomalies is not well understood, but often attributed to malformed tracheal cartilage. The development of tracheal cartilage has not been described in detail. In this study, we aimed to investigate the development pattern and timing of normal tracheal cartilage to better understand the etiology of tracheal anomalies. MATERIALS AND METHODS: The development of tracheal cartilage was examined by studying the trachea in histological sections of 14 healthy human embryos from the Carnegie collection. Two specimens for Carnegie Stages 17-23 (42-60 days of embryological development) were studied. RESULTS: At Carnegie Stages 17-19 (42-51 days), a continuous mesenchymal condensation was observed ventral to the tracheal lumen. At Stages 20 and 21 (51-54 days), this pre-tracheal mesenchyme showed sites of increased condensation indicative of future tracheal rings. Furthermore, growth centers were identified both proximally and distally in the trachea. Characteristic horseshoe shaped tracheal rings were apparent at Carnegie Stages 22 and 23 (54-60 days). CONCLUSIONS: In human embryos, tracheal rings arise from growth centers in the ventral mesenchyme at approximately 51-54 days of embryological development. The observation of proximal and distal growth centers suggests a centripetal growth gradient, potentially contributing to occurrence of complete tracheal ring deformity (CTRD). Although this study shows new insights on tracheal cartilage development, the exact origin of congenital tracheal defects has yet to be elucidated.

The RNA helicase DDX3 induces neural crest by promoting AKT activity.

Mutations in the RNA helicase DDX3 have emerged as a frequent cause of intellectual disability in humans. Because many individuals carrying DDX3 mutations have additional defects in craniofacial structures and other tissues containing neural crest (NC)-derived cells, we hypothesized that DDX3 is also important for NC development. Using Xenopus tropicalis as a model, we show that DDX3 is required for normal NC induction and craniofacial morphogenesis by regulating AKT kinase activity. Depletion of DDX3 decreases AKT activity and AKT-dependent inhibitory phosphorylation of GSK3ß, leading to reduced levels of ß-catenin and Snai1: two GSK3ß substrates that are crucial for NC induction. DDX3 function in regulating these downstream signaling events during NC induction is likely mediated by RAC1, a small GTPase whose translation depends on the RNA helicase activity of DDX3. These results suggest an evolutionarily conserved role of DDX3 in NC development by promoting AKT activity, and provide a potential mechanism for the NC-related birth defects displayed by individuals harboring mutations in DDX3 and its downstream effectors in this signaling cascade.

An in situ hybridization study of the Syndecan family in the developing condylar cartilage of fetal mouse mandible.

Mandibular condylar cartilage is a representative secondary cartilage, differing from primary cartilage in various ways. Syndecan is a cell-surface heparan sulfate proteoglycan and speculated to be involved in chondrogenesis and osteogenesis. This study aimed to investigate the expression patterns of the syndecan family in the developing mouse mandibular condylar cartilage. At embryonic day (E)13.0 and E14.0, syndecan-1 and -2 mRNAs were expressed in the mesenchymal cell condensation of the condylar anlage. When condylar cartilage was formed at E15.0, syndecan-1 mRNA was expressed in the embryonic zone, wherein the mesenchymal cell condensation is located. Syndecan-2 mRNA was mainly expressed in the perichondrium. At E16.0, syndecan-1 was expressed from fibrous to flattened cell zones and syndecans-2 was expressed in the lower hypertrophic cell zone. Syndecan-3 mRNA was expressed in the condylar anlage at E13.0 and E13.5 but was not expressed in the condylar cartilage at E15.0. It was later expressed in the lower hypertrophic cell zone at E16.0. Syndecan-4 mRNA was expressed in the condylar anlage at E14.0 and the condylar cartilage at E15.0 and E16.0. These findings indicated that syndecans-1 and -2 could be involved in the formation from mesenchymal cell condensation to condylar cartilage. The different expression patterns of the syndecan family in the condylar and limb bud cartilage suggest the functional heterogeneity of chondrocytes in the primary and secondary cartilage.

Using an aquatic model, Xenopus laevis, to uncover the role of chromodomain 1 in craniofacial disorders.

The chromodomain family member chromodomain 1 (CHD1) has been shown to have numerous critical molecular functions including transcriptional regulation, splicing, and DNA repair. Complete loss of function of this gene is not compatible with life. On the other hand, missense and copy number variants of CHD1 can result in intellectual disabilities and craniofacial malformations in human patients including cleft palate and Pilarowski-Bjornsson Syndrome. We have used the aquatic developmental model organism Xenopus laevis, to determine a specific role for Chd1 in such cranioafcial disorders. Protein and gene knockdown techniques in Xenopus, including antisense oligos and mosaic Crispr/Cas9-mediated mutagenesis, recapitulated the craniofacial defects observed in humans. Further analysis indicated that embryos deficient in Chd1 had defects in cranial neural crest development and jaw cartilage morphology. Additionally, flow cytometry and immunohistochemistry revealed that decreased Chd1 resulted in increased in apoptosis in the developing head. Together, these experiments demonstrate that Chd1 is critical for fundamental processes and cell survival in craniofacial development. We also presented evidence that Chd1 is regulated by retinoic acid signaling during craniofacial development. Expression levels of chd1 mRNA, specifically in the head, were increased by RAR agonist exposure and decreased upon antagonist treatment. Subphenotypic levels of an RAR antagonist and Chd1 morpholinos synergized to result in orofacial defects. Further, RAR DNA binding sequences (RAREs) were detected in chd1 regulatory regions by bioinformatic analysis. In summary, by combining human genetics and experiments in an aquatic model we now have a better understanding of the role of CHD1 in craniofacial disorders.

A protocol for differential staining of cartilages and ossified bones in fetal and adult mouse skeletons using alcian blue and alizarin red S.

The technique for clearing and staining whole specimens consists of many steps. This study discusses the alcian blue/alizarin red S staining method and aims to provide a useful reference and review for users who intend to do this staining. To specifically address the influences of tissue removal on staining results, the mouse fetuses at embryonic stage E18.5 and adult mice at 12 weeks of age were used in this study. The fetuses were divided into three groups: Group 1 skin, muscle, and viscera removed, Group 2 skin and muscle removed, and Group 3 viscera removed. For successful skeletal staining, it was concluded that (1) skin removal from fetuses was necessary for alcian blue staining but unnecessary for alizarin red S staining, (2) removal of muscle surrounding thorax and neck of fetuses could improve transparency effects, (3) retaining fetal viscera would not significantly affect transparency but might avoid the tissue damage, and (4) complete skin, muscle, and viscera removal were essential for good staining of adult mice. The representative images and detailed staining procedures might be good for researchers presently using alcian blue and alizarin red S staining to differentiate cartilages and ossified bones.

Aggrecan, the Primary Weight-Bearing Cartilage Proteoglycan, Has Context-Dependent, Cell-Directive Properties in Embryonic Development and Neurogenesis: Aggrecan Glycan Side Chain Modifications Convey Interactive Biodiversity.

This review examines aggrecan's roles in developmental embryonic tissues, in tissues undergoing morphogenetic transition and in mature weight-bearing tissues. Aggrecan is a remarkably versatile and capable proteoglycan (PG) with diverse tissue context-dependent functional attributes beyond its established role as a weight-bearing PG. The aggrecan core protein provides a template which can be variably decorated with a number of glycosaminoglycan (GAG) side chains including keratan sulphate (KS), human natural killer trisaccharide (HNK-1) and chondroitin sulphate (CS). These convey unique tissue-specific functional properties in water imbibition, space-filling, matrix stabilisation or embryonic cellular regulation. Aggrecan also interacts with morphogens and growth factors directing tissue morphogenesis, remodelling and metaplasia. HNK-1 aggrecan glycoforms direct neural crest cell migration in embryonic development and is neuroprotective in perineuronal nets in the brain. The ability of the aggrecan core protein to assemble CS and KS chains at high density equips cartilage aggrecan with its well-known water-imbibing and weight-bearing properties. The importance of specific arrangements of GAG chains on aggrecan in all its forms is also a primary morphogenetic functional determinant providing aggrecan with unique tissue context dependent regulatory properties. The versatility displayed by aggrecan in biodiverse contexts is a function of its GAG side chains.

Pdgfra regulates multipotent cell differentiation towards chondrocytes via inhibiting Wnt9a/beta-catenin pathway during chondrocranial cartilage development.

The mammalian skull is composed of the calvarial bones and cartilages. Malformation of craniofacial cartilage has been identified in multiple human syndromes. However, the mechanisms of their development remain largely unknown. In the present study, we identified Pdgfra as a novel player of chondrocranial cartilage development. Our data show that Pdgfra is required for normal chondrocranial cartilage development. Using tissue-specific genetic tools, we demonstrated that Pdgfra is essential for chondrocyte progenitors formation, but not in mature chondrocytes. Further analysis revealed that Pdgfra regulates chondrocytes progenitors development at two stages: in embryonic mesenchymal stem cells (eMSCs), Pdgfra directs their differentiation toward chondrocyte progenitors; in chondrocytes progenitors, Pdgfra activation promotes cell proliferation. We also found that excessive Pdgfra activity causes ectopic cartilage formation. Our data show that Pdgfra directs eMSCs differentiation via inhibiting Wnt9a transcription and its downstream signaling, and activating Wnt signaling rescues ectopic cartilage phenotype caused by excessive Pdgfra activity. In summary, our study dissected the role of Pdgfra signaling in chondrocranial cartilage formation, and illustrated the underlying mechanisms at multiple stages.

The involvement of cytokine-like 1 (Cytl1) in chondrogenesis and cartilage metabolism.

The Cytokine-like 1 (Cytl1) is first identified in bone marrow cells and preferentially expressed in cartilaginous tissue, and showed chondrogenic effects in mesenchymal cells, not essential for cartilage or bone development as in Cytl1 knock-out mice but associated with cartilage inflammatory and destruction. Here, we show the involvement of Cytl1 in chondrogenesis. Using specified chondrogenic embryonic skeleton and adult cartilage, the Cytl1 gene expression was investigated with associated chondrogenic factors by quantitative RT-PCR. The effect of Cytl1 protein (rCytl1) on cultured chondrocytes to regulate expression of key factors and phenotypic markers was studied. The results revealed that Cytl1 was highly expressed in chondrogenic process in embryos and adult cartilage. The rCytl1 increased the expression of Sox9 and Col2α1 with stabilized Col1α1 in cultured chondrocytes (redifferentiation). The Cytl1 was expressed and involved at all stages of cartilage development. Furthermore, Cytl1 expression shared similar patterns as other chondrogenic factors, implying interactions with other factors in chondrogenic process. Cytl1 is involved in cartilage development and matrix homeostasis, which defines the dedifferentiation phenotype of chondrocytes, essential to forming of functional cartilage in both physiologic remodeling and pathologic 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.