Differentiation Of Sclerotome Research Articles

Antagonistic regulation of homeologous uncx.L and uncx.S genes orchestrates myotome and sclerotome differentiation in the evolutionarily divergent vertebral column of Xenopus laevis.

In anurans, the vertebral column diverges widely from that of other tetrapods; yet the molecular mechanisms underlying its morphogenesis remain largely unexplored. In this study, we investigate the role of the homeologous uncx.L and uncx.S genes in the vertebral column morphogenesis of the allotetraploid frog Xenopus laevis. We initiated our study by cloning the uncx orthologous genes in the anuran Xenopus and determining their spatial expression patterns using in situ hybridization. Additionally, we employed gain-of-function and loss-of-function approaches through dexamethasone-inducible uncx constructs and antisense morpholino oligonucleotides, respectively. Comparative analysis of the messenger RNA sequences of homeologous uncx genes revealed that the uncx.L variant lacks the eh1-like repressor domain. Our spatial expression analysis indicated that in the presomitic mesoderm and somites, the transcripts of uncx.L and uncx.S are located in overlapping domains. Alterations in the function of uncx genes significantly impact the development and differentiation of the sclerotome and myotome, resulting in axial skeleton malformations. Our findings suggest a scenario where the homeologous genes uncx.L and uncx.S exhibit antagonistic functions during somitogenesis. Specifically, uncx.S appears to be crucial for sclerotome development and differentiation, while uncx.L primarily influences myotome development. Postallotetraploidization, the uncx.L gene in X. laevis evolved to lose its eh1-like repressor domain, transforming into a "native dominant negative" variant that potentially competes with uncx.S for the same target genes. Finally, the histological analysis revealed that uncx.S expression is necessary for the correct formation of pedicles and neural arch of the vertebrae, and uncx.L is required for trunk muscle development.

Canonical and noncanonical TGF-\u03b2 signaling regulate fibrous tissue differentiation in the axial skeleton

Previously, we showed that embryonic deletion of TGF-β type 2 receptor in mouse sclerotome resulted in defects in fibrous connective tissues in the spine. Here we investigated how TGF-β regulates expression of fibrous markers: Scleraxis, Fibromodulin and Adamtsl2. We showed that TGF-β stimulated expression of Scleraxis mRNA by 2 h and Fibromodulin and Adamtsl2 mRNAs by 8 h of treatment. Regulation of Scleraxis by TGF-β did not require new protein synthesis; however, protein synthesis was required for expression of Fibromodulin and Adamtsl2 indicating the necessity of an intermediate. We subsequently showed Scleraxis was a potential intermediate for TGF-β-regulated expression of Fibromodulin and Adamtsl2. The canonical effector Smad3 was not necessary for TGF-β-mediated regulation of Scleraxis. Smad3 was necessary for regulation of Fibromodulin and Adamtsl2, but not sufficient to super-induce expression with TGF-β treatment. Next, the role of several noncanonical TGF-β pathways were tested. We found that ERK1/2 was activated by TGF-β and required to regulate expression of Scleraxis, Fibromodulin, and Adamtsl2. Based on these results, we propose a model in which TGF-β regulates Scleraxis via ERK1/2 and then Scleraxis and Smad3 cooperate to regulate Fibromodulin and Adamtsl2. These results define a novel signaling mechanism for TGFβ-mediated fibrous differentiation in sclerotome.

Antagonism of BMP signaling is insufficient to induce fibrous differentiation in primary sclerotome

Sclerotome is the embryonic progenitor of the axial skeleton. It was previously shown that Tgfbr2 is required in sclerotome for differentiation of fibrous skeletal tissues including the annulus fibrosus of the intervertebral disc. Alternatively, BMP signaling is required to form the vertebral body through chondrogenesis. In addition, TGFβ added to sclerotome cultures induces expression of markers for fibrous tissue differentiation but not cartilage or bone. The mechanism of how TGFβ signaling regulates this lineage decision in sclerotome is not known and could be due to the production of instructive or inhibitory signals or a combination of the two. Here we show that TGFβ antagonizes BMP/ Smad1/5 signaling in primary sclerotome likely through regulation of Noggin, an extracellular BMP antagonist, to prevent chondrogenesis. We then tested whether inhibition of BMP signaling, and inhibition of chondrogenesis, is sufficient to push cells toward the fibrous cell fate. While Noggin inhibited BMP/ Smad1/5 signaling and the formation of chondrogenic nodules in sclerotome cultures; Noggin and inhibition of BMP signaling through Gremlin or DMH2 were insufficient to induce fibrous tissue differentiation. The results suggest inhibition of BMP signaling is not sufficient to stimulate fibrous tissue differentiation and additional signals are likely required. We propose that TGFβ has a dual role in regulating sclerotome fate. First, it inhibits BMP signaling potentially through Noggin to prevent chondrogenesis and, second, it provides an unknown instructive signal to promote fibrous tissue differentiation in sclerotome. The results have implications for the design of stem cell-based therapies for skeletal diseases.

Paraxis is required for somite morphogenesis and differentiation in Xenopus laevis.

In most vertebrates, the segmentation of the paraxial mesoderm involves the formation of metameric units called somites through a mesenchymal-epithelial transition. However, this process is different in Xenopus laevis because it does not form an epithelial somite. Xenopus somitogenesis is characterized by a complex cells rearrangement that requires the coordinated regulation of cell shape, adhesion, and motility. The molecular mechanisms that control these cell behaviors underlying somite formation are little known. Although the Paraxis has been implicated in the epithelialization of somite in chick and mouse, its role in Xenopus somite morphogenesis has not been determined. Using a morpholino and hormone-inducible construction approaches, we showed that both gain and loss of function of paraxis affect somite elongation, rotation and alignment, causing a severe disorganization of somitic tissue. We further found that depletion or overexpression of paraxis in the somite led to the downregulation or upregulation, respectively, of cell adhesion expression markers. Finally, we demonstrated that paraxis is necessary for the proper expression of myotomal and sclerotomal differentiation markers. Our results demonstrate that paraxis regulates the cell rearrangements that take place during the somitogenesis of Xenopus by regulating cell adhesion. Furthermore, paraxis is also required for somite differentiation.

Adverse effects of high glucose levels on somite and limb development in avian embryos

Gestational diabetes has an adverse impact on fetal musculoskeletal development, but the mechanism involved is still not completely understood. In this study, we investigated the effects of high glucose on the developing somites and their derivate using the chick embryo as a model. We demonstrated that under high glucose, the number of generated somites was reduced and their morphology altered in 2-day old chick embryos. In addition, high glucose repressed the development of the limb buds in 5.5-day old chick embryos. We also demonstrated that high glucose abridged the development of the sclerotome and the cartilage in the developing limb bud. The sonic hedgehog (Shh) gene has been reported to play a crucial role in the development and differentiation of sclerotome. Hence, we examined how Shh expression in the sclerotome was affected under high glucose. We found that high glucose treatment significantly inhibited Shh expression. The high glucose also impaired myotome formation at trunk level – as revealed by immunofluorescent staining with MF20 antibodies. In the neural tube, we established that Wnt3a expression was also significantly repressed. In summary, our study demonstrates that high glucose concentrations impair somite and limb bud development in chick embryos, and suggests that Shh and Wnt genes may play a role in the underlying mechanism.

Characterization of zebrafish Pax1b and Pax9 in fin bud development.

Both Pax1 and Pax9 belong to the important paired box gene family (PAX), which mainly participates in animal development and sclerotome differentiation. To date, the precise molecular mechanism and related signaling pathway of Pax1 remain unclear. In our study, microinjection of morpholino- (MO-) modified antisense oligonucleotides against pax1b induced pectoral fin bud defects. Furthermore, we demonstrate that the phenotypes caused by the knockdown of Pax1b in zebrafish could not be phenocopied by pax9 MO and could not be rescued by either Pax1a or Pax9 overexpression. We further find that Pax1b affects the expression of col2a1, Uncx4.1, Noggin3, and aggrecan, confirming the role of Pax1b in chondrocyte differentiation and bone maturation. Moreover, we identify an interaction between PAX1 and FOXO1 and find that the interaction was enhanced under hypoxia stress. Together, this evidence for cell death caused by pax1b knockdown provides new insight into the role of the Pax protein family in cell fate determination and tissue specification.

Notch pathway regulation of chondrocyte differentiation and proliferation during appendicular and axial skeleton development

The role of Notch signaling in cartilage differentiation and maturation in vivo was examined. Conditional Notch pathway gain and loss of function was achieved using a Cre/loxP approach to manipulate Notch signaling in cartilage precursors and chondrocytes of the developing mouse embryo. Conditional overexpression of activated Notch intracellular domain (NICD) in the chondrocyte lineage results in skeletal malformations with decreased cartilage precursor proliferation and inhibited hypertrophic chondrocyte differentiation. Likewise, expression of NICD in cartilage precursors inhibits sclerotome differentiation, resulting in severe axial skeleton abnormalities. Furthermore, conditional loss of Notch signaling via RBP-J gene deletion in the chondrocyte lineage results in increased chondrocyte proliferation and skeletal malformations consistent with the observed increase in hypertrophic chondrocytes. In addition, the Notch pathway inhibits expression of Sox9 and its target genes required for normal chondrogenic cell proliferation and differentiation. Together, our results demonstrate that appropriate Notch pathway signaling is essential for proper chondrocyte progenitor proliferation and for the normal progression of hypertrophic chondrocyte differentiation into bone in the developing appendicular and axial skeletal elements.

A gradient of Shh establishes mutually repressing somitic cell fates induced by Nkx3.2 and Pax3

Wnt and Sonic Hedgehog (Shh) signals are known to pattern the somite into dermomyotomal, myotomal and sclerotomal cell fates. By employing explants of presomitic mesoderm cultured with constant levels of Wnt3a conditioned medium and increasing levels of Shh, we found that differing levels of Shh signaling elicit differing responses from somitic cells: the lowest level of Shh signaling allows dermomyotomal gene expression, intermediate levels induce loss of dermomyotomal markers and activation of myogenic differentiation, and higher levels induce loss of myotomal markers and activation of sclerotomal gene expression. In addition, we have found that in the presence of high levels of Wnt signaling, instead of inducing sclerotomal markers, Shh signals act to maintain the expression of dermomyotomal and myotomal markers. One of the sclerotomal genes induced by high levels of Shh signaling is Nkx3.2. Forced expression of Nkx3.2 blocks somitic expression of the dermomyotomal marker Pax3 both in vitro and in vivo. Conversely, forced expression of Pax3 in somites can block Shh-mediated induction of sclerotomal gene expression and chondrocyte differentiation in vitro. Thus we propose that varying levels of Shh signaling act in a morphogen-like manner to elicit differing responses from somitic cells, and that Pax3 and Nkx3.2 set up mutually repressing cell fates that promote either dermomyotome/myotome or sclerotome differentiation, respectively.

Meox homeodomain proteins are required for Bapx1 expression in the sclerotome and activate its transcription by direct binding to its promoter.

The axial skeleton of vertebrates derives from the sclerotomal compartment of the somites. Genetic analysis has demonstrated that the transcription factors Pax1, Pax9, Meox1, Meox2, and Bapx1 are all required for sclerotomal differentiation. Their hierarchical relationship is, however, poorly understood. Because Bapx1 expression in the somites starts slightly later than that of the Meox genes, we asked whether Bapx1 is one of their downstream targets. Our analysis of Meox1; Meox2 mutant mice supports this hypothesis, as Bapx1 expression in the sclerotome is lost in the absence of both Meox proteins. Using transient-transfection assays, we show that Meox1 activates the Bapx1 promoter in a dose-dependent manner and that this activity is enhanced in the presence of Pax1 and/or Pax9. Furthermore, by electrophoretic mobility shift and chromatin immunoprecipitation experiments, we demonstrate that Meox1 can bind the Bapx1 promoter. The palindromic sequence TAATTA, present in the Bapx1 promoter, binds the Meox1 protein in vitro and is necessary for Meox1-induced transactivation of the Bapx1 promoter. Our data demonstrate that the Meox genes are required for Bapx1 expression in the sclerotome and suggest that the mechanism by which the Meox proteins exert this function is through direct activation of the Bapx1 gene.

Epaxial-adaxial-hypaxial regionalisation of the vertebrate somite: evidence for a somitic organiser and a mirror-image duplication.

During vertebrate neural tube formation, the initially lateral borders between the neural and epidermal ectoderm fuse to form the definitive dorsal region of the embryo, while the initially dorsally located notochord-floor plate complex is being internalised. Along the definitive dorso-ventral body axis, one can distinguish an epaxial (dorsal to the notochord) and a hypaxial (ventral to the notochord) body region. The mesodermal somites on both sides of the notochord and neural tube give rise to the trunk skeleton and skeletal muscle. Muscle forms from the somite-derived dermomyotomes and myotomes that elongate dorsally and ventrally. Based on gene expression patterns and comparative embryology, it is proposed here that the epaxial (dermo)myotome region in amniote embryos is subdivided into a dorsalmost and a centrally intercalated subregion. The intercalated subregion abuts to the hypaxial (dermo)myotome region that elongates ventrally via the hypaxial somitic bud. The dorsalmost subregion elongates towards the dorsal neural tube and is proposed to derive from an epaxial somitic bud. The dorsalmost and hypaxial somite derivatives share specific gene expression patterns which are distinct from those of the intercalated somite derivatives. The intercalated somite derivatives develop adaxially, i.e. at the level of the notochord-floor plate complex. Thus, the dorsalmost and intercalated (dermo)myotome subregions may be influenced preferentially by signals from the dorsal neural tube and from the notochord-floor plate complex, respectively. These (dermo)myotome subregions are sharply delimited from each other by molecular boundary markers, including Engrailed and Wnts. It thus appears that the molecular network that polarises borders in Drosophila and vertebrate embryogenesis is redeployed during subregionalisation of the (dermo)myotome. It is proposed here that cells within the amniote (dermo)myotome establish polarised borders with organising capacity, and that the epaxial somitic bud represents a mirror-image duplication of the hypaxial somitic bud along such a border. The resulting epaxial-intercalated/adaxial-hypaxial regionalisation of somite derivatives is conserved in vertebrates although the differentiation of sclerotome and myotome starts heterochronically in embryos of different vertebrate groups.

Maintenance of somite borders in mice requires the Delta homologue Dll1

During vertebrate embryonic development, the paraxial mesoderm is subdivided into metameric subunits called somites. The arrangement and cranio-caudal polarity of the somites governs the metamerism of all somite-derived tissues and spinal ganglia. Little is known about the molecular mechanisms underlying somite formation, segment polarity, maintenance of segment borders, and the interdependency of these processes. The mouse Delta homologue Dll1, a member of the DSL gene family, is expressed in the presomitic mesoderm and posterior halves of somites. Here we report that, in Dll1-deficient mouse embryos, a primary metameric pattern is established in mesoderm, and cytodifferentiation is apparently normal, but the segments have no cranio-caudal polarity, and no epithelial somites form. Caudal sclerotome halves do not condense, and the pattern of spinal ganglia and nerves is perturbed, indicating loss of segment polarity. Myoblasts span segment borders, demonstrating that these borders are not maintained. These results show that Dll1 is involved in compartmentalization of somites, that dermomyotome and sclerotome differentiation are independent of formation of epithelia and subdivision of somites in cranial and caudal halves, and that compartmentalization is essential for the maintenance of segment borders in paraxial mesoderm-derived structures.

Chicken Pax-1 gene: structure and expression during embryonic somite development

Recent mouse genetic studies have implicated Pax-1, a paired-box-containing gene, in sclerotomal differentiation and vertebral body formation. To investigate Pax-1 function in somitic sclerotomal differentiation in the chick embryo, we have cloned the chicken Pax-1 gene, and its full length cDNA, and characterized its temporal and spatial expression pattern during somite development. Sequence analysis shows that chicken Pax-1 is highly homologous to murine and human Pax-1 genes with respect to the putative DNA-binding paired-box domain and the octapeptide domain. Northern analysis using probes derived from the paired-box domain and a unique non-paired box sequence of chicken Pax-1 detected 2-kb mRNA transcript. The expression profiles of Pax-1 were examined by in situ hybridization and Northern analysis. The first detectable expression of Pax-1 is seen in the most caudal epithelial somite. As the somite matures, Pax-1 expression takes on a medial distribution, thus corresponding to but preceding the emergence of the sclerotome. In the more mature, rostral somites (stage V and older), Pax-1 expression is found to be progressively localized first to the ventral-medial regions, and then to the caudal-ventral-medial quadrant of the mature somite. This pattern strongly supports the notion that Pax-1 expression is involved in somitogenesis and sclerotomal differentiation, and that it is subsequently a characteristic of the caudal half of the sclerotome, the presumptive precursor of vertebral cartilage. Northern analysis substantiated this expression profile and further revealed that the level of somitic Pax-1 expression increases as a function of embryonic development. Finally, we subjected chicken embryos to controlled heat shock treatment to perturb somite formation and segmentation. The pattern of Pax-1 expression in the anomalous somitic structures generated by controlled heat shock further supports a functional role for Pax-1 in somite development.

Pax genes and sclerotome development

Differentiation of somites leads to the development of dermomyotome and sclerotome with the latter forming after an epithelial-mesenchymal transition of the ventral part of the somite. Inductive effects of the notochord are required for differentiation of the sclerotome and for proper formation of the axial skeleton, the inductive effect of the notochord being mediated bysonic hedgehog(SHH), the mammalian homologue of theDrosophilasegment polarity genehedgehog. Shh acts by antagonizing the activity of the cAMP dependent protein kinase A (Pka).Pax1andPax9are paired box containing genes expressed in the developing sclerotome that may be involved in mediating the inductive effects of the notochord. Loss ofPax1function in mice leads to severe abnormalities in sclerotome differentiation and vertebral column formation.

The Biochemically and Immunologically Distinct CSPG of Notochord Is a Product of the Aggrecan Gene

Using the monoclonal antibody S103L, which reacts specifically with an epitope in the chondroitin sulfate-rich domain of the chick cartilage chondroitin sulfate proteoglycan (CSPG) core protein, we have identified the predominant CSPG expressed by notochord. This large notochord CSPG is first detected immunohistochemically as early as stage 16, long before chondrogenesis occurs, and is expressed continuously during the time of active neural crest migration and through the onset of sclerotomal differentiation. Because of the cross-reactivity of both notochord and cartilage CSPGs with the S103L antibody, extensive molecular and biochemical analysis of the two CSPGs was carried out. Striking differences distinguish the notochord and cartilage (aggrecan) CSPGs at the level of posttranslational modification. Notably, cartilage aggrecan carries a significant content of keratan sulfate (KS) chains, while the notochord CSPG is devoid of KS. In contrast, cartilage aggrecan lacks the HNK-1 epitope, while the notochord CSPG has a high content of HNK-1. Three different approaches were used to establish the relationship of the two CSPGs at the molecular level. Northern blot analysis, using aggrecan probes, detected same-sized messages from notochord and cartilage RNA. Overlapping fragments, generated by RT-PCR using primers covering 98% of the entire coding sequence from the known cartilage structure, were of identical size in notochord and cartilage. Taking advantage of our recent studies, which demonstrated a single base change in the aggrecan gene resulting in conversion of Glu to a STOP codon in exon 12 of chick aggrecan as the molecular basis of the defect nanomelia, we demonstrated that the same mutation was present in notochord mRNA from nanomelic chicks. These results provide evidence that the chick aggrecan gene is expressed very early in development in notochord and confirm that the core proteins expressed in chick notochord and cartilage are derived from the same gene. These findings strongly support the hypothesis that the final structural characteristics of each proteoglycan are determined not only by the core protein but also by tissue-specific, developmentally regulated posttranslational mechanisms, functioning within the context of the requirement for specific extracellular matrices.

Expression and function of Pax 1 during development of the pectoral girdle.

Pax 1 is a member of the paired-box containing gene family. Expression has previously been observed in the developing sclerotomes and later in the anlagen of the intervertebral discs. Analysis of Pax 1-deficient undulated mice revealed an important role for this gene in the development of the axial skeleton, in which Pax 1 apparently functions as a mediator of notochordal signals during sclerotome differentiation. Here we demonstrate that Pax 1 is also transiently expressed in the developing limb buds. A comparative phenotypic analysis of different undulated alleles shows that this expression is of functional significance. In mice that are mutant for the Pax 1 gene severe developmental abnormalities are found in the pectoral girdle. These include fusions of skeletal elements which would normally remain separate, and failures in the differentiation of blastemas into cartilaginous structures. Although Pax 1 is also expressed in the developing hindlimb buds and Wolffian ridge, no malformations could be detected in the corresponding regions of Pax 1 mutant mice. These findings show that, in addition to its role in the developing vertebral column, Pax 1 has an important function in the development of parts of the appendicular skeleton.

The role of Pax-1 in axial skeleton development

Previous studies have identified a single amino-acid substitution in the transcriptional regulator Pax-1 as the cause of the mouse skeletal mutant undulated (un). To evaluate the role of Pax-1 in the formation of the axial skeleton we have studied Pax-1 protein expression in early sclerotome cells and during subsequent embryonic development, and we have characterized the phenotype of three different Pax-1 mouse mutants, un, undulated-extensive (unex) and Undulated short-tail (Uns). In the Uns mutation the whole Pax-1 locus is deleted, resulting in the complete absence of Pax-1 protein in these mice. The other two genotypes are interpreted as hypomorphs. We conclude that Pax-1 is necessary for normal vertebral column formation along the entire axis, although the severity of the phenotype is strongest in the lumbar region and the tail. Pax-1-deficient mice lack vertebral bodies and intervertebral discs. The proximal part of the ribs and the rib homologues are also missing or severely malformed, whereas neural arches are nearly normal. Pax-1 is thus required for the development of the ventral parts of vertebrae. Embryonic analyses reveal that although sclerotomes are formed in mutant embryos, abnormalities can be detected from day 10.5 p.c. onwards. The phenotypic analyses also suggest that the notochord still influences vertebral body formation some days after the sclerotomes are formed. Furthermore, the notochord diameter is larger in mutant embryos from day 12 p.c., due to increased cell proliferation. In the strongly affected genotypes the notochord persists as a rod-like structure and the nucleus pulposus is never properly formed. Since the notochord is Pax-1-negative these findings suggest a bidirectional interaction between notochord and paraxial mesoderm. The availability of these Pax-1 mutant alleles permitted us to define an early role for Pax-1 in sclerotome patterning as well as a late role in intervertebral disc development. Our observations suggest that Pax-1 function is required for essential steps in ventral sclerotome differentiation, i.e. for the transition from the mesenchymal stage to the onset of chondrogenesis.

Reproduction of radicular symptoms by paraspinal muscle EMG needling

Wnt and Sonic Hedgehog (Shh) signals are known to pattern the somite into dermomyotomal, myotomal and sclerotomal cell fates. By employing explants of presomitic mesoderm cultured with constant levels of Wnt3a conditioned medium and increasing levels of Shh, we found that differing levels of Shh signaling elicit differing responses from somitic cells: the lowest level of Shh signaling allows dermomyotomal gene expression, intermediate levels induce loss of dermomyotomal markers and activation of myogenic differentiation, and higher levels induce loss of myotomal markers and activation of sclerotomal gene expression. In addition, we have found that in the presence of high levels of Wnt signaling, instead of inducing sclerotomal markers, Shh signals act to maintain the expression of dermomyotomal and myotomal markers. One of the sclerotomal genes induced by high levels of Shh signaling is Nkx3.2. Forced expression of Nkx3.2 blocks somitic expression of the dermomyotomal marker Pax3 both in vitro and in vivo. Conversely, forced expression of Pax3 in somites can block Shh-mediated induction of sclerotomal gene expression and chondrocyte differentiation in vitro. Thus we propose that varying levels of Shh signaling act in a morphogen-like manner to elicit differing responses from somitic cells, and that Pax3 and Nkx3.2 set up mutually repressing cell fates that promote either dermomyotome/myotome or sclerotome differentiation, respectively.