Expression of Sulf1 and Sulf2 in cartilage, bone and endochondral fracture healing.

SULF1/SULF2 enzymes regulate cell signalling that impacts the growth and differentiation of many tissues. To determine their possible role in cartilage and bone growth or repair, their expression was examined during development and bone fracture healing using RT-PCR and immunochemical analyses. Examination of epiphyseal growth plates revealed differential, inverse patterns of SULF1 and SULF2 expressions, with the former enriched in quiescent and the latter in hypertrophic chondrocyte zones. Markedly higher levels of both SULFs, however, were expressed in osteoblasts actively forming bone when compared with proliferating pre-osteoblasts in the periosteum or the entombed osteocytes which express the lowest levels. The increased expression of Sulf1 and Sulf2 in differentiating osteoblasts was further confirmed by RT-PCR analysis of mRNA levels in rat calvarial osteoblast cultures. SULF1 and SULF2 were expressed in most foetal articular chondrocytes but down-regulated in a larger subset of cells in the post-natal articular cartilage. Unlike adult articular chondrocytes, SULF1/SULF2 expression varied markedly in post-natal hypertrophic chondrocytes in the growth plate, with very high SULF2 expression compared with SULF1 apparent during neonatal growth in both primary and secondary centres of ossification. Similarly, hypertrophic chondrocytes expressed greatly higher levels of SULF2 but not SULF1 during bone fracture healing. SULF2 expression unlike SULF1 also spread to the calcifying matrix around the hypertrophic chondrocytes indicating its possible ligand inhibiting role through HSPG desulphation. Higher levels of SULF2 in both developing and healing bone closely correlated with parallel increases in hedgehog signalling analysed by ptc1 receptor expression.

Regulation of Wnt signaling and embryo patterning by an extracellular sulfatase.

The developmental signaling functions of cell surface heparan sulfate proteoglycans (HSPGs) are dependent on their sulfation states. Here, we report the identification of QSulf1, the avian ortholog of an evolutionarily conserved protein family related to heparan-specific N-acetyl glucosamine sulfatases. QSulf1 expression is induced by Sonic hedgehog in myogenic somite progenitors in quail embryos and is required for the activation of MyoD, a Wnt-induced regulator of muscle specification. QSulf1 is localized on the cell surface and regulates heparan-dependent Wnt signaling in C2C12 myogenic progenitor cells through a mechanism that requires its catalytic activity, providing evidence that QSulf1 regulates Wnt signaling through desulfation of cell surface HSPGs.

Heterogeneity of Atlantic salmon troponin-I.

Three non-identical, full length troponin-I (Tn-I) clones were isolated from an Atlantic salmon myotomal (trunk) muscle cDNA library. The primary structures, which are predicted to range from 172 to 180 amino acids in length, exhibit similar percent identity scores when compared with fast, slow and cardiac specific Tn-Is from higher vertebrates. When the sequence data are considered along with the results of Western blotting it is evident that Tn-I is more heterogeneous in Atlantic salmon than has been previously shown in higher vertebrates.

The effect of cross-innervation on the tropomyosin composition of rabbit skeletal muscle.

Soleus, semitendinosus and crureus muscles of the rabbit were found to contain alpha- and beta-tropomyosin subunits and additional forms that have been provisionally designated gamma and delta. Extensor digitorum longus and psoas muscles contained only alpha and beta subunits, the relative proportions of which varied between single fibres of psoas muscle. On cross-innervation of rabbit soleus and extensor digitorum longus muscles, the fraction of the total tropomyosin present as the beta subunit remained constant. The relative proportions of alpha, gamma and delta subunits changed as would be expected from the change in speed that occurred.

Developmental expression of troponin I isoforms in fetal human heart.

We have used antibodies specific for troponin I proteins to examine human cardiac development and have detected a transiently expressed developmental isoform. This isoform is distinct from adult cardiac troponin I (TnIc) but is indistinguishable, on the basis of electrophoretic mobility and antibody reactivity, from the isoform found in slow skeletal muscle (TnIs). Furthermore, we show that mRNA for TnIs is present in fetal, but not adult, heart. Analysis of a developmental series of fetal samples indicates that there is a transition in expression from TnIs to TnIc which occurs between 20 weeks fetal and 9 months postnatal development.

Changes in the distribution of fast and slow forms of troponin I in some neuromuscular disorders.

As judged from the atrophy of the muscle cells, the distribution of slow and fast troponin I in type I and type II cells in peripheral neuropathies in the adults (24-67 years of age) was unaffected for long periods after denervation. Reinnervation by fast or slow nerve in chronic neuropathies usually resulted in typical fibre type grouping with complete segregation of slow and fast troponin I in type I and type II cells, respectively. Intermediate fibres (slow and fast troponin I in the same cell) were present during the course of transformation. Unlike peripheral neuropathies, the number of cells staining for slow troponin I in the very atrophic fascicles in spinal muscular atrophy was considerably reduced. The synthesis of fast troponin I was stimulated in original type I cells containing slow troponin I. These observations support the experimental data (Dhoot and Perry 1982b) that the suppression of fast troponin I in type I or presumptive type I cells requires innervation by the slow nerve. Its absence results in an increased expression of fast troponin I in cells that originally contained only slow troponin I. Once suppressed, fast troponin I is absent in type I cells even after long periods of experimental denervation or cases of human peripheral neuropathies but not so in the cases of spinal muscular atrophy.

Transformation of fibre types in muscular dystrophies.

In the normal human skeletal muscle, slow and fast forms of troponin I are segregated in type I and type II cells, respectively. Muscle biopsies from different dystrophies showed a large number of intermediate cells that stained with antibodies to both fast and slow troponin I. Intermediate cells of variable size were scattered at random and did not show a motor unit distribution as generally seen in some neuromuscular disorders. The preponderance of a particular cell type depended on the type of dystrophy. Except for dystrophia myotonica, all cases showed presence of small cells that looked like regenerating cells.

Role of Sulf1A in Wnt1- and Wnt6-induced growth regulation and myoblast hyper-elongation.

Sulf1A expression, which is a characteristic of embryonic muscle, is undetectable in mature muscle fibres and quiescent satellite cells, but is re-activated in vivo upon injury and in vitro following activation of satellite cells. Sulf1A is known to enhance canonical Wnt signalling, and its association with Wnt1-induced satellite cell proliferation in vitro in the present study further confirmed this. However, exogenous Wnt6 decreased satellite cell proliferation but promoted the adoption of a hyper-elongated cell morphology in myoblasts on isolated single fibres in culture. Such Wnt6-induced cellular hyper-elongation and inhibition of proliferation was found to be dependent upon Sulf1A, as treatment with Sulf1A neutralising antibodies abolished both these effects. This indicates that Sulf1A can regulate Wnt6 signalling and cellular differentiation in skeletal muscle.

Transient expression of fast troponin C transcripts in embryonic quail heart.

Most myofibrillar proteins, including troponin I and troponin T subunits of troponin complex, undergo developmental stage-specific isoform transitions in vertebrate heart before attaining adult contractile and regulatory characteristics. Only the cardiac/slow skeletal muscle type isoform of troponin C, however, has been shown to be expressed in both adult and developing heart. The changes in troponin C could be functionally important as the TnC isoforms vary in their affinities for Ca(2+). For example, fast troponin C has two Ca(2+) binding sites while slow/cardiac troponin C has a single regulatory site. This study demonstrates the co-expression of both fast and slow transcripts of troponin C in not only quail embryonic skeletal muscle but also embryonic heart using two different analytical techniques of polymerase chain reaction and in situ hybridisation procedure. Fast troponin C expression in the quail heart using in situ hybridisation procedure was first observed at embryonic day 3, with maximum expression at day 5 after which its level in the developing heart was gradually down regulated. In situ hybridisation staining of sections at these developmental stages demonstrated the expression of both fast and slow transcripts of troponin C in all cardiomyocytes.

Dynamic Id2 expression in the medial and lateral domains of avian dermamyotome.

Id2 cDNA was isolated from a subtractive screen of stage-12 quail caudal somites. In situ hybridisation analysis identified the previously un-described expression of Id2 mRNA in distinct medial and lateral domains of the somitic dermamyotome in both quail and chick embryos. Id2 expression in somites was highly dynamic being first initiated in the lateral domain of the dermamyotome of stage-8-10 embryos, followed by expression in a separate medial domain. Id2 mRNA during subsequent embryonic development could be detected in both medial and lateral domains in the anterior to mid regions while the posterior, recently segmented somites, showed expression only in the lateral domain, which was eventually down regulated in the anterior-most somites. Tissue manipulation studies revealed that Id2 expression in somites required positive signalling from not only axial structures and lateral plate mesoderm but also surface ectoderm. In addition, Id2 expression was also observed in anterior and posterior domains of developing avian limb buds and interdigital tissue.

Mammalian myoblasts become fast or slow myocytes within the somitic myotome.

A monoclonal antibody has been prepared to slow skeletal muscle type myosin heavy chain which in the rat distinguishes fast and slow myocytes within the somitic myotome at 11.5 days in utero. The distribution of slow myotubes identified by this antibody in developing limb buds is also restricted to presumptive slow muscle cell type regions only. No slow myoblasts in hindlimb buds, however, were detected at 14.5 day in utero when a small number of fast muscle cells were already present. The presence of slow muscle cell population detected by this specific antibody became apparent a day later. This study thus demonstrated the diversification into different muscle cell types during both early embryonic and foetal development.

Selective synthesis and degradation of slow skeletal myosin heavy chains in developing muscle fibers.

During fetal development of fast skeletal muscles in the rat, three types of cells could be identified using a monoclonal antibody to slow skeletal muscle myosin heavy chain. Presumptive type I cells stained positive for slow forms of skeletal myosin heavy chain and a previously described protein of an apparent molecular weight of 100 KD, whereas presumptive type 2B cells did not stain for either of these peptides. Presumptive type 2A cells, on the other hand, did not stain for slow isoform of 100-K protein, but did stain positive for slow skeletal myosin heavy chain. There was a progressive suppression or degradation of slow skeletal myosin heavy chain in presumptive type 2A cells during subsequent fetal development, so that it was almost undetectable in most animals at birth. Soleus, a slow muscle, however, did not show clear differentiation into presumptive type I and type 2 cells until 4 days after birth.

Initiation of differentiation into skeletal muscle fiber types.

A monoclonal antibody directed against a slow isoform of a 100-K myofibrillar protein has been used to study the differentiation of muscle fibers in the fast and slow muscles of the rat. Immunohistochemical studies have shown that initiation of differentiation into fast and slow muscle fibers occurs very early during development. The distribution pattern of cell types in different muscles is unique and is also determined very early during fetal muscle development. It is concluded that motor innervation is probably not essential to initiate differentiation into distinct muscle fiber types.

Neural regulation of differentiation of rat skeletal muscle cell types.

Three monoclonal antibodies (LM5, F2 and F39) to the fast class of myosin heavy chain (MHC) were used to study the effect of denervation on the differentiation of muscle cell types in some rat skeletal muscles. Antibody LM5 in immunocytochemical investigations did not stain any myotubes during early fetal development but presumptive fast muscle cells started to stain during later fetal development. Unlike antibody LM5, antibodies F2 and F39 stained all myotubes during fetal development. The suppression of fast myosin heavy chains recognised in presumptive slow muscle cells was observed within 1-2 days after birth with antibody F39 but not until 10-14 days after birth with antibody F2. The emergence of subsets of fast muscle fibre types in rat extensor digitorum longus (EDL) and tibialis anteri (TA) detectable by F39 and F2 antibodies was not observed until 2-3 weeks after birth. Denervation of developing muscles led to marked changes in the expression of myosins identified by these antibodies.

Changes in the distribution of slow skeletal myosin heavy chain SM1 in developing avian muscle fibres.

The use of monoclonal antibodies against fast skeletal and slow skeletal myosin heavy chains (MHC) has shown the presence of significant amounts of slow skeletal type MHC in embryonic skeletal muscles of white leghorn chickens. The presence of this slow skeletal myosin heavy chain (SMHC) was not restricted to presumptive slow muscles only, as it was also observed in presumptive fast skeletal muscles. As was the case for embryonic MHC reactive with the antibody against fast skeletal myosin heavy chain (FMHC), the presence of SMHC could be detected at the earliest stages of myogenesis. It appeared to be present in most muscle cells during early embryonic development. The changes in its cellular distribution during subsequent embryonic and post-hatch period indicated its suppression in a certain proportion of the cells in both presumptive fast and slow skeletal muscles. Its time course of suppression, however, was much prolonged, not synchronized, and varied in fast and slow skeletal muscles during both embryonic and post-hatch development.

Evidence for the presence of a distinct embryonic isoform of myosin heavy chain in chicken skeletal muscle.

Immunochemical studies have identified a distinct myosin heavy chain (MHC) in the chicken embryonic skeletal muscle that was undetectable in this muscle in the posthatch period by both immunocytochemical and the immunoblotting procedures. This embryonic isoform, identified by antibody 96J, which also recognises the cardiac and SM1 myosin heavy chains, differs from the embryonic myosin heavy chain belonging to the fast class described previously. Although the fast embryonic isoform is a major species present in the leg and pectoral embryonic muscles, slow embryonic isoform was present in significant amounts during early embryonic development. Immunocytochemical studies using another monoclonal antibody designated 9812, which is specific for SM1 MHC, showed this isoform to be restricted to only presumptive slow muscle cells. From these studies and those reported on the changes in SM2 MHC, it is proposed that as is the case for the fast class, there also exists a slow class of myosin heavy chains composed of slow embryonic, SM1 and SM2 isoforms. The differentiation of a muscle cell involves transitions in a series of myosin isozymes in both presumptive fast and slow skeletal muscle cells.

Identification and distribution of the fast class of troponin T in the adult and developing avian skeletal muscle.

In the adult chicken, two groups of fast troponin T, the higher molecular weight (B1 and B2) present only in the pectoral muscle and the lower molecular weight (L1-L5) the only group present in the leg muscle, were identified by the immunoblotting procedure using monoclonal antibodies against fast skeletal troponin T. The presence of significant amounts of three major variants of leg muscle type troponin T (L3-L5), however, could also be detected in the adult chicken pectoral muscle. Although none of the antibodies cross-reacted with slow troponin T itself, the proportions of both leg and pectoral type troponin T variants belonging to the fast class varied in fast muscles that contained slow muscle fibres or fast muscles devoid of slow muscle cells. The troponin T present in the early embryonic skeletal muscles did not react with any of the antibodies raised against adult fast isoforms. The gradual expression of some of the adult isoforms of troponin T was detected at about day 13 in ovo. However, the adult isoforms did not all appear simultaneously and their full complement was not achieved until after hatching. In addition to the increased number of bands in the leg type troponin T region, the presence of two other protein bands (neonatal forms) with slower electrophoretic mobility than the other fast isoforms of troponin T, was detected in post-hatch pectoral muscle tested at 1-12 weeks of age. These neonatal forms (N1 and N2) in the pectoral muscle were undetectable at eight months of age. The presence of breast type troponin T in the leg muscle was not detected with these antibodies at any stage of development.

Identification and changes in the pattern of expression of slow-skeletal-muscle-like myosin heavy chains in a developing fast muscle.

Immunochemical studies of chicken pectoralis major, a fast muscle, have demonstrated large amounts of myosin heavy chains (MHCs) of the slow-skeletal-muscle type during early stages of embryonic development. A large majority of the myotubes present in early embryonic muscle stained for this class of MHC. As development progressed, its synthesis was suppressed in most of the muscle, except in the deeper presumptive red-strip region. The level of this MHC in the embryonic muscle appeared to be reduced by its suppression in a proportion of the existing cells, by the addition of many presumptive fast cells that never expressed this MHC, and by atrophy or degeneration of a small proportion of the slow MHC-positive cells. Further suppression of this MHC in a proportion of the histochemically typed slow cells present in the red-strip region did not occur until quite late in the post-hatch period.