Effects of Myostatin on Nuclear Morphology at the Myotendinous Junction.

Myostatin (Myo) is known to suppress skeletal muscle growth, and was recently reported to control tendon homeostasis. The purpose of the present study was to investigate the regulatory involvement of Myo in the myotendinous junction (MTJ) in vivo and in vitro. After Achilles tendon injury in mice, we identified unexpected cell accumulation on the tendon side of the MTJ. At postoperative day 7 (POD7), the nuclei had an egg-like profile, whereas at POD28 they were spindle-shaped. The aspect ratio of nuclei on the tendon side of the MTJ differed significantly between POD7 and POD28 (p = 4.67 × 10-34). We then investigated Myo expression in the injured Achilles tendon. At the MTJ, Myo expression was significantly increased at POD28 relative to POD7 (p = 0.0309). To investigate the action of Myo in vitro, we then prepared laminated sheets of myoblasts (C2C12) and fibroblasts (NIH3T3) (a pseudo MTJ model). Myo did not affect the expression of Pax7 and desmin (markers of muscle development), scleraxis and temonodulin (markers of tendon development), or Sox9 (a common marker of muscle and tendon development) in the cell sheets. However, Myo changed the nuclear morphology of scleraxis-positive cells arrayed at the boundary between the myoblast sheet and the fibroblast sheet (aspect ratio of the cell nuclei, myostatin(+) vs. myostatin(-): p = 0.000134). Myo may strengthen the connection at the MTJ in the initial stages of growth and wound healing.

Morphology and relationships of the biceps brachii and brachialis with the musculocutaneous nerve.

INTRODUCTION: Major anatomical textbooks generally state that the biceps brachii muscle (BB) is composed of long and short heads, whereas the brachialis muscle (BR) consists of a single head. However, the numbers of heads comprising the BB and the BR are very variable. The purpose of this study was to investigate how the branching patterns of the musculocutaneous nerve (MC) influence the number of heads of the BB and the BR. MATERIALS AND METHODS: Morphological examinations of the BB and MC were conducted using cadavers of 22 Japanese individuals, and morphological examinations of the BR and the MC were conducted in 9 of those 22 individuals. RESULTS: A three-headed BB was observed in 7 of the 22 specimens (31.8%). Most of these specimens showed a Type III branch pattern (after penetrating the long head or the short head, the MC innervated the supernumerary head or communicated with the main root again). The number of BR heads was categorized into three types: Type A, two heads (superficial and deep heads, 22.2%); Type B, three or four heads (two or three superficial heads and one deep head, 44.4%); and Type C, multiple heads (33.3%). Among these categories, branches of the MC in Type A specimens were most simple. CONCLUSION: A supernumerary head of the BB seemed to be present if the MC penetrates it. The BR basically consists of superficial and deep heads, and the number of superficial heads is affected by branches of the MC.

Midline sensory nerve supply to the anoscrotal junction: a study using human male fetuses.

The origin of the posterior scrotal nerve is considered to be the bilateral pudendal nerves but the course to the midline is still obscure. Using 5 late-stage human male fetuses, we identified the single nerve through the intramuscular midline septum of the bulbospongiosus and the bilateral nerves along the left and right sides of the septum. Thus, the posterior scrotal nerve showed a variation: a single midline trunk or bilateral nerves. Branches of the bilateral pudendal nerves ran medially between the muscle and Cowper's gland and, at the midline area, they joined or associated closely. During the proximal course, much or less, the nerve penetrated the superior part of the muscle. The nerve entered the subcutaneous tissue at and near the perineal raphe. The communication with intrapelvic autonomic nerves were suggested behind Cowper's gland. Notably, the midline skin immediately anterior to the anus carried a considerable dense supply of thin sensory nerves. However, these nerves seemed to come from a space between the rectal smooth muscle and the external anal sphincter, not from the posterior scrotal nerve. Therefore, surgical treatment of the intersphincteric layer was likely to injure the original sensory nerve supply to the anterior anal skin.

Teres major and latissimus dorsi muscles in human embryos: A reconsideration of the so-called brother muscles.

The teres major and latissimus dorsi muscles (TM, LD) are considered to be the so-called brother muscles. Actually, being similar to the TM, an uppermost part of the LD usually arises from the scapular plate. In embryos of 11 mm CRL, anlagen of the TM and LD appeared to be fused to provide a single mass at an angle between the axillary and radial nerves. However, splitting had already finished in not only the TM and LD but also the other muscles at and around the shoulder in specimens of 14 mm CRL. Thus, muscle splitting at the region appeared to occur simultaneously at a short stage of 12-13 mm CRL.The TM and LD carried a common tendon still at 6 weeks (14-16 mm CRL), but their muscle bellies were separated clearly. A concept of brother muscles might be applied to the TM and LD according to a bias from the gross and comparative anatomy, not from the embryological view.

Regressing vitelline vein and the initial development of the superior mesenteric vein in human embryos.

The superior mesenteric vein was considered to develop in situ in the midgut mesentery secondary to regression of the left vitelline vein. We revisited the morphology using serial sections of 20 embryos at 5-6 weeks (CRL 9-15 mm). The regressing vitelline vein provided a long peritoneal fold in the immediately superior side of the midgut mesentery containing the thick superior mesenteric artery. Notably, in a half of specimens, there were tissue clefts along the superior mesenteric artery in the mesentery and they were communicated with the left vitelline vein at the superior end of the peritoneal fold. The tissue clefts appeared not to carry the endothelial lining. We considered the cleft as the initial superior mesenteric vein. Conversely, the initial vein seemed not to develop from budding or venous plexus.

The expression of embryonic globin mRNA in a severely anemic mouse model induced by treatment with nitrogen-containing bisphosphonate.

BACKGROUND: Mammalian erythropoiesis can be divided into two distinct types, primitive and definitive, in which new cells are derived from the yolk sac and hematopoietic stem cells, respectively. Primitive erythropoiesis occurs within a restricted period during embryogenesis. Primitive erythrocytes remain nucleated, and their hemoglobins are different from those in definitive erythrocytes. Embryonic type hemoglobin is expressed in adult animals under genetically abnormal condition, but its later expression has not been reported in genetically normal adult animals, even under anemic conditions. We previously reported that injecting animals with nitrogen-containing bisphosphonate (NBP) decreased erythropoiesis in bone marrow (BM). Here, we induced severe anemia in a mouse model by injecting NBP injection in combination with phenylhydrazine (PHZ), and then we analyzed erythropoiesis and the levels of different types of hemoglobin. METHODS: Splenectomized mice were treated with NBP to inhibit erythropoiesis in BM, and with PHZ to induce hemolytic anemia. We analyzed hematopoietic sites and peripheral blood using morphological and molecular biological methods. RESULTS: Combined treatment of splenectomized mice with NBP and PHZ induced critical anemia compared to treatment with PHZ alone, and numerous nucleated erythrocytes appeared in the peripheral blood. In the BM, immature CD71-positive erythroblasts were increased, and extramedullary erythropoiesis occurred in the liver. Furthermore, embryonic type globin mRNA was detected in both the BM and the liver. In peripheral blood, spots that did not correspond to control hemoglobin were observed in 2D electrophoresis. ChIP analyses showed that KLF1 and KLF2 bind to the promoter regions of ß-like globin. Wine-colored capsuled structures were unexpectedly observed in the abdominal cavity, and active erythropoiesis was also observed in these structures. CONCLUSION: These results indicate that primitive erythropoiesis occurs in adult mice to rescue critical anemia because primitive erythropoiesis does not require macrophages as stroma whereas macrophages play a pivotal role in definitive erythropoiesis even outside the medulla. The cells expressing embryonic hemoglobin in this study were similar to primitive erythrocytes, indicating the possibility that yolk sac-derived primitive erythroid cells may persist into adulthood in mice.

Regulation of Osteoclast Multinucleation by the Actin Cytoskeleton Signaling Network.

Although it is known that osteoclasts are multinucleated cells that are responsible for bone resorption, the mechanism by which their size is regulated is unclear. We previously reported that an actin-rich superstructure, termed the zipper-like structure, specifically appears during the fusion of large osteoclast-like cells (OCLs). Actin cytoskeleton reorganization in osteoclasts is regulated by a signaling network that includes the macrophage colony-stimulating factor (M-CSF) receptor, a proto-oncogene, Src, and small GTPases. Here, we examined the role of actin reorganization in the multinucleation of OCLs differentiated from RAW 264.7 cells using various pharmacological agents. Jasplakinolide, which stabilizes actin stress fibers, induced the development of small OCLs, and the Src inhibitor SU6656 and the dynamin inhibitor dynasore impaired the maintenance of the podosome belt and the zipper-like structure. These inhibitors decreased the formation of large OCLs but increased the number of small OCLs. M-CSF is known to stimulate osteoclast fusion. M-CSF signaling via Src up-regulated Rac1 activity but down-regulated Rho activity. Rac1 and Rho localized to the center of the zipper-like structure. Rho activator II promoted the formation of small OCLs, whereas the Rho inhibitor Y27632 promoted the generation of large OCLs. These results suggest that the status of the actin cytoskeleton signaling network determines the size of OCLs during cell fusion.

In situ proliferation and differentiation of macrophages in dental pulp.

The presence of macrophages in dental pulp is well known. However, whether these macrophages proliferate and differentiate in the dental pulp in situ, or whether they constantly migrate from the blood stream into the dental pulp remains unknown. We have examined and compared the development of dental pulp macrophages in an organ culture system with in vivo tooth organs to clarify the developmental mechanism of these macrophages. The first mandibular molar tooth organs from ICR mice aged between 16 days of gestation (E16) to 5 days postnatally were used for in vivo experiments. Those from E16 were cultured for up to 14 days with or without 10% fetal bovine serum. Dental pulp tissues were analyzed with immunohistochemistry to detect the macrophages and with reverse transcription and the polymerase chain reaction (RT-PCR) for the detection of factors related to macrophage development. The growth curves for the in vivo and in vitro cultured cells revealed similar numbers of F4/80-positive macrophages in the dental pulp. RT-PCR analysis indicated the constant expression of myeloid colony-stimulating factor (M-CSF) in both in-vivo- and in-vitro-cultured dental pulp tissues. Anti-M-CSF antibodies significantly inhibited the increase in the number of macrophages in the dental pulp. These results suggest that (1) most of the dental pulp macrophages proliferate and differentiate in the dental pulp without a supply of precursor cells from the blood stream, (2) M-CSF might be a candidate molecule for dental pulp macrophage development, and (3) serum factors might not directly affect the development of macrophages.

A comparative study of myostatin, follistatin and decorin expression in muscle of different origin.

Muscle regeneration supports muscle function in aging, and plays a role in the functional impairment caused by progressive neuromuscular diseases. Major substances controlling this process are growth factors and the extracellular matrix (ECM). Thus, follistatin is known to antagonize the function of several members of the TGF-ß family of secreted signaling factors, including myostatin-the most powerful inhibitor of muscle growth characterized to date. Decorin-a small leucine-rich proteoglycan-traps myostatin and modulates its activity towards myogenic cells in the ECM. In addition, there are few reports concerning the regenerative muscle process of masseter muscles, which are of branchial arch origin, in mdx mice. Thus, in order to clarify the muscle regenerative process of masseter muscle, gene and protein expression of myostatin, follistatin and decorin were examined using the tibialis anterior (TA)muscle as a positive control. In both muscles, a gradual increase in mRNA myostatin, follistatin and decorin expression was detected, with the increase being greater in TA muscle than in masseter muscle. At 2 weeks, both muscles exhibited normal skeletal muscle cells. At 3 weeks, masseter muscle demonstrated scant areas of necrosis, whereas large necrotic zones were seen in TA muscle. At 4 weeks, the formation of necrotic tissue and presence of follistatin protein was observed clearly in masseter muscle. This result indicates that follistatin production is stimulated in the presence of necrosis. Interestingly, both muscles showed the same process of muscular formation, but with different time frames, which could be related to muscle origin.

Increased expression of decorin during the regeneration stage of mdx mouse.

Satellite cells exist in postnatal muscle tissue and constitute the main source of muscle precursor cells for growth and repair. These cells carry out important roles for skeletal muscle formation postnatally during growth of muscle mass as well as damage-induced regenerative processes. Muscle regeneration supports muscle function in aging and has a role in the functional impairment caused by progressive neuromuscular diseases. Major substances controlling this process are growth factors and extracellular matrix. Myostatin, a member of TGF-beta family, was mainly expressed in muscle tissue. Decorin, a member of the small leucine-rich proteoglycan gene family, is composed of a core protein and a dermatan/chondroitin sulfate chain. Recent studies have shown that decorin enhanced the proliferation and differentiation of myogenic cells by suppressing myostatin activity. Thus, decorin appears to be a new molecule in the myostatin signaling pathway and a promising target for treatment of progressive neuromuscular diseases. Therefore, in this study, we examined the localization of decorin as well as myostatin in a muscular dystrophy model in mdx mice and B10 Scott Snells mice as a control to elucidate the differences between decorin and myostatin messages as well as protein distribution. This study revealed increased expression of decorin protein as well as mRNA at the regenerative stage of mdx mice compared to early stages, while only weak expression of decorin was detected in the control mice. Our study contributes to identifying the relationship between decorin and myostatin as well as the development of a therapeutic strategy for progressive neuromuscular diseases.

Myosin heavy chain composition of tongue muscle in microphthalmic (mi/mi) mice before and after weaning.

To elucidate the effects of teeth on muscle fibers in the tongue during the developmental process, we examined the expression of muscle contractile proteins and the genes for those proteins in normal mice and microphthalmic (mi/mi) mice with impaired tooth eruption. The mice were observed during the growth period, including weaning, which is when feeding movements undergo major changes. Expression of the myosin heavy chain (MyHC)-2a protein, whose contraction speed is relatively slow, disappeared after weaning in normal mice, while it remained in high concentrations even after weaning in mi/mi mice. The presence of MyHC-2a after weaning in mice with no tooth eruption was attributed to a compensation for lack of proper masticatory function and sucking-like movements, as MyHC-2a is necessary for these movements.