Initiation of primary myogenesis in amniote limb muscles.

BACKGROUND: Vertebrate muscles are defined and patterned at the stage of primary myotube formation, but there is no clear description of how these cells form in vivo. Of particular interest is whether primary myotubes are "seeded" by a unique myoblast population that differentiates as mononucleated myocytes, similar to the founder myoblasts of insects. RESULTS: We analyzed the cell populations and processes leading to initiation of primary myogenesis in limb buds of rats and mice. Pax3(+ve) myogenic precursors migrate into the limb bud and initially consolidate into dorsal and ventral muscle masses in the absence of Pax7 expression. Approximately a day later, Pax7(+ve) cells appear in the central aspect of the limb base and subsequently throughout the limb muscle masses. Primary myogenesis is initiated within each muscle mass at a time when only Pax3, and not Pax7, protein can be detected. Primary myotubes form initially as elongate mononucleated myocytes, well before cleavage of the muscle masses has occurred. Multinucleate myotubes appear approximately a day later. A similar process is seen during initiation of chick limb primary myogenesis. CONCLUSIONS: Primary myotubes of vertebrate limb muscles are initiated by mononucleated myocytes, that appear structurally analogous to the founder myoblasts of insects.

Methylguanine DNA methyltransferase-mediated drug resistance-based selective enrichment and engraftment of transplanted stem cells in skeletal muscle.

Cell replacement therapy using stem cell transplantation holds much promise in the field of regenerative medicine. In the area of hematopoietic stem cell transplantation, O(6)-methylguanine-DNA methyltransferase MGMT (P140K) gene-mediated drug resistance-based in vivo enrichment strategy of donor stem cells has been shown to achieve up to 75%-100% donor cell engraftment in the host's hematopoietic stem cell compartment following repeated rounds of selection. This strategy, however, has not been applied in any other organ system. We tested the feasibility of using this MGMT (P140K)-mediated enrichment strategy for cell transplantation in skeletal muscles of mice. We demonstrate that muscle cells expressing an MGMT (P140K) drug resistance gene can be protected and selectively enriched in response to alkylating chemotherapy both in vitro and in vivo. Upon transplantation of MGMT (P140K)-expressing male CD34(+ve) donor stem cells isolated from regenerating skeletal muscle into injured female muscle treated with alkylating chemotherapy, donor cells showed enhanced engraftment in the recipient muscle 7 days following transplantation as examined by quantitative-polymerase chain reaction using Y-chromosome specific primers. Fluorescent in situ hybridization analysis using a Y-chromosome paint probe revealed donor-derived de novo muscle fiber formation in the recipient muscle 14 days following transplantation, with approximately 12.5% of total nuclei within the regenerated recipient muscle being of donor origin. Following engraftment, the chemo-protected donor CD34(+ve) cells induced substantial endogenous regeneration of the chemo-ablated host muscle that is otherwise unable to self-regenerate. We conclude that the MGMT (P140K)-mediated enrichment strategy can be successfully implemented in muscle.

Dynamic patterns of mononucleated myogenic cell populations in the developing rat hindlimb.

Formation of an organ is governed by both the genetic programming of individual cells and dynamic interactions amongst different cell communities or the 'community effect'. Using the developing vertebrate limb muscle, we identified myogenic stem cell communities derived from migratory somitic cells. These cells express Pax3, a gene from the paired box (PAX) family of transcription factors and Pax7, a paralog of Pax3. Both Pax genes act upstream of myogenic regulatory factor (MRF) whose activation marks a specified myogenic lineage and subsequent differentiation. Quantitative analyses on the size of the individual cell populations revealed that Pax3 and MRF compartments remained constant. Further analysis showed that the size of the Pax7 cell population increased significantly. The pool of foetal MRF populations contained decreasing Pax3 and increasing Pax7 proportions. This increase is dynamic at the developmental stage. Upon abrupt disruption of the p38 regulatory pathway for myogenic differentiation, established kinetic patterns were significantly altered. Changes in the proportions of these myogenic subpopulations imply that a community effect involving dynamic interactions among differentiating cell communities may play a crucial role in correct maintenance and propagation of myogenic stem cells.

Changes in the proportion and number of Pax(7+ve) and MF20(+ve) myoblasts during chick myogenesis in the head and limb.

Previous studies suggested that all myoblasts are present in the head and limb prior to the commencement of primary myotube formation. As a consequence, these myoblasts must be in various developmental states during myogenesis, i.e. proliferating, differentiating or terminally differentiated. There are few in vivo studies investigating dynamic quantitative changes of subgroups of these myoblasts during myogenesis. In this report, using anti-Pax7 and anti-myosin heavy chain antibodies, we examined the quantitative change of proliferating (Pax7(+ve)) and terminally differentiated (MF20(+ve)) myoblasts during primary and secondary myogenesis in the chick head and limb. Our results show that during primary myogenesis, less than 30% of myoblasts are in the proliferating phase, but as soon as secondary myogenesis begins, over 95% of myoblasts start to proliferate. Moreover, we have found that the proportion of terminally differentiated myoblasts is maintained at a low level (less than 3%) during primary and secondary myogenesis.

The investing layer of the deep cervical fascia does not exist between the sternocleidomastoid and trapezius muscles.

OBJECTIVE: We sought to describe the 3-dimensional organization of connective tissues in the suboccipital region. STUDY DESIGN AND SETTING: We conducted a sectional anatomic investigation with the use of E12 sheet plastination. SUBJECTS: Six human adult cadavers (2 male and 4 female; age range, 54 to 86 years) were used in this study. Five of them were sectioned as 2.5-mm-thick coronal (1 cadaver), transverse (2 cadavers), or sagittal (2 cadavers) sections. RESULTS: No aggregation of fibrous connective tissue was seen between the sternocleidomastoid and trapezius muscles. The intervening space was fully occupied by fatty tissue that was indistinguishable from the subcutaneous tissue. CONCLUSIONS: The investing layer of the deep cervical fascia is incomplete so that the carotid sheath is directly exposed to the subcutaneous tissue via a gap between the sternocleidomastoid and trapezius muscle. SIGNIFICANCE: This anatomic feature should be considered when designing a minimally invasive endoscopic approach to the carotid sheath and the surrounding deep cervical structures.

Aged skeletal muscle retains the ability to fully regenerate functional architecture.

While the general understanding of muscle regenerative capacity is that it declines with increasing age due to impairments in the number of muscle progenitor cells and interaction with their niche, studies vary in their model of choice, indices of myogenic repair, muscle of interest and duration of studies. We focused on the net outcome of regeneration, functional architecture, compared across three models of acute muscle injury to test the hypothesis that satellite cells maintain their capacity for effective myogenic regeneration with age. Muscle regeneration in extensor digitorum longus muscle (EDL) of young (3 mo-old), old (22 mo-old) and senescent female mice (28 mo-old) was evaluated for architectural features, fiber number and central nucleation, weight, collagen and fat deposition. The 3 injury paradigms were: a myotoxin (notexin) which leaves the blood vessels and nerves intact, freezing (FI) that damages local muscle, nerve and blood vessels and denervation-devascularization (DD) which dissociates the nerves and blood vessels from the whole muscle. Histological analyses revealed successful architectural regeneration following notexin injury with negligible fibrosis and fully restored function, regardless of age. In comparison, the regenerative response to injuries that damaged the neurovascular supply (FI and DD) was less effective, but similar across the ages. The focus on net regenerative outcome demonstrated that old and senescent muscle has a robust capacity to regenerate functional architecture.

Configuration of the connective tissue in the posterior atlanto-occipital interspace: a sheet plastination and confocal microscopy study.

STUDY DESIGN: The connective tissue structures in the posterior atlanto-occipital region were investigated using E12 sheet plastinations and confocal microscopy. OBJECTIVES: To define the relationship between rectus capitis posterior minor (RCPm), posterior atlanto-occipital (PAO) membrane, nuchal ligament, and the spinal dura in the PAO interspace. SUMMARY OF BACKGROUND DATA: It has been speculated that connections between the dura and muscles and/or ligaments in the PAO interspace may transmit forces from the cervical spine joint complexes to the pain-sensitive dura, generating cervicogenic headaches. Anatomic structures involved in these connections include the RCPm, PAO membrane, and nuchal ligament. However, there is little information about the nature of these connections and the relationships between these anatomic structures. METHODS: The study used a combined approach, consisting of the gross anatomic dissection of nine cadavers and the E12 sheet plastination method for thirteen adult human cadavers, five of which were further examined using confocal microscopy. RESULTS: The study demonstrates that (1) the tendinous fibers from the medial and deep part of the RCPm muscle are continuous antero-inferiorly with the spinal dura; (2) the PAO membrane is part of the RCPm fascia and tendon and the perivascular sheathes; (3) antero-inferiorly the PAO membrane fuses with the spinal dura rather than the atlas; and (4) the nuchal ligament does not exist in the PAO interspace. CONCLUSIONS: The connective tissue structures that connect the spinal dura to the RCPm muscle in the PAO interspace are the RCPm fascia and tendinous fibers and perivascular sheathes.