Jaw myogenesis in the monk parakeet: evidence of developmental reprogramming in the emergence of novel muscles in Psittaciformes (Aves).

Psittaciformes have apomorphies in the muscles of the jaw that include both the adductors m. ethmomandibularis (EM) and m. pseudomasseter (PM), which are responsible for the generation of strong bite forces. While the EM is present in all Psittaciformes, the PM can be absent or present, and even underdeveloped or well-developed. The aim of this study is to identify developmental reprogramming processes by comparing the myogenesis of the jaw of the monk parakeet Myiopsitta monachus with the information available about other species of Psittaciformes. Seventeen specimens including embryos at different developmental stages, and nestlings of different ages were studied through the analysis of serial histological sections. At embryonic stage 24 (S24) the muscle precursor was observed in the first pharyngeal arch. At S27 the muscle precursor was found to be divided into lateral, intermediate and medial portions. At S31 the independent development of the EM as a rostro-dorsal projection of the mm. pterygoidei could be observed. At S36 the individualization of all muscles was complete. Finally, the PM was detected two days after hatching as an aponeurotic dorsal projection of the m. adductor mandibulae externus superficialis, located lateral to the arcus jugalis. Our results suggest that in M. monachus the muscles EM and PM emerge as a result of a process of heterotipy, and variations in the degree of development of the PM are associated to a heterochronic process of post-displacement, with M. monachus having an underdeveloped PM with respect to basal Psittaciformes.

Expression of CGRP in embryonic mouse masseter muscle.

Neuropeptide calcitonin gene-related peptide (CGRP) is a mediator of inflammation and head pain that influences the functional vascular blood supply. The CGRP also regulate myoblast and acetylcholine receptors on neuromuscular junctions in development. However, little is known about its appearance and location during mouse masseter muscle (MM) development. We detected the mRNA abundance of CGRP, vascular genesis markers (Vascular endothelial growth factor A (VEGF-A), PECAM (CD31), lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1)) and embryonic and adult myosin heavy chain (MyHCs) (embryonic, IIa, IIb, and IIx) using real-time RT-PCR during development from the embryonic stage to after birth (E12.5, E14.5, E17.5, E18.5, P0, P1 and P5). We also endeavored to analyze the expression and localization of CGRP in situ hybridization in the developing mouse MM during development from the embryonic stage to after birth (E12.5, E14.5, E17.5, and P1). The antisense probe for CGRP was detected by in situ hybridization at E12.5, E14.5 E17.5 and then no longer detected after birth. The CGRP, CD31, embryonic MyHC abundance levels are highest at E17.5 (p<0.001) and they show a pattern similar to that of the other markers from E12.5 to P5. PCA analysis indicates a specific relation between CGRP and embryonic MyHC, CD31, and LYVE-1 in MM development. Cluster analyses identified the following distinct clusters for mRNA abundance in the MM: cluster 1, P5; cluster 2, E12.5, E14.5, E17.5, E18.5, P0, and P1. The positive correlation between CGRP and embryonic MyHC (Pearson's r>0.65; p<0.01) was analyzed. These data suggested that CGRP may have an influence on embryonic MyHC during mouse MM development. CGRP also affects the angiogenesis markers at embryonic stages.

Morphometric aspects of the facial and skeletal muscles in fetuses.

OBJECTIVES: There are few research reports providing a comparison of the muscle fiber morphometry between human fetuses and adults. Data on fetal and adult muscle fibers would be valuable in understanding muscle development and a variety of muscle diseases. This study investigated human muscle fiber growth to clarify the difference between the facial muscles and other skeletal muscles. METHODS: The materials were obtained from three male fetuses (6-month-old) and 11 Japanese male cadavers aged 43-86 years (average: 71.8). Human buccinator muscles (facial muscles), masseter and biceps brachii muscles (skeletal muscles) were resected. We counted the muscle fibers and measured their transverse area. We also calculated the number of muscle fibers per mm(2) (NMF) and the average transverse area of the muscle fibers (TAMFs). RESULTS: The average of the NMF of the buccinator, masseter and biceps brachii muscles in fetuses had, respectively, 19, 37, and 22 times as many fibers as those in adults. The average fetus/adult ratios of the TAMF of the buccinator, masseter and biceps brachii muscles were 4.0%, 2.4%, 4.1%, respectively. CONCLUSIONS: The average NMF for all kinds of muscles decreased after birth; however, the peak in life-span or decreases with the aging process tended to vary with the kind of muscles examined. The average TAMF for all kinds of muscles enlarged after birth. We considered that the enlargement of the TAMF was connected with the emergence of fetal movements and functional demands after birth.

Tenomodulin regulated the compartments of embryonic and early postnatal mouse masseter muscle.

The masseter muscle (MM) is a complex tendinous laminar structure during development; however, the stage of the laminar structure formation is unknown. Tenomodulin (TeM) is a useful marker of tendons and has an anti-angiogenic cysteine-rich C-terminal domain. Therefore, we analyzed mRNA of TeM and angiogenesis markers (CD31 and vascular endothelial growth factor (VEGF)) and performed in situ hybridization for the TeM genes in MM from on embryonic day 12.5 (E12.5) to postnatal day 5 (P5). The TeM expression is at first detectable in the middle region of the mesenchymal connective tissue in the MM at E 12.5. The expression domains of the TeM during development typically include the middle region of the MM, particularly surrounding the vascular regions. The level of TeM mRNA in the MM increased from E12.5 to E17.5 and decreased after birth. In contrast, the levels of CD31 and VEGF mRNAs were almost constant from E12.5 to E18.5 and then low from birth onward. Therefore, the development of the laminar tendinous structure in the middle region between superficial and deeper regions of the MM first occurs during the process of tendon formation at embryonic day 12.5. In our study of MM development, the laminar structure regulating TeM also prevents vascular invasion during the formation of compartment of the MM. The tendon may relate to the components of muscle mass of MM.

Maturation of the neuromuscular junction in masseters of human fetus.

OBJECTIVES: The aim of the present investigation is to examine if the histological maturation of the neuromuscular junction in the masseters of human fetuses has already begun by the 12-th week of gestation or not. MATERIAL AND METHODS: Twenty-four masseter muscles from 14 human fetuses at gestational age 12 weeks were divided into two groups. In the first group, muscle sections were stained with Bielschowsky and Holzer stains for examination of neurofibrils and glial cells respectively. In the second group, rhodamine and fluorescein conjugated alpha-bungarotoxin were used to detect nicotinic receptors and anti-GAD for neuronal terminals. RESULTS: It was observed the presence of one axon for each end-plate and glial cells spread over a branched axon. The nicotinic receptors clustered in the neuromuscular junction, neuronal terminals and large oval nucleus were detected. CONCLUSIONS: These observations suggest that the maturation of the neuromuscular junctions of the masseter muscles in the human fetuses has already begun at the 12-th week of gestation.

Developmental and functional considerations of masseter muscle partitioning.

The masseter muscle participates in a wide variety of activities including mastication, swallowing and speech. The functional demands for accurate mandibular positioning and generation of forces during incising or a power stroke require a diverse set of forces that are determined by the innate muscle form. The complex internal tendon architecture subdivides the masseter into multiple partitions that can be further subdivided into neuromuscular compartments representing small motor unit territories. Individual masseter compartments have unique biomechanical properties that, when activated individually or in groups, can generate a wide range of sagittal and off-sagittal torques about the temporomandibular joint. The myosin heavy chain (MyHC) fibre-type distribution in the adult masseter is sexually dimorphic and is influenced by hormones such as testosterone. These testosterone-dependent changes cause a phenotype switch from slower to faster fibre-types in the male. The development of the complex organization of the masseter muscle, the MyHC fibre-type message and protein expression, and the formation of endplates appear to be pre-programmed and not under control of the muscle nerve. However, secondary myotube generation and endplate maturation are nerve dependent. The delayed development of the masseter muscle compared with the facial, tongue and jaw-opening muscles may be related to the delayed functional requirements for chewing. In summary, masseter muscle form is pre-programmed prior to birth while muscle fibre contractile characteristics are refined postnatally in response to functional requirements. The motor control mechanisms that are required to coordinate the activation of discrete functional elements of this muscle remain to be determined.

Observation of the relationship between the shape of skeletal muscles and their nerve distribution patterns: a transparent and microanatomic study.

BACKGROUND: There are many gaps in the understanding of the neuroanatomy of skeletal muscles with regards to the nerve distribution pattern and shape of the muscles. This study was designed to examine the entire intramuscular nerve-distribution patterns of various human skeletal muscles. METHODS: The relationships among nine skeletal muscles with various architecture (rhomboid major, biceps brachii, flexor pollicis longus, rectus femoris, sternohyoid, trapezius, masseter, digastric muscles) and their nerve-distribution patterns were investigated in four fetal cadavers using the Sihler staining method. The diameter and number of extramuscular (main) and major nerve branches, the number of minor nerve branches, and anastomoses were examined and evaluated statistically. RESULTS: With regards to the number of extramuscular (main) nerve branches, the rhomboid major muscle resembled the flexor pollicis longus, trapezius, masseter, and sternohyoid muscles, and the anterior belly of the digastricus muscle (p > 0.05), whereas it was significantly different from the rectus femoris, the posterior belly of digastricus, and the long and short heads of the biceps brachii (p < 0.05). Trapezius and masseter muscles were different from all of the skeletal muscles that were studied with regards to the diameter of main branches (p < 0.05). The masseter muscle had the largest diameter (p < 0.05). With regards to the number of minor nerve branches, the sternohyoid muscle was significantly different from all the skeletal muscles that were studied (p < 0.05) except the short head of the biceps brachii, rectus femoris, and the posterior belly of digastricus (p > 0.05). As for the number of neural anastomoses, the sternohyoid muscle was statistically different from all skeletal muscles that were studied (p < 0.05) except the masseter and trapezius muscles (p > 0.005). CONCLUSIONS: A surgeon's thorough knowledge of the relationship between the shape and nerve distribution pattern of skeletal muscles is important in successful reinnervation and regeneration of these muscles. It might also be useful in the field of muscle transplantation.

Mesencephalic trigeminal nucleus development is dependent on Krox-20 expression.

Krox-20, a C2H2-type zinc-finger transcription factor, plays an important role in rhombomere development. This study reveals that the Krox-20 null mutation impacts the development of mesencephalic trigeminal (Me5) neurons, a cell group traditionally thought to emerge from the mesencephalon. Based on cell counting studies, we show that Krox-20 null mutants have twice as many Me5 neurons relative to wildtypes at E15, but by birth have half the number of Me5 cells as wildtypes. TUNEL studies reveal a period of increased apoptosis from E17-P0 in mutants. The mutation does not result in differences in Me5 cell size, morphology, gene expression or peripheral projection patterns between genotypes, as demonstrated by retrograde tracing and Brn3a immunohistochemistry. The data suggest that Krox-20 regulates the period and extent of Me5 apoptosis, impacting the final number of Me5 neurons. The loss of Me5 in Krox-20-/- mice may highlight species-specific differences in the origin of these cells.

Masticatory function and properties of masseter muscle fibers in microphthalmic (mi/mi) mice during postnatal development.

It is well known that teeth do not erupt in microphthalmic (mi/mi) mice, a type of osteopetrotic mice, due to bone resorption failure. Therefore, we surmise that the masticatory function of these mice is different from that of normal mice. In this study, the differences to the properties of masseter muscle fibers were clarified morphologically and immunohistochemically in the mi/mi and normal mice. Morphological observations revealed that the muscle fibers in the mi/mi mice were smaller than those in normal mice at 9 weeks of age. However, no marked differences between mi/mi and normal mice at 2 and 4 weeks of age. Immunohistochemical observations showed myosin heavy chain (MHC) slow type fibers, which were usually seen at only early stages of development, in 4-week old mi/mi mice. There were also differences in isoform compositions between the mi/mi and normal mice. These results suggest that differences in masticatory function affect the properties of its muscle fibers.

Myosin heavy chain isoforms of the murine masseter muscle during pre- and post-natal development.

Masticatory muscles that are derived from the branchial arches express different compositions of myosin heavy chain (MHC) isoforms during the transitional phase from suckling to mastication. To clarify the developmental changes of murine masseter muscle, the composition of MHC isoforms was examined using immunohistochemical staining and competitive reverse transcription PCR. We found that MHC1 was expressed transiently in the pre and post-natal stages. In the compositional change of isoforms, the embryonic type MHCp was expressed consistently, whereas the adult isoforms increased with the developmental process. In particular, a significant change was observed between embryonic days 14 and 16, a stage when murine facial development is conspicuous. This suggests that the development of murine masseter muscle is closely associated with facial development.

Changes in mRNA expression of nicotinic acetylcholine receptor subunits during embryonic development of mouse masseter muscle.

Nicotinic acetylcholine receptors (nAChRs) switch from the embryonic-type (alpha 2 beta gamma delta subunits) to the adult-type (alpha 2 beta epsilon delta subunits), and disappear besides the neuromuscular junctions with the development of trunk and limb skeletal muscles. However, little is known about this process during the embryonic development of masseter muscle. To identify the time course of the nAChR transition from embryonic day (E) 11 to the newborn stage in mouse masseter muscle, we analyzed the expression level of delta, epsilon, and gamma subunit mRNAs by competitive polymerase chain reaction in combination with reverse transcription as well as distribution of delta subunit protein by immunohistochemistry. The nAChR delta subunit mRNA was initially detected at E11, showed an approximately 25-fold increase (p < 0.0001) between E11 and E17, and plateaued thereafter until the newborn stage. Immunostaining for delta subunit was observed in the whole portions of masseter myofibers at E17 and birth, suggesting that the nAChR elimination does not begin even at the newborn stage. The epsilon subunit mRNA initially appeared at E17, and increased in quantity by 144% (p < 0.0001) up to the newborn stage. The quantity of gamma subunit mRNA increased by approximately 240% (p < 0.0001) between E11 and E17, and then decreased by 22% (p < 0.05) from E17 value at the newborn stage. The beginning of the expression of the epsilon subunit mRNA was coincident with the beginning of the decrease in the quantity of the gamma subunit mRNA, suggesting that the nAChR subunit switch begins at E17.

[The persistence of ontogenic characteristics in the adult masseter muscle]. / Persistance de caractères ontogéniques dans le muscle masséter adulte.

During embryonic and foetal development, the masseter is formed from two successive generations of muscle fibers in a manner which is very similar to that which has been previously described for other skeletal muscles. This phenotype is characterised by the persistence of ontogenic myosin isoforms (embryonic and foetal myosin heavy chains, embryonic light chain) and by the presence of two distinct populations of fibers: small diameter fibers which coexpress the embryonic, foetal and fast isoforms of the myosin heavy chains but never express the slow isoform; large diameter fibers which express the slow myosin heavy chain either exclusively or in variable associations with the other isoforms. These characteristics of the human masseter muscle probably correspond not only to its embryological origin and its special innervation, but also to the functional constraints to which it is submitted after birth.

Computerized 3-dimensional study of the embryologic development of the human masticatory muscles and temporomandibular joint.

PURPOSE: In this study, the development of human embryonic temporomandibular joint (TMJ) and masticatory muscles were investigated by using computed 3-dimensional reconstructions. MATERIALS AND METHODS: Sixteen human embryos and fetuses, ranging from 6.5 to 107 mm crown-rump length, were examined. RESULTS: At 10 weeks, a band of mesenchyme extending from the attachment of the lateral pterygoid muscle to the condylar process was observed to pass through the medial side of the condylar process to attach to the malleus. The temporal, masseter, and pterygoid muscles develop from the so called "temporal muscle" primordium, and the temporal muscle was in continuity with the masseter muscle until 14 weeks of fetal life. CONCLUSIONS: The study shows that the muscles of mastication arise from a single primordium. It also confirms the presence of a ligamentous attachment between the lateral pterygoid muscle and the malleus.

Mandibular deformities in parathyroid hormone-related protein (PTHrP) deficient mice: possible involvement of masseter muscle.

Previous studies using parathyroid hormone-related protein (PTHrP) null mutant mice have indicated severe abnormalities in the endochondral ossification, suggesting that PTHrP affects chondrocyte differentiation. In this study, we found in newborn PTHrP-deficient mice some deformities in the mandible that is formed via intramembranous ossification. The mandibular ramus was bent downwards and a prominent bone crest to which the deep layer of masseter muscle was tendinously attached was observed in the mandibular body. Transmission electron microscopic studies showed that active bone formation was progressing along the tendon fibers of the masseter muscle. The examination of 3-D reconstruction models indicated that the mandibular ramus was bent at the site of muscle attachment, which was shifted in the direction of the muscle fibers. Muscle fiber type analysis using myosin ATPase staining showed that the masseter muscle in the newborn PTHrP-deficient mice contained numerous type 2B fibers, demonstrating premature maturation of this muscle. Based on these findings, we speculated that premature maturation of the masseter muscle leads, probably due to increased tensile forces, to accelerated bone crest formation and subsequent bending of the mandibular ramus. These results further suggest that PTHrP is involved in the regulation of muscle development in normal animals.

Delayed embryonic development of mouse masseter muscle correlates with delayed MyoD family expression.

While the masseter muscle is known to have several unique developmental characteristics as compared with other skeletal muscles, little is known about its myogenesis. Thus, we examined the expression of myogenic marker and of myoD family gene mRNA from embryonic day (E) 11 to birth. The obtained results were compared with our earlier results of the mouse tongue muscle, which is also involved in oral functions. The mRNA quantities were determined by means of the reverse-transcription and competitive-polymerase chain-reaction techniques. The expression of myogenic marker mRNA indicated that differentiation and maturation in the masseter began at E13 as in the tongue, and were not yet completed at birth, although they were completed in the tongue. The expression of myoD, myogenin, and myf5 mRNA peaked later in the masseter (E17) than in the tongue (E13). The expression of MRF4 mRNA began later in the masseter (E15) than in the tongue (E13). These results suggest that the delayed expression of the myoD family genes in the masseter correlates with delayed differentiation and maturation, probably due to the later functional requirements of the masseter than of the tongue.

Development of the motor endplates in the masseter muscle in the human fetus.

The development of the motor endplate (MEP) and the structure of the masseter muscle in the human fetus were examined by light and electron microscopy. At 12 weeks of gestation, the masseter muscle was composed mostly of irregularly-arranged myotubes. The number of muscle fibers increased while that of the myotubes decreases during development. After 28 weeks of gestation, the masseter muscle was composed only of muscle fibers, and these fibers rapidly increased in size in comparison with those prior to 28 weeks of gestation. The MEP appeared during the first 12 weeks of gestation, and were of an undeveloped simple type, with only one axon branch. After 20 weeks of gestation, MEPs were classified as complex or simple types in terms of the branching of the axons. The complex type of MEP was found in muscle fibers of large diameter, while the simple type was found in muscle fibers of small diameter. Schwann-like cells appeared at the surface of the MEP. After 28 weeks of gestation, only the complex type was found in the masseter muscle. These results suggest that, the development of the MEP is closely related to the development of muscle, and that the timing of the development of the masticatory muscles differs from that of other skeletal muscles, such as those in the trunk and limb.

Patterns of abnormal myogenesis in human cleft palates.

To test the hypothesis that soft palate muscles are abnormal in cleft palate, we compared soft palate morphogenesis in fetuses with cleft palate (n = 4) to age-matched (n = 3) and nonmatched (n = 1) control specimens. The morphologic status of all soft palate and masticatory structures were classified into one of six stages based on the level of histogenesis. At 54 mm crown-rump length (CRL), the levator veli palatini (L), palatopharyngeus (PP), and palatoglossus (PG) in cleft subjects demonstrated mesenchymal condensation into myoblastic fields, lagging behind the control specimens (97 mm CRL), which displayed definitive fields of myoblasts and myotube formation. In the 175 mm and 225 mm cleft and the 170 mm and 192 mm control specimens, muscular morphology was similar and had reached its postnatal appearance for the tensor veli palatini (175 m only) and L, PP, PG (225 mm only). Muscle fiber directions were, however, disoriented and disorganized, especially close to the medial epithelial edge of the cleft. The levator veli palatini, could not be distinguished as a discrete muscle in the cleft specimens, and what we believed to be the PP and PG seemed "normal" at the level of light microscopy, but malpositioned in a superior direction. This preliminary study demonstrates for the first time that early myogenesis in cleft palates differs from normal.

[The characteristic phenotype of the masseter muscle fibers is established after birth]. / Le phénotype particulier des fibres du muscle masséter s'établit apres la naissance.

Muscle biopsies were taken from the superficial portion of the M. masseter in 10 foetuses (aged between 12 and 38 weeks), in a child of 18 months and in an adult without any neuromuscular disease. Serial frozen sections were processed for immunocytochemistry using antibodies specific for the embryonic, foetal, slow and fast myosin heavy chains (MHCs). Diameter of the different types of fibers were measured with a Leitz ASM 68 K; the results have been expressed as average diameters and histograms. During foetal development, the masseter is formed from two successive generations of muscle fibers in a manner very similar to that which has been previously described for other skeletal muscles. After birth, a particular phenotype appears. This phenotype is characterised by the persistence of embryonic and foetal MHCs and by the presence of two distinct populations of fibers: small diameter fibers which coexpress embryonic, foetal and fast myosin isoforms but never express the slow MHC; large diameter fibers which express slow myosin either exclusively or in variable associations with the other isoforms.

Transition of myosin isozymes during development of human masseter muscle. Persistence of developmental isoforms during postnatal stage.

Previous results have shown that the adult human masseter muscle contains myosin isoforms that are specific to early stages of development in trunk and limb muscles, i.e. embryonic and fetal (neonatal) myosin heavy chains (MHC) and embryonic myosin light chain (MLC1emb). We wanted to know if this specific pattern is the result of a late maturation or of a distinct evolution during development. We show here that the embryonic and the fetal MHC and the MLC1emb are expressed throughout perinatal and postnatal masseter development. Our results also demonstrate that MLC1emb accumulation increases considerably during the postnatal period. In addition, both the slow MLCs and the slow isoform of tropomyosin are expressed later in the masseter than quadriceps and the fast skeletal muscle isoform MLC3 is not detected during fetal and early postnatal development in the masseter whereas it is expressed throughout fetal development in the quadriceps. Our results thus confirm previous histochemical data and demonstrate that the masseter muscle displays a pattern of myosin and tropomyosin isoform transitions different to that previously described in trunk and limb muscles. This suggests that control of masseter muscle development involves mechanisms distinct from other body muscles, possibly as a result of either its craniofacial innervation or of a possibly different embryonic origin.

Immunocytochemical analysis of the perinatal development of cat masseter muscle using anti-myosin antibodies.

The developmental changes in myosin gene expression in the masseter muscle of embryonic and juvenile kittens were examined immunocytochemically using anti-myosin heavy chain antibodies of various specificities. In the mature cat, this muscle contains only two phenotypes, the majority of fibres are superfast, the rest being slow fibres. In foetal tissues, the histological appearance of bundles of myotubes, comprising a large central myotube surrounded by a rosette of smaller myotubes, strongly suggest the existence in the jaw muscle of primary and secondary fibres during development. Immunocytochemical data are consistent with the hypothesis that there are four types of fibre; two types of primary fibre as well as two types of secondary fibre. (1) Slow primaries stain strongly with an anti-slow myosin antibody throughout the period under study. These fibres transiently express embryonic but not foetal myosin. (2) Superfast primaries stain for embryonic/foetal and slow myosins in the perinatal period but progressively replace these myosins with superfast myosin during postnatal development. (3) Superfast secondaries initially express embryonic/foetal myosins, but later, beginning around the time of birth progressively replace these myosins with superfast myosin. These fibres do not express slow myosin. (4) Slow secondaries, which initially also express embryonic/foetal myosins, but which postnatally express slow or slow and superfast myosins and express only slow myosin in the adult. These four types of fibres are homologous to the four isotypes of limb muscle fibres and may be derived from distinct lineages of myoblasts.