Etv1 Controls the Establishment of Non-overlapping Motor Innervation of Neighboring Facial Muscles during Development.

The somatotopic motor-neuron projections onto their cognate target muscles are essential for coordinated movement, but how that occurs for facial motor circuits, which have critical roles in respiratory and interactive behaviors, is poorly understood. We report extensive molecular heterogeneity in developing facial motor neurons in the mouse and identify markers of subnuclei and the motor pools innervating specific facial muscles. Facial subnuclei differentiate during migration to the ventral hindbrain, where neurons with progressively later birth dates-and evolutionarily more recent functions-settle in more-lateral positions. One subpopulation marker, ETV1, determines both positional and target muscle identity for neurons of the dorsolateral (DL) subnucleus. In Etv1 mutants, many markers of DL differentiation are lost, and individual motor pools project indifferently to their own and neighboring muscle targets. The resulting aberrant activation patterns are reminiscent of the facial synkinesis observed in humans after facial nerve injury.

Neural crest and the patterning of vertebrate craniofacial muscles.

Patterning of craniofacial muscles overtly begins with the activation of lineage-specific markers at precise, evolutionarily conserved locations within prechordal, lateral, and both unsegmented and somitic paraxial mesoderm populations. Although these initial programming events occur without influence of neural crest cells, the subsequent movements and differentiation stages of most head muscles are neural crest-dependent. Incorporating both descriptive and experimental studies, this review examines each stage of myogenesis up through the formation of attachments to their skeletal partners. We present the similarities among developing muscle groups, including comparisons with trunk myogenesis, but emphasize the morphogenetic processes that are unique to each group and sometimes subsets of muscles within a group. These groups include branchial (pharyngeal) arches, which encompass both those with clear homologues in all vertebrate classes and those unique to one, for example, mammalian facial muscles, and also extraocular, laryngeal, tongue, and neck muscles. The presence of several distinct processes underlying neural crest:myoblast/myocyte interactions and behaviors is not surprising, given the wide range of both quantitative and qualitative variations in craniofacial muscle organization achieved during vertebrate evolution.

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.

Divergent and conserved roles of Dll1 signaling in development of craniofacial and trunk muscle.

Craniofacial and trunk skeletal muscles are evolutionarily distinct and derive from cranial and somitic mesoderm, respectively. Different regulatory hierarchies act upstream of myogenic regulatory factors in cranial and somitic mesoderm, but the same core regulatory network - MyoD, Myf5 and Mrf4 - executes the myogenic differentiation program. Notch signaling controls self-renewal of myogenic progenitors as well as satellite cell homing during formation of trunk muscle, but its role in craniofacial muscles has been little investigated. We show here that the pool of myogenic progenitor cells in craniofacial muscle of Dll1(LacZ/Ki) mutant mice is depleted in early fetal development, which is accompanied by a major deficit in muscle growth. At the expense of progenitor cells, supernumerary differentiating myoblasts appear transiently and these express MyoD. The progenitor pool in craniofacial muscle of Dll1(LacZ/Ki) mutants is largely rescued by an additional mutation of MyoD. We conclude from this that Notch exerts its decisive role in craniofacial myogenesis by repression of MyoD. This function is similar to the one previously observed in trunk myogenesis, and is thus conserved in cranial and trunk muscle. However, in cranial mesoderm-derived progenitors, Notch signaling is not required for Pax7 expression and impinges little on the homing of satellite cells. Thus, Dll1 functions in satellite cell homing and Pax7 expression diverge in cranial- and somite-derived muscle.

ALK5-mediated transforming growth factor ß signaling in neural crest cells controls craniofacial muscle development via tissue-tissue interactions.

The development of the craniofacial muscles requires reciprocal interactions with surrounding craniofacial tissues that originate from cranial neural crest cells (CNCCs). However, the molecular mechanism involved in the tissue-tissue interactions between CNCCs and muscle progenitors during craniofacial muscle development is largely unknown. In the current study, we address how CNCCs regulate the development of the tongue and other craniofacial muscles using Wnt1-Cre; Alk5(fl/fl) mice, in which loss of Alk5 in CNCCs results in severely disrupted muscle formation. We found that Bmp4 is responsible for reduced proliferation of the myogenic progenitor cells in Wnt1-Cre; Alk5(fl/fl) mice during early myogenesis. In addition, Fgf4 and Fgf6 ligands were reduced in Wnt1-Cre; Alk5(fl/fl) mice and are critical for differentiation of the myogenic cells. Addition of Bmp4 or Fgf ligands rescues the proliferation and differentiation defects in the craniofacial muscles of Alk5 mutant mice in vitro. Taken together, our results indicate that CNCCs play critical roles in controlling craniofacial myogenic proliferation and differentiation through tissue-tissue interactions.

Anatomy research of nasolabial muscle structure in fetus with cleft lip: an iodine staining technique based on microcomputed tomography.

A thorough knowledge of the anatomic structure of the orbicularis oris of the upper lip and the nasalis in fetus with cleft lip is the key for the success of cleft lip repair. To understand the anatomic structure of the muscles of nasolabial region in fetus with cleft lip, the nasolabial tissues in 4 aborted fetuses with cleft lip were soaked for 7 days with iodine solution (Lugol solution of 3.75%) and were given micro-computed tomography. After the iodine solution permeated into the soft tissues, a good contrast was showed between muscle fibers and other fibrillar connective tissues. Through the observation of the obtained images, we found that most orbicularis oris fibers gathered into bundles with clear outline and only had slight deformation and displacement on the health side of the cleft of the unilateral incomplete cleft lip; however, in the lateral cleft, the muscle fibers not only had deformation and displacement but also were immature, disorganized, and not gathered into bundles. After being restored in Digital Imaging and Communications in Medicine format, the obtained images were then transferred into Materialise's interactive medical image control system, edited, and reconstructed into three-dimensional models. The models clearly showed the spatial relationship between the muscular tissues of the nasolabial region and the nasolabial outline in fetus with cleft lip.

Development of the platysma muscle and the superficial musculoaponeurotic system (human specimens at 8-17 weeks of development).

There is controversy regarding the description of the different regions of the face of the superficial musculoaponeurotic system (SMAS) and its relationship with the superficial mimetic muscles. The purpose of this study is to analyze the development of the platysma muscle and the SMAS in human specimens at 8-17 weeks of development using an optical microscope. Furthermore, we propose to study the relationship of the anlage of the SMAS and the neighbouring superficial mimetic muscles. The facial musculature derives from the mesenchyme of the second arch and migrates towards the different regions of the face while forming premuscular laminae. During the 8th week of development, the cervical, infraorbital, mandibular, and temporal laminae are observed to be on the same plane. The platysma muscle derives from the cervical lamina and its mandibular extension enclosing the lower part of the parotid region and the cheek, while the SMAS derives from the upper region. During the period of development analyzed in this study, we have observed no continuity between the anlage of the SMAS and that of the superficial layer of the temporal fascia and the zygomaticus major muscle. Nor have we observed any structure similar to the SMAS in the labial region.

A zebrafish model of axenfeld-rieger syndrome reveals that pitx2 regulation by retinoic acid is essential for ocular and craniofacial development.

PURPOSE: The homeobox transcription factor PITX2 is a known regulator of mammalian ocular development, and human PITX2 mutations are associated with Axenfeld-Rieger syndrome (ARS). However, the treatment of patients with ARS remains mostly supportive and palliative. METHODS: The authors used molecular genetic, pharmacologic, and embryologic techniques to study the biology of ARS in a zebrafish model that uses transgenes to mark neural crest and muscle cells in the head. RESULTS: The authors demonstrated in vivo that pitx2 is a key downstream target of retinoic acid (RA) in craniofacial development, and this pathway is required for coordinating neural crest, mesoderm, and ocular development. pitx2a knockdown using morpholino oligonucleotides disrupts jaw and pharyngeal arch formation and recapitulates ocular characteristics of ARS, including corneal and iris stroma maldevelopment. These phenotypes could be rescued with human PITX2A mRNA, demonstrating the specificity of the knockdown and evolutionary conservation of pitx2a function. Expression of the ARS dominant negative human PITX2A K50E allele also caused ARS-like phenotypes. Similarly, inhibition of RA synthesis in the developing eye (genetic or pharmacologic) disrupted craniofacial and ocular development, and human PITX2A mRNA partially rescued these defects. CONCLUSIONS: RA regulation of pitx2 is essential for coordinating interactions among neural crest, mesoderm, and developing eye. The marked evolutionary conservation of Pitx2 function in eye and craniofacial development makes zebrafish a potentially powerful model of ARS, amenable to in vivo experimentation and development of potential therapies.

Importance of muscle movement for normal craniofacial development.

After the craniofacial structures have completed embryologic development, movement of facial muscles begins. Paraxial mesoderm of the first (mastication) and second pharyngeal (facial expression) arches gives rise to the muscles of the craniofacial area. Muscles derived from the third and fourth pharyngeal arches are involved in swallowing and vocalization. For the human newborn face to have a normal morphologic appearance, contractions of these muscles must occur to stimulate forward growth of bone, cartilage growth, and facial muscle bulk. Facial muscles begin to contract between 6 and 8 weeks of embryonic development and can be observed on prenatal ultrasound by 9 weeks after fertilization. Lack of craniofacial muscle contractions may lead to ocular hypertelorism, flat zygoma and midface, high bridge of the nose, depressed tip of the nose, small and open mouth, trismus, microretrognathia, small tongue, and abnormal palate (high arch, bifid uvula, submucous cleft, and cleft palate).

[Age dynamics of the human maxillofacial apparatus development in the early period of prenatal ontogenesis].

The literature review discusses the debatable problems on terms of separation of different anlages of human maxillo-facial apparatus, chronology of histo- and organogenetic remodeling of hard and soft tissues during the period of their formation in the first trimester of pregnancy. It is suggested that these controversies are most likely determined by imperfection of current embryogenesis periodization systems and of criteria of human embryos and fetuses age definition; therefore further research in this direction is required.

Core issues in craniofacial myogenesis.

Branchiomeric craniofacial muscles control feeding, breathing and facial expression. These muscles differ on multiple counts from all other skeletal muscles and originate in a progenitor cell population in pharyngeal mesoderm characterized by a common genetic program with an adjacent population of cardiac progenitor cells, the second heart field, that gives rise to much of the heart. The transcription factors and signaling molecules that trigger the myogenic program at sites of branchiomeric muscle formation are correspondingly distinct from those in somite-derived muscle progenitor cells. Here new insights into the regulatory hierarchies controlling branchiomeric myogenesis are discussed. Differences in embryological origin are reflected in the lineage, transcriptional program and proliferative and differentiation properties of branchiomeric muscle satellite cells. These recent findings have important implications for our understanding of the diverse myogenic strategies operative both in the embryo and adult and are of direct biomedical relevance to deciphering the mechanisms underlying the cause and progression of muscle restricted myopathies.

The transcription factor Six1a plays an essential role in the craniofacial myogenesis of zebrafish.

Transcription factor Six1a plays important roles in morphogenesis, organogenesis, and cell differentiation. However, the role of Six1a during zebrafish cranial muscle development is still unclear. Here, we demonstrated that Six1a was required for sternohyoideus, medial rectus, inferior rectus, and all pharyngeal arch muscle development. Although Six1a was also necessary for myod and myogenin expression in head muscles, it did not affect myf5 expression in cranial muscles that originate from head mesoderm. Overexpression of myod enabled embryos to rescue all the defects in cranial muscles induced by injection of six1a-morpholino (MO), suggesting that myod is directly downstream of six1a in controlling craniofacial myogenesis. However, overexpression of six1a was unable to rescue arch muscle defects in the tbx1- and myf5-morphants, suggesting that six1a is only involved in myogenic maintenance, not its initiation, during arch muscle myogenesis. Although the craniofacial muscle defects caused by pax3-MO phenocopied those induced by six1a-MO, injection of six1a, myod or myf5 mRNA did not rescue the cranial muscle defects in pax3 morphants, suggesting that six1a and pax3 do not function in the same regulatory network. Therefore, we proposed four putative regulatory pathways to understand how six1a distinctly interacts with either myf5 or myod during zebrafish craniofacial muscle development.

Developmental origins of species-specific muscle pattern.

Vertebrate jaw muscle anatomy is conspicuously diverse but developmental processes that generate such variation remain relatively obscure. To identify mechanisms that produce species-specific jaw muscle pattern we conducted transplant experiments using Japanese quail and White Pekin duck, which exhibit considerably different jaw morphologies in association with their particular modes of feeding. Previous work indicates that cranial muscle formation requires interactions with adjacent skeletal and muscular connective tissues, which arise from neural crest mesenchyme. We transplanted neural crest mesenchyme from quail to duck embryos, to test if quail donor-derived skeletal and muscular connective tissues could confer species-specific identity to duck host jaw muscles. Our results show that duck host jaw muscles acquire quail-like shape and attachment sites due to the presence of quail donor neural crest-derived skeletal and muscular connective tissues. Further, we find that these species-specific transformations are preceded by spatiotemporal changes in expression of genes within skeletal and muscular connective tissues including Sox9, Runx2, Scx, and Tcf4, but not by alterations to histogenic or molecular programs underlying muscle differentiation or specification. Thus, neural crest mesenchyme plays an essential role in generating species-specific jaw muscle pattern and in promoting structural and functional integration of the musculoskeletal system during evolution.

Heart and craniofacial muscle development: a new developmental theme of distinct myogenic fields.

Head muscle development has been studied less intensively than myogenesis in the trunk, although this situation is gradually changing, as embryological and genetic insights accumulate. This review focuses on novel studies of the origins, composition and evolution of distinct craniofacial muscles. Cellular and molecular parallels are drawn between cardiac and branchiomeric muscle developmental programs, both of which utilize multiple lineages with distinct developmental histories, and argue for the tissues' common evolutionary origin. In addition, there is increasing evidence that the specification of skeletal muscles in the head appears to be distinct from that operating in the trunk: considerable variation among the different craniofacial muscle groups is seen, in a manner resembling myogenic specification in lower organisms.

The facial expression musculature in primates and its evolutionary significance.

Facial expression is a mode of close-proximity non-vocal communication used by primates and is produced by mimetic/facial musculature. Arguably, primates make the most-intricate facial displays and have some of the most-complex facial musculature of all mammals. Most of the earlier ideas of primate mimetic musculature, involving its function in facial displays and its evolution, were essentially linear "scala natural" models of increasing complexity. More-recent work has challenged these ideas, suggesting that ecological factors and social systems have played a much larger role in explaining the diversity of structures than previously believed. The present review synthesizes the evidence from gross muscular, microanatomical, behavioral and neurobiological studies in order to provide a preliminary analysis of the factors responsible for the evolution of primate facial musculature with comparisons to general mammals. In addition, the unique structure, function and evolution of human mimetic musculature are discussed, along with the potential influential roles of human speech and eye gaze.

[Genes, forces and forms: mechanical aspects of prenatal craniofacial development]. / Gènes, forces et formes: aspects mécaniques du développement cranio-facial prénatal.

Current knowledge of molecular signaling during craniofacial development is advancing rapidly. We know that cells can respond to mechanical stimuli by biochemical signaling. Thus, the link between mechanical stimuli and gene expression has become a new and important area of the morphological sciences. This field of research seems to be a revival of the old approach of developmental mechanics, which goes back to the embryologists His [36], Carey [13, 14], and Blechschmidt [5]. These researchers argued that forces play a fundamental role in tissue differentiation and morphogenesis. They understood morphogenesis as a closed system with living cells as the active part and biological, chemical, and physical laws as the rules. This review reports on linking mechanical aspects of developmental biology with the contemporary knowledge of tissue differentiation. We focus on the formation of cartilage (in relation to pressure), bone (in relation to shearing forces), and muscles (in relation to dilation forces). The cascade of molecules may be triggered by forces, which arise during physical cell and tissue interaction. Detailed morphological knowledge is mandatory to elucidate the exact location and timing of the regions where forces are exerted. Because this finding also holds true for the exact timing and location of signals, more 3D images of the developmental processes are required. Further research is also required to create methods for measuring forces within a tissue. The molecules whose presence and indispensability we are investigating appear to be mediators rather than creators of form.

Cranial muscle defects of Pitx2 mutants result from specification defects in the first branchial arch.

Pitx2 expression is observed during all states of the myogenic progression in embryonic muscle anlagen and persists in adult muscle. Pitx2 mutant mice form all but a few muscle anlagen. Loss or degeneration in muscle anlagen could generally be attributed to the loss of a muscle attachment site induced by some other aspect of the Pitx2 phenotype. Muscles derived from the first branchial arch were absent, whereas muscles derived from the second branchial arch were merely distorted in Pitx2 mutants at midgestation. Pitx2 was expressed well before, and was required for, initiation of the myogenic progression in the first, but not second, branchial arch mesoderm. Pitx2 was also required for expression of premyoblast specification markers Tbx1, Tcf21, and Msc in the first, but not second, branchial arch. First, but not second, arch mesoderm of Pitx2 mutants failed to enlarge after embryonic day 9.5, well before the onset of the myogenic progression. Thus, Pitx2 contributes to specification of first, but not second, arch mesoderm. The jaw of Pitx2 mutants was vestigial by midgestation, but significant size reductions were observed as early as embryonic day 10.5. The diminutive first branchial arch of mutants could not be explained by loss of mesoderm alone, suggesting that Pitx2 contributes to the earliest specification of jaw itself.

Laser burn wound healing in naso-labial region of fetal and neonatal mice.

OBJECTIVE: To investigate the characteristics of wound healing in the mouse naso-labial region in both the fetal and neonatal stages, histological and immunohistochemical analyses were performed using a newly established laser burn wound healing system. MATERIALS AND METHODS: Fetal mice at embryonic day 14 (E 14) were wounded as a model of fetal wound healing. To compare it, neonatal mice at day 5 after birth (d 5) were adopted as a model of neonatal wound healing. The healing process was examined by van Gieson staining and immunohistochemistry for fibronectin and tenascin. RESULTS: Relatively large damage remained after wound healing even in fetal mice. In both types of wound healing, rapid regeneration of muscle tissues were observed. Fibronectin and tenascin immunostaining was detected not only in wound healing region, but also in the endomysium of regenerating muscle tissues. Especially, tenascin showed a restricted expression pattern. CONCLUSIONS: Rapid regeneration of muscle tissues in the naso-labial region in both the fetal and neonatal mice seemed to leave relatively large damage even in the fetal wound healing. Contracted force exerted by muscle tissues may be a reason for this phenomenon. Fibronectin and tenascin were closely related to the wound healing process including muscle regeneration in this region.

Tbx1 regulation of myogenic differentiation in the limb and cranial mesoderm.

The T-box transcription factor Tbx1 has been implicated in DiGeorge syndrome, the most frequent syndrome due to a chromosomal deletion. Gene inactivation of Tbx1 in mice results in craniofacial and branchial arch defects, including myogenic defects in the first and second branchial arches. A T-box binding site has been identified in the Xenopus Myf5 promoter, and in other species, T-box genes have been implicated in myogenic fate. Here we analyze Tbx1 expression in the developing chick embryo relating its expression to the onset of myogenic differentiation and cellular fate within the craniofacial mesoderm. We show that Tbx1 is expressed before capsulin, the first known marker of branchial arch 1 and 2 muscles. We also show that, as in the mouse, Tbx1 is expressed in endothelial cells, another mesodermal derivative, and, therefore, Tbx1 alone cannot specify the myogenic lineage. In addition, Tbx1 expression was identified in both chick and mouse limb myogenic cells, initially being restricted to the dorsal muscle mass, but in contrast, to the head, here Tbx1 is expressed after the onset of myogenic commitment. Functional studies revealed that loss of Tbx1 function reduces the number of myocytes in the head and limb, whereas increasing Tbx1 activity has the converse effect. Finally, analysis of the Tbx1-mesoderm-specific knockout mouse demonstrated the cell autonomous requirement for Tbx1 during myocyte development in the cranial mesoderm.