Combinatorial chromatin dynamics foster accurate cardiopharyngeal fate choices.

During embryogenesis, chromatin accessibility profiles control lineage-specific gene expression by modulating transcription, thus impacting multipotent progenitor states and subsequent fate choices. Subsets of cardiac and pharyngeal/head muscles share a common origin in the cardiopharyngeal mesoderm, but the chromatin landscapes that govern multipotent progenitors competence and early fate choices remain largely elusive. Here, we leveraged the simplicity of the chordate model Ciona to profile chromatin accessibility through stereotyped transitions from naive Mesp+ mesoderm to distinct fate-restricted heart and pharyngeal muscle precursors. An FGF-Foxf pathway acts in multipotent progenitors to establish cardiopharyngeal-specific patterns of accessibility, which govern later heart vs. pharyngeal muscle-specific expression profiles, demonstrating extensive spatiotemporal decoupling between early cardiopharyngeal enhancer accessibility and late cell-type-specific activity. We found that multiple cis-regulatory elements, with distinct chromatin accessibility profiles and motif compositions, are required to activate Ebf and Tbx1/10, two key determinants of cardiopharyngeal fate choices. We propose that these 'combined enhancers' foster spatially and temporally accurate fate choices, by increasing the repertoire of regulatory inputs that control gene expression, through either accessibility and/or activity.

A single-cell transcriptional roadmap for cardiopharyngeal fate diversification.

In vertebrates, multipotent progenitors located in the pharyngeal mesoderm form cardiomyocytes and branchiomeric head muscles, but the dynamic gene expression programmes and mechanisms underlying cardiopharyngeal multipotency and heart versus head muscle fate choices remain elusive. Here, we used single-cell genomics in the simple chordate model Ciona to reconstruct developmental trajectories forming first and second heart lineages and pharyngeal muscle precursors and characterize the molecular underpinnings of cardiopharyngeal fate choices. We show that FGF-MAPK signalling maintains multipotency and promotes the pharyngeal muscle fate, whereas signal termination permits the deployment of a pan-cardiac programme, shared by the first and second heart lineages, to define heart identity. In the second heart lineage, a Tbx1/10-Dach pathway actively suppresses the first heart lineage programme, conditioning later cell diversity in the beating heart. Finally, cross-species comparisons between Ciona and the mouse evoke the deep evolutionary origins of cardiopharyngeal networks in chordates.

Growth Effects on Velopharyngeal Anatomy From Childhood to Adulthood.

Purpose The observed sexual dimorphism of velopharyngeal structures among adult populations has not been observed in the young child (4- to 9-year-old) population. The purpose of this study was to examine the age at which sexual dimorphism of velopharyngeal structures become apparent and to examine how growth trends vary between boys and girls. Method Static 3-dimensional magnetic resonance imaging velopharyngeal data were collected among 202 participants ranging from 4 to 21 years of age. Participants were divided into 3 groups based on age, including Group 1: 4-10 years of age, Group 2: 11-17 years of age, and Group 3: 18-21 years of age. Nine velopharyngeal measures were obtained and compared between groups. Results Significant sex effects were evident for levator length ( p = .011), origin to origin ( p = .018), and velopharyngeal ratio ( p = .036) for those in Group 2 (11-17 years of age). Sex effects became increasingly apparent with age, with 7 of 9 variables becoming significantly different between male and female participants in Group 3. Boys, in general, displayed a delayed growth peak in velopharyngeal growth compared to girls. Conclusion Results from this study demonstrate the growth of velopharyngeal anatomy with sexual dimorphism becoming apparent predominantly after 18 years of age. However, velopharyngeal variables displayed variable growth trends with some variables presenting sexual dimorphism at an earlier age compared to other velopharyngeal variables.

Developmental finite element analysis of cichlid pharyngeal jaws: Quantifying the generation of a key innovation.

Advances in imaging and modeling facilitate the calculation of biomechanical forces in biological specimens. These factors play a significant role during ontogenetic development of cichlid pharyngeal jaws, a key innovation responsible for one of the most prolific species diversifications in recent times. MicroCT imaging of radiopaque-stained vertebrate embryos were used to accurately capture the spatial relationships of the pharyngeal jaw apparatus in two cichlid species (Haplochromis elegans and Amatitlania nigrofasciata) for the purpose of creating a time series of developmental stages using finite element models, which can be used to assess the effects of biomechanical forces present in a system at multiple points of its ontogeny. Changes in muscle vector orientations, bite forces, force on the neurocranium where cartilage originates, and stress on upper pharyngeal jaws are analyzed in a comparative context. In addition, microCT scanning revealed the presence of previously unreported cement glands in A. nigrofasciata. The data obtained provide an underrepresented dimension of information on physical forces present in developmental processes and assist in interpreting the role of developmental dynamics in evolution.

Fgf receptors Fgfr1a and Fgfr2 control the function of pharyngeal endoderm in late cranial cartilage development.

Cranial cartilage derives mainly from cranial neural crest cells and its formation requires fibroblast growth factor (Fgf) signaling for early differentiation and survival of developing chondrocytes as well as patterning of the endodermal pouches. Here, we investigate the role of Fgf receptors in chondrocyte maturation at later stages, beyond 24 hpf. Using inducible expression of a dominant-negative Fgf receptor, we show that Fgf signaling is required around 30 hpf for correct cartilage formation. The receptor genes fgfr1a and fgr2 are expressed in pharyngeal endodermal pouches after 24 hpf or 26 hpf, respectively. Depletion of any of these two receptors by microinjection of antisense morpholinos results in severe defects in cartilage formation at 4 dpf and a decrease in expression of the late chondrocyte markers barx1 and runx2b. Although endodermal pouches are correctly formed and patterned, receptor knock down leads to decreased expression of runx3, egr1 and sox9b in this tissue, while expression of fsta, coding for a secreted BMP/Tgfß inhibitor, is clearly increased. Rescue experiments revealed that each Fgfr1a or Fgfr2 receptor is able to compensate for the loss of the other. Thus, we show that minimal amounts of Fgfr1a or Fgfr2 are required to initiate a regulatory cascade in pharyngeal endoderm reducing expression of fsta, thereby allowing correct BMP signaling to the maturing chondrocytes of the head cartilage.

Sternohyoid and diaphragm muscle form and function during postnatal development in the rat.

NEW FINDINGS: What is the central question of this study? Co-ordinated activity of the thoracic pump and pharyngeal dilator muscles is critical for maintaining airway calibre and respiratory homeostasis. Whilst postnatal maturation of the diaphragm has been well characterized, surprisingly little is known about the developmental programme in the airway dilator muscles. What is the main finding and its importance? Developmental increases in force-generating capacity and fatigue in the sternohyoid and diaphragm muscles are attributed to a maturational shift in muscle myosin heavy chain phenotype. This maturation is accelerated in the sternohyoid muscle relative to the diaphragm and may have implications for the control of airway calibre in vivo. The striated muscles of breathing, including the thoracic pump and pharyngeal dilator muscles, play a critical role in maintaining respiratory homeostasis. Whilst postnatal maturation of the diaphragm has been well characterized, surprisingly little is known about the developmental programme in airway dilator muscles given that co-ordinated activity of both sets of muscles is needed for the maintenance of airway calibre and effective pulmonary ventilation. The form and function of sternohyoid and diaphragm muscles from Wistar rat pups [postnatal day (PD) 10, 20 and 30] was determined. Isometric contractile and endurance properties were examined in tissue baths containing Krebs solution at 35°C. Myosin heavy chain (MHC) isoform composition was determined using immunofluorescence. Muscle oxidative and glycolytic capacity was assessed by measuring the activities of succinate dehydrogenase and glycerol-3-phosphate dehydrogenase using semi-quantitative histochemistry. Sternohyoid and diaphragm peak isometric force and fatigue increased significantly with postnatal maturation. Developmental myosin disappeared by PD20, whereas MHC2B areal density increased significantly from PD10 to PD30, emerging earlier and to a much greater extent in the sternohyoid muscle. The numerical density of fibres expressing MHC2X and MHC2B increased significantly during development in the sternohyoid. Diaphragm succinate dehydrogenase activity and sternohyoid glycerol-3-phosphate dehydrogenase activity increased significantly with age. Developmental increases in force-generating capacity and fatigue in the sternohyoid and diaphragm muscles are attributed to a postnatal shift in muscle MHC phenotype. The accelerated maturation of the sternohyoid muscle relative to the diaphragm may have implications for the control of airway calibre in vivo.

Implications of peripheral muscular and anatomical development for the acquisition of lingual control for speech production: a review.

OBJECTIVES: Normally developing children learn to produce intelligible speech during rapid, non-uniform growth of their articulators and other vocal tract structures. The purpose of this review is to focus attention on the consequences of peripheral growth and development for the acquisition of lingual control for speech production. This paper (1) reviews physiological underpinnings of tongue shaping and movements that are likely to be changing in young children; (2) estimates, from previously published studies, the net consequences of growth of multiple vocal tract structures on lingual control; (3) integrates our findings with the example of [R] production, and (4) highlights areas where further investigations would be most helpful. PATIENTS AND METHODS: The authors searched the literature, including the PubMed database, for studies of the development of muscle proteins, muscle fibers, and motor units of the tongue, and of the growth of the tongue, jaw, adenoids, soft and hard palates, oral and pharyngeal cavities, and the vocal tract as a whole. CONCLUSIONS: Substantial anatomical and muscular data sets focused on children from 1-4 years of age, and rigorous definitions of the tongue boundaries are needed.

Respiratory control and sternohyoid muscle structure and function in aged male rats: decreased susceptibility to chronic intermittent hypoxia.

Obstructive sleep apnoea syndrome (OSAS) is a common respiratory disorder characterized by chronic intermittent hypoxia (CIH). We have shown that CIH causes upper airway muscle dysfunction in the rat due to oxidative stress. Ageing is an independent risk factor for the development of OSAS perhaps due to respiratory muscle remodelling and increased susceptibility to hypoxia. We sought to examine the effects of CIH on breathing and pharyngeal dilator muscle structure and function in aged rats. Aged (18-20 months), male Wistar rats were exposed to alternating cycles of normoxia and hypoxia (90 s each; F(I)O(2)=5% O(2) at nadir) or sham treatment for 8h/day for 9 days. Following CIH exposure, breathing was assessed by whole-body plethysmography. In addition, sternohyoid muscle contractile and endurance properties were examined in vitro. Muscle fibre type and cross-sectional area, and the activity of key oxidative and glycolytic enzymes were determined. CIH had no effect on basal breathing or ventilatory responses to hypoxia or hypercapnia. CIH did not alter succinate dehydrogenase or glycerol phosphate dehydrogenase enzyme activities, myosin heavy chain fibre areal density or cross-sectional area. Sternohyoid muscle force and endurance were unaffected by CIH exposure. Since we have established that this CIH paradigm causes sternohyoid muscle weakness in adult male rats, we conclude that aged rats have decreased susceptibility to CIH-induced stress. We suggest that structural remodelling with improved hypoxic tolerance in upper airway muscles may partly compensate for impaired neural regulation of the upper airway and increased propensity for airway collapse in aged mammals.

Characterization of the effects of methylmercury on Caenorhabditis elegans.

The rising prevalence of methylmercury (MeHg) in seafood and in the global environment provides an impetus for delineating the mechanism of the toxicity of MeHg. Deleterious effects of MeHg have been widely observed in humans and in other mammals, the most striking of which occur in the nervous system. Here we test the model organism, Caenorhabditis elegans (C. elegans), for MeHg toxicity. The simple, well-defined anatomy of the C. elegans nervous system and its ready visualization with green fluorescent protein (GFP) markers facilitated our study of the effects of methylmercuric chloride (MeHgCl) on neural development. Although MeHgCl was lethal to C. elegans, induced a developmental delay, and decreased pharyngeal pumping, other traits including lifespan, brood size, swimming rate, and nervous system morphology were not obviously perturbed in animals that survived MeHgCl exposure. Despite the limited effects of MeHgCl on C. elegans development and behavior, intracellular mercury (Hg) concentrations (

Lifespan extension and increased pumping rate accompany pharyngeal muscle-specific expression of nfi-1 in C. elegans.

Caenorhabditis elegans nfi-1 belongs to the Nuclear Factor I (NFI) family of transcription factors known to regulate metazoan gene expression and development. We showed previously that loss of nfi-1 in worms results in multiple behavioral defects; slower pharyngeal pumping rate, impaired egg laying, defective motility, and a shortened life span. Here, we generated cell-type specific transgenic worms to determine the cells in which nfi-1 must be expressed to rescue the pharyngeal pumping defect. Expression of nfi-1 from the pharyngeal muscle-specific myo-2 promoter, but not from the F25B3.3 or myo-3 promoters, rescued the pharyngeal pumping defect of nfi-1 worms. Surprisingly, myo-2-driven nfi-1 expression also rescued the shortened lifespan of nfi-1 worms, demonstrating a possible cell-autonomous role of nfi-1 in pharyngeal muscle for both phenotypes. We propose some relationships between the pharyngeal pumping and lifespan phenotypes and potential mechanisms of nfi-1 function.

Histological analysis of palatopharyngeal muscle from children with snoring and obstructive sleep apnea syndrome.

Obstructive sleep apnea syndrome (OSAS) is an upper airway obstruction that occurs during the sleep. One of the suggested mechanisms involved in this process is a neuromuscular abnormality of the palatal muscles. Whether children with OSAS develop into OSAS adults, or children and adult OSAS are two distinct disorders occurring at different ages are questions to be answered. Here, we made the histological analysis of palatophryngeal muscle in 34 oral-breathing children of both genders, aged 5-12 years old, with hypertrophic tonsils and adenoids. According to the polysomnographic study the participants were divided into children without sleeping disorders (group I) and children with primary snoring (group II) or apnea (group III). The main histological findings were fiber size variability in 70% cases from groups II and III and in 71% from group I; perimysial connective tissue infiltration in 48% children from groups II and III and in 71% from group I; intracytoplasmatic mitochondrial proliferation in 63% cases from groups II and III and in 57% cases from group I. Muscle necrosis was only observed in one case, in association with subglandular inflammation. Others findings observed in all groups included fibers with internal architecture alteration, such as moth-eaten and lobulated fibers, type 2 fiber predominance, and small areas of fiber type grouping. The presence of similar histological findings in the palatopharyngeal muscle in children with primary snoring or apnea but also in children without sleeping disorders indicate that such changes could be a normal histological feature of this muscle rather than a neurogenic or myopathic pathology.

Hyperactivation of the G12-mediated signaling pathway in Caenorhabditis elegans induces a developmental growth arrest via protein kinase C.

The G(12) type of heterotrimeric G-proteins play an important role in development and behave as potent oncogenes in cultured cells. However, little is known about the molecular nature of the components that act in the G(12)-signaling pathway in an organism. We characterized a C. elegans Galpha subunit gene, gpa-12, which is a homolog of mammalian G(12)/G(13)alpha, and found that animals defective in gpa-12 are viable. Expression of activated GPA-12 (G(12)QL) results in a developmental growth arrest caused by a feeding behavior defect that is due to a dramatic reduction in pharyngeal pumping. To elucidate the molecular nature of the signaling pathways in which G(12) participates, we screened for suppressors of the G(12)QL phenotype. We isolated 50 suppressors that contain mutations in tpa-1, which encodes two protein kinase C isoforms, TPA-1A and TPA-1B, most similar to PKCtheta/delta. TPA-1 mediates the action of the tumor promoter PMA. Expression of G(12)QL and treatment of wild-type animals with PMA induce an identical growth arrest caused by inhibition of larval feeding, which is dependent on TPA-1A and TPA-1B function. These results suggest that TPA-1 is a downstream target of both G(12) signaling and PMA in modulating feeding and growth in C. elegans. Taken together, our findings provide a potential molecular mechanism for the transforming capability of G(12) proteins.

Hyoid apparatus and pharynx in the lion (Panthera leo), jaguar (Panthera onca), tiger (Panthera tigris), cheetah (Acinonyxjubatus) and domestic cat (Felis silvestris f. catus).

Structures of the hyoid apparatus, the pharynx and their topographical positions in the lion, tiger, jaguar, cheetah and domestic cat were described in order to determine morphological differences between species or subfamilies of the Felidae. In the lion, tiger and jaguar (species of the subfamily Pantherinae) the Epihyoideum is an elastic ligament lying between the lateral pharyngeal muscles and the Musculus (M.) thyroglossus rather than a bony element like in the cheetah or the domestic cat. The M. thyroglossus was only present in the species of the Pantherinae studied. In the lion and the jaguar the Thyrohyoideum and the thyroid cartilage are connected by an elastic ligament, whereas in the tiger there is a synovial articulation. In adult individuals of the lion, tiger and jaguar the ventral end of the tympanohyal cartilage is rotated and therefore the ventral end of the attached Stylohyoideum lies caudal to the Tympanohyoideum and the cranial base. In newborn jaguars the Apparatus hyoideus shows a similar topographical position as in adult cheetahs or domestic cats. In adult Pantherinae, the Basihyoideum and the attached larynx occupy a descended position: they are situated near the cranial thoracic aperture, the pharyngeal wall and the soft palate are caudally elongated accordingly. In the Pantherinae examined the caudal end of the soft palate lies dorsal to the glottis. Differences in these morphological features between the subfamilies of the Felidae have an influence on specific structural characters of their vocalizations.

Postnatal changes in the types of muscle fibre in the canine inferior pharyngeal constrictor.

Deglutition is considered to be immature in infants and to mature postnatally. We evaluated postnatal changes in muscle fibre type composition in the canine inferior pharyngeal constrictor muscle, which consists of the thyropharyngeal (TP) and cricopharyngeal (CP) muscles, using ATPase staining with respect to the maturation of deglutition. In the TP muscle type IIA and type IIB fibres, the main components in the adult, were already predominant at 1 week postnatally. The percentage of primitive type IIC fibre showed a rapid reduction and reached the adult level within 6 weeks. In the CP muscle, the majority of fibres were type IIC at 2 weeks. At 2 months, more than 20% of the fibres were still type IIC and the proportion of type I fibres as a main component in the adult was smaller than that of the adult. None of the puppies younger than 9 weeks old had a fibre type composition similar to that of the adult. In the extensor digitorum longus and flexor digitorum superficialis, the compositions of muscle fibre types became similar to that of the adult at 6 and 9 weeks of age, respectively. Thus, the TP muscle matured more rapidly than the limb muscles, while the CP muscle matured more slowly. We speculated that the TP and CP muscles have specific individual differentiation patterns associated with their functional roles before and after birth, compared with the limb muscles.