Rnf149-related is an FGF/MAPK-independent regulator of pharyngeal muscle fate specification

During embryonic development, cell fate specification gives rise to dedicated lineages that underlie tissue formation. In olfactores, which comprise tunicates and vertebrates, the cardiopharyngeal field is formed by multipotent progenitors to both cardiac and branchiomeric muscles. The ascidian Ciona is a powerful model to study the cardiopharyngeal fate specification with cellular resolution, as only 2 pairs of cardiopharyngeal multipotent progenitors give rise to the heart and to pharyngeal muscles (aka atrial siphon muscles, ASM). These progenitors are multilineage primed, in as much as they express a combination of early ASM- and heart-specific transcripts that become restricted to their corresponding precursors, following oriented asymmetric divisions. Here, we identify the primed gene Rnf149-related (Rnf149-r), which becomes restricted to the heart progenitors, but appears to regulate pharyngeal muscle fate specification in the cardiopharyngeal lineage. CRISPR/Cas9-mediated loss knock-out of Rnf149-r function impairs atrial siphon muscle morphogenesis, and down-regulates Tbx1/10 and Ebf, two key determinants of the pharyngeal muscle fate, while upregulating heart-specific gene expression. These phenotypes are reminiscent of loss of FGF-MAPK signaling in the cardiopharyngeal lineage, and integrated analysis of lineage-specific bulk RNA-seq profiling of loss-of-function perturbations identified a significant overlap between FGF-MAPK and Rnf149-r targets. However, functional interaction assays suggested the Rnf149-r does not directly modulate the activity of the FGF-MAPK-Ets1/2 pathway. Instead, we propose that Rnf149-r acts both in parallel to the FGF-MAPK signaling on shared targets, as well as on FGF-MAPK-independent targets through (a) separate pathway(s).

Parachute Technique for Head and Neck Free-Flap Inset

The treatment outcome of advanced squamous cell carcinoma involving the head and neck is well known to be dire and usually needs multimodality treatment even including optimal reconstruction after ablative surgery. When a significant area of the soft tissue is resected, reconstruction of oral cavity or pharynx needs to minimize morbidities while achieving adequate functional outcomes. For the better functional outcome, invasive approaching procedures such as lip and jaw splitting, or extensive floor of mouth or pharyngeal muscle ablation should be avoided. Without these surgical procedures, reconstructive surgeons may encounter technical difficulties in flap inset due to deep and narrow space after head and neck cancer resection. In a deep and narrow surgical defect, accurate approximation and suture is extremely difficult. Eventually, repeated flap manipulation and stretch might be inevitable, and even pedicle kinking or injury could happen. Herein, we suggested the “parachute” technique, which was generally used in blood vessels or aortic valve suturing in a narrow surgical field and for avoiding mismatched suture. We applied this “parachute” technique for free-flap inset to head and neck defect, and we herein report our successful outcomes.

Olfactory Stimulation Successfully Improves Swallowing Function of Aged Rats through Activating Central Neuronal Networks and Downstream DHPR-RyR-mediated Neuromuscular Activities

Presbyphagia is age-related changes in swallowing function, which imposes a high risk of aspiration in older adults. Considering olfactory stimulation (OS) can influence behavioral activities by modulating neuronal excitability, the present study aims to determine whether OS could improve the swallowing function of aged rats through activating the central neuronal networks and downstream muscular activities participated in the control of swallowing. Aged male Wistar rats received OS by inhaling a mixture of plant-based volatile molecules twice a day for 12 days were subjected to functional magnetic resonance imaging (fMRI) and c-fos, choline acetyltransferase (ChAT) immunostaining to detect the neuronal activities of the orbitofrontal cortex (OFC) and medullary nuclei engaged in swallowing control, respectively. The functional effects of OS on downstream pharyngeal muscle activity were examined by evaluating the dihydropyridine receptor-ryanodine receptor (DHPR-RyR) mediated intra-muscular Ca2 + expression, and analyzing the amplitude/frequency of muscle contraction, respectively. In untreated rats, only moderate signal of fMRI and mild c-fos/ChAT expression was detected in the OFC and medullary nuclei, respectively. However, following OS, intense signals of fMRI and immunostaining were clearly expressed in the orbitofronto-medullary networks. Functional data corresponded well with above findings in which OS significantly enhanced DHPR-RyR-mediated intra-muscular Ca2 + expression, effectively facilitated a larger amplitude of pharyngeal muscle contraction, and exhibited better performance in consuming larger amounts of daily dietary. As OS successfully activates the neuromuscular activities participated in the control of swallowing, applying OS may serve as an effective, easy, and safe strategy to greatly improve the swallow function of aging populations.

The Influence on Airflow Characteristics In Upper Airway In Sleep Apnea Hypopnea By The Disturbance of Pharyngeal Muscle Group Based on High Simulation of The Wall Flow Field

Background: To observe the influence on morphology and internal airflow characteristics in upper airway in sleep apnea hypopnea by the disturbance of pharyngeal muscle group based on high simulation of the wall flow field. Methods: One goat for experimental purpose bought by our hospital in December 2018 was included as the research object. This experimental goat received injection of hardener and submucosal injection edema into pharyngeal muscle group of upper respiratory tract. The goat received CT scan before and after injection. Computational fluid dynamics(CFD) model was built on the base of CT scans by 3Dslicer 4.5 and MeshLab and ANSYS ICEM CFD 14.0. The internal flow of upper respiratory tract was simulated by ANSYS-FLUENT 14.0 and the results were analyzed by ANSYS-CFD-POST 14.0. Results: Soft palate and uvula were elongated and thickened and pharyngeal muscle group was disturbed after injection of hardener and submucosal injection edema into the goat. The area that changed the most of upper airway was located in the lower bound of pharyngopalatiae, and it reduced area from 0.3602cm2 to 0.1699cm2; the air velocity was elevated from 3.53009m/s to 7.24478m/s, the negative pressure was elevated from -28.6184 Pa to -66.4510 Pa, while the resistance of cavum pharyngis elevated from 3396.09Pas/L to 3813.65Pas/L. Conclusion: Injection of hardener and submucosal injection edema into pharyngeal muscle group of the goat would result in the disturbance of pharyngeal muscle group, narrow the upper respiratory tract, elevate the negative pressure and resistance of the upper respiratory tract in this area, bring about the collapsibility of the upper airway, obstruct the patency of the airflow and form the pathological model of OSAHS at last.

Cardiopharyngeal deconstruction and ancestral tunicate sessility

A key problem in understanding chordate evolution has been the origin of sessility of ascidians, and whether the appendicularian free-living style represents a primitive or derived condition of tunicates. To address this problem, we performed comprehensive developmental and genomic comparative analyses of the cardiopharyngeal gene regulatory network (GRN) between appendicularians and ascidians. Our results reveal that the cardiopharyngeal GRN has suffered a process of evolutionary deconstruction with massive ancestral losses of genes (Mesp, Ets1/2, Gata4/5/6, Mek1/2, Tbx1/10, and RA- and FGF-signaling related genes) and subfunctions (e.g. FoxF, Islet, Ebf, Mrf, Dach and Bmp signaling). These losses have led to the deconstruction of two modules of the cardiopharyngeal GRN that in ascidians are related to early and late multipotent state cells involved in lineage fate determination towards first and secondary heart fields, and siphon muscle. Our results allow us to propose an evolutionary scenario, in which the evolutionary deconstruction of the cardiopharyngeal GRN has had an adaptive impact on the acceleration of the developmental cardiac program, the redesign of the cardiac architecture into an open-wide laminar structure, and the loss of pharyngeal muscle. Our findings, therefore, provide evidence supporting that the ancestral tunicate had a sessile ascidian-like lifestyle, and points to the deconstruction of the cardiopharyngeal GRN in appendicularians as a key event that facilitated the evolution of their pelagic free-living style connected to the innovation of the house.

Intrinsic and extrinsic factors collaborate to activate pharyngeal satellite cells without muscle injury

Satellite cells (SCs), adult muscle stem cells in craniofacial muscles proliferate and differentiate/fuse without injury, unlike quiescent SCs in uninjured limb muscle. However, whether intrinsic or extrinsic factors driving their increased basal activity are largely unknown. We compared SCs from the pharynx, which contains constrictor muscles critical for swallowing, to SCs from limb muscle. Pharyngeal SCs are intrinsically more proliferative and contain higher mitochondrial content relative to limb SCs. Pharyngeal SCs occupy less quiescent microenvironments containing collagen V and pharyngeal muscles provide a distinctive SC niche enriched with neighboring resident macrophages and fibroadipogenic progenitors. Loss of SCs impacts pharyngeal myofiber cross-sectional area and the number of neighboring cells, suggesting that SCs are required to maintain pharyngeal muscle homeostasis and its unique niche. Taken together, this study gives new insights to explain the distinctive SC activity of craniofacial muscles, which may explain their unique susceptibility to various muscular dystrophies.

Morphine alters respiratory control but not other key obstructive sleep apnoea phenotypes: a randomised trial

Accidental opioid-related deaths are increasing. These often occur during sleep. Opioids such as morphine may worsen obstructive sleep apnoea (OSA). Thus, people with OSA may be at greater risk of harm from morphine. Possible mechanisms include respiratory depression and reductions in drive to the pharyngeal muscles to increase upper airway collapsibility. However, the effects of morphine on the four key phenotypic causes of OSA (upper airway collapsibility (pharyngeal critical closure pressure; Pcrit), pharyngeal muscle responsiveness, respiratory arousal threshold and ventilatory control (loop gain) during sleep) are unknown.21 males with OSA (apnoea–hypopnoea index range 7–67 events·h−1) were studied on two nights (1-week washout) according to a double-blind, randomised, cross-over design (ACTRN12613000858796). Participants received 40 mg of MS-Contin on one visit and placebo on the other. Brief reductions in continuous positive airway pressure (CPAP) from the therapeutic level were delivered to induce airflow limitation during non-rapid eye movement (REM) sleep to quantify the four phenotypic traits. Carbon dioxide was delivered via nasal mask on therapeutic CPAP to quantify hypercapnic ventilatory responses during non-REM sleep.Compared to placebo, 40 mg of morphine did not change Pcrit (−0.1±2.4 versus −0.4±2.2 cmH2O, p=0.58), genioglossus muscle responsiveness (−2.2 (−0.87 to −5.4) versus −1.2 (−0.3 to −3.5) μV·cmH2O−1, p=0.22) or arousal threshold (−16.7±6.8 versus −15.4±6.0 cmH2O, p=0.41), but did reduce loop gain (−10.1±2.6 versus −4.4±2.1, p=0.04) and hypercapnic ventilatory responses (7.3±1.2 versus 6.1±1.5 L·min−1, p=0.006).Concordant with recent clinical findings, 40 mg of MS-Contin does not systematically impair airway collapsibility, pharyngeal muscle responsiveness or the arousal threshold in moderately severe OSA patients. However, consistent with blunted chemosensitivity, ventilatory control is altered.

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.