Embryonic evidence uncovers convergent origins of laryngeal echolocation in bats.

Bats are the second-most speciose group of mammals, comprising 20% of species diversity today. Their global explosion, representing one of the greatest adaptive radiations in mammalian history, is largely attributed to their ability of laryngeal echolocation and powered flight, which enabled them to conquer the night sky, a vast and hitherto unoccupied ecological niche. While there is consensus that powered flight evolved only once in the lineage, whether laryngeal echolocation has a single origin in bats or evolved multiple times independently remains disputed. Here, we present developmental evidence in support of laryngeal echolocation having multiple origins in bats. This is consistent with a non-echolocating bat ancestor and independent gain of echolocation in Yinpterochiroptera and Yangochiroptera, as well as the gain of primitive echolocation in the bat ancestor, followed by convergent evolution of laryngeal echolocation in Yinpterochiroptera and Yangochiroptera, with loss of primitive echolocation in pteropodids. Our comparative embryological investigations found that there is no developmental difference in the hearing apparatus between non-laryngeal echolocating bats (pteropodids) and terrestrial non-bat mammals. In contrast, the echolocation system is developed heterotopically and heterochronically in the two phylogenetically distant laryngeal echolocating bats (rhinolophoids and yangochiropterans), providing the first embryological evidence that the echolocation system evolved independently in these bats.

Descent of the human larynx: An unrecognized factor in airway distress in babies with cleft palate?

The intranarial epiglottis, a feature of all newborn mammals, allows suckling and breathing to continue almost simultaneously by separating an oral food stream from a nasal airstream. In contrast to other mammals, the human larynx descends in the neck between birth and six months, extending the distance between the caudal aspect of the soft palate and the cephalic tip of the epiglottis. The mechanism of airway protection changes from a pattern in which an upright epiglottis is grasped by an intact palatopharyngeal sphincter to one in which the epiglottis folds down over the laryngeal aditus and the adducted vocal folds. The comparative anatomy and anthropological literature describing laryngeal descent was reviewed. A series of MRI images were used to illustrate the normal descent of the human larynx, which take place in infants in the first six months of life. Based on this information, we hypothesize that a cleft palate, by interrupting the sphincter function of palatopharyngeus on a high neonatal epiglottis, precipitates a need for premature and rapid maturation of the neonate's airway protection pattern, particularly during feeding. This may explain why, even in the absence of Robin sequence, some babies with cleft palates suffer respiratory distress during feeding.

The development of the human hyoid-larynx complex revisited.

OBJECTIVES/HYPOTHESIS: The hyoid-larynx complex is highly prone to anatomical variation. The etiology of anatomical variants such as Eagle's syndrome and the aberrant hyoid apparatus can be explained from embryonic development. Modern textbooks state that the hyoid bone body develops from the second and third pharyngeal arch cartilages, and that thyroid cartilage derives from the fourth and sixth arch cartilages. This description, however, is incompatible with various anatomical variants, and it is unclear whether it was based on observations in human embryos or on comparative embryology. STUDY DESIGN: 14 human embryos from the Carnegie collection between Carnegie stage 17 and 23 (42-60 days) were selected based on their histological quality. METHODS: Histological sections of the selected embryos were examined. Three-dimensional models were prepared in an interactive format. These anatomical models provide crucial spatial information and facilitate interpretation. RESULTS: We observed a less-complicated development of the hyoid-larynx complex than is currently described in textbooks. The body of the hyoid bone originates from a single growth center, without overt contributions from second and third pharyngeal arch cartilages. The fourth and sixth arch cartilages were not detected in human embryos; the thyroid and cricoid cartilages develop as mesenchymal condensations in the neck region. CONCLUSIONS: Despite new research techniques, theories about hyoid-larynx complex development from the beginning of the 20th century have not been refuted properly and can still be found in modern literature. Based on observations in human embryos, we propose a new and relatively simple description of the development of the hyoid-larynx complex to facilitate better understanding of the etiology of anatomical variants. LEVEL OF EVIDENCE: NA Laryngoscope, 1829-1834, 2018.

Evolution of Shh endoderm enhancers during morphological transition from ventral lungs to dorsal gas bladder.

Shh signalling plays a crucial role for endoderm development. A Shh endoderm enhancer, MACS1, is well conserved across terrestrial animals with lungs. Here, we first show that eliminating mouse MACS1 causes severe defects in laryngeal development, indicating that MACS1-directed Shh signalling is indispensable for respiratory organogenesis. Extensive phylogenetic analyses revealed that MACS1 emerged prior to the divergence of cartilaginous and bony fishes, and even euteleost fishes have a MACS1 orthologue. Meanwhile, ray-finned fishes evolved a novel conserved non-coding sequence in the neighbouring region. Transgenic assays showed that MACS1 drives reporter expression ventrally in laryngeal epithelium. This activity has been lost in the euteleost lineage, and instead, the conserved non-coding sequence of euteleosts acquired an enhancer activity to elicit dorsal epithelial expression in the posterior pharynx and oesophagus. These results implicate that evolution of these two enhancers is relevant to the morphological transition from ventral lungs to dorsal gas bladder.

Cilia-mediated Hedgehog signaling controls form and function in the mammalian larynx.

Acoustic communication is fundamental to social interactions among animals, including humans. In fact, deficits in voice impair the quality of life for a large and diverse population of patients. Understanding the molecular genetic mechanisms of development and function in the vocal apparatus is thus an important challenge with relevance both to the basic biology of animal communication and to biomedicine. However, surprisingly little is known about the developmental biology of the mammalian larynx. Here, we used genetic fate mapping to chart the embryological origins of the tissues in the mouse larynx, and we describe the developmental etiology of laryngeal defects in mice with disruptions in cilia-mediated Hedgehog signaling. In addition, we show that mild laryngeal defects correlate with changes in the acoustic structure of vocalizations. Together, these data provide key new insights into the molecular genetics of form and function in the mammalian vocal apparatus.

Pediatric airway surgery.

Pediatric airway surgery is a challenging field in pediatric surgery. Laryngotracheal stenosis has a variety of congenital and acquired conditions that require precise assessment and tailored treatment for each individual patient. About 90% of acquired conditions are represented by subglottic stenosis (SGS) resulting as a complication of tracheal intubation. Congenital tracheal stenosis (CTS) is a rare and life-threatening malformation, usually associated with complete tracheal rings along a variable length of the trachea. Tracheomalacia (TM) is a process characterized by flaccidity of the supporting tracheal cartilage, widening of the posterior membranous wall, and reduced anterior-posterior airway caliber. The clinical presentation can vary from almost asymptomatic patients to near fatal airway obstruction. There is considerable variation in both the morphologic subtypes and the prognosis of pediatric airway. The patients are divided into three clinical groups (mild, moderate, and severe). A further division was proposed according to the presence or absence of associated anomalies. The definitive diagnosis of pediatric airway was made by means of rigid bronchoscope and computed tomography scan with three-dimensional reconstruction (3D-CT). Rigid bronchoscopy and 3D-CT confirmed the diagnosis in all the cases. Other associated anomalies include congenital heart disease, vascular anomalies, and BPFM (maldevelopment of aerodigestive tract). After definitive diagnosis of pediatric airway lesions, surgical intervention should be considered. Surgical strategy was presented on each lesion.

Switching of the Laryngeal Cavity From the Respiratory Diverticulum to the Vestibular Recess: A Study Using Serial Sagittal Sections of Human Embryos and Fetuses.

A cecum-like protrusion of the pharynx (the laryngeal cecum or vestibular recess [VR]) develops immediately anterior to the laryngeal part of the respiratory diverticulum. An expansion of the VR has been well described, whereas the fate of the diverticulum is still obscure, although its pharyngeal opening corresponds to the glottis. We observed sagittal sections of 10 embryos (five specimens at 5-6 weeks and another five at 7-8 weeks) and eight fetuses at 25-30 weeks. At 5-6 weeks, a lumen of the laryngeal part of the respiratory diverticulum appeared, and subsequently, the VR opened into the epithelial lamina. Because of this discrete separation, it seemed unlikely that the pharyngeal pouches contributed to the laryngeal epithelium. At 6-7 weeks, the VR exhibited a high boot-shaped lumen with canalization to the diverticular lumen at the level of the cricoid cartilage. Thus, in a midline area between the bilateral arytenoid cartilages, double laryngeal lumina were evident, separated by the thick midline epithelial lamina. At 25-30 weeks, the inferior part of the VR lumen had become enlarged because of the destruction of the epithelial lamina along the arytenoid and corniculate cartilages. In contrast, candidates for the initial diverticular lumen remained as epithelial slits in the anterosuperior side of the transverse arytenoid muscle. Therefore, the final anterior and lateral laryngeal walls seemed to originate from the VR with canalization, in contrast to the part of the posterior wall derived from the initial diverticular wall.

Management of congenital tracheal anomalies and laryngotracheoesophageal clefts.

Congenital obstructions and anomalies of the pediatric airway are rare problems that may be associated with mild symptoms or critical stenoses that may be life threatening in the first few days of life. This review provides an overview of the embryologic development of the airway, different congenital anomalies associated with airway development, and surgical correction that may be associated with good long-term outcome.

Development of the rat larynx: a histological study.

OBJECTIVES/HYPOTHESIS: To evaluate and describe the cartilaginous and muscular development of the rat larynx. STUDY DESIGN: Histologic evaluation. METHODS: The larynges of Sprague Dawley rats of embryonic day (E) 13, 15, 17, 19, 21, postnatal day 0, 14, and adult of 250 gm were collected. Four larynges of each age were harvested, cut into 15-µm serial sections, stained with hematoxylin and eosin, and evaluated under light microscopy. Representative digital images were recorded and evaluated at the preglottic (supraglottic in humans), glottic, and postglottic (subglottic in humans) levels. RESULTS: Brachial arches were observed at E13. At E17, immature structures of the larynx, including skeletal muscle, cartilage, and the lumen were identifiable. Chondrification and muscle formation were clearly seen by E19. The muscular and cartilagenous components of the larynx were well established by E21. During the span between birth and adult maturation, the size of the larynx increased from a height of 1.10 mm to 2.90 mm, and from a width of 1.80 mm to 5.40 mm, and from a length of 1.38 mm to 4.77 mm in the stained section. Although developed at E21, the laryngeal structures continued to grow by approximately 30%. CONCLUSION: Rat laryngeal development parallels that in mice and humans. In the rat, at E17 immature structures of the larynx are identifiable, they are well developed at birth and grow by approximately 30% into adulthood. Understanding the chronology and morphology of the embryogenesis of the rat laryngeal musculature is essential and will allow for further evaluation of the embryologic innervation of these muscles.

Embryologic innervation of the rat laryngeal musculature--a model for investigation of recurrent laryngeal nerve reinnervation.

OBJECTIVES/HYPOTHESIS: Optimal management of vocal fold paralysis would entail recurrent laryngeal nerve (RLN) reinnervation resulting in normal vocal fold motion. Unfortunately, RLN reinnervation currently results in a nonfunctional vocal fold due to synkinetic reinnervation. Therapeutic interventions that guide regenerating axons back to the appropriate muscle would prevent synkinesis and restore vocal fold and glottal function. The initial step toward developing these therapies is the elucidation of the embryologic innervation of the larynx. This study aimed to identify the age of occurrence, timing, and pattern of embryologic innervation of the rat larynx, hypothesizing that differences in these parameters exist between distinct laryngeal muscles. STUDY DESIGN: Descriptive anatomic study. METHODS: The larynx of rats aged embryologic day (E) 15, 16, 17, 19, and 21 were harvested and then sectioned. Two rats were used for each age. Sections were colabeled with neuronal class III ß-tubulin polyclonal antibody to identify the presence of axons and alexa 488 conjugate α-bungarotoxin to identify the presence of motor endplates. The age at which axons and motor endplates were first present was noted. The position and pattern of the axons and motor endplates was recorded in relation to each other as well as the musculoskeletal anatomy of the larynx. The time at which axons appeared to innervate the medial thyroarytenoid (MTA) muscle, lateral thyroarytenoid (LTA) muscle, and the posterior cricoarytenoid (PCA) muscle was documented. RESULTS: Findings in the rat suggest the RLN reaches the larynx and begins branching by E15. Axons branch dorsally first and reach the PCA muscle before the other muscles. Branching toward the MTA muscle occurs only after axons have reached the LTA muscle. By E19, RLN axons have been guided to and selected their respective muscles with formation of neuromuscular junctions (NMJs) in the PCA, LTA and MTA muscles, though the formation of NMJs in the MTA muscle was comparatively delayed. CONCLUSIONS: This study describes the embryologic innervation of the rat larynx and suggests that there are distinct differences in the age of occurrence, timing, and pattern of innervation of the PCA, LTA, and MTA muscles of the rat. These findings lay the foundation for studies investigating the role of guidance cues in RLN axon guidance and the utility of these cues in the treatment of RLN injury via the stimulation of functional, nonsynkinetic reinnervation.

Morphogenesis of the human laryngeal ventricles.

BACKGROUND: Two theories explain the origin of human laryngeal ventricles: (1) ventricles derive from the fifth pharyngeal pouches; (2) development independent from the pouches. METHODS: In all, there were 21 serially sectioned human embryos from stages 15 to 23, and 11 fetuses of 9 to 18 weeks. Computer-aided 3-dimentional reconstructions were made. RESULTS: The cranial part of the laryngeal sulcus and future vestibule expands from the pharyngeal floor between the third and fourth pharyngeal pouches during stages 15 and 16 (33-37 days). The primordia of the ventricles appeared at stage 18 (44 days) as 2 lateral swellings in the caudal end of the future vestibule, limited by the third pharyngeal pouches. Active epithelial expansion and subsequent canalization during late embryonic and early fetal periods finalizes their development. CONCLUSIONS: The laryngeal ventricles do not derive from the pharyngeal pouches but the median region of the pharyngeal floor between the third and fourth pharyngeal pouches and arches at the caudal end of the future vestibule.

The fetal larynx and pharynx: structure and development on two- and three-dimensional ultrasound.

OBJECTIVES: To present a systematic approach for evaluating the fetal pharynx and larynx based on two- and three-dimensional ultrasound (2D-US and 3D-US) modalities, describing the sonographic appearance and function of the fetal upper respiratory tract and measuring the anatomical components of the pharynx and larynx. METHODS: Gravidae presenting from the late first trimester to mid-gestation for routine booked examinations with structurally normal singleton fetuses of confirmed gestational age were enrolled. Transabdominal 2D-US was performed for anatomical and functional evaluation of the pharynx and larynx. Color Doppler was used to show fluid motion in the target area. 3D-US (Voluson® E6 with RAB-4-8-D transducer) scans of the fetal neck were acquired during fetal quiescence and in the absence of movements of the pharynx and larynx. Multiplanar reconstruction (MPR) in post-processing allowed adjustment of the volume to obtain the coronal plane. After a learning period to understand the sonographic anatomy of the target area, we measured the pharynx width and height, the upper, middle and lower larynx width and the larynx height. Render mode was applied for spatial evaluation of the target area. We developed a new methodological approach for structured evaluation of the fetal pharynx and larynx based on five spatial planes: posterior and anterior coronal planes and high, mid and low axial planes. RESULTS: We examined 582 fetuses during the second trimester of pregnancy; target anatomy was imaged successfully in 218 patients at 11-24 gestational weeks. Acquisition added approximately 1 min to examination time. Rates of successful visualization and measurements increased significantly as pregnancy progressed, being 23% (46/194) at 11-13 weeks, 29% (69/240) at 14-16 weeks, 35% (18/51) at 17-19 weeks and 88% (85/97) at 20-24 weeks (P < 0.01). Pharynx components identified were: the sphenoid bone, pterygoid processes, constrictor muscles, piriform recesses and uvula. Larynx components identified were: the epiglottis, aryepiglottic folds, corniculate cartilages, arytenoid cartilages, cricoid cartilage, thyroid cartilage and vocal cords. MPR showed the biconcave shape of the uvula, which may explain the 'equals sign' observed on 2D-US. We observed the bilateral mode of movements of the constrictor muscles, aryepiglottic folds and vocal cords, and the bidirectional fluid jet flows through the larynx. Scatterplots of measured structures vs gestational age were created. Pharynx width ranged from 0.11 to 0.93 (mean ± SD, 0.48 ± 0.17) cm; pharynx height ranged from 0.23 to 2.01 (mean ± SD, 0.94 ± 0.34) cm; upper larynx width ranged from 0.04 to 0.37 (mean ± SD, 0.15 ± 0.07) cm; middle larynx width ranged from 0.08 to 0.77 (mean ± SD, 0.34 ± 0.16) cm; lower larynx width ranged from 0.05 to 0.64 (mean ± SD, 0.24 ± 0.11) cm; and larynx height ranged from 0.20 to 1.83 (mean ± SD, 0.71 ± 0.31) cm. All measurements were positively correlated with gestational age. CONCLUSIONS: The fetal larynx and pharynx can be evaluated thoroughly using 2D- and 3D-US modalities. Knowledge of normal anatomy, function and biometry may prove useful in the evaluation of anatomical or functional pathology involving the fetal upper respiratory tract. Recognition of anatomical anomalies may enhance fetal intervention such as balloon placement in cases of diaphragmatic hernia.

Long-term outcomes after fetal therapy for congenital high airway obstructive syndrome.

BACKGROUND/PURPOSE: Congenital high airway obstructive syndrome (CHAOS) is a rare and devastating condition that is uniformly fatal without fetal intervention. We sought to describe fetal treatment and long-term outcomes of CHAOS at a single referral center. METHODS: The medical records of patients with fetal CHAOS evaluated at our center between 1993 and 2011 were reviewed. Maternal history, radiographic findings, antenatal management, and postnatal outcomes were compared. RESULTS: Twelve fetuses with CHAOS were identified. Eleven had concomitant hydrops at diagnosis. Six were electively terminated, and 2 had intra- or peripartum demise. Four patients underwent fetal intervention. Two underwent delivery via ex utero intrapartum treatment (EXIT) procedure with tracheostomy placement only, and 2 underwent fetal bronchoscopy with attempted wire tracheoplasty followed by EXIT with tracheostomy at delivery. All 4 patients who underwent EXIT were alive at last follow-up. One patient was ventilator and tracheostomy free and feeding by mouth. CONCLUSION: Long-term and tracheostomy-free survival is possible with appropriate fetal intervention even in the presence of hydrops. Fetal intervention earlier in pregnancy may improve long-term outcomes, but patient selection for intervention remains challenging. Magnetic resonance imaging may help select those patients for whom fetal intervention before EXIT delivery may be beneficial.

Changes of laryngeal parameters during intrauterine life.

OBJECTIVE: Increased care of fetal and neonatal airways has led to advances in neonatal medicine. The early diagnosis and treatment of respiratory diseases require a detailed knowledge of fetal airway anatomy and development. The aim of this study was to determine the anatomical development of the thyroid and cricoid cartilages and their structural variability during fetal life. MATERIAL AND METHODS: The study was performed on the thyroid and cricoid cartilages of 55 human fetal larynges of both sexes, between the ages of 13 and 27 weeks of intrauterine life. Numerous measurements of the thyroid and cricoid cartilages were performed. RESULTS: Correlations between the obtained results were calculated in relation to the crown-rump (C-R) length of human fetuses and to sex. The structural variability of the thyroid and cricoid cartilages of human male and female fetuses in subsequent weeks of intrauterine life was observed. In both genders a correlation between laryngeal size and fetal crown-rump length, regardless of sex, was found. The thyroid cartilage presents a sexual dimorphism. CONCLUSIONS: The results of this study can be useful in the analysis of prenatal examinations, and in planning the treatment of airway emergencies.

Anatomical dimensions of larynx, epiglottis and cricoid cartilage in foetuses and their relationship with crown rump length.

BACKGROUND: Advances in perinatal care and increased use of diagnostic and interventional procedures on foetal airway demand a clear understanding of their normal anatomy and development. This study is aimed at determining the normal dimensions of larynx, cricoid cartilage and epiglottis and their correlation to age and crown rump length (CRL). METHOD: After approval from institutional ethics committee and written parental informed consent, laryngeal measurements were taken from 79 foetuses of varying CRL from 30 to 299 mm. The entire larynx from the tip of epiglottis to lowest extension of thyroid gland was isolated and preserved. In small foetuses (30-165 mm) the measurements were taken under a dissecting microscope. In foetuses above the CRL of 165 mm, the measurements were taken with help of Vernier Calliper. RESULTS: Foetuses were analysed for dimensions based on CRL. The length of the larynx significantly increased in comparison to CRL (P < 0.001). Similar measurements were observed for transverse diameter, anteroposterior diameter and inter-cartilaginous distance of larynx, the anterior height of arch and posterior height of lamina of cricoid cartilage and the length and breadth of epiglottis. The internal anteroposterior and transverse diameter of cricoid cartilage showed no correlation with CRL. CONCLUSION: All the measured parameters of larynx, epiglottis and cricoid cartilage showed positive correlation with the CRL except the internal diameters of cricoid.