Tracheal Development Research Articles

Micro-CT Imaging of Tracheal Development in Down Syndrome and Non-Down Syndrome Fetuses.

Down syndrome (DS) is associated with airway abnormalities including a narrowed trachea. It is uncertain whether this narrowed trachea in DS is a consequence of deviant fetal development or an acquired disorder following endotracheal intubation after birth. This study aimed to compare the tracheal morphology in DS and non-DS fetuses using microfocus computed tomography (micro-CT). Twenty fetal samples were obtained from the Dutch Fetal Biobank and divided into groups based on gestational age. Micro-CT images were processed to analyze tracheal length, volume, and cross-sectional area (CSA). Mean tracheal length and tracheal volume were similar in DS and non-DS fetuses for all gestational age groups. Mean, minimum, and maximal tracheal CSA were statistically significantly increased in the single DS fetus in the group of 21-24 weeks of gestation, but not in other gestational age groups. In 90% of all studied fetuses, the minimum tracheal CSA was located in the middle third of the trachea. Tracheal development in DS fetuses was similar to non-DS fetuses between 13 and 21 weeks of gestation. This suggests that the narrowed tracheal diameter in DS children may occur later in fetal development or results from postnatal intubation trauma. The narrowest part of the trachea is in majority of DS and non-DS fetuses the middle third. 3 Laryngoscope, 134:4389-4395, 2024.

A single-cell atlas of Drosophila trachea reveals glycosylation-mediated Notch signaling in cell fate specification

The Drosophila tracheal system is a favorable model for investigating the program of tubular morphogenesis. This system is established in the embryo by post-mitotic cells, but also undergoes remodeling by adult stem cells. Here, we provide a comprehensive cell atlas of Drosophila trachea using the single-cell RNA-sequencing (scRNA-seq) technique. The atlas documents transcriptional profiles of tracheoblasts within the Drosophila airway, delineating 9 major subtypes. Further evidence gained from in silico as well as genetic investigations highlight a set of transcription factors characterized by their capacity to switch cell fate. Notably, the transcription factors Pebbled, Blistered, Knirps, Spalt and Cut are influenced by Notch signaling and determine tracheal cell identity. Moreover, Notch signaling orchestrates transcriptional activities essential for tracheoblast differentiation and responds to protein glycosylation that is induced by high sugar diet. Therefore, our study yields a single-cell transcriptomic atlas of tracheal development and regeneration, and suggests a glycosylation-responsive Notch signaling in cell fate determination.

Synergistic Effects of SDS and H2O2 Combinations on Tracheal Scaffold Development: An In Vitro Study Using Goat Trachea.

Currently, a tissue-engineered trachea has been popularly used as a biological graft for tracheal replacement in severe respiratory diseases. In the development of tissue-engineered tracheal scaffolds, in vitro studies play a crucial role in allowing researchers to evaluate the efficacy and safety of scaffold designs and fabrication techniques before progressing to in vivo or clinical trials. This research involved the decellularization of goat trachea using SDS, H2O2, and their combinations. Various quantitative and qualitative assessments were performed, including histological analysis, immunohistochemistry, and biomechanical testing. Hematoxylin and eosin staining evaluated the cellular content, while safranin O-fast green and Masson's trichrome staining assessed glycosaminoglycan content and collagen distribution, respectively. The immunohistochemical analysis focused on detecting MHC-1 antigen presence. Tensile strength measurements were conducted to evaluate the biomechanical properties of the decellularized scaffolds. The results demonstrated that the combination of SDS and H2O2 for goat tracheal decellularization yielded scaffolds with minimal cellular remnants, low toxicity, preserved ECM, and high tensile strength and elasticity. This method holds promise for developing functional tracheal scaffolds to address severe respiratory diseases effectively.

A trade-off in evolution: the adaptive landscape of spiders without venom glands.

Venom glands play a key role in the predation and defense strategies of almost all spider groups. However, the spider family Uloboridae lacks venom glands and has evolved an adaptive strategy: they excessively wrap their prey directly with spider silk instead of paralyzing it first with toxins. This shift in survival strategy is very fascinating, but the genetic underpinnings behind it are poorly understood. Spanning multiple spider groups, we conducted multiomics analyses on Octonoba sinensis and described the adaptive evolution of the Uloboridae family at the genome level. We observed the coding genes of myosin and twitchin in muscles are under positive selection, energy metabolism functions are enhanced, and gene families related to tracheal development and tissue mechanical strength are expanded or emerged, all of which are related to the unique anatomical structure and predatory behavior of spiders in the family Uloboridae. In addition, we also scanned the elements that are absent or under relaxed purifying selection, as well as toxin gene homologs in the genomes of 2 species in this family. The results show that the absence of regions and regions under relaxed selection in these spiders' genomes are concentrated in areas related to development and neurosystem. The search for toxin homologs reveals possible gene function shift between toxins and nontoxins and confirms that there are no reliable toxin genes in the genome of this group. This study demonstrates the trade-off between different predation strategies in spiders, using either chemical or physical strategy, and provides insights into the possible mechanism underlying this trade-off. Venomless spiders need to mobilize multiple developmental and metabolic pathways related to motor function and limb mechanical strength to cover the decline in adaptability caused by the absence of venom glands.

Tracheal ulcer development from high volume, low-pressure cuffed endotracheal tubes

IntroductionIatrogenic injury to the larynx, particularly the vocal cords from prolonged intubation, has been well-studied; however, tracheal injuries are rarely reported. This study investigates the effectiveness of cuffed, high-volume, low-pressure endotracheal tubes in preventing the development of tracheal ulcers in intubated subjects. MethodsA retrospective, IRB-approved review was performed on 1355 subjects who underwent percutaneous tracheostomy from 2002 to 2018. The presence and severity of tracheal ulcers were collected using documentation and photos during percutaneous tracheostomy placement. Primary outcome measures included: the length of time on a ventilator until tracheostomy (LOVT), length of hospitalization (LOH), and mortality in relationship to the severity of the tracheal injury. Data was reported as n (%) and median (IQR). The differences in means between groups were analyzed by ANOVA and Chi-square test with an alpha of 0.05. Results206 subjects met the inclusion criteria; 65 subjects had an absence of tracheal injury, and 141 subjects developed tracheal ulcers. Subjects with tracheal ulcers were grouped by the following severity scale: no ulcer; mild ulcer (minimal mucosal erosion with exudate); moderate ulcer (mucosal erosion); and severe (tracheal ring exposure). There were no statistically significant differences in age (p = 0.99), gender (p = 0.83), BMI (p = 0.44), LOH (p = 0.88), LOVT (p = 0.93), and mortality (p = 0.306) between subjects with differing severity of ulcers. The average annual incidence of clinically significant ulcers (moderate and severe) was 2.2 %. ConclusionsThe lack of statistical correlation between the duration of intubation and tracheal ulcer severity, along with a low annual incidence of tracheal ulcers, supports the improved safety of high-volume, low-pressure cylindrical, cuffed endotracheal tubes. This study is among the first to specifically focus on injuries at the level of the cuff and tip of endotracheal tubes with implications in preventive measures and potential product design changes.

Wnt signaling regulates ion channel expression to promote smooth muscle and cartilage formation in developing mouse trachea.

Ion channels play critical roles in the physiology and function of the nervous system and contractile tissue; however, their role in noncontractile tissue and embryonic development has yet to be understood. Tracheobronchomalacia (TBM) and complete tracheal rings (CTR) are disorders affecting the muscle and cartilage of the trachea and bronchi, whose etiology remains poorly understood. We demonstrated that trachealis muscle organization and polarity are disrupted after epithelial ablation of Wntless (Wls), a cargo receptor critical for the Wnt signaling pathway, in developing trachea. The phenotype resembles the anomalous trachealis muscle observed after deletion of ion channel encoding genes in developing mouse trachea. We sought to investigate whether and how the deletion of Wls affects ion channels during tracheal development. We hypothesize that Wnt signaling influences the expression of ion channels to promote trachealis muscle cell assembly and patterning. Deleting Wls in developing trachea causes differential regulation of genes mediating actin binding, cytoskeleton organization, and potassium ion channel activity. Wnt signaling regulates the expression of Kcnj13, Kcnd3, Kcnj8, and Abcc9 as demonstrated by in vitro studies and in vivo analysis in Wnt5a and β-catenin-deficient tracheas. Pharmacological inhibition of potassium ion channels and Wnt signaling impaired contractility of developing trachealis smooth muscle and formation of cartilaginous mesenchymal condensation. Thus, in mice, epithelial-induced Wnt/β-catenin signaling mediates trachealis muscle and cartilage development via modulation of ion channel expression, promoting trachealis muscle architecture, contractility, and cartilaginous extracellular matrix. In turn, ion channel activity may influence tracheal morphogenesis underlying TBM and CTR.NEW & NOTEWORTHY Ion channels play critical roles in the physiology and function of the nervous system and contractile tissue; however, their role in noncontractile tissue and embryonic development has yet to be understood. In this study, we focused on the role of ion channels in the differentiation and patterning of the large airways of the developing respiratory tract. We identify a mechanism by which Wnt-beta-catenin signaling controls levels of ion channel-encoding genes to promote tracheal differentiation.

Live-bearing cockroach genome reveals convergent evolutionary mechanisms linked to viviparity in insects and beyond

Live birth (viviparity) has arisen repeatedly and independently among animals. We sequenced the genome and transcriptome of the viviparous Pacific beetle-mimic cockroach and performed comparative analyses with two other viviparous insect lineages, tsetse flies and aphids, to unravel the basis underlying the transition to viviparity in insects. We identified pathways undergoing adaptive evolution for insects, involved in urogenital remodeling, tracheal system, heart development, and nutrient metabolism. Transcriptomic analysis of cockroach and tsetse flies revealed that uterine remodeling and nutrient production are increased and the immune response is altered during pregnancy, facilitating structural and physiological changes to accommodate and nourish the progeny. These patterns of convergent evolution of viviparity among insects, together with similar adaptive mechanisms identified among vertebrates, highlight that the transition to viviparity requires changes in urogenital remodeling, enhanced tracheal and heart development (corresponding to angiogenesis in vertebrates), altered nutrient metabolism, and shifted immunity in animal systems.

The Lipid Phosphate Phosphatase Wunen Promotes Eggshell Formation and Is Essential for Fertility in Drosophila.

The eggshell that surrounds insect eggs acts as a barrier, protecting against biotic factors and desiccation. The eggshell is a multi-layered structure which is synthesised by the somatic follicle cells that surround the developing oocyte. Although the temporal order of expression of the protein eggshell components goes someway to explaining how the different layers are built up, but how the precise three-dimensional structure is achieved and how lipid components responsible for desiccation resistance are incorporated are poorly understood. In this paper, we demonstrate that wunen, which encodes a lipid phosphate phosphatase, is necessary for fertility in Drosophila females. Compared to sibling controls, females null for wunen lay fewer eggs which subsequently collapse such that no larvae emerge. We show that this is due to a requirement for wunen in the ovarian follicle cells which is needed to produce an ordered and functional eggshell. Knockdown of a septate junction component also results in collapsed eggs, supporting the idea that similar to its role in embryonic tracheal development, Wunen in follicle cells also promotes septate junction function.

Serpentine and Vermiform Are Produced Autonomously to Fulfill Their Function in Drosophila Wings

Group I chitin deacetylases (CDAs), CDA1 and CDA2, play an essential role in cuticle formation and molting in the process of insect wing development. A recent report showed that trachea are able to take up a secreted CDA1 (serpentine, serp) produced in the fat body to support normal tracheal development in the fruit fly Drosophila melanogaster. However, whether CDAs in wing tissue were produced locally or derived from the fat body remains an open question. To address this question, we applied tissue-specific RNAi against DmCDA1 (serpentine, serp) and DmCDA2 (vermiform, verm) in the fat body or the wing and analyzed the resulting phenotypes. We found that repression of serp and verm in the fat body had no effect on wing morphogenesis. RT-qPCR showed that RNAi against serp or verm in the fat body autonomously reduced their expression levels of serp or verm in the fat body but had no non-autonomous effect on the expression in wings. Furthermore, we showed that inhibition of serp or verm in the developing wing caused wing morphology and permeability deficiency. Taken together, the production of Serp and Verm in the wing was autonomous and independent of the fat body.

Development of the foregut and the formation of the trachea and esophagus in rat embryos. A symphony of confusion.

Introduction: During embryonic development, the trachea emerges from an area of the foregut, which is often referred to as "anterior" or "common" foregut tube or simply foregut. To explain this process of differentiation, four competing models exist to date. The outgrowth and watershed models propose a foregut that remains constant in length. In the outgrowth model, the trachea buds off and elongates from the foregut, while in the watershed model, a mesenchymal wedge splits the growing foregut into the trachea and esophagus. In contrast, the septation model proposes a cranial splitting and thus a shortening of the "common" foregut tube into the trachea and esophagus by an emerging septum. Finally, the splitting and extension model describes an interaction of cranial splitting of the foregut and simultaneous caudal tracheal and esophageal growth. Methods: Here we examine the development of the undifferentiated foregut by micro computed tomography, which allows precise measurements. Results: Our results show that this area of the foregut transforms into the larynx, a process, which is independent from tracheal and esophageal development. Discussion: These observations are only consistent with the outgrowth model.

NDR kinase tricornered genetically interacts with Ccm3 and metabolic enzymes in Drosophila melanogaster tracheal development.

The Germinal Center Kinase III (GckIII) pathway is a Hippo-like kinase module defined by sequential activation of Ste20 kinases Thousand and One (Tao) and GckIII, followed by nuclear dbf2-related (NDR) kinase Tricornered (Trc). We previously uncovered a role for the GckIII pathway in Drosophila melanogaster tracheal (respiratory) tube morphology. The trachea form a network of branched epithelial tubes essential for oxygen transport, and are structurally analogous to branched tubular organs in vertebrates, such as the vascular system. In the absence of GckIII pathway function, aberrant dilations form in tracheal tubes characterized by mislocalized junctional and apical proteins, suggesting that the pathway is important in maintaining tube integrity in development. Here, we observed a genetic interaction between trc and Cerebral cavernous malformations 3 (Ccm3), the Drosophila ortholog of a human vascular disease gene, supporting our hypothesis that the GckIII pathway functions downstream of Ccm3 in trachea, and potentially in the vertebrate cerebral vasculature. However, how GckIII pathway signaling is regulated and the mechanisms that underpin its function in tracheal development are unknown. We undertook biochemical and genetic approaches to identify proteins that interact with Trc, the most downstream GckIII pathway kinase. We found that known GckIII and NDR scaffold proteins are likely to control GckIII pathway signaling in tracheal development, consistent with their conserved roles in Hippo-like modules. Furthermore, we show genetic interactions between trc and multiple enzymes in glycolysis and oxidative phosphorylation, suggesting a potential function of the GckIII pathway in integrating cellular energy requirements with maintenance of tube integrity.

Systematic Analysis of Smooth Muscle and Cartilage Ring Formation during Mouse Tracheal Tubulogenesis.

The trachea tube is the exclusive route to allow gas exchange between the external environment and the lungs. Recent studies have shown the critical role of mesenchymal cells in tracheal tubulogenesis. Improved methods for studying the dynamics of the tracheal mesenchyme development are needed to investigate the cellular and molecular mechanisms during tracheal tubulogenesis. Here, we describe a detailed protocol for a systematic analysis of tracheal tube development to enable observing tracheal smooth muscle (SM) and cartilage ring formation. We describe immunostaining, confocal and stereomicroscopy imaging, and quantitative methods to study the process of tracheal SM and cartilage ring development, including SM cell alignment, polarization, and changes in cell shape as well as mesenchymal condensation. The technologies and approaches described here not only improve analysis of the patterning of the developing trachea but also help uncover the mechanisms underlying airway disease. This protocol also provides a useful technique to analyze cell organization, polarity, and nuclear shape in other organ systems.

Time-Lapse Imaging and Morphometric Analysis of Tracheal Development in Drosophila.

Detailed and quantitative analyses of the cellular events underlying the formation of specific organs or tissues is essential to understand the general mechanisms of morphogenesis and pattern formation. Observation of live tissues or whole-mount fixed specimens has emerged as the method of choice for identifying and quantifying specific cellular and tissular structures within the organism. In both cases, cell and subcellular structure identification and good quality image acquisition for these analyses are essential. Many markers for live imaging and fixed tissue are now available for detecting cell membranes, subcellular structures, and extracellular structures like the extracellular matrix (ECM). Combination of live imaging and analysis of fixed tissue is ideal to obtain a general and detailed picture of the events underlying embryonic development. By applying morphometric methods to both approaches, we can, in addition, obtain a quantitative evaluation of the specific parameters under investigation in morphogenetic and cell biological studies. In this chapter, we focus on the development of the tracheal system of Drosophila melanogaster, which provides an ideal paradigm to understand the formation of branched tubular organs. We describe the most used methods of imaging and morphometric analysis in tubulogenesis using mainly (but not exclusively) examples from embryonic development. We cover embryo preparation for fixed and live analysis of tubulogenesis, together with methods to visualize larval tracheal terminal cell branching and lumen formation. Finally, we describe morphometric analysis and quantification methods using fluorescent images of tracheal cells.

Expansion of ring-shaped supracellular contractile cables induces epithelial sheet folding.

The folding of epithelial cell sheets is a fundamental process that sculpts developing tissues and organs into their proper shapes required for normal physiological functions. In the absence of detailed biochemical regulations, the epithelial sheet folding may simply proceed through buckling due to mechanical compression arising extrinsically from the surroundings or intrinsically within the sheets. Previous studies hypothesized that the formation of an expanding supracellular actomyosin ring within epithelial sheets could result in compression that ultimately leads to epithelial folding during tracheal development in the Drosophila (fruit fly) embryo. However, the exact mechanism by which the formation of epithelial folds is coordinated by the ring expansion remains unclear. Using a vertex-based mechanical model, here I systematically study the dependence of epithelial fold formation on the physical properties of expanding supracellular contractile rings. The simulations show that depending on the contractile strength, epithelial cell sheets can undergo distinct patterns of folding during ring expansion. The formation of folds in particular is robust against fluctuations in the ring properties such as ring numbers and tensions. These findings provide a systematic view to understand how the expansion of supracellular contractile rings in epithelial sheets mediates epithelial folding morphogenesis.

Developmental basis of evolutionary lung loss in plethodontid salamanders.

One or more members of four living amphibian clades have independently dispensed with pulmonary respiration and lack lungs, but little is known of the developmental basis of lung loss in any taxon. We use morphological, molecular, and experimental approaches to examine the Plethodontidae, a dominant family of salamanders, all of which are lungless as adults. We confirm an early anecdotal report that plethodontids complete early stages of lung morphogenesis: Transient embryonic lung primordia form but regress by apoptosis before hatching. Initiation of pulmonary development coincides with expression of the lung-specification gene Wnt2b in adjacent mesoderm, and the lung rudiment expresses pulmonary markers Nkx2.1 and Sox9. Lung developmental-genetic pathways are at least partially conserved despite the absence of functional adult lungs for at least 25 and possibly exceeding 60 million years. Adult lung loss appears associated with altered expression of signaling molecules that mediate later stages of tracheal and pulmonary development.

Bi-allelic variants in WNT7B disrupt the development of multiple organs in humans

BackgroundPulmonary hypoplasia, Diaphragmatic anomalies, Anophthalmia/microphthalmia and Cardiac defects delineate the PDAC syndrome. We aim to identify the cause of PDAC syndrome in patients who do not carry pathogenic variants in...

Investigating Cell Fate Specification upon Nkx2‐1Loss

Nkx2‐1 has long been known to be essential for proper respiratory development, as its loss in mice results in an unseparated trachea with underdeveloped cystic lungs. However, how NKX2‐1 regulates distinct cell fates in the proximal airways is unknown. While Nkx2‐1 has been extensively studied for its role in lung adenocarcinomas, few studies have described the effects of Nkx2‐1 loss on the trachea beyond its role in early tracheal separation morphogenesis. In previously published work we have shown that the majority of genes differentially expressed in the trachea and esophagus, during early foregut development are not regulated by NKX2‐1 and maintain their expected dorsoventral expression patterns in the unseparated foregut upon NKX2‐1 loss. These results suggested that Nkx2‐1 may not be a master regulator of early respiratory fate, but may instead regulate specific cell fate programs during tracheal development. To address this question, we examined E16.5 and E18.5 Nkx2‐1Δ/Δand control mouse tracheas by immunofluorescent staining for tracheal cell types. Secretory cells (MUC5AC), multiciliated cells (acetylated α‐TUBULIN and FOXJ1), and Club cells (SCGB1A1) were all found in Nkx2‐1Δ/Δventral foreguts by immunofluorescence. While all expected proximal airway cell types were present in Nkx2‐1Δ/Δ tracheas, a disproportionate number of multiciliated cells were present in the Nkx2‐1Δ/Δforegut compared to control. This was not a consequence of effects on early respiratory induction, because tamoxifen‐induced loss of NKX2‐1 in Nkx2‐1CreER/loxembryos at E12.5 also resulted in overabundance of FOXJ1 positive cells at E16.5. Based on these results, while we conclude that NKX2‐1 is not required to define the early proximal respiratory epithelial lineage, it may instead be required for specification of proper proportions and distribution of respiratory cell types.

Evolution of holometaboly revealed by developmental transformation of internal thoracic structures in a green lacewing Chrysopa pallens (Neuroptera: Chrysopidae).

Despite extensive studies on the morphology of holometabolous insects and their larvae, the morphological transformations of internal structures during metamorphosis have been rarely documented. Here, we used micro-computed tomography to investigate the developmental transformations of thoracic structures in the green lacewing Chrysopa pallens (Rambur, 1838) (Neuroptera: Chrysopidae), with emphasis on the development of the digestive, tracheal, and thoracic skeleto-muscular system. All the adult organs were modified during the prepupal or early pupal stage. The histolysis and remodeling began with the skeletal elements, followed by changes in the digestive system before it concluded with modifications of the musculature. Similar to the tracheal system's development, the digestive system did not disappear completely throughout metamorphosis but underwent a dramatic morphological change, which included the midgut significantly decreasing in size during the pupal stage. Our results provide important evidence for understanding the evolutionary pattern of developmental transformations in different major lineages of Holometabola.

Roles of matrix metalloproteinases in development, immunology, and ovulation in fruit Fly (Drosophila).

Matrix metalloproteinase (MMP), a protease enzyme, participates in proteolytic cleavage of extracellular matrix proteins from Drosophila and mammals. But, recent studies have revealed other physiologically important roles of MMP in Drosophila. MMP contributes to cardioblast movement and distribution of collagen proteins during cardiogenesis in developing Drosophila. Tissue remodeling, especially tracheal development is also maintained by MMP. MMP regulates certain immunological functions in Drosophila such as wound repairing, plasmatocyte assemblage at the injured site of the basement membrane and glial response to axon degeneration in Drosophila nervous system. But, the contribution of MMP to tumor formation and metastasis in Drosophila has made it an interesting topic among researchers. Ovulation and egg laying are also found to be affected positively by MMP in Drosophila.

The History of Engineered Tracheal Replacements: Interpreting the Past and Guiding the Future.

The development of a tracheal graft to replace long-segment defects has thwarted clinicians and engineers alike for over 100 years. To better understand the challenges facing this field today, we have consolidated all published reports of engineered tracheal grafts used to repair long-segment circumferential defects in humans, from the first in 1898 to the most recent in 2018, totaling 290 clinical cases. Distinct trends emerge in the types of grafts used over time, including repair using autologous fascia, rigid tubes of various inert materials, and pretreated cadaveric allografts. Our analysis of maximum clinical follow-up, as a proxy for graft performance, revealed that the Leuven protocol has a significantly longer clinical follow-up time than all other methods of airway reconstruction. This method involves transplanting a cadaveric tracheal allograft that is first prevascularized heterotopically in the recipient. We further quantified graft-related causes of mortality, revealing failure modes that have been resolved, and those that remain a hurdle, such as graft mechanics. Finally, we briefly summarize recent preclinical work in tracheal graft development. In conclusion, we synthesized top clinical care priorities and design criteria to inform and inspire collaboration between engineers and clinicians toward the development of a functional tracheal replacement graft.Impact statementThe field of tracheal engineering has floundered in recent years due to multiple article retractions. However, with recent advances in biofabrication and tissue analysis techniques, the field remains ripe for advancement through collaboration between engineers and clinicians. With a long history of clinical application of tracheal replacements, engineered tracheas are arguably the regenerative technology with the greatest potential for translation. This work describes the many phases of engineered tracheal replacements that have been applied in human patients over the past 100 years with the goal of carrying forward critical lessons into development of the next generation of engineered tracheal graft.