Tracheal Branches Research Articles

Coupled membranes: a mechanism of frequency filtering and transmission in the field cricket ear evidenced by micro-computed tomography, laser Doppler vibrometry and finite element analysis.

Many animals employ a second frequency filter beyond the initial filtering of the eardrum (or tympanal membrane). In the field cricket ear, both the filtering mechanism and the transmission path from the posterior tympanal membrane (PTM) have remained unclear. A mismatch between PTM vibrations and sensilla tuning has prompted speculations of a second filter. PTM coupling to the tracheal branches is suggested to support a transmission pathway. Here, we present three independent lines of evidence converging on the same conclusion: the existence of a series of linked membranes with distinct resonant frequencies serving both filtering and transmission functions. Micro-computed tomography (µ-CT) highlighted the 'dividing membrane (DivM)', separating the tracheal branches and connected to the PTM via the dorsal membrane of the posterior tracheal branch (DM-PTB). Thickness analysis showed the DivM to share significant thinness similarity with the PTM. Laser Doppler vibrometry indicated the first of two PTM vibrational peaks, at 6 and 14 kHz, originates not from the PTM but from the coupled DM-PTB. This result was corroborated by µ-CT-based finite element analysis. These findings clarify further the biophysical source of neuroethological pathways in what is an important model of behavioural neuroscience. Tuned microscale coupled membranes may also hold biomimetic relevance.

The sex of organ geometry

Organs have a distinctive yet often overlooked spatial arrangement in the body1–5. We propose that there is a logic to the shape of an organ and its proximity to its neighbours. Here, by using volumetric scans of many Drosophila melanogaster flies, we develop methods to quantify three-dimensional features of organ shape, position and interindividual variability. We find that both the shapes of organs and their relative arrangement are consistent yet differ between the sexes, and identify unexpected interorgan adjacencies and left–right organ asymmetries. Focusing on the intestine, which traverses the entire body, we investigate how sex differences in three-dimensional organ geometry arise. The configuration of the adult intestine is only partially determined by physical constraints imposed by adjacent organs; its sex-specific shape is actively maintained by mechanochemical crosstalk between gut muscles and vascular-like trachea. Indeed, sex-biased expression of a muscle-derived fibroblast growth factor-like ligand renders trachea sexually dimorphic. In turn, tracheal branches hold gut loops together into a male or female shape, with physiological consequences. Interorgan geometry represents a previously unrecognized level of biological complexity which might enable or confine communication across organs and could help explain sex or species differences in organ function.

Real-time study of spatio-temporal dynamics (4D) of physiological activities in alive biological specimens with different FOVs and resolutions simultaneously

This article reports the development of a microscopy imaging system that gives feasibility for studying spatio-temporal dynamics of physiological activities of alive biological specimens (over entire volume not only for a particular section, i.e., in 4D). The imaging technology facilitates to obtain two image frames of a section of the larger specimen (∼mm\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$\\sim \ ext {mm}$$\\end{document}) with different FOVs at different resolutions or magnifications simultaneously in real-time (in addition to recovery of 3D (volume) information). Again, this imaging system addresses the longstanding challenges of housing multiple light sources (6 at the maximum till date) in microscopy (in general) and light sheet fluorescence microscopy (LSFM) (in particular), by using a tuneable pulsed laser source (with an operating wavelength in the range ∼420\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$\\sim 420$$\\end{document}–670 nm) in contrast to the conventional CW laser source being adopted for inducing photo-excitation of tagged fluorophores. In the present study, we employ four wavelengths (∼\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$\\sim$$\\end{document} 488 nm, 585 nm, 590 nm, and 594 nm). Our study also demonstrates quantitative characterization of spatio-temporal dynamics (velocity—both amplitude and direction) of organelles (mitochondria) and their mutual correlationships. Mitochondria close to the nucleus (or in clustered cells) are observed to possess a lower degree of freedom in comparison to that at the cellular periphery (or isolated cells). In addition, the study demonstrates real-time observation and recording of the development and growth of all tracheal branches during the entire period (∼95\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$\\sim 95$$\\end{document} min) of embryonic development (Drosophila). The experimental results—with experiments being conducted in various and diversified biological specimens (Drosophila melanogaster, mouse embryo, and HeLa cells)—demonstrate that the study is of great scientific impact both from the aspects of technology and biological sciences.

Detection of tracheal branching with computerized tomography: The relationship between the angles and age-gender.

The data obtained on the anatomical knowledge of the tracheobronchial system can be used for diagnosis, treatment and interventional interventions in areas such as anesthesia, thoracic surgery, pulmonary physiology. To determine the tracheobronchial branching angles in pediatric and adult populations by using the multislice computed tomography (CT) and minimum intensity projection (MinIP) technique, which is a non-invasive method. Our study was carried out retrospectively. Patients who underwent contrast and non-contrast CT examination, whose anatomically and pathophysiologically good tracheobronchial system and lung parenchyma images were obtained, were included in the study. Measurements were made in the coronal plane of the lung parenchyma. In the coronal plane, right main bronchus-left main bronchus angle, right upper lobe bronchus-intermedius bronchus angle, right middle lobe bronchus-right lower lobe bronchus angle, left upper lobe bronchus-left lower lobe bronchus angle were measured. The study population consisted of 1511 patients, 753 pediatric (mean age: 13.4 ± 4.3; range: 1-18 years) and 758 adults (mean age: 54.3 ± 17.3; range: 19-94 years). In our study, tracheal bifurcation angle was found to be 73.3° ± 13.7° (59.6°-87°) in the whole population. In the pediatric group, the right-left main coronal level was found to be higher in boys compared to girls (74.6° ± 12.9° vs 71.2° ± 13.9°, P = 0.001). In the adult group, the right-left main coronal level was found to be lower in males compared to females (71.9° ± 12.9° vs 75.8° ± 14.7°, P < 0.001). Our study, with the number of 1511 patients, is the first study in the literature with the largest number of patient populations including pediatric and adult demographic data, measuring the angle values of the tracheobronchial system using multislice CT and MinIP technique. Study data will not only be a guide during invasive procedures, but it can also guide studies to be done with imaging methods.

Comparative Anatomy of the Insect Tracheal System Part 1: Introduction, Apterygotes, Paleoptera, Polyneoptera

A broad comparative study of insect respiratory morphology is presented. Tracheae, epidermal invaginations extending into the body in branching networks of tubes, supply tissues with direct access to air for gas exchange. While previous tracheal studies focused on a handful of taxa and lacked in consistency, here a unified system of tracheal nomenclature is established using visualizations from micro-CT scanning of representatives from apterygotes, Paleoptera, and Polyneoptera, totaling 29 species, 29 genera, and 26 families in 13 insect orders. Three-dimensional visualizations of named tracheal branches establish robust assessments of homology and provide a framework for further studies across class Insecta. Patterns in respiratory architecture are presented along with a discussion of future investigations into phylogenetic and physiological questions.

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.

Sodium dichloroisocyanurate toxicity in rats during a 90-day inhalation toxicity study

Sodium dichloroisocyanurate-96% (NaDCC) is commonly used to treat drinking water, industrial water, and wastewater. However, exposure to NaDCC by inhalation can have toxic pulmonary effects in humans. In the present study, we evaluated the potential toxicity of NaDCC following a 90-day inhalation toxicity study in Sprague-Dawley/Crl:CD (SD) rats. The animals were exposed to 0.4, 2.0, or 10.0 mg/m3 NaDCC for 90 days. In addition, male and female rats from the 10.0 mg/m3 group were set up as the recovery group for 14 days. The bronchoalveolar lavage fluid showed a concentration-dependent increase in the total cell count, with a significant increase in neutrophils in both the sexes in the 10.0 mg/m3 group compared to the negative control group. In the 10.0 mg/m3 group, lung organ weight was significantly increased among the female rats. Histopathological examination showed eosinophilic droplets in the olfactory/respiratory epithelium, mucous cell hyperplasia, atrophy/degeneration of the tracheal branches, and wall thickening of the alveolar ducts in the nasal cavity of both sexes in the 10.0 mg/m3 group. The adverse effects of NaDCC exposure were observed to decrease during the 14-day recovery period in both sexes. Based on pathological observations, the “no observed adverse effect concentration (NOAEC)” of inhaled NaDCC was 2.0 mg/m3 for both sexes. These results are expected to provide a scientific basis for inhalation toxicity data of NaDCC.

Metabolic control of progenitor cell propagation during Drosophila tracheal remodeling

Adult progenitor cells in the trachea of Drosophila larvae are activated and migrate out of niches when metamorphosis induces tracheal remodeling. Here we show that in response to metabolic deficiency in decaying tracheal branches, signaling by the insulin pathway controls the progenitor cells by regulating Yorkie (Yki)-dependent proliferation and migration. Yki, a transcription coactivator that is regulated by Hippo signaling, promotes transcriptional activation of cell cycle regulators and components of the extracellular matrix in tracheal progenitor cells. These findings reveal that regulation of Yki signaling by the insulin pathway governs proliferation and migration of tracheal progenitor cells, thereby identifying the regulatory mechanism by which metabolic depression drives progenitor cell activation and cell division that underlies tracheal remodeling.

A review of the hexapod tracheal system with a focus on the apterygote groups

Respiratory systems are key innovations for the radiation of terrestrial arthropods. It is therefore surprising that there is still a considerable lack of knowledge. In this review of the available information on tracheal systems of hexapods (with a focus on the apterygote lineages Protura, Collembola, Diplura, Archaeognatha and Zygentoma), we summarize available data on the spiracles (number, position and morphology), the shape and variability of tracheal branching patterns including anastomoses, the tracheal fine structure and the respiratory proteins. The available data are strongly fragmented, and information for most subgroups is missing. In various cases, individual observations for one species account for the knowledge of the entire order. The available data show that there are strong differences between but also within apterygote orders. We conclude that the available data are insufficient to derive detailed conclusions on the hexapod ground plan and outline the possible evolutionary scenarios for the tracheal system in this group.

A Matrix Metalloproteinase Mediates Tracheal Development in Bombyx mori.

The trachea of insects is a tubular epithelia tissue that transports oxygen and other gases. It serves as a useful model for the studying of the cellular and molecular events involved in epithelial tube formation. Almost all of the extracellular matrix can be degraded by Matrix metalloproteinases (MMPs), which is closely related to the processes of development and regeneration. The regulation of trachea by MMPs is roughly known in previous studies, but the detailed regulation mechanism and involved gene function are not fully explored. In this article, we found MMP1 expressed highly during tracheal remodeling, and knocked out it makes the tracheal branch number reduced in Bombyx mori. In trachea of transgenic BmMMP1-KO silkworm, the space expanding of taenidium and epidermal cells and the structure of apical membrane were abnormal. To explore the underlying mechanism, we detected that DE-cadherin and Integrin β1 were accumulated in trachea of transgenic BmMMP1-KO silkworm by immunohistochemistry. Moreover, 5-Bromo-2′-Deoxyuridine (BrdU) labeling showed that knockout of BmMMP1 in silkworm inhibited tracheal cell proliferation, and BmMMP1 also regulated the proliferation and migration of BmNS cells. All of the results demonstrated that BmMMP1 regulates the development of the tracheal tissue by expanding the space of tracheal cuticles and increases the number of tracheal branches by degrading DE-cadherin and Integrin β1.

Robotic Intraoperative Tracheobronchial Repair during Minimally Invasive 3-Stage Esophagectomy

Tracheobronchial injury (TBI) is an uncommon but potentially fatal event. Iatrogenic lesions during bronchoscopy, endotracheal intubation, or thoracic surgery are considered the most common causes of TBI. When TBI is detected during surgery, concomitant surgical treatment is recommended. Herein we present a case of successful robotic primary repair of iatrogenic tracheal and left bronchial branch tears during a robot-assisted hybrid 3-stage esophagectomy after neoadjuvant chemoradiotherapy. A robotic approach can facilitate the repair of this injury while reducing both the potential risk of conversion to open surgery and the associated increased risk of postoperative respiratory complications.

Lint, a transmembrane serine protease, regulates growth and metabolism in Drosophila.

Insulin signaling in Drosophila has a significant role in regulating growth, metabolism, fecundity, stress response, and longevity. The molecular mechanism by which insulin signaling regulates these vital processes is dependent on the nutrient status and oxygen availability of the organism. In a genetic screen to identify novel genes that regulate Drosophila insulin signaling, we discovered lumens interrupted (lint), a gene that has previously been shown to act in tracheal development. The knockdown of lint gene expression using a Dilp2Gal4 driver which expresses in the neuronal insulin producing cells (IPCs), led to defects in systemic insulin signaling, metabolic status and growth. However, our analysis of lint knockdown phenotypes revealed that downregulation of lint in the trachea and not IPCs was responsible for the growth phenotypes, as the Gal4 driver is also expressed in the tracheal system. We found various tracheal terminal branch defects, including reduction in the length as well as number of branches in the lint knockdown background. Our study reveals that substantial effects of lint downregulation arose because of tracheal defects, which induced tissue hypoxia, altered systemic insulin/TOR signaling, and resulted in effects on developmental growth regulation.

Unexpected tracheal agenesis with prenatal diagnosis of aortic coarctation, lung hyperecogenicity and polyhydramnios: a case report

BackgroundTracheal agenesis (TA) is a rare disorder usually diagnosed prenatally when a congenital high airway obstruction syndrome (CHAOS) is identified. We present a case of unexpected TA in a neonate without prenatal diagnosis of airway obstruction, with a difficult management at birth. Moreover, we discuss about differential diagnosis, classification and treatment issues.Case presentationA 2280 g female neonate was born at 35 week gestational age (GA) with prenatal diagnosis of aortic coarctation, polyhydramnios and diffuse hyperechogenicity of the right lung. At birth, the neonate had no audible cry, no air entry to the lungs, and hypotonia. Tracheal intubation was unsuccessful, and no visualization of the trachea was obtained when tracheostomy was attempted. Post-mortem examination showed tracheal agenesis associated with tracheoesophageal fistula and revealed no cardiologic malformations. Aortic coarctation had been suspected prenatally because of the first portion of the descendent thoracic aorta being compressed by a fibrous band connecting the proximal and distal tracheal branches. CHAOS had not developed due to the tracheoesophageal fistula (TOF).ConclusionsTA is not always diagnosed in the fetus and it may present unexpectedly making the neonate’s management at birth critical. An effective rescue temporary oxygenation may be obtained with mask ventilation or oesophageal intubation in those cases of TA associated with a TOF. We suggest to consider a fetal magnetic resonance imaging (MRI) when the association polyhydramnios/lung hyperechogenicity occurs, even in the absence of CHAOS or other malformations. Once a diagnosis is provided, the mother should be transferred to selected centres where an ex-utero intrapartum procedure (EXIT) can be attempted. Moreover, despite high mortality, different surgical management are described to improve survival.

Tracheal branching in ants is area-decreasing, violating a central assumption of network transport models.

The structure of tubular transport networks is thought to underlie much of biological regularity, from individuals to ecosystems. A core assumption of transport network models is either area-preserving or area-increasing branching, such that the summed cross-sectional area of all child branches is equal to or greater than the cross-sectional area of their respective parent branch. For insects, the most diverse group of animals, the assumption of area-preserving branching of tracheae is, however, based on measurements of a single individual and an assumption of gas exchange by diffusion. Here we show that ants exhibit neither area-preserving nor area-increasing branching in their abdominal tracheal systems. We find for 20 species of ants that the sum of child tracheal cross-sectional areas is typically less than that of the parent branch (area-decreasing). The radius, rather than the area, of the parent branch is conserved across the sum of child branches. Interpretation of the tracheal system as one optimized for the release of carbon dioxide, while readily catering to oxygen demand, explains the branching pattern. Our results, together with widespread demonstration that gas exchange in insects includes, and is often dominated by, convection, indicate that for generality, network transport models must include consideration of systems with different architectures.

Increased Pulmonary Function Following Administration of a CBD‐Containing Compound NCMB‐1 in Fibrotic Lungs of African Green Monkeys.

The endocannabinoid system is a neuromodulatory system that is conserved among vertebrate species. Cannabidiol (CBD) is a modulator of cannabinoid (CB) receptor action both in the central nervous system and in the periphery. CB receptors are involved in inflammatory responses in peripheral organs, including lung tissue. CBD administration has been shown to reduce airway inflammation and fibrosis in vitro and in experimentally induced rodent models of asthma through modulation of the immune system. However, little is understood regarding the relationship between pulmonary disease and CBD modulation of general CB receptors in vivo. The African Green Monkey (Chlorocebus aethiops sabaeus; AGM) has bronchiolar tissue and tracheal branching remarkably similar to that in human lungs, therefore making it a translational model for the study of CBD treatment on pulmonary fibrosis‐related lung dysfunction. We hypothesize that administration of NCMB‐1, a hemp extract with CBD, will increase the tidal volume, inspiratory flow rate, and respiration rate of animals with previously induced pulmonary fibrosis. Baseline tidal volume, inspiration flow rate, and respiration rate of 16 male AGM with induced lung fibrosis were measured. Animals were divided into a vehicle and 3 treatment groups: 50 μL of undiluted, 5X dilution, and 25X dilution NCMB‐1 administered orally twice daily via banana sections for two weeks. Compared to control (n=4, 206.6 ± 36.4 mL), 5X NCMB‐1 treated animals increased tidal volume (n=4, 484.0 ± 54.6 mL; p&lt;0.05) from baseline at two weeks of treatment. Inspiratory flow rate also increased in 5X treated animals (n=4, 412.0 ± 49.9 mL/s; p&lt;0.05) compared to control (n=4, 212.3 ± 50.7 mL/s) after two weeks of administration. Respiration rates increased from baseline in 1X treated animals (n=4, 33.0 ± 4.5 breaths/min) and 5X treated animals (n=4, 30.8 ± 3.9 breaths/min) compared to control (n=4, 28.5 ± 4.1 breaths/min; p&lt;0.05). Treatment with 25X dilution of NCMB‐1 did not alter pulmonary function at either one or two weeks of treatment compared to vehicle treated animals. Thus, administration of 5X diluted NCMB‐1 to AGM with induced pulmonary fibrosis increased tidal volume, inspiratory flow rate, and respiration rate from compared to vehicle treated AGM. These results indicate CBD as a potential modulator of CB receptors improving pulmonary function and suggest a potential for therapeutic value of CBD in the treatment of restrictive pulmonary disease. Future directions include identification of active CBD compounds within the extract responsible for changes in airway resistance, expression of specific pulmonary genes involved in reduction of inflammation, and immunohistochemistry of CB receptors in the hypothalamus of AGM with induced pulmonary fibrosis. CBD crossing the blood brain barrier in animals treated with NCMB‐1 may cause up or downregulation of CB1 and/or CB2 receptors that could positively impact lung function.Support or Funding InformationFunding provided by Primates Plus, LLC.

Investigating the role of a V‐ATPase in the Development of the Air Sac Primordia

Drosophila melanogaster is a commonly used organism in biological research. Their Air Sac Primordium (ASP) is analogous to the human lungs in that it supplies oxygen to the flight muscles. Furthermore, the ASP has emerged as a good structure to study cellular invasive processes with the potential to aid in our understanding of tumor metastasis. The ASP originates on the outside of the wing imaginal disc and then burrows into the wing imaginal disc as it develops. What genes are involved in this process is not well understood. A genetic screen for genes that are expressed in the ASP identified a proton pump gene encoding a V‐ATPase. The V‐ATPase is a vacuolar proton pump that plays a large array of roles from regulating the pH of cells internal space, to regulating endocytosis, to processing proteins and degradation. In insects, the V‐ATPase are important in establishing a membrane potential to drive K+ secretion. It has also been found to influence tracheal branching. Using RNA interference, the gene for the V‐ATPase was knocked down resulting in a defect in ASP development. Data from these and other experiments will be presented and their significance will be discussed.Support or Funding InformationKentucky Biomedical Research Infrastructure Network (KBRIN) GrantGatton Academy Research Internship Grant (RIG)Faculty Undergraduate Student Engagement (FUSE) Grant

РЕДКОЕ ОСЛОЖНЕНИЕ ХИРУРГИЧЕСКОГО ЛЕЧЕНИЯ РАКА ЩИТОВИДНОЙ ЖЕЛЕЗЫ

Thyroid cancer is the leading cancer in the endocrine system. Over the past few decades, the incidence of this pathology worldwide has increased at a higher rate than other cancers. The main method of treatment for thyroid cancer is surgical. Some of the complications that can result from surgical treatment are tracheomalacia and trachea damage. Tracheomalacia is a pathological softening of tracheal rings resulting from prolonged compression of the trachea resulting in degenerative changes in cartilages. As a result of the removal of the thyroid tumor, a defect of its wall may form. Damage of the trachea can occur when the thyroid isthmus is separated from it. In addition, when separating the thyroid gland from the trachea, performing central lymphodissection, the blood supply of the trachea may be disturbed, which is carried out according to the segmental type, characterized by a lack of the number of collaterals. There is presented a clinical case of successful treatment of a rare complication after thyroid surgery for papillary cancer in the form of trachea in a patient of 49 years. By analyzing the clinical example, taking into account the macroscopic picture of trachea defects, their location and dimensions, it is possible to assume segmental disorder of blood supply of the upper third of the trachea in the postoperative period with the development of ischemic changes and regions of necrosis of ring ligaments. In order to prevent such complications in thyroid operations, it is necessary to preserve the paratracheal vascular anastomotic network by carefully preparing the tracheal branches of the lower thyroid artery.

Unpredictable Effects of the Genetic Background of Transgenic Lines in Physiological Quantitative Traits.

Physiology, fitness and disease phenotypes are complex traits exhibiting continuous variation in natural populations. To understand complex trait gene functions transgenic lines of undefined genetic background are often combined to assess quantitative phenotypes ignoring the impact of genetic polymorphisms. Here, we used inbred wild-type strains of the Drosophila Genetics Reference Panel to assess the phenotypic variation of six physiological and fitness traits, namely, female fecundity, survival and intestinal mitosis upon oral infection, defecation rate and fecal pH upon oral infection, and terminal tracheal cell branching in hypoxia. We found continuous variation in the approximately 150 strains tested for each trait, with extreme values differing by more than four standard deviations for all traits. In addition, we assessed the effects of commonly used Drosophila UAS-RNAi transgenic strains and their backcrossed isogenized counterparts, in the same traits plus baseline intestinal mitosis and tracheal branching in normoxia, in heterozygous conditions, when only half of the genetic background was different among strains. We tested 20 non-isogenic strains (10 KK and 10 GD) from the Vienna Drosophila Resource Center and their isogenized counterparts without Gal4 induction. Survival upon infection and female fecundity exhibited differences in 50% and 40% of the tested isogenic vs. non-isogenic pairs, respectively, whereas all other traits were affected in only 10–25% of the cases. When 11 isogenic and their corresponding non-isogenic UAS-RNAi lines were expressed ubiquitously with Gal4, 4 isogenic vs. non-isogenic pairs exhibited differences in survival to infection. Furthermore, when a single UAS-RNAi line was crossed with the same Gal4 transgene inserted in different genetic backgrounds, the quantitative variations observed were unpredictable on the basis of pure line performance. Thus, irrespective of the trait of interest, the genetic background of commonly used transgenic strains needs to be considered carefully during experimentation.

Glia-derived exosomal miR-274 targets Sprouty in trachea and synaptic boutons to modulate growth and responses to hypoxia

Secreted exosomal microRNAs (miRNAs) mediate interorgan/tissue communications by modulating target gene expression, thereby regulating developmental and physiological functions. However, the source, route, and function in target cells have not been formally established for specific miRNAs. Here, we show that glial miR-274 non-cell-autonomously modulates the growth of synaptic boutons and tracheal branches. Whereas the precursor form of miR-274 is expressed in glia, the mature form of miR-274 distributes broadly, including in synaptic boutons, muscle cells, and tracheal cells. Mature miR-274 is secreted from glia to the circulating hemolymph as an exosomal cargo, a process requiring ESCRT components in exosome biogenesis and Rab11 and Syx1A in exosome release. We further show that miR-274 can function in the neurons or tracheal cells to modulate the growth of synaptic boutons and tracheal branches, respectively. Also, miR-274 uptake into the target cells by AP-2-dependent mechanisms modulates target cell growth. In the target cells, miR-274 down-regulates Sprouty (Sty) through a targeting sequence at the sty 3' untranslated region, thereby enhancing MAPK signaling and promoting cell growth. miR-274 expressed in glia of an mir-274 null mutant is released as an exosomal cargo in the circulating hemolymph, and such glial-specific expression resets normal levels of Sty and MAPK signaling and modulates target cell growth. mir-274 mutant larvae are hypersensitive to hypoxia, which is suppressed by miR-274 expression in glia or by increasing tracheal branches. Thus, glia-derived miR-274 coordinates growth of synaptic boutons and tracheal branches to modulate larval hypoxia responses.

Matrix metalloproteinase 1 modulates invasive behavior of tracheal branches during entry into Drosophila flight muscles.

Tubular networks like the vasculature extend branches throughout animal bodies, but how developing vessels interact with and invade tissues is not well understood. We investigated the underlying mechanisms using the developing tracheal tube network of Drosophila indirect flight muscles (IFMs) as a model. Live imaging revealed that tracheal sprouts invade IFMs directionally with growth-cone-like structures at branch tips. Ramification inside IFMs proceeds until tracheal branches fill the myotube. However, individual tracheal cells occupy largely separate territories, possibly mediated by cell-cell repulsion. Matrix metalloproteinase 1 (MMP1) is required in tracheal cells for normal invasion speed and for the dynamic organization of growth-cone-like branch tips. MMP1 remodels the CollagenIV-containing matrix around branch tips, which show differential matrix composition with low CollagenIV levels, while Laminin is present along tracheal branches. Thus, tracheal-derived MMP1 sustains branch invasion by modulating the dynamic behavior of sprouting branches as well as properties of the surrounding matrix.