Larval Tracheae Research Articles

MiRNA-mediated gene silencing in Drosophila larval development involves GW182-dependent and independent mechanisms.

MicroRNAs (miRNAs) regulate a wide variety of biological processes by silencing their target genes. Argonaute (AGO) proteins load miRNAs to form an RNA-induced silencing complex (RISC), which mediates translational repression and/or mRNA decay of the targets. A scaffold protein called GW182 directly binds AGO and the CCR4-NOT deadenylase complex, initiating the mRNA decay reaction. Although previous studies have demonstrated the critical role of GW182 in cultured cells as well as in cell-free systems, its biological significance in living organisms remains poorly explored, especially in Drosophila melanogaster. Here, we generated gw182-null flies using the CRISPR/Cas9 system and found that, unexpectedly, they can survive until an early second-instar larval stage. Moreover, in vivo miRNA reporters can be effectively repressed in gw182-null first-instar larvae. Nevertheless, gw182-null flies have defects in the expression of chitin-related genes and the formation of the larval trachea system, preventing them from completing larval development. Our results highlight the importance of both GW182-dependent and -independent silencing mechanisms in vivo.

Ectopic expression in commonly used transgenic Drosophila GAL4 driver lines.

Transgenic tools such as the GAL4/UAS system in Drosophila have been used extensively to induce spatiotemporally controlled changes in gene expression and tissue-specific expression of a range of transgenes. We previously discovered unexpected expression of the commonly used dilp2-GAL4 line in tracheal tissue which significantly impacted growth phenotypes. We realized that few GAL4 lines have been thoroughly characterized, particularly when considering transient activity that may have significant impact on phenotypic readouts. Here, we characterized a further subset of 12 reportedly tissue-specific GAL4 lines commonly used in genetic studies of development, growth, endocrine regulation, and metabolism. Ten out of 12 GAL4 lines exhibited ectopic activity in other larval tissues, with seven being active in the larval trachea. Since this ectopic activity may result in phenotypes that do not depend on the manipulation in the intended target tissue, it is recommended to carefully analyze the outcome while taking this aspect into consideration.

Abstract A026: Stochastic phenotypes in RAS-dependent developmental diseases

Abstract Germline mutations upregulating RAS signaling are associated with multiple developmental disorders. A hallmark of these conditions is that the same mutation may present vastly different phenotypes in different individuals, even in monozygotic twins. We demonstrate how the origins of such largely unexplained phenotypic variations may be dissected using highly controlled studies in Drosophila that have been gene edited to carry activating variants of MEK, a core enzyme in the RAS pathway. This allowed us to measure the small but consistent increase in signaling output of such alleles in vivo. The fraction of mutation carriers reaching adulthood was strongly reduced, but most surviving animals had normal RAS-dependent structures. We rationalize these results using a stochastic signaling model and support it by quantifying cell fate specification errors in bilaterally symmetric larval trachea, a RAS-dependent structure that allows us to isolate the effects of mutations from potential contributions of genetic modifiers and environmental differences. We propose that the small increase in signaling output shifts the distribution of phenotypes into a regime, where stochastic variation causes defects in some individuals, but not in others. Our findings shed light on phenotypic heterogeneity of developmental disorders caused by deregulated RAS signaling and offer a framework for investigating causal effects of other pathogenic alleles and mild mutations. Citation Format: Robert A. Marmion, Alison G. Simpkins, Lena A. Barrett, Jodi Schottenfeld-Roames, Trudi Schüpbach, Stanislav Y. Shvartsman. Stochastic phenotypes in RAS-dependent developmental diseases [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr A026.

Lateral adherens junctions mediate a supracellular actomyosin cortex in drosophila trachea

Drosophila trachea is a classical model for analyzing epithelial, especially tubular epithelial biology. We identify lateral E-cadherin mediated junctions that encircle the cells just basal to the zonula adherens in the larval trachea. The lateral junction is associated with downstream adapters, including catenins, and has a distinct junctional actin cortex. The lateral cortex contributes to the development of a supracellular actomyosin mesh in the late larvae. Establishment of this cytoskeletal structure depends on lateral junction associated Rho1 and Cdc42 GTPases, and Arp and WASP pathways. The supracellular network takes the character of stress fibers along the AP axis in the early hours of pupation. It contributes to the shortening of the epithelial tube albeit in a manner redundant to ECM-mediated compression mechanism. In conclusion, we show the invivo existence of functional lateral adherens junction and suggest a role for it in mediating dynamic cytoskeletal events during tissue scale morphogenesis.

Stochastic phenotypes in RAS-dependent developmental diseases

Germline mutations upregulating RAS signaling are associated with multiple developmental disorders. A hallmark of these conditions is that the same mutation may present vastly different phenotypes in different individuals, even in monozygotic twins. Here, we demonstrate how the origins of such largely unexplained phenotypic variations may be dissected using highly controlled studies in Drosophila that have been gene edited to carry activating variants of MEK, a core enzyme in the RAS pathway. This allowed us to measure the small but consistent increase in signaling output of such alleles invivo. The fraction of mutation carriers reaching adulthood was strongly reduced, but most surviving animals had normal RAS-dependent structures. We rationalize these results using a stochastic signaling model and support it by quantifying cell fate specification errors in bilaterally symmetric larval trachea, a RAS-dependent structure that allows us to isolate the effects of mutations from potential contributions of genetic modifiers and environmental differences. We propose that the small increase in signaling output shifts the distribution of phenotypes into a regime, where stochastic variation causes defects in some individuals, but not in others. Our findings shed light on phenotypic heterogeneity of developmental disorders caused by deregulated RAS signaling and offer a framework for investigating causal effects of other pathogenic alleles and mild mutations in general.

Genome-wide in vitro and in vivo RNAi screens reveal Fer3 to be an important regulator of kkv transcription in Drosophila.

Krotzkopf verkehrt (kkv) is a key enzyme that catalyzes the synthesis of chitin, an important component of the Drosophila epidermis, trachea, and other tissues. Here, we report the use of comprehensive RNA interference (RNAi) analyses to search for kkv transcriptional regulators. A cell-based RNAi screen identified 537 candidate kkv regulators on a genome-wide scale. Subsequent use of transgenic Drosophila lines expressing RNAi constructs enabled in vivo validation, and we identified six genes as potential kkv transcriptional regulators. Weakening of the kkvDsRed signal, an in vivo reporter indicating kkv promoter activity, was observed when the expression of Akirin, NFAT, 48 related 3 (Fer3), or Autophagy-related 101(Atg101) was knocked down in Drosophila at the 3rd-instar larval stage; whereas we observed disoriented taenidial folds on larval tracheae when Lines (lin) or Autophagy-related 3 (Atg3) was knocked down in the tracheae. Fer3, in particular, has been shown to be an important factor in the activation of kkv transcription via specific binding with the kkv promoter. The genes involved in the chitin synthesis pathway were widely affected by the downregulation of Fer3. Furthermore, Atg101, Atg3, Akirin, Lin, NFAT, Pnr, and Abd-A showed that the potential complex mechanism of kkv transcription is regulated by an interaction network with bithorax complex components. Our study revealed the hitherto unappreciated diversity of modulators impinging on kkv transcription and opens new avenues in the study of kkv regulation and chitin biosynthesis.

Control of hormone-driven organ disassembly by ECM remodeling and Yorkie-dependent apoptosis

Epithelia grow and shape into functional structures during organogenesis. Although most of the focus on organogenesis has been drawn to the building of biological structures, the disassembly of pre-existing structures is also an important event to reach a functional adult organ. Examples of disassembly processes include the regression of the Müllerian or Wolffian ducts during gonad development and mammary gland involution during the post-lactational period in adult females. To date, it is unclear how organ disassembly is controlled at the cellular level. Here, we follow the Drosophila larval trachea through metamorphosis and show that its disassembly is a hormone-driven and precisely orchestrated process. It occurs in two phases: first, remodeling of the apical extracellular matrix (aECM), mediated by matrix metalloproteases and independent of the actomyosin cytoskeleton, results in a progressive shortening of the entire trachea and a nuclear-to-cytoplasmic relocalization of the Hippo effector Yorkie (Yki). Second, a decreased transcription of the Yki target, Diap1, in the posterior metameres and the activation of caspases result in the apoptotic loss of the posterior half of the trachea while the anterior half escapes cell death. Thus, our work unravels a mechanism by which hormone-driven ECM remodeling controls sequential tissue shortening and apoptotic cell removal through the transcriptional activity of Yki, leading to organ disassembly during animal development.

RNAi for chitin synthase 1 rather than 2 causes growth delay and molting defect in Henosepilachna vigintioctopunctata

Chitin synthase (CHS) plays a critical role in chitin synthesis and excretion. In most insects, CHSs have been segregated into 1 and 2 classes. CHS1 is responsible for chitin production in the ectodermally-derived epidermal cells. CHS2 is dedicated to chitin biosynthesis in the midgut peritrophic matrix (PM). Henosepilachna vigintioctopunctata is a serious pest of Solanaceae and Cucurbitaceae plants. In this study, we identified HvCHS1 and HvCHS2. We found that HvCHS1 was abundantly transcribed in the larval tracheae and epidermis, whereas HvCHS2 was mainly expressed in the guts. Escherichia coli HT115 expressed double stranded RNAs targeting HvCHS1 and HvCHS2 (dsCHS1 and dsCHS2) were used to immerse potato foliage and the treated leaves were provided to the newly-molted fourth- and third-instar larvae. Ingestion of dsCHS1 by the fourth-instar larvae significantly diminished the target mRNA level and had slight influence on the expression of HvCHS2. In contrast, consumption of dsCHS2 significantly lowered the target mRNA level but triggered the transcription of HvCHS1. Knockdown of HvCHS1, rather than HvCHS2, arrested larval development and impaired larva-pupa-adult transition. A large proportion of HvCHS1 hypomorphs became stunting prepupae, deformed pupae or misshapen adults. Moreover, knockdown of HvCHS1 damaged gut integrity, decreased cuticle thickness, and delayed the formation of newly-generated cuticle layer during ecdysis. Furthermore, depletion of HvCHS1 inhibited the development of trachea system and thinned tracheal taenidia. Ingestion of dsCHS1 at the third-instar stage caused similar but severe negative effects. Our results demonstrated that HvCHS1 is responsible for chitin biosynthesis during ecdysis. Moreover, HvCHS1 is a potential amenable target gene and young larvae are more susceptible to dsRNA.

Multiple Wnts act synergistically to induce Chk1/Grapes expression and mediate G2 arrest in Drosophila tracheoblasts.

Larval tracheae of Drosophila harbour progenitors of the adult tracheal system (tracheoblasts). Thoracic tracheoblasts are arrested in the G2 phase of the cell cycle in an ATR (mei-41)-Checkpoint Kinase1 (grapes, Chk1) dependent manner prior to mitotic re-entry. Here we investigate developmental regulation of Chk1 activation. We report that Wnt signaling is high in tracheoblasts and this is necessary for high levels of activated (phosphorylated) Chk1. We find that canonical Wnt signaling facilitates this by transcriptional upregulation of Chk1 expression in cells that have ATR kinase activity. Wnt signaling is dependent on four Wnts (Wg, Wnt5, 6,10) that are expressed at high levels in arrested tracheoblasts and are downregulated at mitotic re-entry. Interestingly, none of the Wnts are dispensable and act synergistically to induce Chk1. Finally, we show that downregulation of Wnt signaling and Chk1 expression leads to mitotic re-entry and the concomitant upregulation of Dpp signaling, driving tracheoblast proliferation.

Long-term in vivo imaging of Drosophila larvae.

The Drosophila larva has been used to investigate many processes in cell biology, including morphogenesis, physiology and responses to drugs and new therapeutic compounds. Despite its enormous potential as a model system, longer-term live imaging has been technically challenging because of a lack of efficient methods for immobilizing larvae for extended periods. We describe here a simple procedure for anesthetization and uninterrupted long-term in vivo imaging of the epidermis and other larval organs, including gut, imaginal discs, neurons, fat body, tracheae, muscles and hemocytes, for up to 8 h. We also include a procedure for probing cell properties by laser ablation. We provide a survey of the effects of different anesthetics, demonstrating that short exposure to diethyl ether is the most effective for long-term immobilization of larvae. This protocol does not require specific expertise beyond basic Drosophila genetics and husbandry, and confocal microscopy. It enables high-resolution studies of many systemic and subcellular processes in larvae.

The novel gene apnoia regulates Drosophila tracheal tube size.

Distinct tube size is critical for the function of human tubular organs such as the lung, vascular system, and kidney. Aberrant tube sizes can lead to devastating human illnesses, including polycystic kidney disease. The Drosophila trachea provides a premier genetic system to investigate the fundamental mechanisms that regulate tube size. Here we describe the function of a novel gene, apnoia, in tube-size regulation. apn encodes an apical membrane protein, Apnoia (Apn), with three helical transmembrane domains. Loss-of-function apn mutants show shorter-tube and air-filling defects in larval trachea, whereas there are no obvious defects in embryonic trachea. Conversely, overexpression of apn in trachea leads to significant tube over-elongation. We analyzed apical luminal matrix and cell polarity in these longer tubes. It is interesting to note that we observed normal establishment of cell polarity, whereas all luminal matrix components are significantly reduced. In addition, we observed that some matrix components are localized in cytoplasmic vesicles, suggesting secretion defects in apn overexpressing trachea. Taken together, these results strongly suggest the possibility that apn is directly or indirectly involved in vesicular trafficking to regulate tube size.

The apical protein Apnoia interacts with Crumbs to regulate tracheal growth and inflation.

Most organs of multicellular organisms are built from epithelial tubes. To exert their functions, tubes rely on apico-basal polarity, on junctions, which form a barrier to separate the inside from the outside, and on a proper lumen, required for gas or liquid transport. Here we identify apnoia (apn), a novel Drosophila gene required for tracheal tube elongation and lumen stability at larval stages. Larvae lacking Apn show abnormal tracheal inflation and twisted airway tubes, but no obvious defects in early steps of tracheal maturation. apn encodes a transmembrane protein, primarily expressed in the tracheae, which exerts its function by controlling the localization of Crumbs (Crb), an evolutionarily conserved apical determinant. Apn physically interacts with Crb to control its localization and maintenance at the apical membrane of developing airways. In apn mutant tracheal cells, Crb fails to localize apically and is trapped in retromer-positive vesicles. Consistent with the role of Crb in apical membrane growth, RNAi-mediated knockdown of Crb results in decreased apical surface growth of tracheal cells and impaired axial elongation of the dorsal trunk. We conclude that Apn is a novel regulator of tracheal tube expansion in larval tracheae, the function of which is mediated by Crb.

Failure to Burrow and Tunnel Reveals Roles for jim lovell in the Growth and Endoreplication of the Drosophila Larval Tracheae

The Drosophila protein Jim Lovell (Lov) is a putative transcription factor of the BTB/POZ (Bric- a-Brac/Tramtrack/Broad/ Pox virus and Zinc finger) domain class that is expressed in many elements of the developing larval nervous system. It has roles in innate behaviors such as larval locomotion and adult courtship. In performing tissue-specific knockdown with the Gal4-UAS system we identified a new behavioral phenotype for lov: larvae failed to burrow into their food during their growth phase and then failed to tunnel into an agarose substratum during their wandering phase. We determined that these phenotypes originate in a previously unrecognized role for lov in the tracheae. By using tracheal-specific Gal4 lines, Lov immunolocalization and a lov enhancer trap line, we established that lov is normally expressed in the tracheae from late in embryogenesis through larval life. Using an assay that monitors food burrowing, substrate tunneling and death we showed that lov tracheal knockdown results in tracheal fluid-filling, producing hypoxia that activates the aberrant behaviors and inhibits development. We investigated the role of lov in the tracheae that initiates this sequence of events. We discovered that when lov levels are reduced, the tracheal cells are smaller, more numerous and show lower levels of endopolyploidization. Together our findings indicate that Lov is necessary for tracheal endoreplicative growth and that its loss in this tissue causes loss of tracheal integrity resulting in chronic hypoxia and abnormal burrowing and tunneling behavior.

FGF coordinates air sac development by activation of the EGF ligand Vein through the transcription factor PntP2.

How several signaling pathways are coordinated to generate complex organs through regulation of tissue growth and patterning is a fundamental question in developmental biology. The larval trachea of Drosophila is composed of differentiated functional cells and groups of imaginal tracheoblasts that build the adult trachea during metamorphosis. Air sac primordium cells (ASP) are tracheal imaginal cells that form the dorsal air sacs that supply oxygen to the flight muscles of the Drosophila adult. The ASP emerges from the tracheal branch that connects to the wing disc by the activation of both Bnl-FGF/Btl and EGFR signaling pathways. Together, these pathways promote cell migration and proliferation. In this study we demonstrate that Vein (vn) is the EGF ligand responsible for the activation of the EGFR pathway in the ASP. We also find that the Bnl-FGF/Btl pathway regulates the expression of vn through the transcription factor PointedP2 (PntP2). Furthermore, we show that the FGF target gene escargot (esg) attenuates EGFR signaling at the tip cells of the developing ASP, reducing their mitotic rate to allow proper migration. Altogether, our results reveal a link between Bnl-FGF/Btl and EGFR signaling and provide novel insight into how the crosstalk of these pathways regulates migration and growth.

Developmental compartments in the larval trachea of Drosophila.

The Drosophila tracheal system is a branched tubular network that forms in the embryo by a post-mitotic program of morphogenesis. In third instar larvae (L3), cells constituting the second tracheal metamere (Tr2) reenter the cell cycle. Clonal analysis of L3 Tr2 revealed that dividing cells in the dorsal trunk, dorsal branch and transverse connective branches respect lineage restriction boundaries near branch junctions. These boundaries corresponded to domains of gene expression, for example where cells expressing Spalt, Delta and Serrate in the dorsal trunk meet vein-expressing cells in the dorsal branch or transverse connective. Notch signaling was activated to one side of these borders and was required for the identity, specializations and segregation of border cells. These findings suggest that Tr2 is comprised of developmental compartments and that developmental compartments are an organizational feature relevant to branched tubular networks.

Insulin- and warts-dependent regulation of tracheal plasticity modulates systemic larval growth during hypoxia in Drosophila melanogaster.

Adaptation to dynamic environmental cues during organismal development requires coordination of tissue growth with available resources. More specifically, the effects of oxygen availability on body size have been well-documented, but the mechanisms through which hypoxia restricts systemic growth have not been fully elucidated. Here, we characterize the larval growth and metabolic defects in Drosophila that result from hypoxia. Hypoxic conditions reduced fat body opacity and increased lipid droplet accumulation in this tissue, without eliciting lipid aggregation in hepatocyte-like cells called oenocytes. Additionally, hypoxia increased the retention of Dilp2 in the insulin-producing cells of the larval brain, associated with a reduction of insulin signaling in peripheral tissues. Overexpression of the wildtype form of the insulin receptor ubiquitously and in the larval trachea rendered larvae resistant to hypoxia-induced growth restriction. Furthermore, Warts downregulation in the trachea was similar to increased insulin receptor signaling during oxygen deprivation, which both rescued hypoxia-induced growth restriction, inhibition of tracheal molting, and developmental delay. Insulin signaling and loss of Warts function increased tracheal growth and augmented tracheal plasticity under hypoxic conditions, enhancing oxygen delivery during periods of oxygen deprivation. Our findings demonstrate a mechanism that coordinates oxygen availability with systemic growth in which hypoxia-induced reduction of insulin receptor signaling decreases plasticity of the larval trachea that is required for the maintenance of systemic growth during times of limiting oxygen availability.

Specification of Differentiated Adult Progenitors via Inhibition of Endocycle Entry in the Drosophila Trachea

A population of Drosophila adult tracheal progenitor cells arises from differentiated cells of the larval main trachea that retain the ability to reenter the cell cycle and give rise to the multiple adult tracheal cell types. These progenitors are unique to the second tracheal metamere as homologous cells from other segments, express fizzy-related (fzr), the Drosophila homolog of CDH1 protein of the APC complex, and enter endocycle and do not contribute to adult trachea. Here, we examine the mechanisms for their quiescence and show that they reenter the cell cycle by expression of string/cdc25 through ecdysone. Furthermore, we show that preventing endocycle entry is both necessary and sufficient for these tracheal cells to exhibit markers of adult progenitors, thus modifying their genetic program. Finally, we show that Hox-mediated regulation of fzr expression is responsible for progenitor identity and thus specifies a group of differentiated cells with facultative stem cell features.

The effects of hypoxia in determining larval size in Drosophila melanogaster (888.1)

In Drosophila, hypoxia systemically restricts larval growth, but the molecular mechanism through which it does so remains poorly characterized. We show that Sima, the alpha subunit of the Drosophila hypoxia‐inducible factor, accumulates in nuclei of larval fat body cells during hypoxia and inhibits systemic growth when overexpressed in this same tissue. Oxygen deprivation and overexpression of Sima in the fat body also cause the retention of Drosophila insulin‐like peptides (Dilps) in the larval brain, suggesting that an endocrine signal emanates from the fat body and is relayed to the brain to regulate Dilp secretion during an oxygen‐deprived state. Additionally, hypoxia and overexpression of Sima in the fat body cause lipid droplets to aggregate in this metabolic tissue but not in the larval oenocytes, a liver‐like cell type, unlike what is observed in starvation. Furthermore, hypoxia reduces total cholesterol, triglyceride, and free fatty acid levels in wild‐type larvae. Finally, ubiquitous overexpression of the wild‐type insulin receptor rescues hypoxic growth restriction, suggesting that hypoxia‐induced insulin resistance in peripheral tissues serves to inhibit systemic growth. Interestingly, one tissue that mediates hypoxic growth restriction is the larval trachea, as both overexpression of the wild‐type insulin receptor and activation of TOR (Target of rapamycin) signaling in this tissue rescue inhibition of larval growth under hypoxic conditions. Rearing wild‐type larvae in hypoxia also decreases the number of lipid droplets that stud the larval trachea, which is suggestive of a novel finding that lipid droplet mobilization to the larval trachea functions to promote growth. Collectively, our results suggest that coordination of larval size is dependent on the communication between many organs through the use of insulin signaling and alterations to lipid metabolism. These findings may be applicable to other instances in which an adaptive response to hypoxia is required.

Proteomic analysis of larval integument, trachea and adult scale from the silkworm,Bombyx mori

Multidimensional LC-tandem MS was used to investigate the protein compositions of three tissues of silkworm, Bombyx mori. A total of 162, 259, and 175 peptides from silkworm larval integument and trachea, and adult scale obtained by database search were matched to 48, 51, and 40 proteins, respectively. Forty-one cuticular proteins were identified from three tissues and covered all five cuticular protein families of silkworm. In the adult scale, all seven cuticular proteins were identified for the first time in the final pellet after SDS extraction. The majority of cuticular proteins were found in each tissue differentially, suggesting that tissue-specific cuticular proteins were involved in the building of the specialized tissues. Seventy-three non-cuticular proteins were also identified in this analysis mainly including muscular proteins, proteinases, inhibitors, transport proteins, and redox-related proteins.

A matrix metalloproteinase mediates airway remodeling in Drosophila

Organ size typically increases dramatically during juvenile growth. This growth presents a fundamental tension, as organs need resiliency to resist stresses while still maintaining plasticity to accommodate growth. The extracellular matrix (ECM) is central to providing resiliency, but how ECM is remodeled to accommodate growth is poorly understood. We investigated remodeling of Drosophila respiratory tubes (tracheae) that elongate continually during larval growth, despite being lined with a rigid cuticular ECM. Cuticle is initially deposited with a characteristic pattern of repeating ridges and valleys known as taenidia. We find that for tubes to elongate, the extracellular protease Mmp1 is required for expansion of ECM between the taenidial ridges during each intermolt period. Mmp1 protein localizes in periodically spaced puncta that are in register with the taenidial spacing. Mmp1 also degrades old cuticle at molts, promotes apical membrane expansion in larval tracheae, and promotes tube elongation in embryonic tracheae. Whereas work in other developmental systems has demonstrated that MMPs are required for axial elongation occurring in localized growth zones, this study demonstrates that MMPs can also mediate interstitial matrix remodeling during growth of an organ system.