Tracheal Morphogenesis Research Articles

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.

NKX2‐1 directly regulates Efnb2 to generate a boundary that allocates tracheal cells and defines the position of tracheoesophageal separation

Tracheoesophageal (TE) fistula is a human congenital condition characterized by an improper connection between the esophagus and trachea. These two tubular organs originate from a single foregut tube and separate from one another beginning at the site of lung bud evagination and progressing rostrally. Genetic studies in mice have identified signaling pathways that initiate dorsoventral patterning of the foregut; NKX2‐1 marks ventral, presumptive tracheal cells whereas high levels of SOX2 expression predominantly mark the dorsal, esophageal‐fated cells. Perturbation of either factor results in loss of that identity, increased expression of the opposing transcription factor, and failure of TE separation. Currently, the downstream mechanisms required to effect morphogenetic separation at the cellular and tissue level remain largely unknown. Here, we show that EPHRIN‐B2, a member of the EPH/EPHRIN signaling family, is restricted to the dorsal, esophageal‐fated cells, and that this domain of expression is required for TE separation and for maintaining the correct allocation of tracheal cells to the nascent trachea. Further, we demonstrate that NKX2‐1 directly binds at the Efnb2 locus and negatively regulates its expression, establishing an NKX2‐1/EPHRIN‐B2 TE boundary. Nkx2‐1 mosaic mutants exhibit ectopic NKX2‐1/EPHRIN‐B2 boundaries which drive ectopic tracheal separation morphogenesis in an EPHRIN‐B2 dependent manner. These results establish NKX2‐1 as a regulator of TE cell sorting and tissue separation, through modulation of EPH/EPHRIN signaling, which then serves to translate tissue identity information into morphogenetic outcomes.

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.

BMP4 and Wnt signaling interact to promote mouse tracheal mesenchyme morphogenesis.

Tracheobronchomalacia and complete tracheal rings are congenital malformations of the trachea associated with morbidity and mortality for which the etiology remains poorly understood. Epithelial expression of Wls (a cargo receptor mediating Wnt ligand secretion) by tracheal cells is essential for patterning the embryonic mouse trachea's cartilage and muscle. RNA sequencing indicated that Wls differentially modulated the expression of BMP signaling molecules. We tested whether BMP signaling, induced by epithelial Wnt ligands, mediates cartilage formation. Deletion of Bmp4 from respiratory tract mesenchyme impaired tracheal cartilage formation that was replaced by ectopic smooth muscle, recapitulating the phenotype observed after epithelial deletion of Wls in the embryonic trachea. Ectopic muscle was caused in part by anomalous differentiation and proliferation of smooth muscle progenitors rather than tracheal cartilage progenitors. Mesenchymal deletion of Bmp4 impaired expression of Wnt/β-catenin target genes, including targets of WNT signaling: Notum and Axin2. In vitro, recombinant (r)BMP4 rescued the expression of Notum in Bmp4-deficient tracheal mesenchymal cells and induced Notum promoter activity via SMAD1/5. RNA sequencing of Bmp4-deficient tracheas identified genes essential for chondrogenesis and muscle development coregulated by BMP and WNT signaling. During tracheal morphogenesis, WNT signaling induces Bmp4 in mesenchymal progenitors to promote cartilage differentiation and restrict trachealis muscle. In turn, Bmp4 differentially regulates the expression of Wnt/β-catenin targets to attenuate mesenchymal WNT signaling and to further support chondrogenesis.

Mammalian tracheal development and reconstruction: insights from in vivo and in vitro studies.

ABSTRACTThe trachea delivers inhaled air into the lungs for gas exchange. Anomalies in tracheal development can result in life-threatening malformations, such as tracheoesophageal fistula and tracheomalacia. Given the limitations of current therapeutic approaches, development of technologies for the reconstitution of a three-dimensional trachea from stem cells is urgently required. Recently, single-cell sequencing technologies and quantitative analyses from cell to tissue scale have been employed to decipher the cellular basis of tracheal morphogenesis. In this Review, recent advances in mammalian tracheal development and the generation of tracheal tissues from pluripotent stem cells are summarized.

Roles of LmCDA1 and LmCDA2 in cuticle formation in the foregut and hindgut of Locusta migratoria.

Chitin deacetylases (CDAs, including CDA1 and CDA2) are considered key enzymes for body cuticle formation and tracheal morphogenesis in various insect species. However, their functions in the formation of the cuticular intima of the foregut and hindgut are unclear. Here, we investigated the roles of their respective genes LmCDA1 and LmCDA2 in this process, in the hemimetabolous insect Locusta migratoria. Transcripts of LmCDA1 and LmCDA2 were highly expressed both before and after molting in the foregut. In the hindgut, their expression was high only before molting. In both the foregut and hindgut, LmCDA1 protein was localized in the basal half of the chitin matrix (procuticle), whereas LmCDA2 was detected in the upper half of the procuticle. Knockdown of LmCDA1 by RNA interference (RNAi) in 5th-instar nymphs caused no visible defects of the hindgut cuticle. By contrast, the chitinous lamellae of the cuticular intima in the foregut of knockdown animals were less compact than in control animals. RNAi against LmCDA2 led to thickening of both the foregut and hindgut cuticles, with a greater number of thinner laminae than in the respective control cuticles. Taken together, our results show that LmCDA1 and LmCDA2 have distinct, but overlapping, functions in chitin organization in the foregut cuticle. However, in the hindgut, this process seems independent of LmCDA1 activity but requires LmCDA2 function. Thus, the CDAs reflect tissue-specific differences in cuticular organization and function, which need further detailed molecular and histological analyses for full comprehension.

The bicistronic gene würmchen encodes two essential components for epithelial development in Drosophila

Epithelial tissues are fundamental for the establishment and maintenance of different body compartments in multicellular animals. To achieve this specific task epithelial sheets secrete an apical extracellular matrix for tissue strength and protection and they organize a transepithelial barrier function, which is mediated by tight junctions in vertebrates or septate junctions in invertebrates.Here, we show that the bicistronic gene würmchen is functionally expressed in epithelial tissues. CRISPR/Cas9-mediated mutations in both coding sequences reveal two essential polypeptides, Würmchen1 and Würmchen2, which are both necessary for normal epithelial tissue development. Würmchen1 represents a genuine septate junction core component. It is required during embryogenesis for septate junction organization, the establishment of a transepithelial barrier function, distinct cellular transport processes and tracheal system morphogenesis. Würmchen2 is localized in the apical membrane region of epithelial tissues and in a central core of the tracheal lumen during embryogenesis. It is essential during the later larval development.

Multiple Requirements for Rab GTPases in the Development of Drosophila Tracheal Dorsal Branches and Terminal Cells.

The tracheal epithelium in fruit fly larvae is a popular model for multi- and unicellular migration and morphogenesis. Like all epithelial cells, tracheal cells use Rab GTPases to organize their internal membrane transport, resulting in the specific localization or secretion of proteins on the apical or basal membrane compartments. Some contributions of Rabs to junctional remodelling and governance of tracheal lumen contents are known, but it is reasonable to assume that they play important further roles in morphogenesis. This pertains in particular to terminal tracheal cells, specialized branch-forming cells that drastically reshape both their apical and basal membrane during the larval stages. We performed a loss-of-function screen in the tracheal system, knocking down endogenously tagged alleles of 26 Rabs by targeting the tag via RNAi. This revealed that at least 14 Rabs are required to ensure proper cell fate specification and migration of the dorsal branches, as well as their epithelial fusion with the contralateral dorsal branch. The screen implicated four Rabs in the subcellular morphogenesis of terminal cells themselves. Further tests suggested residual gene function after knockdown, leading us to discuss the limitations of this approach. We conclude that more Rabs than identified here may be important for tracheal morphogenesis, and that the tracheal system offers great opportunities for studying several Rabs that have barely been characterized so far.

Two-step regulation of trachealess ensures tight coupling of cell fate with morphogenesis in the Drosophila trachea.

During organogenesis, inductive signals cause cell differentiation and morphogenesis. However, how these phenomena are coordinated to form functional organs is poorly understood. Here, we show that cell differentiation of the Drosophila trachea is sequentially determined in two steps and that the second step is synchronous with the invagination of the epithelial sheet. The master gene trachealess is dispensable for the initiation of invagination, while it is essential for maintaining the invaginated structure, suggesting that tracheal morphogenesis and differentiation are separately induced. trachealess expression starts in bipotential tracheal/epidermal placode cells. After invagination, its expression is maintained in the invaginated cells but is extinguished in the remaining sheet cells. A trachealess cis-regulatory module that shows both tracheal enhancer activity and silencer activity in the surface epidermal sheet was identified. We propose that the coupling of trachealess expression with the invaginated structure ensures that only invaginated cells canalize robustly into the tracheal fate.

The putative chitin deacetylases Serpentine and Vermiform have non-redundant functions during Drosophila wing development

The chitin modifying deacetylases (CDA) CDA1 and CDA2 have been reported to play partially redundant roles during insect cuticle formation and molting and tracheal morphogenesis in various insect species. In order to distinguish possible functional differences between these two enzymes, we analyzed their function during wing development in the fruit fly Drosophila melanogaster. In tissue-specific RNA interference experiments, we demonstrate that DmCDA1 (Serpentine, Serp) and DmCDA2 (Vermiform, Verm) have distinct functions during Drosophila adult wing cuticle differentiation. Chitosan staining revealed that Serp is the major enzyme responsible for chitin deacetylation during wing cuticle formation, while Verm does not seem to be needed for this process. Indeed, it is questionable whether Verm is a chitin deacetylase at all. Atomic force microscopy suggested that Serp and Verm have distinct roles in establishing the shape of nanoscale bumps at the wing surface. Moreover, our data indicate that Verm but not Serp is required for the laminar arrangement of chitin. Both enzymes participate in the establishment of the cuticular inward barrier against penetration of xenobiotics. Taken together, correct differentiation of the wing cuticle involves both Serp and Verm in parallel in largely non-overlapping functions.

FGF10 is an essential regulator of tracheal submucosal gland morphogenesis

Mucus secretion and mucociliary clearance are crucial processes required to maintain pulmonary homeostasis. In the trachea and nasal passages, mucus is secreted by submucosal glands (SMGs) that line the airway, with an additional contribution from goblet cells of the surface airway epithelium. The SMG mucus is rich in mucins and antimicrobial enzymes. Defective tracheal SMGs contribute to hyper-secretory respiratory diseases, such as cystic fibrosis, asthma, and chronic obstructive pulmonary disease, however little is known about the signals that regulate their morphogenesis and patterning. Here, we show that Fgf10 is essential for the normal development of murine tracheal SMGs, with gland development arresting at the early bud stage in the absence of FGF10 signalling. As Fgf10 knockout mice are lethal at birth, inducible knockdown of Fgf10 at late embryonic stages was used to follow postnatal gland formation, confirming the essential role of FGF10 in SMG development. In heterozygous Fgf10 mice the tracheal glands formed but with altered morphology and restricted distribution. The reduction in SMG branching in Fgf10 heterozygous mice was not rescued with time and resulted in a reduction in overall tracheal mucus secretion. Fgf10 is therefore a key signal in SMG development, influencing both the number of glands and extent of branching morphogenesis, and is likely, therefore, to play a role in aspects of SMG-dependent respiratory health.

The Caspase-3 homolog DrICE regulates endocytic trafficking during Drosophila tracheal morphogenesis

Although well known for its role in apoptosis, the executioner caspase DrICE has a non-apoptotic function that is required for elongation of the epithelial tubes of the Drosophila tracheal system. Here, we show that DrICE acts downstream of the Hippo Network to regulate endocytic trafficking of at least four cell polarity, cell junction and apical extracellular matrix proteins involved in tracheal tube size control: Crumbs, Uninflatable, Kune-Kune and Serpentine. We further show that tracheal cells are competent to undergo apoptosis, even though developmentally-regulated DrICE function rarely kills tracheal cells. Our results reveal a developmental role for caspases, a pool of DrICE that co-localizes with Clathrin, and a mechanism by which the Hippo Network controls endocytic trafficking. Given reports of in vitro regulation of endocytosis by mammalian caspases during apoptosis, we propose that caspase-mediated regulation of endocytic trafficking is an evolutionarily conserved function of caspases that can be deployed during morphogenesis.

A Hippo-like Signaling Pathway Controls Tracheal Morphogenesis in Drosophila melanogaster

Hippo-like pathways are ancient signaling modules first identified in yeasts. The best-defined metazoan module forms the core of the Hippo pathway, which regulates organ size and cell fate. Hippo-like kinase modules consist of a Sterile 20-like kinase, an NDR kinase, and non-catalytic protein scaffolds. In the Hippo pathway, the upstream kinase Hippo can be activated by another kinase, Tao-1. Here, we delineate a related Hippo-like signaling module that Tao-1 regulates to control tracheal morphogenesis in Drosophila melanogaster. Tao-1 activates the Sterile 20-like kinase GckIII by phosphorylating its activation loop, a mode of regulation that is conserved in humans. Tao-1 and GckIII act upstream of the NDR kinase Tricornered to ensure proper tube formation in trachea. Our study reveals that Tao-1 activates two related kinase modules to control both growth and morphogenesis. The Hippo-like signaling pathway we have delineated has a potential role inthe human vascular disease cerebral cavernous malformation.

Development and Function of the Drosophila Tracheal System.

The tracheal system of insects is a network of epithelial tubules that functions as a respiratory organ to supply oxygen to various target organs. Target-derived signaling inputs regulate stereotyped modes of cell specification, branching morphogenesis, and collective cell migration in the embryonic stage. In the postembryonic stages, the same set of signaling pathways controls highly plastic regulation of size increase and pattern elaboration during larval stages, and cell proliferation and reprograming during metamorphosis. Tracheal tube morphogenesis is also regulated by physicochemical interaction of the cell and apical extracellular matrix to regulate optimal geometry suitable for air flow. The trachea system senses both the external oxygen level and the metabolic activity of internal organs, and helps organismal adaptation to changes in environmental oxygen level. Cellular and molecular mechanisms underlying the high plasticity of tracheal development and physiology uncovered through research on Drosophila are discussed.

Notum attenuates Wnt/β–catenin signaling to promote tracheal cartilage patterning

Tracheobronchomalacia (TBM) is a common congenital disorder in which the cartilaginous rings of the trachea are weakened or missing. Despite the high prevalence and clinical issues associated with TBM, the etiology is largely unknown. Our previous studies demonstrated that Wntless (Wls) and its associated Wnt pathways are critical for patterning of the upper airways. Deletion of Wls in respiratory endoderm caused TBM and ectopic trachealis muscle. To understand mechanisms by which Wls mediates tracheal patterning, we performed RNA sequencing in prechondrogenic tracheal tissue of Wlsf/f;ShhCre/wt embryos. Chondrogenic Bmp4, and Sox9 were decreased, while expression of myogenic genes was increased. We identified Notum, a deacylase that inactivates Wnt ligands, as a target of Wls induced Wnt signaling. Notum’s mesenchymal ventral expression in prechondrogenic trachea overlaps with expression of Axin2, a Wnt/β-catenin target and inhibitor. Notum is induced by Wnt/β-catenin in developing trachea. Deletion of Notum activated mesenchymal Wnt/β-catenin and caused tracheal mispatterning of trachealis muscle and cartilage as well as tracheal stenosis. Notum is required for tracheal morphogenesis, influencing mesenchymal condensations critical for patterning of tracheal cartilage and muscle. We propose that Notum influences mesenchymal cell differentiation by generating a barrier for Wnt ligands produced and secreted by airway epithelial cells to attenuate Wnt signaling.

Faithful mRNA splicing depends on the Prp19 complex subunit faint sausage and is required for tracheal branching morphogenesis in Drosophila.

Morphogenesis requires the dynamic regulation of gene expression, including transcription, mRNA maturation and translation. Dysfunction of the general mRNA splicing machinery can cause surprisingly specific cellular phenotypes, but the basis for these effects is not clear. Here, we show that the Drosophila faint sausage (fas) locus, which is implicated in epithelial morphogenesis and has previously been reported to encode a secreted immunoglobulin domain protein, in fact encodes a subunit of the spliceosome-activating Prp19 complex, which is essential for efficient pre-mRNA splicing. Loss of zygotic fas function globally impairs the efficiency of splicing, and is associated with widespread retention of introns in mRNAs and dramatic changes in gene expression. Surprisingly, despite these general effects, zygotic fas mutants show specific defects in tracheal cell migration during mid-embryogenesis when maternally supplied splicing factors have declined. We propose that tracheal branching, which relies on dynamic changes in gene expression, is particularly sensitive for efficient spliceosome function. Our results reveal an entry point to study requirements of the splicing machinery during organogenesis and provide a better understanding of disease phenotypes associated with mutations in general splicing factors.

A fat body-derived apical extracellular matrix enzyme is transported to the tracheal lumen and is required for tube morphogenesis in Drosophila.

The apical extracellular matrix plays a central role in epithelial tube morphogenesis. In the Drosophila tracheal system, Serpentine (Serp), a secreted chitin deacetylase expressed by the tracheal cells plays a key role in regulating tube length. Here, we show that the fly fat body, which is functionally equivalent to the mammalian liver, also contributes to tracheal morphogenesis. Serp was expressed by the fat body, and the secreted Serp was taken up by the tracheal cells and translocated to the lumen to functionally support normal tracheal development. This process was defective in rab9 and shrub/vps32 mutants and in wild-type embryos treated with a secretory pathway inhibitor, leading to an abundant accumulation of Serp in the fat body. We demonstrated that fat body-derived Serp reached the tracheal lumen after establishment of epithelial barrier function and was retained in the lumen in a chitin synthase-dependent manner. Our results thus reveal that the fat body, a mesodermal organ, actively contributes to tracheal development.

IV. Tools and methods for studying cell migration and cell rearrangement in tissue and organ development

A vast diversity of biological systems, ranging from prokaryotes to multicellular organisms, show cell migration behavior. Many of the basic cellular and molecular concepts in cell migration apply to diverse model organisms. Drosophila, with its vast repertoire of tools for imaging and for manipulation, is one of the favorite organisms to study cell migration. Moreover, distinct Drosophila tissues and organs offer diverse cell migration models that are amenable to live imaging and genetic manipulations. In this review, we will provide an overview of the fruit fly toolbox that is of particular interest for the analysis of cell migration. We provide examples to highlight how those tools were used in diverse migration systems, with an emphasis on tracheal morphogenesis, a process that combines morphogenesis with cell migration.

Fascin links Btl/FGFR signalling to the actin cytoskeleton during Drosophilia tracheal morphogenesis

Fascin links Btl/FGFR signalling to the actin cytoskeleton during <i>Drosophilia</i> tracheal morphogenesis

Fascin links Btl/FGFR signalling to the actin cytoskeleton during Drosophila tracheal morphogenesis

A key challenge in normal development and in disease is to elucidate the mechanisms of cell migration. Here we approach this question using the tracheal system of Drosophila as a model. Tracheal cell migration requires the Breathless/FGFR pathway; however, how the pathway induces migration remains poorly understood. We find that the Breathless pathway upregulates singed at the tip of tracheal branches, and that this regulation is functionally relevant. singed encodes Drosophila Fascin, which belongs to a conserved family of actin-bundling proteins involved in cancer progression and metastasis upon misregulation. We show that singed is required for filopodia stiffness and proper morphology of tracheal tip cells, defects that correlate with an abnormal actin organisation. We propose that singed-regulated filopodia and cell fronts are required for timely and guided branch migration and for terminal branching and branch fusion. We find that singed requirements rely on its actin-bundling activity controlled by phosphorylation, and that active Singed can promote tip cell features. Furthermore, we find that singed acts in concert with forked, another actin cross-linker. The absence of both cross-linkers further stresses the relevance of tip cell morphology and filopodia for tracheal development. In summary, our results on the one hand reveal a previously undescribed role for forked in the organisation of transient actin structures such as filopodia, and on the other hand identify singed as a new target of Breathless signal, establishing a link between guidance cues, the actin cytoskeleton and tracheal morphogenesis.