Molecular mechanisms of de novo lumen formation.

Many organs contain networks of epithelial tubes that transport gases or fluids. A lumen can be generated by tissue that enwraps a pre-existing extracellular space or it can arise de novo either between cells or within a single cell in a position where there was no space previously. Apparently distinct mechanisms of de novo lumen formation observed in vitro - in three-dimensional cultures of endothelial and Madin-Darby canine kidney (MDCK) cells - and in vivo - in zebrafish vasculature, Caenorhabditis elegans excretory cells and the Drosophila melanogaster trachea - in fact share many common features. In all systems, lumen formation involves the structured expansion of the apical plasma membrane through general mechanisms of vesicle transport and of microtubule and actin cytoskeleton regulation.

Bronchial tree of the human embryo: Examination based on a mammalian model.

The symmetry of the right and left bronchi, proposed in a previous comparative anatomical study as the basic model of the mammalian bronchial tree, was examined to determine if it applied to the embryonic human bronchial tree. Imaging data of 41 human embryo specimens at Carnegie stages (CS) 16-23 (equivalent to 6-8 weeks after fertilization) belonging to the Kyoto collection were obtained using phase-contrast X-ray computed tomography. Three-dimensional bronchial trees were then reconstructed from these images. Bronchi branching from both main bronchi were labeled as dorsal, ventral, medial, or lateral systems based on the branching position with numbering starting cranially. The length from the tracheal bifurcation to the branching point of the labeled bronchus was measured, and the right-to-left ratio of the same labeled bronchus in both lungs was calculated. In both lungs, the human embryonic bronchial tree showed symmetry with an alternating pattern of dorsal and lateral systems up to segmental bronchus B9 as the basic shape, with a more peripheral variation. This pattern is similar to that described in adult human lungs. Bronchial length increased with the CS in all labeled bronchi, whereas the right-to-left ratio was constant at approximately 1.0. The data demonstrated that the prototype of the human adult bronchial branching structure is formed and maintained in the embryonic stage. The morphology and branching position of all lobar bronchi and B6, B8, B9, and the subsegmental bronchus of B10 may be genetically determined. On the other hand, no common structures between individual embryos were found in the peripheral branches after the subsegmental bronchus of B10, suggesting that branch formation in this region is influenced more by environmental factors than by genetic factors.

E3 ubiquitin ligase FBXW7 balances airway cell fates.

The airway epithelium is composed of multiple cell types each with designated roles. A stereotyped ratio of these cells is essential for proper airway function. Imbalance of airway cell types underlies many lung diseases, including chronic obstructive pulmonary disease (COPD) and asthma. While a number of signals and transcription factors have been implicated in airway cell specification, how cell numbers are coordinated, especially at the protein level is poorly understood. Here we show that in the mouse trachea which contain epithelial cell types similar to human airway, epithelium-specific inactivation of Fbxw7, which encodes an E3 ubiquitin ligase, led to reduced club and ciliated cells, increased goblet cells, and ectopic P63-negative, Keratin5-positive transitory basal cells in the luminal layer. The protein levels of FBXW7 targets including NOTCH1, KLF5 and TGIF were increased. Inactivation of either Notch1, Klf5 but not Tgif genes in the mutant background led to attenuation of selected aspects of the phenotypes, suggesting that FBXW7 acts through different targets to control different cell fates. These findings demonstrate that protein-level regulation by the ubiquitin proteasome system is critical for balancing airway cell fates.

The Drosophila NCAM homolog Fas2 signals independently of adhesion.

The development of tissues and organs requires close interaction of cells. To achieve this, cells express adhesion proteins such as the neural cell adhesion molecule (NCAM) or its Drosophila ortholog Fasciclin 2 (Fas2). Both are members of the Ig-domain superfamily of proteins that mediate homophilic adhesion. These proteins are expressed as isoforms differing in their membrane anchorage and their cytoplasmic domains. To study the function of single isoforms, we have conducted a comprehensive genetic analysis of Fas2 We reveal the expression pattern of all major Fas2 isoforms, two of which are GPI anchored. The remaining five isoforms carry transmembrane domains with variable cytoplasmic tails. We generated Fas2 mutants expressing only single isoforms. In contrast to the null mutation, which causes embryonic lethality, these mutants are viable, indicating redundancy among the different isoforms. Cell type-specific rescue experiments showed that glial-secreted Fas2 can rescue the Fas2 mutant phenotype to viability. This demonstrates that cytoplasmic Fas2 domains have no apparent essential functions and indicate that Fas2 has function(s) other than homophilic adhesion. In conclusion, our data suggest novel mechanistic aspects of a long-studied adhesion protein.

CFAP53 regulates mammalian cilia-type motility patterns through differential localization and recruitment of axonemal dynein components.

Motile cilia can beat with distinct patterns, but how motility variations are regulated remain obscure. Here, we have studied the role of the coiled-coil protein CFAP53 in the motility of different cilia-types in the mouse. While node (9+0) cilia of Cfap53 mutants were immotile, tracheal and ependymal (9+2) cilia retained motility, albeit with an altered beat pattern. In node cilia, CFAP53 mainly localized at the base (centriolar satellites), whereas it was also present along the entire axoneme in tracheal cilia. CFAP53 associated tightly with microtubules and interacted with axonemal dyneins and TTC25, a dynein docking complex component. TTC25 and outer dynein arms (ODAs) were lost from node cilia, but were largely maintained in tracheal cilia of Cfap53-/- mice. Thus, CFAP53 at the base of node cilia facilitates axonemal transport of TTC25 and dyneins, while axonemal CFAP53 in 9+2 cilia stabilizes dynein binding to microtubules. Our study establishes how differential localization and function of CFAP53 contributes to the unique motion patterns of two important mammalian cilia-types.

Cytoskeletal players in single-cell branching morphogenesis.

Branching networks are a very common feature of multicellular animals and underlie the formation and function of numerous organs including the nervous system, the respiratory system, the vasculature and many internal glands. These networks range from subcellular structures such as dendritic trees to large multicellular tissues such as the lungs. The production of branched structures by single cells, so called subcellular branching, which has been better described in neurons and in cells of the respiratory and vascular systems, involves complex cytoskeletal remodelling events. In Drosophila, tracheal system terminal cells (TCs) and nervous system dendritic arborisation (da) neurons are good model systems for these subcellular branching processes. During development, the generation of subcellular branches by single-cells is characterized by extensive remodelling of the microtubule (MT) network and actin cytoskeleton, followed by vesicular transport and membrane dynamics. In this review, we describe the current knowledge on cytoskeletal regulation of subcellular branching, based on the terminal cells of the Drosophila tracheal system, but drawing parallels with dendritic branching and vertebrate vascular subcellular branching.

Developmental basis of trachea-esophageal birth defects.

Trachea-esophageal defects (TEDs), including esophageal atresia (EA), tracheoesophageal fistula (TEF), and laryngeal-tracheoesophageal clefts (LTEC), are a spectrum of life-threatening congenital anomalies in which the trachea and esophagus do not form properly. Up until recently, the developmental basis of these conditions and how the trachea and esophagus arise from a common fetal foregut was poorly understood. However, with significant advances in human genetics, organoids, and animal models, and integrating single cell genomics with high resolution imaging, we are revealing the molecular and cellular mechanisms that orchestrate tracheoesophageal morphogenesis and how disruption in these processes leads to birth defects. Here we review the current understanding of the genetic and developmental basis of TEDs. We suggest future opportunities for integrating developmental mechanisms elucidated from animals and organoids with human genetics and clinical data to gain insight into the genotype-phenotype basis of these heterogeneous birth defects. Finally, we envision how this will enhance diagnosis, improve treatment, and perhaps one day, lead to new tissue replacement therapy.

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/ß-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/ß-catenin targets to attenuate mesenchymal WNT signaling and to further support chondrogenesis.

QuBiT: a quantitative tool for analyzing epithelial tubes reveals unexpected patterns of organization in the Drosophila trachea.

Biological tubes are essential for animal survival, and their functions are dependent on tube shape. Analyzing the contributions of cell shape and organization to the morphogenesis of small tubes has been hampered by the limitations of existing programs in quantifying cell geometry on highly curved tubular surfaces and calculating tube-specific parameters. We therefore developed QuBiT (Quantitative Tool for Biological Tubes) and used it to analyze morphogenesis of the embryonic Drosophila trachea (airway). In the main tube, we find previously unknown anterior-to-posterior (A-P) gradients of cell apical orientation and aspect ratio, and periodicity in the organization of apical cell surfaces. Inferred cell intercalation during development dampens an A-P gradient of the number of cells per cross-section of the tube, but does not change the patterns of cell connectivity. Computationally 'unrolling' the apical surface of wild-type trachea and the hindgut reveals previously unrecognized spatial patterns of the apical marker Uninflatable and a non-redundant role for the Na+/K+ ATPase in apical marker organization. These unexpected findings demonstrate the importance of a computational tool for analyzing small diameter biological tubes.

Wnt/Fgf crosstalk is required for the specification of basal cells in the mouse trachea.

Basal progenitor cells are crucial for the establishment and maintenance of the tracheal epithelium. However, it remains unclear how these progenitor cells are specified during foregut development. Here, we found that ablation of the Wnt chaperone protein Gpr177 (also known as Wntless) in mouse tracheal epithelium causes a significant reduction in the number of basal progenitor cells accompanied by cartilage loss in Shh-Cre;Gpr177loxp/loxp mutants. Consistent with the association between cartilage and basal cell development, Nkx2.1+p63+ basal cells are co-present with cartilage nodules in Shh-Cre;Ctnnb1DM/loxp mutants, which maintain partial cell-cell adhesion but not the transcription regulation function of ß-catenin. More importantly, deletion of Ctnnb1 in the mesenchyme leads to the loss of basal cells and cartilage, concomitant with reduced transcript levels of Fgf10 in Dermo1-Cre;Ctnnb1loxp/loxp mutants. Furthermore, deletion of Fgf receptor 2 (Fgfr2) in the epithelium also leads to significantly reduced numbers of basal cells, supporting the importance of Wnt/Fgf crosstalk in early tracheal development.

polished rice mediates ecdysone-dependent control of Drosophila embryonic organogenesis.

In many animals, progression of developmental stages is temporally controlled by steroid hormones. In Drosophila, the level of ecdysone titer oscillates and developmental stage transitions, such as larval molting and metamorphosis, are induced at each of ecdysone peaks. Ecdysone titer also peaks at the stage of mid-embryogenesis and the embryonic ecdysone is necessary for morphogenesis of several organs, although the regulatory mechanisms of embryonic organogenesis dependent on ecdysone signaling are still open questions. In this study, we find that absence or interruption of embryonic ecdysone signaling caused multiple defects in the tracheal system, including decrease in luminal protein deposition, uneven dilation of the dorsal trunk and loss of terminal branches. We also reveal that an ecdysone-inducible gene polished rice (pri) is essential for tip cell fate decision in dorsal branches. As over-expression of pri can restore the defects caused by disturbance of ecdysone biosynthesis, pri functions as one of the major mediators of embryonic ecdysone signal in tracheogenesis. These results demonstrate that ecdysone and its downstream target pri play essential roles in tracheal development by modulating cell fate decision.

Blastocyst complementation reveals that NKX2-1 establishes the proximal-peripheral boundary of the airway epithelium.

BACKGROUND: Distinct boundaries between the proximal conducting airways and more peripheral-bronchial regions of the lung are established early in foregut embryogenesis, demarcated in part by the distribution of SOX family and NKX2-1 transcription factors along the cephalo-caudal axis of the lung. We used blastocyst complementation to identify the role of NKX2-1 in the formation of the proximal-peripheral boundary of the airways in mouse chimeric embryos. RESULTS: While Nkx2-1-/- mouse embryos form primordial tracheal cysts, peripheral pulmonary structures are entirely lacking in Nkx2-1-/- mice. Complementation of Nkx2-1-/- embryos with NKX2-1-sufficient embryonic stem cells (ESCs) enabled the formation of all tissue components of the peripheral lung but did not enhance ESC colonization of the most proximal regions of the airways. In chimeric mice, a precise boundary was formed between NKX2-1-deficient basal cells co-expressing SOX2 and SOX9 in large airways and ESC-derived NKX2-1+ SOX9+ epithelial cells of smaller airways. NKX2-1-sufficient ESCs were able to selectively complement peripheral, rather than most proximal regions of the airways. ESC complementation did not prevent ectopic expression of SOX9 but restored ß-catenin signaling in Nkx2-1-/- basal cells of large airways. CONCLUSIONS: NKX2-1 and ß-catenin function in an epithelial cell-autonomous manner to establish the proximal-peripheral boundary along developing airways.

Space colonization by branching trachea explains the morphospace of a simple respiratory organ.

Branching morphogenesis helps increase the efficiency of gas and liquid transport in many animal organs. Studies in several model organisms have highlighted the molecular and cellular complexity behind branching morphogenesis. To understand this complexity, computational models have been developed with the goal of identifying the "major rules" that globally explain the branching patterns. These models also guide further experimental exploration of the biological processes that execute and maintain these rules. In this paper we introduce the tracheal gills of mayfly (Ephemeroptera) larvae as a model system to study the generation of branched respiratory patterns. First, we describe the gills of the mayfly Cloeon dipterum, and quantitatively characterize the geometry of its branching trachea. We next extend this characterization to those of related species to generate the morphospace of branching patterns. Then, we show how an algorithm based on the "space colonization" concept (SCA) can generate this branching morphospace via growth towards a hypothetical attractor molecule (M). SCA differs from other branch-generating algorithms in that the geometry generated depends to a great extent on its perception of the "external" space available for branching, uses few rules and, importantly, can be easily translated into a realistic "biological patterning algorithm". We identified a gene in the C. dipterum genome (Cd-bnl) that is orthologous to the fibroblast growth factor branchless (bnl), which stimulates growth and branching of embryonic trachea in Drosophila. In C. dipterum, this gene is expressed in the gill margins and areas of finer tracheolar branching from thicker trachea. Thus, Cd-bnl may perform the function of M in our model. Finally, we discuss this general mechanism in the context of other branching pattern-generating algorithms.

Fetal endoscopic tracheal occlusion reverses the natural history of right-sided congenital diaphragmatic hernia: European multicenter experience.

OBJECTIVE: To evaluate the neonatal outcome of fetuses with isolated right-sided congenital diaphragmatic hernia (iRCDH) based on prenatal severity indicators and antenatal management. METHODS: This was a retrospective review of prospectively collected data on consecutive cases diagnosed with iRCDH before 30 weeks' gestation in four fetal therapy centers, between January 2008 and December 2018. Data on prenatal severity assessment, antenatal management and perinatal outcome were retrieved. Univariate and multivariate logistic regression analysis were used to identify predictors of survival at discharge and early neonatal morbidity. RESULTS: Of 265 patients assessed during the study period, we excluded 40 (15%) who underwent termination of pregnancy, two cases of unexplained fetal death, two that were lost to follow-up, one for which antenatal assessment of lung hypoplasia was not available and six cases which were found to have major associated anomalies or syndromes after birth. Of the 214 fetuses with iRCDH included in the neonatal outcome analysis, 86 were managed expectantly during pregnancy and 128 underwent fetal endoscopic tracheal occlusion (FETO) with a balloon. In the expectant-management group, lung size measured by ultrasound or by magnetic resonance imaging was the only independent predictor of survival (observed-to-expected lung-to-head ratio (o/e-LHR) odds ratio (OR), 1.06 (95% CI, 1.02-1.11); P = 0.003). Until now, stratification for severe lung hypoplasia has been based on an o/e-LHR cut-off of 45%. In cases managed expectantly, the survival rate was 15% (4/27) in those with o/e-LHR ≤ 45% and 61% (36/59) for o/e-LHR > 45% (P = 0.001). However, the best o/e-LHR cut-off for the prediction of survival at discharge was 50%, with a sensitivity of 78% and specificity of 72%. In the expectantly managed group, survivors with severe pulmonary hypoplasia stayed longer in the neonatal intensive care unit than did those with mildly hypoplastic lungs. In fetuses with an o/e-LHR ≤ 45% treated with FETO, survival rate was higher than in those with similar lung size managed expectantly (49/120 (41%) vs 4/27 (15%); P = 0.014), despite higher prematurity rates (gestational age at birth: 34.4 ± 2.7 weeks vs 36.8 ± 3.0 weeks; P < 0.0001). In fetuses treated with FETO, gestational age at birth was the only predictor of survival (OR, 1.25 (95% CI, 1.04-1.50); P = 0.02). CONCLUSIONS: Antenatal measurement of lung size can predict survival in iRCDH. In fetuses with severe lung hypoplasia, FETO was associated with a significant increase in survival without an associated increase in neonatal morbidity. © 2020 International Society of Ultrasound in Obstetrics and Gynecology.

An Ichor-dependent apical extracellular matrix regulates seamless tube shape and integrity.

During sprouting angiogenesis in the vertebrate vascular system, and primary branching in the Drosophila tracheal system, specialized tip cells direct branch outgrowth and network formation. When tip cells lumenize, they form subcellular (seamless) tubes. How these seamless tubes are made, shaped and maintained remains poorly understood. Here we characterize a Drosophila mutant called ichor (ich), and show that ich is essential for the integrity and shape of seamless tubes in tracheal terminal cells. We find that Ich regulates seamless tubulogenesis via its role in promoting the formation of a mature apical extracellular matrix (aECM) lining the lumen of the seamless tubes. We determined that ich encodes a zinc finger protein (CG11966) that acts, as a transcriptional activator required for the expression of multiple aECM factors, including a novel membrane-anchored trypsin protease (CG8213). Thus, the integrity and shape of seamless tubes are regulated by the aECM that lines their lumens.

Anisotropic Crb accumulation, modulated by Src42A, is coupled to polarised epithelial tube growth in Drosophila.

The control of the size of internal tubular organs, such as the lungs or vascular system, is critical for proper physiological activity and to prevent disease or malformations. This control incorporates the intrinsic physical anisotropy of tubes to generate proportionate organs that match their function. The exact mechanisms underlying tube size control and how tubular anisotropy is translated at the cellular level are still not fully understood. Here we investigate these mechanisms using the Drosophila tracheal system. We show that the apical polarity protein Crumbs transiently accumulates anisotropically at longitudinal cell junctions during tube elongation. We provide evidence indicating that the accumulation of Crumbs in specific apical domains correlates with apical surface expansion, suggesting a link between the anisotropic accumulation of Crumbs at the cellular level and membrane expansion. We find that Src42A is required for the anisotropic accumulation of Crumbs, thereby identifying the first polarised cell behaviour downstream of Src42A. Our results indicate that Src42A regulates a mechanism that increases the fraction of Crb protein at longitudinal junctions, and genetic interaction experiments are consistent with Crb acting downstream of Src42A in controlling tube size. Collectively, our results suggest a model in which Src42A would sense the inherent anisotropic mechanical tension of the tube and translate it into a polarised Crumbs accumulation, which may promote a bias towards longitudinal membrane expansion, orienting cell elongation and, as a consequence, longitudinal growth at the tissue level. This work provides new insights into the key question of how organ growth is controlled and polarised and unveils the function of two conserved proteins, Crumbs and Src42A, with important roles in development and homeostasis as well as in disease, in this biological process.

Development pattern of tracheal cartilage in human embryos.

INTRODUCTION: Congenital tracheal anomalies are associated with high morbidity and mortality. The etiology of congenital tracheal anomalies is not well understood, but often attributed to malformed tracheal cartilage. The development of tracheal cartilage has not been described in detail. In this study, we aimed to investigate the development pattern and timing of normal tracheal cartilage to better understand the etiology of tracheal anomalies. MATERIALS AND METHODS: The development of tracheal cartilage was examined by studying the trachea in histological sections of 14 healthy human embryos from the Carnegie collection. Two specimens for Carnegie Stages 17-23 (42-60 days of embryological development) were studied. RESULTS: At Carnegie Stages 17-19 (42-51 days), a continuous mesenchymal condensation was observed ventral to the tracheal lumen. At Stages 20 and 21 (51-54 days), this pre-tracheal mesenchyme showed sites of increased condensation indicative of future tracheal rings. Furthermore, growth centers were identified both proximally and distally in the trachea. Characteristic horseshoe shaped tracheal rings were apparent at Carnegie Stages 22 and 23 (54-60 days). CONCLUSIONS: In human embryos, tracheal rings arise from growth centers in the ventral mesenchyme at approximately 51-54 days of embryological development. The observation of proximal and distal growth centers suggests a centripetal growth gradient, potentially contributing to occurrence of complete tracheal ring deformity (CTRD). Although this study shows new insights on tracheal cartilage development, the exact origin of congenital tracheal defects has yet to be elucidated.

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.

Single-cell branching morphogenesis in the Drosophila trachea.

The terminal cells of the tracheal epithelium in Drosophila melanogaster are one of the few known cell types that undergo subcellular morphogenesis to achieve a stable, branched shape. During the animal's larval stages, the cells repeatedly sprout new cytoplasmic processes. These grow very long, wrapping around target tissues to which the terminal cells adhere, and are hollowed by a gas-filled subcellular tube for oxygen delivery. Our understanding of this ramification process remains rudimentary. This review aims to provide a comprehensive summary of studies on terminal cells to date, and attempts to extrapolate how terminal branches might be formed based on the known genetic and molecular components. Next to this cell-intrinsic branching mechanism, we examine the extrinsic regulation of terminal branching by the target tissue and the animal's environment. Finally, we assess the degree of similarity between the patterns established by the branching programs of terminal cells and other branched cells and tissues from a mathematical and conceptual point of view.