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.

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.

Extracellular Vesicles and Their miRNA Content in Amniotic and Tracheal Fluids of Fetuses with Severe Congenital Diaphragmatic Hernia Undergoing Fetal Intervention.

Infants with congenital diaphragmatic hernia (CDH) are at high risk of postnatal mortality due to lung hypoplasia and arterial pulmonary hypertension. In severe cases, prenatal intervention by fetal endoscopic tracheal occlusion (FETO) can improve survival by accelerating lung growth. However, postnatal mortality remains in the range of about 50% despite fetal treatment, and there is currently no clear explanation for this different clinical response to FETO. We evaluated the concentration of extracellular vesicles (EVs) and associated microRNA expression in amniotic and tracheal fluids of fetuses with CDH undergoing FETO, and we examined the association between molecular findings and postnatal survival. We observed a higher count of EVs in the amniotic fluid of non-survivors and in the tracheal fluid sampled in utero at the time of reversal of tracheal occlusion, suggesting a pro-inflammatory lung reactivity that is already established in utero and that could be associated with a worse postnatal clinical course. In addition, we observed differential regulation of four EV-enclosed miRNAs (miR-379-5p, miR-889-3p; miR-223-3p; miR-503-5p) in relation to postnatal survival, with target genes possibly involved in altered lung development. Future research should investigate molecular therapeutic agents targeting differentially regulated miRNAs to normalize their expression and potentially improve clinical outcomes.

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.

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.

Prenatal diagnosis of persistent left superior vena cava, polyhydramnios and a small gastric bubble in a fetus with VACTERL association.

OBJECTIVE: We reported a fetus that presenting with persistent left superior vena cava (PLSVC), polyhydramnios, and a small gastric bubble during prenatal examination and identified VACTERL association after birth. CASE REPORT: A 34-year-old woman underwent amniocentesis at 18 weeks of gestation because of advanced maternal age and the result was normal. Subsequently, an ultrasound revealed single umbilical artery (SUA) at 21 weeks of gestation. She received a detailed fetal anatomy survey that presented the same findings and PLSVC. A small visible gastric bubble was noted at that time, and the other organs were unremarkable. Polyhydramnios was identified at 30 weeks of gestation and amnioreduction was subsequently performed at 32 weeks of gestation. However, polyhydramnios was persisted despite amnioreduction and intrauterine growth restriction was also detected. A cesarean section was performed because of fetal distress at 36 + 2 weeks, and a 1832-g female baby was delivered. Pre-axial polydactyly at left thumb, SUA and esophageal atresia with distal tracheoesophageal fistula (TEF) were identified after birth. The neonate died at age of 4 days because of surgical complication following esophageal anastomosis. CONCLUSION: Prenatal diagnosis of PLSVC associated with polyhydramnios and a small gastric bubble may indicate esophageal atresia with TEF, and further examination for associated syndromes such as VACTERL association is warranted.

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.

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.

Genotype-Phenotype Correlation of Tracheal Cartilaginous Sleeves and Fgfr2 Mutations in Mice.

OBJECTIVES: To characterize tracheal cartilage morphology in mouse models of fibroblast growth factor receptor (Fgfr2)-related craniosynostosis syndromes. To establish relationships between specific Fgfr2 mutations and tracheal cartilaginous sleeve (TCS) phenotypes in these mouse models. METHODS: Postnatal day 0 knock-in mouse lines with disease-specific genetic variations in the Fgfr2 gene (Fgfr2C342Y/C342Y , Fgfr2C342Y/+ , Fgfr2+/Y394C , Fgfr2+/S252W , and Fgfr2+/P253R ) as well as line-specific controls were utilized. Tracheal cartilage morphology as measured by gross analyses, microcomputed tomography (µCT), and histopathology were compared using Chi-squared and single-factor analysis of variance statistical tests. RESULTS: A greater proportion of rings per trachea were abnormal in Fgfr2C342Y/+ tracheas (63%) than Fgfr2+/S252W (17%), Fgfr2+/P253R (17%), Fgfr2+/Y394C (12%), and controls (10%) (P < .001 for each vs. Fgfr2C342Y/+ ). TCS segments were found only in Fgfr2C342Y/C342Y (100%) and Fgfr2C342Y/+ (72%) tracheas. Cricoid and first-tracheal ring fusion was noted in all Fgfr2C342Y/C342Y and 94% of Fgfr2C342Y/+ samples. The Fgfr2C342Y/C342Y and Fgfr2C342Y/+ groups were found to have greater areas and volumes of cartilage than other lines on gross analysis and µCT. Histologic analyses confirmed TCS among the Fgfr2C342Y/C342Y and Fgfr2C342Y/+ groups, without appreciable differences in cartilage morphology, cell size, or density; no histologic differences were observed among other Fgfr2 lines compared to controls. CONCLUSION: This study found TCS phenotypes only in the Fgfr2C342Y mouse lines. These lines also had increased tracheal cartilage compared to other mutant lines and controls. These data support further study of the Fgfr2 mouse lines and the investigation of other Fgfr2 variants to better understand their role in tracheal development and TCS formation. LEVEL OF EVIDENCE: NA Laryngoscope, 131:E1349-E1356, 2021.

Global pattern of interkinetic nuclear migration in tracheoesophageal epithelia of the mouse embryo: Interorgan and intraorgan regional differences.

Interkinetic nuclear migration (INM) is an apicobasal (AB) polarity-based regulatory mechanism of proliferation/differentiation in epithelial stem/progenitor cells. We previously documented INM in the endoderm-derived tracheal/esophageal epithelia at embryonic day (E) 11.5 and suggested that INM is involved in the development of both organs. We here investigated interorgan (trachea vs esophagus) and intraorgan regional (ventral vs dorsal) differences in the INM mode in the tracheal and esophageal epithelia of the mouse embryo. We also analyzed convergent extension (CE) and planar cell movement (PCM) in the epithelia based on cell distribution. The pregnant C57BL/6J mice were intraperitoneally injected with 5-ethynyl-2'-deoxyuridine at E11.5 and E12.5 and were sacrificed 1, 4, 6, 8, and 12 hours later to obtain the embryos. The distribution of labeled cell nuclei along the AB axis was chronologically analyzed in the total, ventral, and dorsal sides of the epithelia. The percentage distribution of the nuclei population was represented by histogram and the chronological change was analyzed statistically using multidimensional scaling. The interorgan comparison of the INM mode during E11.5-E12.0, but not E12.5-E13.0, showed a significant difference. During E11.5-E12.0 the trachea, but not the esophagus, showed a significant difference between ventral and dorsal sides. During E12.5-E13.0 neither organ showed regional differences. CE appeared to occur in both organs during E11.5-E12.0 while PCM was unclear in both organs. These findings suggest a difference between the trachea and esophagus, and a regional difference in the trachea, not in the esophagus, in the INM mode, which may be related with the later differential organogenesis/histogenesis of these organs.

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.

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.

Unravelling the distinct contribution of cell shape changes and cell intercalation to tissue morphogenesis: the case of the Drosophila trachea.

Intercalation allows cells to exchange positions in a spatially oriented manner in an array of diverse processes, spanning convergent extension in embryonic gastrulation to the formation of tubular organs. However, given the co-occurrence of cell intercalation and changes in cell shape, it is sometimes difficult to ascertain their respective contribution to morphogenesis. A well-established model to analyse intercalation, particularly in tubular organs, is the Drosophila tracheal system. There, fibroblast growth factor (FGF) signalling at the tip of the dorsal branches generates a 'pulling' force believed to promote cell elongation and cell intercalation, which account for the final branch extension. Here, we used a variety of experimental conditions to study the contribution of cell elongation and cell intercalation to morphogenesis and analysed their mutual requirements. We provide evidence that cell intercalation does not require cell elongation and vice versa. We also show that the two cell behaviours are controlled by independent but simultaneous mechanisms, and that cell elongation is sufficient to account for full extension of the dorsal branch, while cell intercalation has a specific role in setting the diameter of this structure. Thus, rather than viewing changes in cell shape and cell intercalation as just redundant events that add robustness to a given morphogenetic process, we find that they can also act by contributing to different features of tissue architecture.

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.

Bidirectional Wnt signaling between endoderm and mesoderm confers tracheal identity in mouse and human cells.

The periodic cartilage and smooth muscle structures in mammalian trachea are derived from tracheal mesoderm, and tracheal malformations result in serious respiratory defects in neonates. Here we show that canonical Wnt signaling in mesoderm is critical to confer trachea mesenchymal identity in human and mouse. At the initiation of tracheal development, endoderm begins to express Nkx2.1, and then mesoderm expresses the Tbx4 gene. Loss of ß-catenin in fetal mouse mesoderm causes loss of Tbx4+ tracheal mesoderm and tracheal cartilage agenesis. The mesenchymal Tbx4 expression relies on endodermal Wnt activation and Wnt ligand secretion but is independent of known Nkx2.1-mediated respiratory development, suggesting that bidirectional Wnt signaling between endoderm and mesoderm promotes trachea development. Activating Wnt, Bmp signaling in mouse embryonic stem cell (ESC)-derived lateral plate mesoderm (LPM) generates tracheal mesoderm containing chondrocytes and smooth muscle cells. For human ESC-derived LPM, SHH activation is required along with WNT to generate proper tracheal mesoderm. Together, these findings may contribute to developing applications for human tracheal tissue repair.

New device permitting non-invasive reversal of fetal endoscopic tracheal occlusion: ex-vivo and in-vivo study.

OBJECTIVE: One of the drawbacks of fetal endoscopic tracheal occlusion (FETO) for congenital diaphragmatic hernia is the need for a second invasive intervention to re-establish airway patency. The 'Smart-TO' device is a new balloon for FETO that deflates spontaneously when placed in a strong magnetic field, therefore overcoming the need for a second procedure. The safety and efficacy of this device have not yet been demonstrated. The aim of this study was to investigate the reversibility, local side effects and occlusiveness of the Smart-TO balloon, both in a simulated in-utero environment and in the fetal lamb model. METHODS: First, the reversibility of tracheal occlusion by the Smart-TO balloon was tested in a high-fidelity simulator. Following videoscopic tracheoscopic balloon insertion, the fetal mannequin was placed within a 1-L water-filled balloon to mimic the amniotic cavity. This was held by an operator in front of their abdomen, and different fetal and maternal positions were simulated to mimic the most common clinical scenarios. Following exposure to the magnetic field generated by a 1.5-T magnetic resonance (MR) machine, deflation of the Smart-TO balloon was assessed by tracheoscopy. In cases of failed deflation, the mannequin was reinserted into a water-filled balloon for additional MR exposure, up to a maximum of three times. Secondly, reversibility, occlusiveness and local effects of the Smart-TO balloon were tested in vivo in fetal lambs. Tracheal occlusion was performed in fetal lambs on gestational day 95 (term, 145 days), either using the balloon currently used in clinical practice (Goldbal2) (n = 5) or the Smart-TO balloon (n = 5). On gestational day 116, the presence of the balloon was assessed by tracheoscopy. Deflation was performed by puncture (Goldbal2) or MR exposure (Smart-TO). Six unoccluded fetal lambs served as controls. Following euthanasia, the lung-to-body-weight ratio (LBWR), lung morphometry and tracheal circumference were assessed. Local tracheal changes were measured using a hierarchical histologic scoring system. RESULTS: Ex vivo, Smart-TO balloon deflation occurred after a single MR exposure in 100% of cases in a maternal standing position with the mannequin at a height of 95 cm (n = 32), 55 cm (n = 8) or 125 cm (n = 8), as well as when the maternal position was 'lying on a stretcher' (n = 8). Three out of eight (37.5%) balloons failed to deflate at first exposure when the maternal position was 'sitting in a wheelchair'. Of these, two balloons deflated after a second MR exposure, but one balloon remained inflated after a third exposure. In vivo, all Smart-TO balloons deflated successfully. The LBWR in fetal lambs with tracheal occlusion by a Smart-TO balloon was significantly higher than that in unoccluded controls, and was comparable with that in the Goldbal2 group. There were no differences in lung morphometry and tracheal circumference between the two balloon types. Tracheal histology showed minimal changes for both balloons. CONCLUSIONS: In a simulated in-utero environment, the Smart-TO balloon was effectively deflated by exposure of the fetus in different positions to the magnetic field of a 1.5-T MR system. There was only one failure, which occurred when the mother was sitting in a wheelchair. In healthy fetal lambs, the Smart-TO balloon is as occlusive as the clinical standard Goldbal2 system and has only limited local side effects. © 2020 International Society of Ultrasound in Obstetrics and Gynecology.

Genetic Mouse Models and Induced Pluripotent Stem Cells for Studying Tracheal-Esophageal Separation and Esophageal Development.

Esophagus and trachea arise from a common origin, the anterior foregut tube. The compartmentalization process of the foregut into the esophagus and trachea is still poorly understood. Esophageal atresia/tracheoesophageal fistula (EA/TEF) is one of the most common gastrointestinal congenital defects with an incidence rate of 1 in 2,500 births. EA/TEF is linked to the disruption of the compartmentalization process of the foregut tube. In EA/TEF patients, other organ anomalies and disorders have also been reported. Over the last two decades, animal models have shown the involvement of multiple signaling pathways and transcription factors in the development of the esophagus and trachea. Use of induced pluripotent stem cells (iPSCs) to understand organogenesis has been a valuable tool for mimicking gastrointestinal and respiratory organs. This review focuses on the signaling mechanisms involved in esophageal development and the use of iPSCs to model and understand it.