iPSCs derived from esophageal atresia patients reveal SOX2 dysregulation at the anterior foregut stage.

A series of well-regulated cellular and molecular events result in the compartmentalization of the anterior foregut into the esophagus and trachea. Disruption of the compartmentalization process leads to esophageal atresia/tracheoesophageal fistula (EA/TEF). The cause of EA/TEF remains largely unknown. Therefore, to mimic the early development of the esophagus and trachea, we differentiated induced pluripotent stem cells (iPSCs) from EA/TEF patients, and iPSCs and embryonic stem cells from healthy individuals into mature three-dimensional esophageal organoids. CXCR4, SOX17 and GATA4 expression was similar in both patient-derived and healthy endodermal cells. The expression of the key transcription factor SOX2 was significantly lower in the patient-derived anterior foregut. We also observed an abnormal expression of NKX2.1 (or NKX2-1) in the patient-derived mature esophageal organoids. At the anterior foregut stage, RNA sequencing revealed the critical genes GSTM1 and RAB37 to be significantly lower in the patient-derived anterior foregut. We therefore hypothesize that a transient dysregulation of SOX2 and the abnormal expression of NKX2.1 in patient-derived cells could be responsible for the abnormal foregut compartmentalization.

Histological, immunohistochemical and transcriptomic characterization of human tracheoesophageal fistulas.

Esophageal atresia (EA) and tracheoesophageal fistula (TEF) are relatively frequently occurring foregut malformations. EA/TEF is thought to have a strong genetic component. Not much is known regarding the biological processes disturbed or which cell type is affected in patients. This hampers the detection of the responsible culprits (genetic or environmental) for the origin of these congenital anatomical malformations. Therefore, we examined gene expression patterns in the TEF and compared them to the patterns in esophageal, tracheal and lung control samples. We studied tissue organization and key proteins using immunohistochemistry. There were clear differences between TEF and control samples. Based on the number of differentially expressed genes as well as histological characteristics, TEFs were most similar to normal esophagus. The BMP-signaling pathway, actin cytoskeleton and extracellular matrix pathways are downregulated in TEF. Genes involved in smooth muscle contraction are overexpressed in TEF compared to esophagus as well as trachea. These enriched pathways indicate myofibroblast activated fibrosis. TEF represents a specific tissue type with large contributions of intestinal smooth muscle cells and neurons. All major cell types present in esophagus are present-albeit often structurally disorganized-in TEF, indicating that its etiology should not be sought in cell fate specification.

Transition Zone in Total Colonic Aganglionosis and Colorectal Hirschsprung's Disease Shows a Similar Trend of Mucosal Innervation: Image Processing and Analysis Study.

The aganglionic segment of bowel in Hirschsprung's disease (HD) varies in length. It is not clear whether total colonic aganglionosis (TCA) merely represents a long form of HD or a different phenotype of the disease. Animal model studies suggest that TCA may have a longer transition zone (TZ) than conventional colorectal HD. We compared mucosal innervation of TZ in 2 TCA cases and 10 conventional colorectal HD cases by quantifying calretinin-positive mucosal nerve fibers using image processing and analysis. One TCA was associated with esophageal atresia-tracheoesophageal fistula, the other with trisomy 21. The gradients of calretinin-stained pixel count increase per distance from the beginning of TZ (slope) for TCA were not significantly different from those for the conventional HD group. Given this observation, it is speculated that the length of TZ in TCA may fall within the range of and may not be much longer than conventional colorectal HD.

Distinct molecular pathways mediate Mycn and Myc-regulated miR-17-92 microRNA action in Feingold syndrome mouse models.

Feingold syndrome is a skeletal dysplasia caused by loss-of-function mutations of either MYCN (type 1) or MIR17HG that encodes miR-17-92 microRNAs (type 2). Since miR-17-92 expression is transcriptionally regulated by MYC transcription factors, it has been postulated that Feingold syndrome type 1 and 2 may be caused by a common molecular mechanism. Here we show that Mir17-92 deficiency upregulates TGF-ß signaling, whereas Mycn-deficiency downregulates PI3K signaling in limb mesenchymal cells. Genetic or pharmacological inhibition of TGF-ß signaling efficiently rescues the skeletal defects caused by Mir17-92 deficiency, suggesting that upregulation of TGF-ß signaling is responsible for the skeletal defect of Feingold syndrome type 2. By contrast, the skeletal phenotype of Mycn-deficiency is partially rescued by Pten heterozygosity, but not by TGF-ß inhibition. These results strongly suggest that despite the phenotypical similarity, distinct molecular mechanisms underlie the pathoetiology for Feingold syndrome type 1 and 2.

Interkinetic nuclear migration in the tracheal and esophageal epithelia of the mouse embryo: Possible implications for tracheo-esophageal anomalies.

Interkinetic nuclear migration (INM) is a cell polarity-based phenomenon in which progenitor cell nuclei migrate along the apico-basal axis of the pseudostratified epithelium in synchrony with the cell cycle. INM is suggested to be at least partially cytoskeleton-dependent and to regulate not only the proliferation/differentiation of stem/progenitor cells but also the localized/overall size and shape of organs/tissues. INM occurs in all three of the germ-layer derived epithelia, including the endoderm-derived gut. However, INM has not been documented in the esophagus and respiratory tube arising from the anterior foregut. Esophageal atresia with or without trachea-esophageal fistula (EA/TEF) is a relatively common developmental defect. Transcription factors and signaling molecules have been implicated in EA/TEF, but the etiology of EA/TEF-which has been suggested to involve cell polarity-related mechanisms-remains highly controversial. In the present study, we first examined whether INM exists in the trachea and esophagus of mouse embryos at embryonic day 11.5 (E11.5), just after separation of the two tubes from the anterior foregut. By labeling the DNA-synthesizing stem cell nuclei with 5-ethynyl-2'-deoxyuridine, a nucleotide analogue, and statistically analyzing chronological changes in the distribution pattern of the labeled nuclei by using multidimensional scaling, we showed the existence of INM in both the esophagus and trachea, with differences in the INM magnitude and cycle pattern. We further showed morphological changes from the INM-based pseudostratified single layer to the stratified multilayer in the esophageal epithelium in association with a temporal loss/perturbation of AB polarity, suggesting a possible relation with the pathogenesis of EA/TEF.

Assessment of Clinical Symptoms and Multichannel Intraluminal Impedance and pH Monitoring in Children After Thoracoscopic Repair of Esophageal Atresia and Distal Tracheoesophageal Fistula.

BACKGROUND: Motility disturbances of the esophagus and gastroesophageal reflux disease (GERD) are observed frequently in children after repair of congenital esophageal atresia with distal tracheoesophageal fistula (EA/TEF). Recently, in many pediatric surgical centers, thoracoscopic repair was introduced, which theoretically would change the postoperative course. OBJECTIVES: The goal of the study was to assess physical development, disease symptoms, and GERD symptom frequency in children who underwent thoracoscopic surgery of congenital EA/TEF. MATERIAL AND METHODS: The study comprised 22 children (14 boys and 8 girls), aged 16 to 79 months (average age 47.3 months) after surgery of EA/TEF. Clinical symptoms and physical development were analyzed. In 19 children, multichannel intraluminal esophageal impedance connected with pH-metry (MII/pH) was performed. In 11 patients, esophagogastroduodenoscopy with a histological study of mucosa samples was done. RESULTS: The most frequent symptoms were dysphagia, belching, cough and recurrent bronchitis. In 31.5% of the children, body mass deficiency was observed and in 28.6%, low body mass and short stature. Prematurity was present in half of the patients. Depending on the result of the MII/pH study, the children were divided into two groups: 10 children with GERD and 9 children without diagnosis of gastroesophageal reflux. In the 10 with GERD, acid reflux was diagnosed in 9 and non-acid reflux was diagnosed in one. MII/pH demonstrated statistically significant differences in the number of reflux episodes, reflux index, bolus exposure index, mean time of esophageal exposure and acid exposure and mean acid clearance time in children with GERD in comparison to children without this disease. In 36.4% of children who underwent endoscopy, esophagitis was diagnosed, esophageal stricture in 18% and gastric metaplasia in 9.1%. CONCLUSIONS: In children who underwent corrective thoracoscopic surgery of EA/TEF, GERD caused by motility disorders of the esophagus was frequently observed. These children require constant monitoring and early treatment of the complication.

Lung maturity in esophageal atresia: Experimental and clinical study.

INTRODUCTION: Esophageal atresia and tracheoesophageal fistula (EA-TEF) survivors suffer respiratory morbidity of unclear pathogenesis. Defective lung morphogenesis has been described in the rat model. This study examined fetal lung growth and maturity in rats and patients with EA-TEF. METHODS: Pregnant rats received either adriamycin or vehicle. Control and adriamycin-exposed lungs, with and without EA-TEF, were weighed and processed for RT-PCR, DNA quantification, immunofluorescence and immunoblot analysis of TTF1, VEGF, Sp-B, and α-sma. Twenty human lungs were also processed for immunofluorescence and Alcian-blue staining. RESULTS: Lungs from fetuses with EA-TEF (E21) showed decreased total DNA; FGF7 and TTF1 mRNA expressions were upregulated at E15 and E18, respectively. Protein expression and immunofluorescent distribution of maturity markers were similar. Lungs from stillborns with EA-TEF showed decreased epithelial expression of Sp-B and VEGF whereas those from newborns tended to have less Sp-B and more VEGF and mucous glands. DISCUSSION: The lungs of rats with EA-TEF were hypoplastic but achieved near-normal maturity. Stillborns with EA-TEF exhibited an apparently disturbed differentiation of the airway epithelium. Newborns with EA-TEF demonstrated subtle differences in the expression of differentiation markers, and increased number of mucous glands that could influence postnatal respiratory adaptation and explain some respiratory symptoms of EA-TEF survivors.

De novo GLI3 mutation in esophageal atresia: reproducing the phenotypic spectrum of Gli3 defects in murine models.

Esophageal atresia is a common and life-threatening birth defect with a poorly understood etiology. In this study, we analyzed the sequence variants of coding regions for a set of esophageal atresia-related genes including MYCN, SOX2, CHD7, GLI3, FGFR2 and PTEN for mutations using PCR-based target enrichment and next-generation sequencing in 27 patients with esophageal atresia. Genomic copy number variation analysis was performed using Affymetrix SNP 6.0. We found a de novo heterozygous mutation in the N-terminal region of the GLI3 gene (c.332T>C, p.M111T) in a patient with esophageal atresia and hemivertebrae. The N-terminal region (amino acids 1-397) of GLI3 contains the repressor domain, which interacts with SKI family proteins. Using the co-immunoprecipitation assay, we found that interaction of GLI3 with the SKI family protein SKIL was significantly compromised by the p.M111T mutation of GLI3. Thus far, all the identified mutations mapped within the repressor domain of GLI3 were nonsense and frame-shift mutations. In this study, a missense mutation was initially detected in this region. Our finding is the first to link this GLI3 gene mutation with esophageal atresia in humans, which was previously suggested in an animal model.

Genetic and cellular mechanisms of the formation of esophageal atresia and tracheoesophageal fistula.

Foregut separation involves dynamic changes in the activities of signaling pathways and transcription factors. Recent mouse genetic studies demonstrate that some of these pathways interact with each other to form a complex network, leading to a unique dorsal-ventral patterning in the early foregut. In this review, it is discussed how this unique dorsal-ventral patterning is set prior to the foregut separation and how disruption of this patterning affects the separation process. Roles of downstream targets of these pathways in regulating separation at cellular and molecular levels would be discussed further. Understanding the mechanism of normal separation process will provide insights into the pathobiology of a relatively common birth defect, esophageal atresia with/without tracheoesophageal fistula.

Analysis of saliva pepsin level in patients with tracheoesophageal fistula and voice prosthesis complications.

The aim of this crossectional study was to investigate the relationship between pepsin concentration in saliva and the occurrence of tracheoesophageal fistula (TEF) complications and voice prosthesis (VP) complications, after total laryngectomy and VP implantation. We assessed the concentrations of pepsin in the saliva of 41 laryngectomized patients and correlated it with the incidence of TEF complications (periprostethic leakage, atrophy, esophageal mucosa hypertrophy, granulations, fistula enlargement, and VP dislocation), VP complications (transprosthetic leakage, Candida infection) and voice quality. Pepsin levels were measured by enzyme-linked immunoadsorbent assay (ELISA). Voice quality was assessed by Harrison-Robilard--Schultz (HRS) scale. In all, 17 (42%) patients had complications. All of them had TEF complications, whereas VP complication, together with TEF was found in 9 (22%) patients. We found no significant correlation between adjuvant radiotherapy and TEF complications. Most of patients, 30 (73%), had positive pepsin level in saliva. Median value of pepsin concentration in all patients was 4.8 (range 81.7). Median pepsin concentration was higher in patients free of TEF or VP complications (6.6, range 81.7 vs. 3.2, range 19.3) but that difference was not statistically significant (Mann-Whitney test, Z--1.562, p = 0.118). In addition, statistically insignificant negative correlation between pepsin levels and voice quality measured by HRS scale (Spearman's rho, p > 0.05). Although reflux was proposed as cause of TEF complications and pepsin has been proven as a most sensitive and specific marker of ekstraesophageal reflux, we did not find any statistically significant correlation between pepsin levels and occurrence of TEF or VP complications.

Fgf10 gene expression is delayed in the embryonic lung mesenchyme in the adriamycin mouse model.

BACKGROUND: The adriamycin mouse model is a well-established teratogenic model of esophageal atresia/tracheoesophageal fistula. Fibroblast growth factor 10 (Fgf10) plays a key role in branching of the lung buds during lung morphogenesis. Fgf10 knockout mice exhibit the absence of the lungs. Optical projection tomography (OPT) is a technique that allows three-dimensional (3D) imaging of gene expression in small tissue specimens in an anatomical context. The aim of this study was to investigate the temporo-spatial expression of Fgf10 during the critical period of separation of the trachea and esophagus in normal and adriamycin-treated embryos using OPT. METHODS: Time-mated CBA/Ca mice received intraperitoneal injections of adriamycin (6 mg/kg) or saline on days 7 and 8 of gestation. Embryos were harvested on days 10-13, stained after whole mount in situ hybridization with labeled RNA probes to detect Fgf10 transcripts (n = 5 for each treatment/day of gestation). Immunolocalization with endoderm marker Hnf3 beta was used to visualize morphology. Embryos were scanned by OPT to obtain 3D representations of gene expression domains. RESULTS: Computer reconstructed specimens allowed precise staging of developing embryos according to Theiler Staging (TS) criteria. OPT elegantly displayed Fgf10 gene expression in the pulmonary mesenchyme around the tip of the lung buds in both controls and treated embryos in the same spatial territory. Fgf10 gene expression was first detected in the control embryos at TS17. However, Fgf10 gene expression in adriamycin-treated embryos was first only observed at TS18 in 67% of the specimens. CONCLUSION: Delayed Fgf10 gene expression during the critical period of separation of the trachea and esophagus may affect lung bud formation in the adriamycin model leading to tracheoesophageal malformations.

Visualizing expression patterns of Shh and Foxf1 genes in the foregut and lung buds by optical projection tomography.

Congenital malformations of the foregut are common in humans. The respiratory and digestive tubes are both derived by division of the foregut primordium. Sonic hedgehog (Shh) and Fork head box F1 (Foxf1) genes encode regulatory molecules that play a pivotal role in gut and lung morphogenesis and are therefore important candidate genes to be examined in models of foregut developmental disruption. Optical projection tomography (OPT) is a new, rapid and non-invasive technique for three-dimensional (3D) imaging of small biological tissue specimens that allows visualization of the tissue distribution of RNA in developing organs while also recording morphology. To explore the application of OPT in this context, we visualized Shh and Foxf1 gene expression patterns in the mouse foregut and lung buds at several stages of development. Time-mated CBA/Ca mice were harvested on embryonic days 9-12. The embryos were stained following whole mount in situ hybridization with labelled RNA probes to detect Shh and Foxf1 transcripts at each stage. The embryos were scanned by OPT to obtain 3D representations of gene expression domains in the context of the changing morphology of the embryo. OPT analysis of Shh and Foxf1 expression in the foregut and lung buds revealed extra details of the patterns not previously reported, particularly in the case of Foxf1 where gene expression was revealed in a changing pattern in the mesenchyme around the developing lung. Shh expression was also revealed in the epithelium of the lung bud itself. Both genes were detected in complementary patterns in the developing bronchi as late as E12, showing successful penetration of molecular probes and imaging at later stages. OPT is a valuable tool for revealing gene expression in an anatomical context even in internal tissues like the foregut and lung buds across stages of development, at least until E12. This provides the possibility of visualizing altered gene expression in an in vivo context in genetic or teratogenic models of congenital malformations.

The structural characteristics and expression of neuropeptides in the esophagus of patients with congenital esophageal atresia and tracheoesophageal fistula.

PURPOSE: The aim of this study was to investigate the structural characteristics and the expression of a group of neuropeptides in the esophagus of patients with congenital esophageal atresia and tracheoesophageal fistula (EA-TEF), as well to elucidate the roles of these neuropeptides in the pathogenesis of postoperative incoordination of esophagus after successful surgical repair of EA-TEF. METHODS: Twenty-four specimens from distal tracheoesophageal fistulas of patients with EA-TEF (EA-TEF group) and 10 esophageal specimens from neonates who died of nonesophageal diseases (control group) were studied. All of the specimens were subjected to routine pathologic study, ultrastructural observation, and immunohistochemical staining for neuron-specific enolase, substance P, vasoactive intestinal polypeptide, and nitric oxide synthase. RESULTS: In the EA-TEF group, mitochondria were distributed along the membrane of smooth muscle cell, whereas mitochondria in the control group were distributed along the karyotheca of the smooth muscle cells. The ratio of granulated vesicles to clear vesicles in the varicosity of the intramuscular motor nerve ending of the EA-TEF group (0.520 +/- 0.137) was much higher than that in the control group (0.192 +/- 0.020, P < .05). The percentages of specimens shown to have positive expression of neuron-specific enolase and substance P in the EA-TEF group (20.8% and 12.5%, respectively) were significantly lower than those in the control group (90% and 80% respectively, P < .05). The percentages of specimens shown to have positive expression of vasoactive intestinal polypeptide and nitric oxide synthase in the EA-TEF group (83.3% and 75%, respectively) were significantly higher than that in the control group (30% and 10% respectively, P < .05). CONCLUSION: Imbalance of neurotransmitters excretion in nerve vesicle, abnormal intrinsic dysplasia of nerve plexus and increased expression of certain neuropeptides were the main characteristics of esophagus with abnormal intrinsic innervation, which may be responsible for the postoperative esophageal dysfunction of EA-TEF.

Multiple dose-dependent roles for Sox2 in the patterning and differentiation of anterior foregut endoderm.

Sox2 is expressed in developing foregut endoderm, with highest levels in the future esophagus and anterior stomach. By contrast, Nkx2.1 (Titf1) is expressed ventrally, in the future trachea. In humans, heterozygosity for SOX2 is associated with anopthalmia-esophageal-genital syndrome (OMIM 600992), a condition including esophageal atresia (EA) and tracheoesophageal fistula (TEF), in which the trachea and esophagus fail to separate. Mouse embryos heterozygous for the null allele, Sox2(EGFP), appear normal. However, further reductions in Sox2, using Sox2(LP) and Sox2(COND) hypomorphic alleles, result in multiple abnormalities. Approximately 60% of Sox2(EGFP/COND) embryos have EA with distal TEF in which Sox2 is undetectable by immunohistochemistry or western blot. The mutant esophagus morphologically resembles the trachea, with ectopic expression of Nkx2.1, a columnar, ciliated epithelium, and very few p63(+) basal cells. By contrast, the abnormal foregut of Nkx2.1-null embryos expresses elevated Sox2 and p63, suggesting reciprocal regulation of Sox2 and Nkx2.1 during early dorsal/ventral foregut patterning. Organ culture experiments further suggest that FGF signaling from the ventral mesenchyme regulates Sox2 expression in the endoderm. In the 40% Sox2(EGFP/COND) embryos in which Sox2 levels are approximately 18% of wild type there is no TEF. However, the esophagus is still abnormal, with luminal mucus-producing cells, fewer p63(+) cells, and ectopic expression of genes normally expressed in glandular stomach and intestine. In all hypomorphic embryos the forestomach has an abnormal phenotype, with reduced keratinization, ectopic mucus cells and columnar epithelium. These findings suggest that Sox2 plays a second role in establishing the boundary between the keratinized, squamous esophagus/forestomach and glandular hindstomach.

Genetic players in esophageal atresia and tracheoesophageal fistula.

Esophageal atresia is a common and serious developmental anomaly, of which the causes remain largely unknown. Studies in vertebrate models indicate the importance of the sonic hedgehog pathway in esophageal atresia, but its relevance to the human condition remains to be defined. Now, three genes have been identified that cause syndromic forms of esophageal atresia when mutated. NMYC and SOX2 are transcription factors, whereas CHD7 is encoded by a chromodomain helicase DNA-binding gene, important for chromatin structure and gene expression. These new genes broaden our view of human foregut development.

Aberrant fibroblast growth factor receptor 2 signalling in esophageal atresia with tracheoesophageal fistula.

BACKGROUND: Although the pathogenesis of esophageal atresia with tracheoesophageal fistula (EA/TEF) remains unknown, it has been shown that despite its esophageal appearance, the fistula tract originates from respiratory epithelium. The authors now hypothesize that defects in fibroblast growth factor (FGF) signaling contribute to the esophaguslike phenotype of the fistula tract. FGF2R is critical to normal lung morphogenesis and occurs in 2 isoforms (FGF2RIIIb and FGF2RIIIc), each with different ligand-binding specificity. To characterize FGF signaling in the developing EA/TEF, the authors analyzed levels of FGF2R splice variants in experimental EA/TEF. METHODS: The standard Adriamycin-induced EA/TEF model in rats was used. Individual foregut components from Adriamycin-treated and control embryos were processed for real-time, fluorescence-activated semiquantitative reverse transcriptase polymerase chain reaction on gestational days 12.5 and 13.5. RESULTS: Both fistula tract and Adriamycin-treated or normal esophagus showed significantly lower levels of FGF2RIIIb than either Adriamycin-treated lung buds (E12.5, P =.02; E13.5, P <.005) or normal lung buds (E12.5, P <.005; E13.5, P <.01). At E13.5, the fistula tract had lower levels of FGF2RIIIc than either treated (P <.01) or normal lung (P <.05). CONCLUSIONS: Levels of FGF2R in the developing fistula tract resemble that of distal esophagus rather than developing lung. This defect in FGF2RIIIb signaling may account for the nonbranching, esophaguslike phenotype of the fistula, despite its respiratory origin.

Defective fibroblast growth factor signaling allows for nonbranching growth of the respiratory-derived fistula tract in esophageal atresia with tracheoesophageal fistula.

BACKGROUND/PURPOSE: The fistula tract in esophageal atresia with tracheoesophageal fistula (EA-TEF) appears to arise from a trifurcation of the embryonic lung bud. Subsequently, it does not branch like the other bronchi, which also arise from the lung bud. Previous results have implied that aberrant mesenchymal-epithelial signaling in the developing foregut, possibly involving fibroblast growth factors, may allow for the nonbranching growth of the fistula, and the ultimate development of the fistula tract in TEF. METHODS: Adriamycin injections into pregnant rat dams induced EA-TEF formation in rat embryos. Control and Adriamycin-exposed embryos were harvested on the 13th gestational day, and the developing foregut was isolated with microdissection. mRNA was isolated from the developing fistula tract, embryonic lung, and normal embryonic esophagus. Reverse transcription-polymerase chain reaction (RT-PCR) for the IIIb splice variant of the FGF2R receptor was performed. Foregut specimens also were processed for histologic analysis, and immunofluorescence for FGF1 was performed. RESULTS: FGF2R-IIIb is specifically absent from the developing fistula tract in TEF, whereas it is present in the normal developing lung and esophagus. FGF1 also is uniquely absent from the developing fistula tract, but it is present in the normal lung mesenchyme. CONCLUSIONS: FGF1, FGF7, and FGF10 are critical mesenchymal factors that mediate proliferation and branching morphogenesis by the developing respiratory epithelium. The absence of FGF2R-IIIb, the obligate common receptor for FGF7 and FGF10, from the fistula tract, and the absence of FGF1 in the fistula tract mesenchyme, collectively imply the absence of a specific FGF signaling pathway in the developing fistula tract. This absence of FGF signaling could explain the lack of branching by the developing fistula tract as it grows caudally in the abnormally developing embryo. Downregulation of these components of the FGF signaling pathways may allow for a patterned compensation by the embryo for the proximal foregut atresia in this anomaly. This compensation may then reestablish gastrointestinal continuity as the fistula tract connects to the developing stomach.