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.

Use of hPSC-derived 3D organoids and mouse genetics to define the roles of YAP in the development of the esophagus.

Balanced progenitor activities are crucial for the development and maintenance of high turn-over organs such as the esophagus. However, the molecular mechanisms regulating these progenitor activities in the esophagus remain to be elucidated. Here, we demonstrated that Yap is required for the proliferation of esophageal progenitor cells (EPCs) in the developing murine esophagus. We found that Yap deficiency reduces EPC proliferation and stratification whereas persistent Yap activation increases cell proliferation and causes aberrant stratification of the developing esophagus. We further demonstrated that the role of YAP signaling is conserved in the developing human esophagus by utilizing 3D human pluripotent stem cell (hPSC)-derived esophageal organoid culture. Taken together, our studies combining loss/gain-of-function murine models and hPSC differentiation support a key role for YAP in the self-renewal of EPCs and stratification of the esophageal epithelium.

Esophageal atresia and tracheoesophageal fistula: prenatal sonographic manifestation from early to late pregnancy.

OBJECTIVE: Esophageal atresia and/or tracheoesophageal fistula (EA/TEF) remains one of the most frequently missed congenital anomalies prenatally. The aim of our study was to elucidate the sonographic manifestation of EA/TEF throughout pregnancy. METHODS: This was a retrospective study of data obtained from a tertiary center over a 12-year period. The prenatal ultrasound scans of fetuses with EA/TEF were assessed to determine the presence and timing of detection of three principal signs: small/absent stomach and worsening polyhydramnios, both of which were considered as 'suspected' EA/TEF, and esophageal pouch, which was considered as 'detected' EA/TEF. We assessed the yield of the early (14-16 weeks' gestation), routine mid-trimester (19-26 weeks) and third-trimester (≥ 27 weeks) anomaly scans in the prenatal diagnosis of EA/TEF. RESULTS: Seventy-five cases of EA/TEF with available ultrasound images were included in the study. A small/absent stomach was detected on the early anomaly scan in 3.6% of fetuses scanned, without a definitive diagnosis. On the mid-trimester scan, 19.4% of scanned cases were suspected and 4.3% were detected. On the third-trimester anomaly scan, 43.9% of scanned cases were suspected and 33.9% were detected. An additional case with an esophageal pouch was detected on magnetic resonance imaging (MRI) in the mid-trimester and a further two were detected on MRI in the third trimester. In total, 44.0% of cases of EA/TEF in our cohort were suspected, 33.3% were detected and 10.7% were suspected but, eventually, not detected prenatally. CONCLUSIONS: Prenatal diagnosis of EA/TEF on ultrasound is not feasible before the late second trimester. A small/absent stomach may be visualized as early as 15 weeks' gestation. Polyhydramnios does not develop before the mid-trimester. An esophageal pouch can be detected as early as 22 weeks on a targeted scan in suspected cases. The detection rates of all three signs increase with advancing pregnancy, peaking in the third trimester. The early and mid-trimester anomaly scans perform poorly as a screening and diagnostic test for EA/TEF. © 2020 International Society of Ultrasound in Obstetrics and Gynecology.

A Simple and Easy Technique for Imaging the Fetal Esophagus in the First, Second, and Third Trimesters Using the Transverse Section of the Esophagus in the Area Behind the Heart as a Reference Point.

OBJECTIVE: To describe and evaluate a simple technique of imaging the fetal esophagus, using the echogenic transverse section of the esophagus in the area behind the heart as a reference point, in all the 3 trimesters of pregnancy. METHOD: This was a prospective cross-sectional study of ultrasound imaging of the esophagus in 2 groups of patients: the first group comprised women at 18 to 30 weeks' gestation (208 fetuses); the second group comprised women at 11 to 14 weeks' gestation (102 fetuses). Using a 3- to 5-MHz curvilinear transducer, the transverse section of the collapsed esophagus was imaged in the area behind the heart. The probe was rotated 90 degrees to identify the longitudinal section of the esophagus which was then traced along its entire length. RESULT: This study shows that the collapsed, echogenic, transverse section of the esophagus was persistently seen in the area behind the heart, in more than 99% of fetuses in both the groups. It was a useful starting point to image the longitudinal section of the esophagus in both the groups, particularly in the 18 - 30 week group. Using this technique, the entire length of the esophagus could be traced in 92.3% of 18 - 30 week fetuses and 88.23% of the 11 - 14 week fetuses. CONCLUSION: A collapsed transverse section of the esophagus was persistently and easily seen as a bright echogenic structure in the area behind the heart in more than 99% of fetuses in all 3 trimesters (in both the groups) and, therefore, was an ideal starting point to begin tracing the esophagus in its longitudinal axis.

Severe type IV hiatal hernia secondary to congenital shortened esophagus.

In patients with severe type IV hiatal hernias, clinicians should consider congenital shortened esophagus. This article reviews the causes of shortened esophagus and its clinical manifestations.

Oesophageal and sternohyal muscle fibres are novel Pax3-dependent migratory somite derivatives essential for ingestion.

Striated muscles that enable mouth opening and swallowing during feeding are essential for efficient energy acquisition, and are likely to have played a fundamental role in the success of early jawed vertebrates. The developmental origins and genetic requirements of these muscles are uncertain. Here, we determine by indelible lineage tracing in mouse that fibres of sternohyoid muscle (SHM), which is essential for mouth opening during feeding, and oesophageal striated muscle (OSM), which is crucial for voluntary swallowing, arise from Pax3-expressing somite cells. In vivo Kaede lineage tracing in zebrafish reveals the migratory route of cells from the anteriormost somites to OSM and SHM destinations. Expression of pax3b, a zebrafish duplicate of Pax3, is restricted to the hypaxial region of anterior somites that generate migratory muscle precursors (MMPs), suggesting that Pax3b plays a role in generating OSM and SHM. Indeed, loss of pax3b function led to defective MMP migration and OSM formation, disorganised SHM differentiation, and inefficient ingestion and swallowing of microspheres. Together, our data demonstrate Pax3-expressing somite cells as a source of OSM and SHM fibres, and highlight a conserved role of Pax3 genes in the genesis of these feeding muscles of vertebrates.

Loss of JAM-C leads to impaired esophageal innervations and megaesophagus in mice.

Megaesophagus is a disease where peristalsis fails to occur properly and esophagus is enlarged. The etiology and mechanism of megaesophagus are not well understood. In this study, we reported that junctional adhesion molecule C (JAM-C) knockout mice on a C57/B6 background developed progressive megaesophagus from embryonic day (E) 15.5 onward with complete penetrance. JAM-C knockout mice exhibited a significant reduction in the number of nerve fibers/ganglia in the wall of the esophagus. However, histological analysis revealed that the esophageal wall thickness and structure of JAM-C knockout mice at embryonic stages and young adult were comparable to that of control littermates. Thus, megaesophagus observed in JAM-C knockout mice could be attributed, at least in part, to impaired esophageal innervations. Our data suggest JAM-C as a potential candidate gene for human megaesophagus, and JAM-C knockout mice might serve as a model for the study of human megaesophagus.

Notochord manipulation does not impact oesophageal and tracheal formation from isolated foregut in 3D explant culture.

BACKGROUND: Tracheo-oesophageal malformations result from disturbed foregut separation during early development. The notochord, a specialised embryonic structure, forms immediately adjacent to the dividing foregut. In the Adriamycin mouse model of oesophageal atresia, foregut and notochord abnormalities co-exist, and the site and severity of foregut malformations closely correlate to the position and extent of the notochord defects. Notochord and foregut abnormalities also co-exist in the Noggin Knockout mouse as well in a small number of human cases. The notochord is a source of powerful molecular signals during early embryogenesis, being particularly important for neural crest development. The influence of notochord signaling on the adjacent foregut is not known. The purpose of this study was to examine the impact of notochord manipulation on foregut separation using a robust 3D explant method for culturing isolated foregut which permits oeosphageal and tracheal formation in vitro. METHODS: Foregut was micro-dissected from embryonic day 9 mice (License B100/4447 Irish Medicines Board), embedded in collagen and cultured for 48 h with native notochord intact (n = 6), notochord removed (n = 10) or additional notochord transplanted from stage matched controls (n = 8). Specimens were analysed for foregut morphology and molecular patterning using immunohistochemistry for Hnf3b (an endoderm marker) and Sox2 (a notochord and oesophageal marker) on cryosections. RESULTS: Foregut separation into distinct oesophagus and trachea was observed in isolated foregut specimens with or without their native notochord. In specimens with additional notochord transplants, foregut morphology and molecular patterning were comparable to controls whether or not the native notochord was maintained. In particular foregut separation was not disrupted by the transplantation of additional notochord at the dorsal foregut endoderm. CONCLUSION: The relationship between the embryonic foregut and notochord is complex and ill-defined; however, the notochord does not contribute essentially to oesophagus and trachea formation beyond E9 in the mouse, and the transplantation of additional notochord does not disrupt foregut separation in 3D explant culture.

One shall become two: Separation of the esophagus and trachea from the common foregut tube.

The alimentary and respiratory organ systems arise from a common endodermal origin, the anterior foregut tube. Formation of the esophagus from the dorsal region and the trachea from the ventral region of the foregut primordium occurs by means of a poorly understood compartmentalization process. Disruption of this process can result in severe birth defects, such as esophageal atresia and tracheo-esphageal fistula (EA/TEF), in which the lumina of the trachea and esophagus remain connected. Here we summarize the signaling networks known to be necessary for regulating dorsoventral patterning within the common foregut tube and cellular behaviors that may occur during normal foregut compartmentalization. We propose that dorsoventral patterning serves to establish a lateral region of the foregut tube that is capable of undergoing specialized cellular rearrangements, culminating in compartmentalization. We review established as well as new rodent models that may be useful in addressing this hypothesis. Finally, we discuss new experimental models that could help elucidate the mechanism behind foregut compartmentalization. An integrated approach to future foregut morphogenesis research will allow for a better understanding of this complex process.

BMP antagonism by Noggin is required in presumptive notochord cells for mammalian foregut morphogenesis.

Esophageal atresia with tracheoesophageal fistula (EA/TEF) is a serious human birth defect, in which the esophagus ends before reaching the stomach, and is aberrantly connected with the trachea. Several mouse models of EA/TEF have recently demonstrated that proper dorsal/ventral (D/V) patterning of the primitive anterior foregut endoderm is essential for correct compartmentalization of the trachea and esophagus. Here we elucidate the pathogenic mechanisms underlying the EA/TEF that occurs in mice lacking the BMP antagonist Noggin, which display correct dorsal/ventral patterning. To clarify the mechanism of this malformation, we use spatiotemporal manipulation of Noggin and BMP receptor 1A conditional alleles during foregut development. Surprisingly, we find that the expression of Noggin in the compartmentalizing endoderm is not required to generate distinct tracheal and esophageal tubes. Instead, we show that Noggin and BMP signaling attenuation are required in the early notochord to correctly resolve notochord cells from the dorsal foregut endoderm, which in turn, appears to be a prerequisite for foregut compartmentalization. Collectively, our findings support an emerging model for a mechanism underlying EA/TEF in which impaired notochord resolution from the early endoderm causes the foregut to be hypo-cellular just prior to the critical period of compartmentalization. Our further characterizations suggest that Noggin may regulate a cell rearrangement process that involves reciprocal E-cadherin and Zeb1 expression in the resolving notochord cells.

Esophageal development and epithelial homeostasis.

The esophagus is a relatively simple organ that evolved to transport food and liquids through the thoracic cavity. It is the only part of the gastrointestinal tract that lacks any metabolic, digestive, or absorptive function. The mucosa of the adult esophagus is covered by a multilayered squamous epithelium with a remarkable similarity to the epithelium of the skin despite the fact that these tissues originate from two different germ layers. Here we review the developmental pathways involved in the establishment of the esophagus and the way these pathways regulate gut-airway separation. We summarize current knowledge of the mechanisms that maintain homeostasis in esophageal epithelial renewal in the adult and the molecular mechanism of the development of Barrett's metaplasia, the precursor lesion to esophageal adenocarcinoma. Finally, we examine the ongoing debate on the hierarchy of esophageal epithelial precursor cells and on the presence or absence of a specific esophageal stem cell population. Together the recent insights into esophageal development and homeostasis suggest that the pathways that establish the esophagus during development also play a role in the maintenance of the adult epithelium. We are beginning to understand how reflux of gastric content and the resulting chronic inflammation can transform the squamous esophageal epithelium to columnar intestinal type metaplasia in Barrett's esophagus.

[Expression of neuron-specific enolase and synaptophysin in esophageal development of human embryos].

OBJECTIVE: To investigate the expression of neuron-specific enolase (NSE) and synaptophysin(SYN) proteins in different developmental stages of human embryonic esophagus. METHODS: Immunohistochemistry was used to detect the expressions of NSE and SYN proteins in embryonic esophagus tissues of fetuses of 2, 3 and 4 month gestational age (n=16). One-way ANOVA and LSD-t test were employed to compare the staining intensity and number of positive expression cells in embryonic esophageal tissues of different gestational age. RESULTS: In fetuses with 2, 3 and 4 months of gestation, the number of NSE-positive nerve cells in the myenteric nerve plexus and submucosa of human embryonic esophageal tissues were 18.38 ± 8.37, 25.00 ± 11.54 and 38.00 ± 15.09, respectively; the staining intensity of NSE-positive nerve cells and nerve fibers in myenteric nerve plexus and submucosa of embryonic esophageal tissues were 74.38 ± 14.93, 62.25 ± 18.59 and 56.44 ± 14.70, respectively. NSE-positive cells were detected in the esophageal epithelium only at the third month. In the fetuses at 2, 3 and 4 months of gestation, SYN in all layers of esophageal tissue were positively or strong positively expressed, especially in the myenteric plexus and submucosal plexus. The staining intensity of SYN-positive cells in embryonic esophagus tissues of 2, 3 and 4 month gestation were 54.69 ± 9.34, 51.84 ± 6.10 and 46.41 ± 6.44, respectively. CONCLUSION: SYN and NSE may be involved in the regulation of nerve system of esophageal tissues during the human embryonic development.

Analysis of the expression of p53 during the morphogenesis of the gastroesophageal mucosa of Gallus gallus domesticus (Linnaeus, 1758).

Ontogenesis comprises a series of events including cell proliferation and apoptosis and resulting in the normal development of the embryo. Protein p53 has been described as being involved in the development of several animal species. The aim of this study was to analyze the expression of protein p53 during the morphogenesis of the gastroesophageal mucosa of Gallus gallus domesticus and to correlate it with the histogenesis of structures present in this tissue. We used 24 embryos (at 12-20 days of incubation) and the thymus of two chickens. Immunohistochemical analysis was performed with the ABC indirect method. The expression of p53 in the gastroesophageal mucosa increased during the formation of the organ, mainly at the stages during which tissue remodeling and cell differentiation began. In the esophagus at stages 42 and 45, we observed immunoreactive (IR) cells in the surface epithelium and in early esophageal glands. In the proventriculus at stages 39-45, IR cells were present in the epithelial mucosa and rarely in the proventricular glands. In the gizzard after stage 42, we found IR cells mainly in the medial and basal epithelial layers of the mucosa and especially within the intercellular spaces that appeared at this phase and formed the tubular gland ducts. Thus, protein p53 occurs at key stages of development: in the esophagus during the remodeling of esophageal glands, in the proventriculus during the differentiation of the epithelium of the mucosa and in the gizzard during the formation of tubular glands.

Regulation of amniotic fluid volume: mathematical model based on intramembranous transport mechanisms.

Experimentation in late-gestation fetal sheep has suggested that regulation of amniotic fluid (AF) volume occurs primarily by modulating the rate of intramembranous transport of water and solutes across the amnion into underlying fetal blood vessels. In order to gain insight into intramembranous transport mechanisms, we developed a computer model that allows simulation of experimentally measured changes in AF volume and composition over time. The model included fetal urine excretion and lung liquid secretion as inflows into the amniotic compartment plus fetal swallowing and intramembranous absorption as outflows. By using experimental flows and solute concentrations for urine, lung liquid, and swallowed fluid in combination with the passive and active transport mechanisms of the intramembranous pathway, we simulated AF responses to basal conditions, intra-amniotic fluid infusions, fetal intravascular infusions, urine replacement, and tracheoesophageal occlusion. The experimental data are consistent with four intramembranous transport mechanisms acting in concert: 1) an active unidirectional bulk transport of AF with all dissolved solutes out of AF into fetal blood presumably by vesicles; 2) passive bidirectional diffusion of solutes, such as sodium and chloride, between fetal blood and AF; 3) passive bidirectional water movement between AF and fetal blood; and 4) unidirectional transport of lactate into the AF. Further, only unidirectional bulk transport is dynamically regulated. The simulations also identified areas for future study: 1) identifying intramembranous stimulators and inhibitors, 2) determining the semipermeability characteristics of the intramembranous pathway, and 3) characterizing the vesicles that are the primary mediators of intramembranous transport.

Altered Tbx1 gene expression is associated with abnormal oesophageal development in the adriamycin mouse model of oesophageal atresia/tracheo-oesophageal fistula.

INTRODUCTION: Oesophageal atresia/tracheo-oesophageal atresia (OA/TOF) frequently arises with associated anomalies and has been clinically linked with 22q11 deletion syndromes, a group of conditions due to Tbx1 gene mutation which include Di George syndrome. Tbx1 and Tbx2 genes modulate pharyngeal and cardiac development, but are also expressed in the developing foregut and are known to interact with key signalling pathways described in oesophageal formation including bone morphogenic proteins. The adriamycin mouse model (AMM) reliably displays OA/TOF-like foregut malformations providing a powerful system for investigating the disturbances in gene regulation and morphology involved in tracheo-oesophageal malformations. We hypothesised that foregut abnormalities observed in the AMM are associated with altered Tbx1 and Tbx2 gene expression. METHODS: Time-mated CBA/Ca mice received intra-peritoneal injection of adriamycin (for treated) or saline (for controls) on embryonic days (E)7 and 8. Untreated Cd1 embryos were used to establish normal expression patterns. Embryos harvested on E9-E11 underwent whole-mount in situ hybridization with labelled RNA probes for Tbx1 and Tbx2. Optical projection tomography was used to visualise expression in whole embryos by 3D imaging. RESULTS: Tbx1 expression was visualised in a highly specific pattern in the proximal oesophageal endoderm in normal and control embryos. In the AMM, extensive ectopic expression of Tbx1 was detected in the dorsal foregut and adjacent to the TOF. The focally restricted oesophageal expression pattern persisted in the AMM, but was posteriorly displaced in relation to the tracheal bifurcation. Tbx2 was widely expressed in the ventral foregut mesoderm of controls, lacking specific endoderm localisation. In the AMM, altered Tbx2 expression in the foregut was only seen in severely affected embryos. CONCLUSION: Highly specific Tbx1 expression in the proximal oesophageal endoderm suggests that Tbx1 may be an important regulator of normal oesophageal development. Altered Tbx1 expression in dorsal foregut and adjacent to the TOF in the AMM suggests that Tbx1 gene disruption may contribute to the pathogenesis of tracheo-oesophageal malformations.

The diagnosis of fetal esophageal atresia and its implications on perinatal outcome.

The current diagnostic accuracy and perinatal outcome of fetuses with esophageal atresia (EA) continues to be debated. In this review, we report on our experience at a tertiary care fetal center with the prenatal ultrasound diagnosis of EA. Enrollment criteria included a small/absent stomach bubble with a normal or elevated amniotic fluid index between 2005 and 2013. Perinatal outcomes were analyzed and compared to postnatally diagnosed EA cases. Of the 22 fetuses evaluated, polyhydramnios occurred in 73%. Three (14%) died in utero or shortly after birth, but none had EA. In the presence of an absent/small stomach and polyhydramnios, the positive predictive value for EA was 67%. In fetal EA cases confirmed postnatally (group 1, n = 11), there were no differences in gestational age, birthweight, or mortality when compared to postnatally diagnosed infants (group 2, n = 59). Group 1 was associated with long-gap EA, need for esophageal replacement, and increased hospital length of stay. When taken in context with the current literature, we conclude that ultrasound findings suggestive of EA continue to be associated with a relatively high rate of false positives. However, among postnatally confirmed cases, there is an increased risk for long-gap EA and prolonged hospitalization.

[The role of genetic and environmental factors in the etiology of esophageal atresia and tracheo-esophageal fistula]. / Udzial czynników genetycznych i srodowiskowych w etiologii wrodzonego zarosniecia przelyku i przetoki tchawiczo-przelykowej

Esophageal atresia and tracheo-esophageal fistula are severe congenital malformations, whose etiology is still poorly understood. So far, numerous genetic and environmental factors that may contribute to the occurrence of these defects have been described and the literature is dominated by the view of their common involvement in the etiology and pathogenesis of congenital esophageal atresia. In this review the authors present current knowledge on the embryogenesis of the esophagus and trachea, discuss environmental risk factors, and also list and describe genetic alterations identified so far in patients with congenital esophageal atresia.

A novel murine allele of Intraflagellar Transport Protein 172 causes a syndrome including VACTERL-like features with hydrocephalus.

The primary cilium is emerging as a crucial regulator of signaling pathways central to vertebrate development and human disease. We identified atrioventricular canal 1 (avc1), a mouse mutation that caused VACTERL association with hydrocephalus, or VACTERL-H. We showed that avc1 is a hypomorphic mutation of intraflagellar transport protein 172 (Ift172), required for ciliogenesis and Hedgehog (Hh) signaling. Phenotypically, avc1 caused VACTERL-H but not abnormalities in left-right (L-R) axis formation. Avc1 resulted in structural cilia defects, including truncated cilia in vivo and in vitro. We observed a dose-dependent requirement for Ift172 in ciliogenesis using an allelic series generated with Ift172(avc1) and Ift172(wim), an Ift172 null allele: cilia were present on 42% of avc1 mouse embryonic fibroblast (MEF) and 28% of avc1/wim MEFs, in contrast to >90% of wild-type MEFs. Furthermore, quantitative cilium length analysis identified two specific cilium populations in mutant MEFS: a normal population with normal IFT and a truncated population, 50% of normal length, with disrupted IFT. Cells from wild-type embryos had predominantly full-length cilia, avc1 embryos, with Hh signaling abnormalities but not L-R abnormalities, had cilia equally divided between full-length and truncated, and avc1/wim embryos, with both Hh signaling and L-R abnormalities, were primarily truncated. Truncated Ift172 mutant cilia showed defects of the distal ciliary axoneme, including disrupted IFT88 localization and Hh-dependent Gli2 localization. We propose a model in which mutation of Ift172 results in a specific class of abnormal cilia, causing disrupted Hh signaling while maintaining L-R axis determination, and resulting in the VACTERL-H phenotype.