Embryology, Esophagus

Article Author:
Jonathan Katz
Article Author (Archived):
Ahmad Malik
Article Editor:
Hajira Basit
Updated:
5/9/2019 11:46:16 AM
PubMed Link:
Embryology, Esophagus

Introduction

Developmental disorders of the esophagus are relatively common, occurring in approximately 1 in 3000 live births. Because of the shared embryologic origins of the esophagus and respiratory system, esophageal defects commonly correlate with concurrent defects in the lungs and trachea. These defects include several combinations of esophageal atresia and tracheoesophageal fistula. The defects, understandably, can lead to difficulties with feeding and in some cases maintaining oxygenation, requiring urgent attention in the perinatal period. In utero, defects of the esophagus can lead to difficult birth conditions stemming from the decreased ability to swallow and resultant polyhydramnios. While the process of organogenesis is immensely complex and the cellular mediators are still being worked out, a basic understanding of the current knowledge on esophageal embryology is important for understanding the link between congenital esophageal and tracheal malformations.

Development

Development of the gastrointestinal system results from a series of highly regulated biochemical processes and folding patterns. Gut and more specifically esophageal development are most easily understood starting at week four. At this stage, the early embryo consists of three distinct layers, in what is known as a trilaminar disc, connected to the yolk sac. The trilaminar disc is composed of outer ectoderm, middle mesoderm, and an inner layer known as the endoderm.[1] The layers orient in such a way that the endoderm layer is in contact with the outer ectoderm layer at the poles of the embryo. At the start of the fourth week, folding occurs such that corresponding cranial, caudal, and lateral edges of the disc come together. This folding occurs through the ventral midline, and the layers fuse allowing for internalization of the endoderm layer, such that the embryo takes on a tube within a tube configuration, an inner tube composed of endoderm and an outer tube consisting of ectoderm, and between the two layers, mesoderm.

Initially, this inner tube is blind-ended at both poles and is the precursor to the final digestive tract.[1] The inner tube itself divides into three anatomical parts, the foregut, midgut, and hindgut. The foregut being the most cranial portion and hindgut the most caudal. The foregut and hindgut delineation is the center component, the midgut, which is continuous with the yolk sac through the vitelline duct. The mechanisms of early folding and tube position have their basis in concentration-dependent signaling which sets up a ventral-dorsal, rostral-caudal, and left-right axis.[1][2] These axes are influenced by and contribute, in a reciprocal manner to local endodermal and mesodermal interactions. The component of the foregut that will give rise to the esophagus also will give rise to the trachea and lungs. From the foregut endoderm will arise the esophageal epithelium as well as mucosal glands. The mesodermal layer surrounding the foregut will give rise to the striated muscular and smooth muscle layers of the esophagus.  These processes are associated with numerous signaling molecules.[3] However, the first step of esophageal organogenesis from the foregut is the differentiation of the foregut cells into the trachea, lung, and esophagus. This process begins with the cellular expression of many genes.[2][4]

After esophageal specification occurs, several notable changes are visible in the developing embryo. At approximately week 6 of development, the circular and longitudinal muscular layers begin to form, and ganglion cells of the myenteric plexus first present. Moving into week 7, cells of mesodermal origin proliferate into the submucosal layer forming the eventual blood supply to the esophagus. The muscular layers which began in week 6, are completed by the 9th week.[5] Rostral-caudally, a distinction occurs in the muscular subtypes found within the esophagus. The cranial third of the esophagus contains mostly striated muscle, the caudal third transitions into mostly smooth muscle, and the middle third being a combination of both muscular subtypes.[6][7] Along with this change in musculature, cranially to caudally, there is hypothesized to be a dual set of innervation of these layers from the enteric nervous system and the vagal nerve, which is a product of branchial arch 6.

Co-innervation of muscle cells is hypothesized to allow for early peristalsis after birth while the nervous system is not fully mature. The process of esophageal innervation occurs throughout the development of the embryo and requires proliferation and migration of neural crest cells which migrate rostrally-caudally through the gut tube starting during the 4th week and ending their migration around the 9th week of development.[8] Setting the precursor cells for innervation along the entire gut. During the 6th week, when the muscular layers have begun to form, cells of neuronal crest origin migrate inward between the muscular layers eventually giving rise to the submucosal plexus.[9] This process which began the neuronal development early in the 4th week continues through a slow maturation process which continues after birth.[8] At around the 4th month of development, the columnar epithelium of the foregut begins to undergo a transition into a squamous epithelium a process which will continue well into the third trimester.[2][10]

Cellular

Esophageal embryogenesis involves complex signaling pathways. The first step of organogenesis of the esophagus from the foregut is the specification of the foregut cells into respiratory lineages or esophageal lineage.[2][4]. The process begins with the cellular expression of the Nkx2-1 gene in the anterior foregut ventral wall, Nkx2-1 being a specific marker of respiratory tract cells. Concurrently, endodermal cells in the posterior aspect of the foregut begin to express Sox2 which seems to guide the dorsal foregut towards esophageal differentiation.[1][2] This sequence indicates that the specification of foregut cells occurs before lung bud outpouching and morphogenic changes. The specification occurs across the ventral-dorsal axis of the foregut. The notochord plays a key role in setting up this ventral-dorsal axis, which allows for the differential expression of Nkx2-1 and Sox2 by releasing Noggin.[1][2][3][11] 

Dose-dependent signaling of Noggin from the notochord as well as specific timed signaling from the surrounding mesoderm via Wingless-related Integration site proteins (Wnt) and fibroblast growth factor (FGF) allows for the progression of the dorsal aspect of the foregut to begin differentiation into the esophagus while the ventral aspect of the foregut. Also, expressing NKx2-1 in conjunction with a lower dose of Noggin begins the specification into the future trachea and lungs.[3] Additional key mesenchymal signaling molecules include the BMP family, which play critical roles in tissue patterning and trachea formation.[12] The activity of one member of the BMP family, BMP4, has been shown by research to be antagonized by Noggin, and thus preferentially acts on the ventral sections of the foregut, which get subjected to decreased Noggin because of the dorsally located notochord.[1][4][12][11] 

Another crucial event in the development of the foregut into the esophagus is the separation of the trachea from the esophagus. While the precise mechanisms of separation have not yet been elucidated, signaling from lateral mesoderm to the ventral foregut allows for differentiation into the two final structures which, once separated, both experience a rapid elongation. Wnt signaling, specifically, Wnt5a and Ror2 (a receptor tyrosine kinase) signaling appears to play an important role in this elongation.[2][3] Knockout of Wnt5a in mice results in an altered short tube esophagus, and Wnt5a-Ror2 signaling promotes straight tube morphogenesis for both esophagus elongation as well as the trachea.[1][2][12]

Another important development is the differentiation of esophagus from the structures that are continuous with the esophagus, namely the pharynx and the stomach. Many cell signaling molecules regulate esophageal differentiation and transition from the pharynx and into the stomach.  Several mediators that have identifiable roles in esophageal and respiratory differentiation also play a part in the differentiation of the pharynx and the stomach from the esophagus, serving to highlight the numerous roles these factors play.[13] The upper esophageal sphincter derives from mesenchymal tissue from brachial arches 4 to 6. And the lower esophageal sphincter is derived from mesenchymal cells of somites located in the region of the foregut.[11][13] The gastroesophageal junction's origin is thought to be a combination of cell signaling as well as cell-cell interactions which occur as a consequence of rotational changes of the stomach.[5] Studies with mice have demonstrated the role of Sonic hedgehog (Shh), BMP, Foxp1, and Foxp2 in the differentiation of the esophagus into the stomach during organogenesis.[14] Shh role seems to play important roles throughout the esophagus and at the level of the lower esophageal sphincter, as defects in Shh result in the decreased formation and the maturation of smooth muscle cells in both the esophagus and the stomach.[15][16] Foxp1 and Foxp2 appear to play similar roles in muscularization, and BMP signaling appears to be important in initiating the change from squamous epithelium into glandular columnar epithelium although the exact roles are not fully understood.[17][14]

Biochemical

Mechanisms of signaling molecule's interactions are a subject of ongoing investigation. This section will serve as a summary of mediators which are discussed above as well as describe other signaling molecules identified in esophageal embryogenesis.

Ventral-Dorsal axis

The notochord is a major structure that participates in the formation of the ventral-dorsal axis. Of the mediators produced by the notochord, Noggin expression plays a well-described role in setting up the ventral-dorsal axis within the embryo. The ventral portion of the foregut begins to express NKx2-1 with BMP signaling, BMP's action gets antagonized by Noggin activity and mediates continued specification and differentiation of the respiratory system from the foregut.[2][3]. BMP inhibits the dorsal aspect of the foregut which expresses Sox2 and plays an important role in initiating esophageal specification. Because of the increase in Noggin concentration in the dorsal epithelium, a greater antagonistic effect on BMP occurs, inhibiting BMP's inhibitory effect on Sox2, allows for increased expression of Sox2 in the portion of the foregut which will eventually give rise to the esophagus.[2] Conversely, the lower concentration of Noggin in the ventral aspect of the foregut allows for increased activity of BMP, specifically BMP4 which then acts to inhibit Sox2 in the ventral foregut leading to tracheal development.[3] These interactions for differential expression of cell signaling molecules and transcription factors are still being studied and are one of the more well-described mediators of ventral-dorsal axis patterning.[2]

Wnt5a-Ror2 Signaling

Wnt5a-Ror2 signaling within subepithelial intercalated smooth muscle cells is thought to be involved in tubulogenesis of the esophagus as well as the trachea. This signal-receptor pair plays key roles in elongation for the trachea and esophagus.

Hedgehog signaling

Hedgehog is another potent mediator that plays critical roles in the development of the esophagus and other structures. Aberrant activation of hedgehog correlates with numerous developmental disorders and cancers of the brain and GI tracts. Of the major components of hedgehog signaling, Sonic hedgehog (Shh) seems to play the most prominent role in the esophagus and tracheal organogenesis.[2][18] Development and differentiation of esophagus and trachea require continual interaction between mesenchymal and endodermal components for proper development to occur. Shh's role is dose-dependent, highly regulated, and positional dependent. Shh initiates cascades which feature several other mediators, most prominently fibroblastic growth factor (FGF), transforming growth factor beta-bone morphogenic protein (TGF beta-BMP), Wnt, retinoic acid, and Notch signaling.[2][3][18] Shh's role appears to be regulated by posttranscriptional modifications including phosphorylations and palmitoylations as well as several different receptors to which Shh can bind on the target cell. Modifications of Shh determine which signaling cascade is activated and thus downstream gene expression.[2][3] A key receptor indicated in foregut differentiation and lung bud formation includes the zinc finger glioma-associated transcription factors which, when bound to Shh activates gene transcription. However, in the absence of Shh, zinc finger glioma-associated transcription factors act as repressors adding an additional dimension to the location dependent effects of Shh on organogenesis.[11][18]  

BMP signaling

Broadly, BMP signaling has been studied in two distinct areas of esophageal embryogenesis. The Noggin inhibits the activity of BMP, and thus in the early foregut is preferentially active on ventral cells. BMP4 appears to play a role in maintaining the specification of foregut cells which have already expressed NKx2-1. Studies in mice have shown that knockout of BMP4 results in loss of expression of NKx2-1 after specification, indicating BMP4 signalings' role is predominantly in the outgrowth of the respiratory tract from the ventral foregut rather than setting up the initial specification for respiratory differentiation. BMP signaling also plays a role in epithelialization of the esophagus. Recall the foregut has a columnar epithelium and must make the transition into a stratified nonkeratinized squamous epithelium, the epithelium of the esophagus. BMP7 plays a major role in this transition.[2] Lack of BMP7 activity, in the early stages of the transition, allows the columnar epithelium to begin to grow in a stratified manner.[10] After stratification of columnar cells,  BMP7 activity can begin to be seen in the upper layers and signals for the final transition into the stratified squamous layer. Concurrently, the basal layer of these cells lacks BMP7 activity allowing the layer to maintain progenitor properties.[1][2][11][10]

Factors of epithelial and mesenchymal development

Studies of knockout mice have revealed numerous other signaling factors for the esophagus. The most prominent of these signaling molecules include Keap1, P63, Foxp1, Foxp2, and HoxC4. These factors seem to play dramatic roles in the differentiation of esophageal epithelium as well as esophageal muscular development. Studies of Foxp1 and Foxp2 indicate that these molecules are critical players in the development of smooth and striated muscle of the esophagus and stomach.[14]The exact mechanisms of how these factors work in conjunction are not understood and our current understanding comes from studies with mice and thus the importance of each of these factors in human esophageal embryogenesis is still under investigation.[2][17]

Molecular

Esophageal embryogenesis through an independent process, is a part of cellular events that leads to formation of lung, trachea and esophagus in a different and specific yet interlinked pathways namely the increased expression of Sox2 in the dorsal portion of the foregut endoderm leading to esophageal differentiation and NKx2-1 expression in the ventral foregut endoderm resulting in differentiation of the respiratory system. These genes are timely expressed and spatially regulated in the foregut endoderm. Therefore, interruption of any of these pathways at any point during development can lead to various trachea-esophageal malformations.[19]

Pathophysiology

Researchers have identified various TE malformations by altering the gene regulation in knockout mice. These include:

Regulatory mediator Noggin, produced by the notochord mediates continued specification and differentiation of the respiratory system from the foregut. Studies in mice highlight this regulatory role, where disruption of the notochord resulted in tracheal-esophageal fistulas and esophageal atresia with excessive endodermal tissue. 

Wnt5a-Ror2 signaling plays critical roles in elongation for the trachea and esophagus. Knockout of wnt5a in mice has shown truncated esophagus development.

Sonic hedgehog (Shh) has a critical role in the development of esophagus and other structures. Studies of Shh knock out mice show decreased rates of cell division and transcription factor expression.[18] Decreased Shh expression has also been associated with lung hypoplasia, abnormalities in smooth muscle migration, esophageal atresia, and stenosis, indicating the important regulatory roles for this protein in the development of the esophagus and suggesting important roles in human foregut defects.[2][11]

Briefly, various other factors resulting in esophageal malformations are as following:

P63 knockout mice show disruption in the stratification of squamous epithelium.[2] 

Loss of  Keap1 results in thick keratinization in the esophagus.[2]

Lastly, knockout HoxC4 results in an obliterated esophageal lumen from over-proliferation, implying a suppression effect of HoxC4.[2]

Clinical Significance

Human foregut malformations are relatively common, at a rate of about 1 in 3000 live births. Foregut malformations include a spectrum of pathologies including, tracheoesophageal fistulas with or without esophageal atresia, and congenital esophageal rings. The types of esophageal atresias and tracheoesophageal fistulas fall into five subtypes; A-E. The most common type being the Type C esophageal atresia /tracheoesophageal fistula (TEF). We will begin with a description of these subtypes and then delve into management. Congenital esophageal rings can be associated with esophageal atresia and TEFs or can occur in isolation.[20]

Esophageal fistulas and TEFs subdivide into five subtypes. Type A is Isolated esophageal atresia without fistula. Type B is Esophageal atresia with the proximal TEF. Type C is esophageal atresia with a distal TEF and is the most common subtype, comprising approximately 85% of cases. Type D is esophageal atresia with both proximal and distal TEF. Lastly, type E is an isolated TEF without esophageal atresia, also known as the H-type. Because the esophagus is still continuous with the stomach, the diagnosis of type E may be delayed as issues with feeding is not readily apparent. The presence of polyhydramnios secondary to impaired swallowing of amniotic fluid may permit prenatal diagnosis. Fetal ultrasounds around 20 weeks gestation can detect esophageal atresia or TEF.[20] However, ultrasound is highly user dependent and can be subjective. Because a prenatal diagnosis of esophageal atresia and TEF is user dependent, undiagnosed esophageal atresia and/or TEF is common. Physical examination of the newborn may show several signs which are indicative of esophageal atresia and TEF.[21] Symptoms may include excessive salivation, poor oral feedings with choking or cyanosis during feeding.

Once suspected, diagnosis can be confirmed by inserting a nasogastric tube with dual lumens (one lumen for continuous suction and another serving as an air vent to prevent the suctioning lumen from attaching to the mucosa). Once inserted, a chest radiograph will show coiling in the atresia portion of the esophagus, which is the gold standard for diagnosis.[22] Once diagnosed, surgical correction is indicated, with urgent repair indicated in infants experiencing respiratory distress, and less urgent repair in those with type E malformation without respiratory compromise or difficulty feeding.

Esophageal atresia is also associated with a syndrome of congenital anomalies known as the VACTERL association. This collection of symptoms includes vertebral, anorectal, cardiac, tracheal, esophageal, renal and limb abnormalities. In order for a diagnosis of VACTERL to be made at least 2 anomalies must be present. Infants with esophageal atresia and TEF should be evaluated for further anomalies because of the association with VACTERL as well as undergo genetic analysis. Treatment is then aimed at correcting associated anomalies.[21]

Congenital esophageal webs can be isolated findings or found in association with atresias and/or fistulas. Formation of esophageal webs is not completely understood. However, they are thought to occur from incomplete cannulization of the esophagus during development. The webs are often located in the ventral wall of the esophagus and can be completely circumferential.[23]  Esophageal webs commonly present with dysphagia or food impaction latter in life and endoscopy reveals the underlying pathology. Treatment is usually conservative with periodic endoscopic dilation.

In summary defects in organogenesis of the esophagus can lead to clinically significant pathology. Because these conditions are common, it is important to understand the various subtypes of esophageal atresia and TEFs in order to recommend proper management. Additionally, because esophageal atresia can be the presenting symptom of the VACTERL association, diagnosis of esophageal atresia should prompt a robust multisystem workup.


References

[1] Gavrilov S,Lacy E, Genetic dissection of ventral folding morphogenesis in mouse: embryonic visceral endoderm-supplied BMP2 positions head and heart. Current opinion in genetics     [PubMed PMID: 23706163]
[2] Jacobs IJ,Ku WY,Que J, Genetic and cellular mechanisms regulating anterior foregut and esophageal development. Developmental biology. 2012 Sep 1;     [PubMed PMID: 22750256]
[3] Morrisey EE,Hogan BL, Preparing for the first breath: genetic and cellular mechanisms in lung development. Developmental cell. 2010 Jan 19;     [PubMed PMID: 20152174]
[4] Serls AE,Doherty S,Parvatiyar P,Wells JM,Deutsch GH, Different thresholds of fibroblast growth factors pattern the ventral foregut into liver and lung. Development (Cambridge, England). 2005 Jan;     [PubMed PMID: 15576401]
[5] Kishimoto K,Tamura M,Nishita M,Minami Y,Yamaoka A,Abe T,Shigeta M,Morimoto M, Synchronized mesenchymal cell polarization and differentiation shape the formation of the murine trachea and esophagus. Nature communications. 2018 Jul 19;     [PubMed PMID: 30026494]
[6] Yamada M,Udagawa J,Matsumoto A,Hashimoto R,Hatta T,Nishita M,Minami Y,Otani H, Ror2 is required for midgut elongation during mouse development. Developmental dynamics : an official publication of the American Association of Anatomists. 2010 Mar;     [PubMed PMID: 20063415]
[7] Kedinger M,Simon-Assmann P,Bouziges F,Arnold C,Alexandre E,Haffen K, Smooth muscle actin expression during rat gut development and induction in fetal skin fibroblastic cells associated with intestinal embryonic epithelium. Differentiation; research in biological diversity. 1990 Apr;     [PubMed PMID: 2197142]
[8] Neuhuber WL,Wörl J, Enteric co-innervation of striated muscle in the esophagus: still enigmatic? Histochemistry and cell biology. 2016 Dec;     [PubMed PMID: 27678007]
[9] Fu M,Tam PK,Sham MH,Lui VC, Embryonic development of the ganglion plexuses and the concentric layer structure of human gut: a topographical study. Anatomy and embryology. 2004 Apr;     [PubMed PMID: 14991401]
[10] Rosekrans SL,Baan B,Muncan V,van den Brink GR, Esophageal development and epithelial homeostasis. American journal of physiology. Gastrointestinal and liver physiology. 2015 Aug 15;     [PubMed PMID: 26138464]
[11] Billmyre KK,Hutson M,Klingensmith J, One shall become two: Separation of the esophagus and trachea from the common foregut tube. Developmental dynamics : an official publication of the American Association of Anatomists. 2015 Mar;     [PubMed PMID: 25329576]
[12] Li Y,Gordon J,Manley NR,Litingtung Y,Chiang C, Bmp4 is required for tracheal formation: a novel mouse model for tracheal agenesis. Developmental biology. 2008 Oct 1;     [PubMed PMID: 18692041]
[13] Rishniw M,Rodriguez P,Que J,Burke ZD,Tosh D,Chen H,Chen X, Molecular aspects of esophageal development. Annals of the New York Academy of Sciences. 2011 Sep     [PubMed PMID: 21950820]
[14] Shu W,Lu MM,Zhang Y,Tucker PW,Zhou D,Morrisey EE, Foxp2 and Foxp1 cooperatively regulate lung and esophagus development. Development (Cambridge, England). 2007 May;     [PubMed PMID: 17428829]
[15] Cardoso WV,Lü J, Regulation of early lung morphogenesis: questions, facts and controversies. Development (Cambridge, England). 2006 May;     [PubMed PMID: 16613830]
[16] Jia X,Min L,Zhu S,Zhang S,Huang X, Loss of sonic hedgehog gene leads to muscle development disorder and megaesophagus in mice. FASEB journal : official publication of the Federation of American Societies for Experimental Biology. 2018 Oct;     [PubMed PMID: 29768039]
[17] Yu WY,Slack JM,Tosh D, Conversion of columnar to stratified squamous epithelium in the developing mouse oesophagus. Developmental biology. 2005 Aug 1;     [PubMed PMID: 15992795]
[18] Fernandes-Silva H,Correia-Pinto J,Moura RS, Canonical Sonic Hedgehog Signaling in Early Lung Development. Journal of developmental biology. 2017 Mar 13;     [PubMed PMID: 29615561]
[19] Faure S,de Santa Barbara P, Molecular embryology of the foregut. Journal of pediatric gastroenterology and nutrition. 2011 May     [PubMed PMID: 21499038]
[20] Lee S, Basic Knowledge of Tracheoesophageal Fistula and Esophageal Atresia. Advances in neonatal care : official journal of the National Association of Neonatal Nurses. 2018 Feb;     [PubMed PMID: 29373345]
[21] Bradshaw CJ,Thakkar H,Knutzen L,Marsh R,Pacilli M,Impey L,Lakhoo K, Accuracy of prenatal detection of tracheoesophageal fistula and oesophageal atresia. Journal of pediatric surgery. 2016 Aug;     [PubMed PMID: 26932255]
[22] Choudhry M,Boyd PA,Chamberlain PF,Lakhoo K, Prenatal diagnosis of tracheo-oesophageal fistula and oesophageal atresia. Prenatal diagnosis. 2007 Jul;     [PubMed PMID: 17457956]
[23] Patel PC,Yates JA,Gibson WS,Wood WE, Congenital esophageal webs. International journal of pediatric otorhinolaryngology. 1997 Dec 10;     [PubMed PMID: 9692624]