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.

The 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 comprises the 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 the 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 that 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 that 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 that will continue well into the third trimester.[2][10]

Esophageal embryogenesis involves complex signaling pathways. The first step of the 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 the 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's 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 the glandular columnar epithelium, although the exact roles are not fully understood.[17][14]

Mechanisms of signaling molecule's interactions are a subject of an 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 the 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 post-transcriptional 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 the 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]

Esophageal embryogenesis, though an independent process, is a part of cellular events that leads to the formation of lung, trachea, and esophagus in 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]