Developmental basis of trachea-esophageal birth defects

Abstract

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.

Keywords: Congenital anomalies; Development; EA/TEF; Esophageal atresia; Esophagus; Foregut; Laryngotracheoesophageal cleft; Trachea; Tracheoesophageal fistula.

Figure 1.. Human TEDs.

A. The trachea is a conduit for air passage from the mouth to the lungs, while the esophagus connects the mouth to the stomach for food passage. Trachea-esophageal birth defects (TEDs) clinically manifest as a spectrum of anatomical anomalies affecting the continuity of the trachea and/or esophagus. B. Esophageal atresia (EA) can present with or without a trachea-esophageal fistula (TEF), with a distal fistula being the most common C. EA results in a blind-ended esophagus with no connection to the stomach. D. TEFs are abnormal connections between the trachea and esophagus. D\E. Incomplete separation of the trachea and esophagus results in laryngotracheal-esophageal clefts. E. Tracheal atresia occurs when the lung buds form directly off of the esophagus with no tracheal tube present. Artwork by Christopher Woods, Department of Pathology, Cincinnati Children’s Hospital Medical Center.

Figure 2.. Locations of TED risk genes and copy number variants on human chromosomes.

De novo copy number variants are associated with cases of isolated and non-isolated esophageal atresia, arising from parents with balanced translocations leading to partial monosomy and partial trisomy (frequently, trisomy 18, 21, 13, and X) (Supplementary Table 1). De novo mutations in TED patients are heterozygous in constrained genes and have been associated with over 35 single gene disorders (Table 1).

Figure 3.. Patterning and morphogenesis of the animal embryonic foregut.

A. The embryonic anterior foregut is first patterned into Sox2+ dorsal (green) and Nkx2-1+ ventral (purple) endoderm domains by reciprocal RA, HH, WNT, and BMP signaling between the foregut endoderm and mesoderm. Nkx2-1 expression is promoted by high levels of WNT and BMP ligands, while the BMP antagonist Noggin and the WNT antagonist Sfrp1/2 maintain Sox2 expression by inhibiting WNT and BMP signaling. B. Morphogenesis initiates with the constriction of the embryonic foregut at the midline Nkx2-1+/Sox2+ epithelial cells. Cells of the opposing epithelial walls contact and adhere to form a transient septum. The septum is then resolved by endosome-mediated epithelial remodeling and extracellular matrix breakdown to separate the epithelium into two tubes and allow the mesenchyme to invade the intervening space. The trachea and esophagus then elongate and differentiate as development proceeds. C. Mouse TED mutant models. Disruptions in the major signaling pathways (RA, WNT, BMP, HH) regulating dorsal-ventral patterning in the embryonic foregut results in the loss of Nkx2-1 patterning and the foregut remaining as a single tube or complete LTEC, often accompanied by tracheal atresia. Hypomorphic loss of Sox2, or Noggin mutations, result in the loss of Sox2 patterning and EA/TEF. Loss of Barx1, a mesenchymal transcription factor regulating WNT signaling, mispatterns the embryonic foregut resulting in a complete LTEC. Loss of Gli2 with one allele of Gli3 (Gli2^−/−;Gli3^+/−) correctly patterns the embryonic foregut but results in a LTEC, suggesting that subsequent cellular mechanisms regulating TE separation are disrupted. Mice harboring Gli3 dominant repressor alleles or mesenchyme-specific Foxf1 knockout have partial LTECs and tracheomalacia.

Figure 4.. Studying EA/TEF causing mutations using hPSCs.

hPSCs can be directly differatiated to definitive endoderm fate, which than can be further differentiated into dorsal anterior foregut (A) or ventral anterior foregut (B) by the manipulation of FGF, Noggin, Wnt and RA. Dorsal anterior foregut organoids will comtinue to grow and differentiate to give rise to human esophageal organoids (HEO), while ventral anterior foregut organoids will differentiate into human airway organoids (HAO).

Figure 5.. Ultrashort echotime MRI of EA/TEF.

An infant with an unrepaired type C (distal TEF) EA/TEF was imaged on the first day of life with ultrashort echotime MRI. This technique removes motion artifact and is able to gather detailed anatomy of the chest to a 0.7 mm resolution in a free breathing, non-sedated infant. The specific anatomy of the trachea, dilated proximal esophageal pouch and distal tracheal esophageal fistula are presented as a three dimensional reconstruction from anterior (A) and posterior (B) perspective.