Disruptions to endosomal trafficking cause congenital trachea-esophageal separation defects

Disrupted endosomal trafficking of the Vangl-Celsr polarity complex underlies congenital anomalies in Xenopus trachea-esophageal morphogenesis.

Dev Cell 2025 May 21; doi: 10.1016/j.devcel.2025.04.026.

Edwards NA, Rankin SA, Kashyap A, Warren A, Agricola ZN, Kenny AP, Kofron M, Shen Y, Chung WK, Zorn AM.

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Edwards et al. investigated how the trachea and esophagus form in Xenopus. They discovered that a specific cellular pathway, endosomal trafficking, is essential for delivering key proteins to the correct locations during development. When this process fails, it can lead to abnormal connections between the trachea and esophagus, known as fistulas. These findings provide broad insights into how tissue morphogenesis events are disrupted during organ development, resulting in congenital birth defects.

Abstract

Disruptions in foregut morphogenesis can result in life-threatening conditions where the trachea and esophagus fail to separate, such as esophageal atresia (EA) and tracheoesophageal fistulas (TEFs). The developmental basis of these congenital anomalies is poorly understood, but recent genome sequencing reveals that de novo variants in intracellular trafficking genes are enriched in EA/TEF patients. Here, we confirm that mutation of orthologous genes in Xenopus disrupts trachea-esophageal separation similar to EA/TEF patients. The Rab11a recycling endosome pathway is required to localize Vangl-Celsr polarity complexes at the luminal cell surface where opposite sides of the foregut tube fuse. Partial loss of endosomal trafficking or Vangl-Celsr complexes disrupts epithelial polarity and cell division orientation. Mutant cells accumulate at the fusion point, fail to relocalize cadherin, and do not separate into distinct trachea and esophagus. These data provide insights into the mechanisms of congenital anomalies and general paradigms of tissue fusion during organogenesis.

Figure 1. Mutating EA/TEF patient endosomal trafficking genes causes TEDs in Xenopus (A) STRING database interactome of endosome-related proteins with potentially pathogenic variants identified in EA/TEF patients and a curated list of core endosome pathway proteins and putative protein cargo. (B) Experimental design of F0 CRISPR-Cas9 X. tropicalis mutagenesis screen to validate candidate risk genes. (C) Confocal images of CRISPR-Cas9 X. tropicalis F0 mutants at NF44 stained for Sox2 (green), Foxf1 (red), and Nkx2-1 (purple). Hashed yellow lines indicate the tracheal (t) and esophageal (e) lumens. Arrows indicate TEFs. Asterisks indicate dysmorphic or occluded esophagus or trachea. Numbers indicate the proportion of mutant tadpoles with TEFs compared with the total mutants screened. Scale bars are 50 m. See also Figure S1 and Tables S1, S2, and S3.

Figure 2. Dynamic endosome localization and epithelial remodeling during tracheoesophageal morphogenesis (AB and IJ) Immunostaining of aPKC, laminin (Lama1), and Cdh1 dynamics in the Xenopus foregut during trachea-esophageal morphogenesis. Scale bars, 50u m. (CH and KP) Immunostaining of Dnm2, Rab5a, and Rab11a in the Xenopus foregut during trachea-esophageal morphogenesis. Scale bar, 50 um. Diagrams depict the temporospatial dynamics of Cdh1 and Rab11a subcellular localization during tracheoesophageal morphogenesis. (Q) Quantification of Rab11a immunostaining intensity during foregut fusion and separation (mean min/max, 2W-ANOVA, p < 0.0001, n = 46 embryos analyzed). (R) Cdh1-Rab11a co-localization at the epithelial interface (mean Pearson co-localization coefficient SEM, p < 0.001, p < 0.0001 1W-ANOVA, n = 513 cells per embryo, N = 68 embryos per stage). (S) A model of how endosomal trafficking may mediate Cdh1 relocalization during epithelial fusion. See also Figure S2.

Figure 3. Endocytosis of Cdh1 is required for trachea-esophageal separation (A) 3D cell-surface rendering of the resolving septum with esophageal cells in green, septal cells in yellow, and tracheal cells in purple. Septal cells significantly decrease surface contact area with each other compared with the contact between tracheal cells and esophageal cells (mean min/max, p < 0.001 1W-ANOVA, n = 6 cells per embryo, N = 9 embryos). (B) Diagrams of the separating foregut at i) the intact bilayer and ii) at the point where the bilayer is separating. The graph quantifies Cdh1/mbGFP intensity at cell surfaces in the bilayer and the adherent vs. separating side of the cells losing contact (mean min/max, p < 0.01 1W-ANOVA, n = 5 cells per embryo, N=6 embryos). (C) Structure of wild-type (WT) and the LL AA Cdh1 mutant that cannot be internalized by endocytosis.25 (D) Quantification of the TEF phenotype in LL-Cdh1 mutants compared with WT-Cdh1 and no DOX control embryos (mean SEM, p < 0.0001 1W-ANOVA, n = 1035 embryos from 3 transgenesis experiments). (E) Cdh1 immunostaining and confocal microscopy of NF44 transgenic embryos and controls. Scale bars, 50 um.

Adapted with permission from Cell Press on behalf of Developmental Cell: Edwards et al. (2025). Disrupted endosomal trafficking of the Vangl-Celsr polarity complex underlies congenital anomalies in Xenopus trachea-esophageal morphogenesis. Dev Cell 2025 May 21; doi: 10.1016/j.devcel.2025.04.026.

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