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Kim E, Jiang M, Huang H, Zhang Y, Tjota N, Gao X, Robert J, Gilmore N, Gan L, Que J.
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The esophagus and trachea arise from the dorsal and ventral aspects of the anteriorforegut, respectively. Abnormal trachea-esophageal separation leads to the common birth defect esophageal atresia with or without trachea-esophageal fistula (EA/TEF). Yet the underlying cellular mechanisms remain unknown. Here, we combine Xenopus and mouse genetic models to identify that the transcription factor Isl1 orchestrates trachea-esophageal separation through modulating a specific epithelial progenitor cell population (midline epithelial cells [MECs], Isl1+ Nkx2.1+ Sox2+) located at the dorsal-ventral boundary of the foregut. Lineage tracing experiments show that MECs contribute to both tracheal and esophageal epithelium, and Isl1 is required for Nkx2.1 transcription in MECs. Deletion of the chromosomal region spanning the ISL1 gene has been found in patients with abnormal trachea-esophageal separation. Our studies thus provide definitive evidence that ISL1 is a critical player in the process of foregut morphogenesis, acting in a small progenitor population of boundary cells.
Figure 1. Isl1 Is Differentially Expressed in the Dorsal-Ventral Region of the Early Mouse Foregut and Required for Trachea-Esophageal Separation in Xenopus laevis
(A) Sox2 and Nkx2.1 maintain differential expression pattern in the early mouse foregut (E9.5-E11.5).
(B) Genes differentially expressed in E11.5 mouse trachea and esophagus are involved in multiple cellular functions as revealed by PANTHER (protein analysis through evolutionary relationships).
(C) p63 and Sox9 are enriched in the E11.5 esophagus and trachea, respectively.
(D) qPCR confirms differentially expressed genes in the E11.5 trachea and esophagus. Data are presented as mean ± SEM; ∗∗∗p < 0.001.
(E) Isl1 expression is enriched in the epithelium (arrows) and mesenchyme of the trachea but not esophagus at E11.5. Note the levels of Isl1 are low in the epithelium compared with the mesenchyme.
(F) Isl1 is enriched in the ventralepithelium and mesenchyme of the mouse foregut at E9.5. Note the co-localization of Sox2 and Isl1 in the dorsal-ventral boundary cells (arrowheads).
(G) Sox2 and Nkx2.1 (arrowheads) are enriched in the esophagus and trachea of X. laevis at stage 41.
(H) Morpholino-mediated knockdown of Isl1 in X. laevis embryos leads to abnormal trachea-esophageal separation (arrow) (n = 8/14). Abbreviations: es, esophagus; tr, trachea; epi, epithelium; mes, mesenchyme. Scale bar: 50 μm. See also Figure S1.
Figure 2. Deletion of Isl1 Leads to EA/TEF and Fusion of RightLung Lobes
(A) Isl1 expression is lost in the foregutepithelium but not mesenchyme of Shh-Cre; Isl1loxp/loxp mutants.
(B) Isl1 deletion causes EA/TEF Formation in Approximately 50% Mutants (n = 18/36)
(C) Isl1 deletion results in fusion of all four rightlung lobes in all Shh-Cre; Isl1loxp/loxp mutants (n = 36/36). Note expression of airway and alveolar epithelium markers seems unperturbed in the mutant lung (n = 5). Abbreviations: Cr, cranial lobe; Ac, accessory lobe; Mi, middle lobe; Ca, caudal lobe. Scale bar: 50 μm. See also Figures S2 and S3.
Figure 3. Nkx2.1+ Lineage-Derived Cells Contribute to the Esophagus
(A) The epithelium at the dorsal-ventral boundary (arrows) co-expresses Sox2 and Nkx2.1.
(B) Sox2+ Nkx2.1+ epithelial cells (arrow) are present in the septum and forming esophagus.
(C) Presence of a few Sox2+ Nkx2.1+ epithelial cells (arrow) in the ventralepithelium of the nascent esophagus.
(D) Sox2+ Isl1+ epithelial cells (arrow) are present in the septum and forming esophagus.
(E) Nkx2.1-CreER lineage labeled cells contribute to the ventralepithelium of the esophagus. A single dose of Tamoxifen induces Xgal+ cells in one location (arrow).
(F) Lineage labeled cells are present in the ventralepithelium (arrows) of the esophagus.
(G) Two doses of Tamoxifen induce Xgal+ cells in two separate locations (arrows). Abbreviations: V, ventral; D, dorsal; Tmx, tamoxifen. Scale bar: 50 μm. See also Figure S4.
Figure 4. Isl1 Regulation of Nkx2.1 in the Boundary Population Is Critical for Trachea-Esophageal Separation
(A) Upon a single low dose of Tmx injection at E8.5, Sox2-CreER lineage labeled cells are present in the dorsal and midline of the foregut at E9.5. At E10.5 lineage labeled cells are present in the esophagus and forming dorsal trachea.
(B) Deletion of Isl1 with Sox2-CreER leads to EA/TEF in all mutants (n = 9). Samples were immunostained with an anti E-cadherin antibody.
(C) Isl1 deletion results in decreased levels of Nkx2.1 in the dorsal-ventral boundary epithelium of Shh-Cre;Isl1loxp/loxp mutants.
(D) Isl1 deletion results in decreased levels of Nkx2.1 in the dorsal-ventral boundary epithelium of Sox2-CreER;Isl1loxp/loxp mutants. Note the decreased levels of Nkx2.1 in the midline cells (arrows and stars) and some ventral epithelial cells also exhibit reduced levels of Nkx2.1.
(E) Luciferase reporter assay shows that ectopic Isl1 expression increases Nkx2.1 promoter activity in two cell lines.
(F) Mutation of the Isl1 binding site abolishes Nkx2.1-driving luciferase activities.
(G) Enrichment of Isl1 binding to the Nkx2.1 locus as shown by ChIP-qPCR. Data are presented as mean ± SEM; ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; NS, not significant. Scale bar: 50 μm. See also Figure S4.
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