Trachea-esophageal Disease Studies Using Xenopus

Back to back papers published in Developmental Cell from Nasr et al. and Kim et al. using Xenopus recapitulate congenital trachea-esophageal birth defects.

Endosome-Mediated Epithelial Remodeling Downstream of Hedgehog-Gli Is Required for Tracheoesophageal Separation

Talia Nasr, Pamela Mancini, Scott A. Rankin, Nicole A. Edwards, Zachary N. Agricola, Alan P. Kenny, Jessica L. Kinney, Keziah Daniels, Jon Vardanyan, Lu Han, Stephen L. Trisno, Sang-Wook Cha, James M. Wells, Matthew J. Kofron, and Aaron M. Zorn

Developmental Cell, Volume 51, Issue 6, P665-674.E6, December 16, 2019
Published online: December 5, 2019
DOI:https://doi.org/10.1016/j.devcel.2019.11.003

Click here to view article at Developmental Cell.

Click here to view article on Pubmed.

The etiology of tracheoesophageal birth defects is unknown. Nasr et al. define the conserved cellular mechanisms of foregut morphogenesis in Xenopus and mouse and show how disruption of Rab11-mediated epithelial remodeling downstream of Hedgehog/Gli signaling results in tracheoesophageal clefts similar to human patients.

Highlights

• The Sox2+ esophagus and Nkx2-1+ trachea arise from the separation of the foregut
• HH/Gli-dependent medial constriction of the foregut initiates morphogenesis
• Rab11-dependent epithelial remodeling and ECM degradation separate the foregut
• HH/Gli mutations reveal the cellular basis of tracheoesophageal birth defects

Summary

The trachea and esophagus arise from the separation of a common foregut tube during early fetal development. Mutations in key signaling pathways such as Hedgehog (HH)/Gli can disrupt tracheoesophageal (TE) morphogenesis and cause life-threatening birth defects (TEDs); however, the underlying cellular mechanisms are unknown. Here, we use mouse and Xenopus to define the HH/Gli-dependent processes orchestrating TE morphogenesis. We show that downstream of Gli the Foxf1+ splanchnic mesenchyme promotes medial constriction of the foregut at the boundary between the presumptive Sox2+ esophageal and Nkx2-1+ tracheal epithelium. We identify a unique boundary epithelium co-expressing Sox2 and Nkx2-1 that fuses to form a transient septum. Septum formation and resolution into distinct trachea and esophagus requires endosome-mediated epithelial remodeling involving the small GTPase Rab11 and localized extracellular matrix degradation. These are disrupted in Gli-deficient embryos. This work provides a new mechanistic framework for TE morphogenesis and informs the cellular basis of human TEDs.

Figure 1. Foxf1+ Mesenchyme Promotes Medial Constriction at the Sox2-Nkx2-1 Boundary
(A–C0) Immunostaining showing TE morphogenesis in Xenopus laevis. (A–C) Surface renderings of whole mount confocal images and (A0–C0) transverse optical sections. dorsal foregut; dfg, ventral foregut; vfg, esophagus; e and trachea; t. Scale bar, 100 mm.
(D) Medial constriction in X. laevis with quantification of the difference in foregut width between NF34 and NF35. Scale bar, 100 mm. Difference of means test, *p < 0.05.
(E) Transgenic membrane-GFP X. laevis show increased medial mesenchyme cell density. Scale bar, 100 mm. Student’s two-tailed t test, *p < 0.05. (F) Medial constriction mouse embryos showing. Dashed yellow lines denote medial mesoderm. Scale bar, 100 mm.
(G) Average change in mesoderm width between E9.5 and E10.0 was not significant. Difference of means test.
(H) Average mesoderm cell density at E9.5 and E10.0 was not significantly different. Student’s two-tailed t test.

Figure 3. Localized ECM Degradation and Mesenchymal Invasion Resolve the TE Septum
(A) Wholemount immunostaining of septum resolution in X. laevis and mouse embryos, quantifying the length of the Sox2+-Nkx2-1+ boundary. Student’s two- tailed t test, *p < 0.05.
(B) Serial optical sections showing laminin, Cdh1, and Foxf1 during TE septum resolution in NF41 X. laevis embryos. Arrowhead indicates localized laminin breakdown.
(C) Immunostaining of transgenic membrane GFP NF41 Xenopus embryo (i) anterior to and (ii) at the septation point showing Cdh1+ epithelial (white in schematic) round up as mesenchymal cells (gray) invade. Scale bar, 100 mm.
(D) Serial optical sections showing laminin and Cdh1 during TE septum resolution in E10.5 mouse embryos. Arrowhead indicates localized laminin breakdown. (E) Immunostaining of Foxf1 and Cdh1 (schematic below) in an E10.5 mouse embryo (i) anterior to and (ii) at the septation point. Scale bar, 100 mm.
(F) Inhibition of MMP activity in Xenopus with GM6001 (from NF32-42) results in impaired laminin breakdown (arrowhead) and a TEC.
(G) Quantification of relative lengths of the laryngotracheal groove (ltg) and trachea (t) in DMSO, GM6001, or 1,10-phenanthroline-treated NF41 X. laevis embryos. One-way ANOVA, *p < 0.05.

Figure 4. HH/Gli Activity Is Required for D-V Patterning, Medial Constriction, and Epithelial Remodeling
(A) E15.5 mouse foregut in Shh/Gli mutant embryos. Esophagus, e; trachea, t; trachea-esophageal cleft, tec; laryngotracheal-esophageal cleft, ltec. Arrows denote distance between cricoid cartilage (yellow) and TE septation point (black). Scale bar, 6.35 mm.
(B) Summary of HH/Gli-regulated events.
(C) Nkx2-1, Foxf1, and Sox2 (or DAPI) immunostaining in E10.0Shh/Gli mutants. Scale bar, 50 mm.
(D) aPKC, laminin, and Cdh1 immunostaining in E11.0 Foxg1Cre;Gli3T and Gli2⁄;Gli3+/ mutants showing a failure epithelial fusion and persistent aPKC. Scale bar, 100 mm.
(E) Immunostaining of control MO and Gli3 MO injected NF41 X. laevis embryos showing mislocalized Rab11 in Gli3 morphants. Scale bar, 50 mm.
(F) Immunostaining of aPKC and Rab11 in Foxg1Cre;Gli3T and Gli2⁄;Gli3+/ E11.0 mutants showing a failure of Rab11 reduction compared to controls.

Adapted with permission from Cell Press on behalf of Developmental Cell: Nasr et al. (2019). Endosome-Mediated Epithelial Remodeling Downstream of Hedgehog-Gli Is Required for Tracheoesophageal Separation. Developmental Cell, Volume 51, Issue 6, P665-674.E6, December 16, 2019. Published online: December 5, 2019. DOI:https://doi.org/10.1016/j.devcel.2019.11.003

Isl1 Regulation of Nkx2.1 in the Early Foregut Epithelium Is Required for Trachea-Esophageal Separation and Lung Lobation

Eugene Kim, Ming Jiang, Huachao Huang, Yongchun Zhang, Natalie Tjota, Xia Gao, Jacques Robert, Nikesha Gilmore, Lin Gan, and Jianwen Que

Developmental Cell, Volume 51, Issue 6, P675-683.E4, December 16, 2019
Published online: December 5, 2019
DOI:https://doi.org/10.1016/j.devcel.2019.11.002 

Click here to view article at Developmental Cell.

Click here to view article on Pubmed.

The disease mechanism of the birth defect esophageal atresia with or without trachea-esophageal fistula remains largely unknown. Kim et al. used Xenopus and mouse genetic models to show that an Isl1-Nkx2.1 axis regulates a midline epithelial progenitor cell population that orchestrates trachea-esophageal separation.

Highlights

• Isl1 disruption leads to abnormal trachea-esophageal separation in frog and mouse
• Nkx2.1 lineage-derived respiratory cells contribute to the esophageal epithelium
• A midline cell population is critical for trachea-esophageal separation
• Isl1 regulates the transcription of Nkx2.1 in the midline epithelial progenitor cells

Summary

The esophagus and trachea arise from the dorsal and ventral aspects of the anterior foregut, 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 ventral epithelium 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 mm. See also Figure S1.

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 ventral epithelium 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 ventral epithelium of the esophagus. A single dose of Tamoxifen induces Xgal+ cells in one loca- tion (arrow).
(F) Lineage labeled cells are present in the ventral epithelium (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 mm. 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 mm. See also Figure S4.

Adapted with permission from Cell Press on behalf of Developmental Cell: Kim et al. (2019). Isl1 Regulation of Nkx2.1 in the Early Foregut Epithelium Is Required for Trachea-Esophageal Separation and Lung Lobation. Developmental Cell, Volume 51, Issue 6, P675-683.E4, December 16, 2019. Published online: December 5, 2019. DOI:https://doi.org/10.1016/j.devcel.2019.11.002