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

Abstract

Highlight bullet points:• The Sox2+ esophagus and Nkx2-1+ trachea arise from the separation of a single foregut tube through a series of cellular events conserved in mouse and Xenopus• Tracheoesophageal morphogenesis initiates with HH/Gli-dependent medial constriction of the gut tube mesenchyme at the Sox2-Nkx2-1 border • The foregut epithelial walls fuse forming a transient septum co-expressing Sox2 and Nkx2-1 • Downstream of HH/Gli Rab11-dependent endosome-mediated epithelial remodeling and localized extracellular matrix degradation separate the esophagus and trachea • 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.

Single cell transcriptomics identifies a signaling network coordinating endoderm and mesoderm diversification during foregut organogenesis

“…Immunostaining confirmed that this was indeed a rare Nkx2-1/Sox2+ cell population at the prospective tracheal-esophageal boundary (Fig. 4k, l), which recent studies have demonstrated to be critical in tracheoesophageal morphogenesis 31,32 . In sum, the foregut lineage trajectories predicted from the single-cell transcriptomes represent a valuable resource for further studies.…”

Disruption of a Hedgehog-Foxf1-Rspo2 Signaling Axis Leads to Tracheomalacia and a Loss of Sox9+ Tracheal Chondrocytes

“…The fact that Sox9 was not upregulated in the Dermo1Cre;Foxf1 f/f mutants indicates that Foxf1 does not repress Sox9, which was one possibility suggested by their reciprocal expression patterns. We also found that the Foxg1Cre;Foxf1 f/f mutant foregut failed to separate into a distinct trachea and esophagus ( Figure 4A), consistent with our recent work suggesting that the early lateral plate mesoderm is required for morphogenesis (Nasr et al, 2019).…”

“…Both Smo f/f and Gli3T Flag/+ mouse models exhibit tracheomalacia and a reduction of Sox9. Previous work showed that Shh -/and Gli2 -/-;Gli3 +/mouse mutant models, which have relatively high levels of Gli3R, displayed tracheomalacia, whereas the Gli2 +/-;Gli3 -/which lack Gli3R, do not (Litingtung et al, 1998;Miller et al, 2004;Nasr et al, 2019;Park et al, 2010). Previous work suggests that some Hedgehog targets may be activated in the loss of GliR; it is also possible that decreased expression of some HH targets is caused by a reduction in GliA rather than an added presence of GliR (Falkenstein and Vokes, 2014).…”

“…Cartilage never forms around the mutant foregut and there is a dramatic reduction in Sox9 expression and proliferation of the ventral foregut mesenchyme (Litingtung et al, 1998;Miller et al, 2004;Park et al, 2010). Gli2 -/-;Gli3 +/mouse embryos that have only one copy of Gli3 also exhibit tracheomalacia, whereas Gli2 +/-;Gli3 -/embryos, which lack Gli3 but have a single copy of Gli2, do not (Motoyama et al, 1998;Nasr et al, 2019). These data suggest that the balance of GliA to GliR is critical for normal tracheal development.…”

Sonic hedgehog signaling directs patterned cell remodeling during cranial neural tube closure

“…Shh signaling has long been recognized to play an important role in controlling positional cell fates in many developing organs, including the limb, the gut, and the spinal cord ( Jessell, 2000 ; Villavicencio et al, 2000 ; McMahon et al, 2003 ; McGlinn and Tabin, 2006 ; Tickle and Towers, 2017 ; Sagner and Briscoe, 2019 ). Although the effects of Shh on cell behavior have received comparatively less attention, Shh has been shown to influence axon guidance ( Zuñiga and Stoeckli, 2017 ), cell migration ( Gordon et al, 2018 ), mesenchymal cell clustering ( Rao-Bhatia et al, 2020 ), and epithelial remodeling in mice, chicks, frogs, and flies ( Corrigall et al, 2007 ; Escudero et al, 2007 ; Nasr et al, 2019 ; Arraf et al, 2020 ). Depending on the context, proteins in the Shh family can have contrasting effects on epithelial cell behavior, promoting apical constriction in the Drosophila eye ( Corrigall et al, 2007 ; Escudero et al, 2007 ), generating short and flat cells in the neural tube ( Fournier-Thibault et al, 2009 and this work), and inducing tall, pseudostratified cells in the chick coelomic cavity ( Arraf et al, 2020 ).…”