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  1. Evaluation Summary:

    The authors present cellular and genetic data, combine mutant analysis and RNA-sequencing, that together support a functional role for Shh in repressing the epithelial-to-mesenchymal transition (EMT) in the developing larynx during larynx-esophageal separation. The authors report that in the absence of Shh, cells undergo EMT and are replaced with a novel epithelial cell population of unknown origin. Given the relative lack of knowledge about how this important structure develops, these results could make a potentially significant contribution to field, but the evidence for EMT needs further support.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.)

  2. Reviewer #1 (Public Review):

    This manuscript addresses the role of HH signaling in larynx development by combining mutant analysis and RNA-sequencing. The authors show that the distinct domain of low Shh expression and induction of N-Cadherin expression at epithelial lamina and esophageal epithelium of the larynx during larynx-esophageal separation. The authors then show that loss of E-Cadherin and upregulation of N-Cadherin accompanied by differentially expressed EMT-related genes in larynx tissues of Shh null embryos, suggesting that Shh regulates larynx morphogenesis through inhibiting cadherin switch and EMT. Notably, Shh-descendant cells undergo transition to a mesenchymal fate in Shh mutant larynx. Additionally, Shh mutant embryos display loss of Nkx2.1 and reduced Sox2 expression in larynx epithelial cells at early laryngeal morphogenesis and disruption of larynx-esophageal separation accompanied by disorganized and thickened epithelium at later stages. The authors find ectopic Pax1 expression in the larynx of Shh mutant, suggesting expansion of pharyngeal pouches in the absence of Shh. Taken together, these characterization studies provide interesting findings and important information for the role of HH signaling in the development of the anterior foregut.

  3. Reviewer #2 (Public Review):

    The authors present genetic and cellular evidence for a role for Shh in repressing EMT, such that in the absence of Shh cells undergo EMT and are replaced with a novel epithelial cell population of unknown origin. The methodology is straightforward and although the paper is essentially a standard mutant phenotype analysis, given the relative lack of knowledge about how this important structure develops these results make a potentially significant contribution to field.

    The first part of the study investigates how Shh signaling normally is deployed during larynx development, using an allele of Shh that expresses both GFP and Cre, allowing both real-time gene expression reporter and lineage trace functions in the presence of normal SHH signaling.They then investigate whether complete loss of Shh impacts these same phenotypes. They show using IHC and RNA-seq the upregulation broadly of EMT-associated markers, and the localized cadherin switch and vimentin expression consistent with EMT, as well as the morphological appearance of what appear to be extruded cells within the lumen. They then document the ontogeny of this cadherin switch, and confirm the origin of these cells using the ShhCre lineage trace, as well as demonstrating they undergo apoptosis at high rates after leaving the epithelium. These cells also downregulate Foxa2, a transcription factor known to suppress EMT. These data are all straightforward, quantified, and reasonably interpreted.

    The more interesting aspect of this mutant analysis is their investigation of why this increase in EMT and apoptosis doesn't result in loss of epithelial integrity. They revisit Figure 4 and point out that only the ventral pharynx is Shh lineage-positive at E10.5, even though it is initially expressed throughout the pharynx. Their conclusion that this result implies "that foregut epithelial cells undergo dynamic regional cellular rearrangements during this timepoint" is a bit vague, and it isn't actually clear what they mean - what kind of rearrangements, and where do these non-Shh lineage cells come from? They then show that there are very few Shh lineage positive cells in the mutants at this stage (although the quantification isn't quite correct - 80% positive to 20% positive is a loss of 75% of the Shh lineage cells - and the image shown only has a couple of TdT+ cells, not consistent with the 20% their quantification in panel L shows).

    The authors then explore where these Shh lineage-negative cells come from. The authors start by suggesting that perhaps they are dorsal-identity cells that spread, and use Sox2 and Nkx2.1 as dorsal and ventral identity markers. However, both of these markers are reduced in the mutants, leading them to conclude that Shh is required for regional identity of both dorsal and ventral cells. However, this doesn't answer the question of whether dorsal cells spread ventrally, and the shape of the pharynx and the epithelium itself are both really abnormal, so it's just unclear what is happening here.

    They then look at RNA-seq data again for a clue and find that these cells have up-regulated pharynx-related genes most of all, the highest of which is Pax1, normally a pharyngeal pouch-specific transcription factor. They propose several possibilities for the origin of these cells, but do not address this question. As written, this is an interesting observation but not really explored.

  4. Reviewer #3 (Public Review):

    The early foregut epithelial cells express Shh, which plays a crucial function in maintaining epithelial integrity. The authors observed that Shh expression is dynamic and speculated that spatio-temporal control over its expression might regulate regionalized remodeling. The domains undergoing remodeling overlap with reduced Shh expression in wild-type tissue. Additionally, these domains were marked by defects associated with disruption of epithelial integrity along with an upregulation in EMT (Cadherin switching and RAB11) markers. To support their findings, the authors demonstrate that Shh-/- epithelial cells undergo premature/ aberrant EMT which also leads to disruption of epithelial integrity. The null mutants showed disruption of basement membrane, extrusion of epithelial cells into the mesenchyme and defects in cell survival. Last, they document that the Shh mutant epithelial cells are replaced by a new population of cells, but while these cells may be of pharyngeal origin this is not demonstrated.