ModelingEA/TEFInHumanPSC-DerivedEmbryonicTissues During development of the vertebrate embryo, a common foregut tube gives rise to the esophagus and respiratory tract and this involves an array of complex molecular and morphological processes. The dorsal foreguttubeformstheesophagusandtheventraldomainformstherespiratorytract,andfailuretodosocan resultintracheaesophagealbirthdefectssuchasesophagealatresiaandtracheoesophagealfistula(EA/TEF). As discussed in project 2, much is known about how Wnt and BMP signaling promote a respiratory fate by activation of the transcription factor Nkx2.1. In contrast, little is known about pro-esophageal factors. Mouse and human studies demonstrate that the HMG-box transcription factor Sox2 is involved in segregation of the esophageal and respiratory lineages, however whether Sox2 promotes an esophageal fate or acts predominantlytorepressrespiratory-inducingpathwaysthedorsalforegutisunclear.Wehypothesizethatboth mechanismsareinvolvedinnormalesophagealdevelopment. In humans, most genes that cause EA/TEF remain unidentified. However, heterozygous mutations in SOX2 can cause of EA and TEF, which is in contrast to mice with heterozygous loss of Sox2, which are normal. CompletelossofSox2fromtheforegutendodermofmouseembryosresultsinesophagealagenesis,however Sox2isalsoexpressedduringdevelopmentoftheentericnervoussystem(ENS)oftheesophagus.Giventhat patients with EA can have motility defects, we hypothesize some EA-associated genes may affect ENS development.However,astudyofhowEA-associatedmutationsdifferentiallyaffecttheepitheliumand/orENS of the esophagus has never been done in any species, let alone humans. We propose several novel PSC- basedapproachestostudyhowSox2andotherEA-associatedgenesimpactHumanesophagusspecification, epithelial morphogenesis, and functional innervation using human pluripotent stem cell-derived esophageal organoidswithanentericnervoussystem. In this project we aim to identify the mechanisms underlying esophageal specification and developmentinhumansbyfirstfocusingonthekeyesophagealfactorSox2.WehypothesizethatSOX2 acts both to repress the respiratory lineage, and promote an esophageal fate via an unidentified gene regulatorynetwork.WewilluseahumanPSC-derivedforegutmodelincombinationwithSOX2gain-andloss- of-function to identify a respiratory GRN that is repressed by SOX2 and an esophageal GRN that is SOX2- dependant.ConverselywewilldetermineifNKX2.1repressestheesophagealfate.Wewilltakeadvantageof theexpandablenatureofhumanforegutculturestoidentifydirecttranscriptionaltargetsofhumanSOX2and NKX2.1usingRNA-seqandChIP-seq.WewilltheninvestigatethediseasemechanismsunderlyingTEF andEAthatarecausedbySox2mutations.WewillgeneratePSClinesharboringpatient-basedmutationsin SOX2 and investigate how these impact the formation of the esophageal and respiratory lineages. We will identify the impact of SOX2 mutations on Wnt and BMP signaling and if Sox2 acts by direct protein-protein interactions with the effector proteins b-catenin/TCF and Smads. Lastly we will investigate how EA mutationsdifferentiallyeffectthedifferentcelltypesoftheesophagus;?theepithelial,smoothmuscle and ENS. Given that some patients with EA have associated motility disorders including achalasia 3, constrictions4andmegaesophagus5,wewillinvestigateifSox2mutationsalsohaveENSdeficits.Wewilluse iPSC lines derived from EA/TEF patients identified in projects 1 and 2 to model the molecular deficits underlyingthisbirthdefectusingourhumanPSC-derivedorganoidmodel.
ModelingEA/TEFinhumanPSC-derivedembryonictissues Theesophagusiscomposedofalayerofstratifiedepithelium,surroundedbylayersofinnervatedmusclethat regulates the unidirectional movement of food from the mouth to the stomach. There are several common pathologies affecting the esophagus including Eosinophilic Esophagitis, GERD, Barrett?s, and Esophageal Cancer,aswellasbirthdefectsincludingtracheoesophagealfistula(TEF)andesophagealatresia(EA).While surgical reconstruction can often restore esophageal continuity, there are often long term physiological deficiencies in esophageal function including motility issues and the formation of strictures that require additionalsurgeries.Incaseswherethereisinsufficientesophagealtissueforreconstruction,suchaslonggap EA or esophageal cancer, surgeons may resort to autologous surrogate tissues like colon, or donated esophagus.Inthisproposalweaimtoidentifythemolecularbasisofhumanesophagealdevelopment,causes of EA/TEF, and use tissue engineering to study germ layer interactions, all by using human pluripotent stem cell-basedapproaches. Inhumans,mostgenesthatcauseEA/TEFremainunidentified,andeventhoseknowntocausethese deficits are poorly understood. For example, heterozygous mutations in SOX2 can cause of EA and TEF, which is in contrast to mice with heterozygous loss of Sox2, which are normal. Biochemical and reporter assayssuggestthathumanmutationsinSOX2mayimpairtranscriptionalactivityandpossiblyhaveinhibitory function, although this has not been studied in human tissues. Complete loss of Sox2 from the foregut endoderm of mouse embryos results in esophageal agenesis, however Sox2 is also expressed during development of the enteric nervous system (ENS) of the esophagus. Given that patients with EA can have motility defects, we hypothesize some EA-associated genes may affect ENS development. However, a study of how EA-associated mutations differentially affect the epithelium and/or ENS of the esophagus has never been done in any species, let alone humans. We propose several novel Pluripotent Stem Cell-based approaches to study how SOX2 and other EA-associated genes impact Human esophagus specification, epithelialmorphogenesis,andfunctionalinnervation.
