Two distributed enhancers of Sox17 additively regulate the developmental fate of bipotential ventral pancreato-biliary progenitor cells

SRY-box transcription factor 17 (Sox17) is critical for hepato-pancreato-biliary (HPB) development. We identified two distributed enhancers of Sox17, Sox17e-13 and Sox17e-231, within the likely murine Sox17 topologically associating domain. Individual CRISPR-mediated enhancer deletions temporally reduce endodermal Sox17 expression, delaying the transition of bipotent ventral pancreato-biliary progenitor cells to a lineage-unipotent ventral pancreatic fate, reducing pancreas size and increasing gallbladder size. Deleting both enhancers further reduces Sox17 expression and delays conversion of bipotent progenitor cells to ventral pancreatic or biliary fates, reducing both pancreas and gallbladder size. Crossing each enhancer mutation to a Sox17 null allele revealed differential and profound sensitivities of the extrahepatic biliary system to alterations of Sox17 expression, as demonstrated by gallbladder hypoplasia, gallbladder agenesis with a cystic duct, and biliary atresia. These studies indicate that Sox17e-13 and Sox17e-231 additively modulate Sox17 expression, the differentiation of bipotential ventral pancreato-biliary cells to unipotent fates is Sox17 dosage dependent, and that thresholds for Sox17 action during pancreato-biliary formation vary by developmental stage, further defining the role of Sox17 in HPB formation and function.