Human organoid modeling of congenital malformations caused by RFX6 mutations reveal an essential role for this transcription factor in establishing and maintaining duodenal identity upstream of PDX1

Abstract

The gastrointestinal (GI) tract consists of highly specialized organs from the proximal esophagus to the distal colon, each with unique functions. Rare congenital malformations of the GI tract, including organ atresia, agenesis or mis-patterning are linked to gene mutations although the molecular basis of these malformations has been poorly studied due to lack of model systems to study human development. We identified a patient with compound heterozygous mutations in the transcription factor RFX6 with pancreatic agenesis as previously described. In addition, the patient had duodenal mal-rotation and atresia suggesting that establishment of the proximal small intestine was impaired in these patients. To identify the molecular basis of the intestinal malformation we generated induced pluripotent stem cell lines from this patient, and derived human intestinal organoid (HIOs) to identify how mutations in RFX6 impact intestinal patterning and function. We identified that the duodenal identity of HIOs and patient tissues had adopted a more distal small intestinal signature, including expression of SATB2, normally expressed in the ileum and colon. CRISPR-mediated correction of RFX6 restored duodenal identity, including expression of PDX1, which is required for duodenal development. Using transcriptomic approaches in HIOs and Xenopus embryos we identified that PDX1 is a downstream transcriptional target of RFX6 and that PDX1 expression in a RFX6 mutant background was sufficient to rescue duodenal identity. However, RFX6 had a PDX1-independent role in regulating expression of components of WNT, HH, and BMP signaling pathways that are critical for establishing early regional identity in the GI tract. In summary, we have identified that RFX6 is one of the most upstream regulators early intestinal patterning in vertebrates and that it acts by regulating key transcriptional and signaling pathways.