Graduate Student Cincinnati Children's Hospital and Medical Center, United States
Abstract: Hepatobiliary (HB) development requires precise left-right (LR) mesenchymal patterning around the e9.5 liver bud to spatially coordinate hepatic and biliary morphogenesis. This laterality interaction potentially drives divergent growth, with liver progenitor cells, known as hepatoblasts, migrating from the left liver bud to proliferate while the biliary system invaginates on the right to tubularize. Disruptions in LR patterning are associated with congenital malformations of the liver like biliary atresia underscoring the need to uncover the mechanisms guiding LR-directed HB development. Here, we investigated the molecular and cellular roles of HB-associated mesenchymal populations—Pitx2hi liver fibroblasts and Pitx2lo mesothelial cells— identified through single-cell RNA sequencing and shown to orient in a LR pattern around mouse liver buds by in situ hybridization. Gene set enrichment analysis between these populations revealed >9-fold enrichment of fluid-sensing, cell migration genes, including Postn, in liver fibroblasts that secrete around the left liver bud. POSTN inhibition by neutralizing antibodies disrupted left liver bud development suggesting its critical role in liver morphogenesis. To model mesenchyme-directed left-patterning in vitro, we developed a microfluidic perfusion device that delivers controlled fluid shear stress on HB organoids containing naïve mesenchyme without LR biased signature. Shear stress increased Msx1 expression, a liver fibroblast-specific marker, by 1.8-fold compared to static culture reinforcing leftward mesenchymal patterning in HB organoids. Furthermore, shear stress was sufficient to trigger hepatoblast migration mediated by transcription factor Prox1 activity. Long-term cultures of shear stress-treated organoids showed ~2-fold production of hepatoblasts that adopted a delaminating morphology similar to e10.5 liver buds as compared to static cultures. These findings reveal a unique role for fluid shear stress in modulating mesenchymal LR patterning to prioritize liver morphogenesis over biliary development. Additionally, this work advances in vitro modeling of early liver development by incorporating axial patterning, offering a powerful platform to explore laterality coordination for HB development in health and disease.
Funding Source: This work was supported by the NIH Director’s New Innovator Award (DP2 DK128799-01)
