Foxa1 and Foxa2 together control developmental gene regulatory networks, and differentiation genes, in both human stem-cell derived liver progenitors and in a human liver cell line: evidence of a collapse of human liver differentiation

Abstract

ABSTRACT:Foxa factors (Foxa1/2/3) play a critical role in liver organogenesis and initiating and maintaining developmental gene regulatory networks (GRN) in murine endoderm, gut, and liver. Nevertheless, it remains to be determined how Foxa2 together with Foxa1 (which compensates for Foxa2 loss) function during human liver differentiation/development. In novel, hypoxic protocols that induce hPSC-derived endoderm and liver progenitors, RNAi (siRNA and shRNA)-mediated knockdown of both Foxa1 and Foxa2 demonstrates widespread regulation of the endoderm and mesendoderm GRN, and albumin, which signifies liver differentiation block. The Foxa1/2 (-/-) phenotype in liver cells demonstrated significant downregulation of albumin, liver GRN (HNF4α, Hex, HNF1ß, and Tbx3), all consistent with a collapse of liver differentiation, and a significant upregulation of germ layer markers like Nanog (pluripotency), Pax6 (neurectoderm), GATA4 (endoderm/mesoderm), Cdx2 (intestine). RNA-seq and bioinformatics analysis of the shRNA Foxa1/2 (-/-) phenotype confirms global downregulation of liver-specific genes and unexpected upregulation of epithelial differentiation, mesodermal (cardiac), and neural genes. These data suggest time-dependent effects on endoderm and liver GRN, widespread effects on the liver transcriptome. Additionally, the Foxa1/2 (-/-) phenotype upregulates of cardiac, neural, and intestinal genes, and this data phenocopies data from human hepatocyte differentiation. These data suggest a novel role for Foxa1/2 in human liver differentiation.