Characterizing the Emergence of Liver and Gallbladder from the Embryonic Endoderm through Single-Cell RNA-Seq

The liver and gallbladder are among the most important internal organs derived from the endoderm. Several inductive signals regulate liver development, yet the pure nascent hepatic and gallbladder cells are unable to be isolated due to limited cell markers and cell numbers. The transcriptome networks of the hepatic lineage in the endoderm, and how the gallbladder differentiates from the adjacent endoderm population, are not fully understood. Using a transgenic Foxa2 reporter mouse line, we performed deep single-cell RNA sequencing on 1,966 individual cells, including nascent hepatic and gallbladder cells, isolated from the endoderm and hepatic regions from ten embryonic stages, ranging from day E7.5 to E15.5. We identified the embryonic liver developmental trajectory from primitive streak to hepatoblasts and characterized the transcriptome of the hepatic lineage. During pre-hepatogenesis (5-6 somite stage), we have identified two groups of foregut endoderm cells, one derived from visceral endoderm and the second derived from primitive streak via a mesenchymal-epithelial transition (MET). During the liver specification stages, liver primordium was identified to share both foregut and liver features. We also documented dynamic gene expression during the epithelial-hepatic transition (EHT). Six gene groups were found to switch on or off at different stages during liver specification. Importantly, we found that RXR complex signaling and newly identified transcription factors associated with liver specification. Moreover, we revealed the gallbladder primordium cells at E9.5 and found genes that transcriptionally distinguish them from the liver primordium. The present data provides a high-resolution resource and critical insights for understanding the emergence of the endoderm, liver and gallbladder development.