Abstract
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 Foxa2eGFP 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.
Footnotes
Grant support The work at Xin-Yuan Fu’s lab was supported by grants from the Singapore National Medical Research Council (R-713-000-181-511), the Ministry of Education (R-713-000-169-112), Cancer Science Institute of Singapore (CSI, Singapore) (to X.-Y.F. grant 713001010271), the Office of Deputy President (DPRT) of National University of Singapore, West China Hospital (Sichuan University, China) (No.139170082), the President Fund of SUSTech (22/Y01226237) and funds from GenEros Biopharma. The work at BGI-Shenzhen was supported by grants from the National Natural Science Foundation of China (No.81672593), the Natural Science Foundation of Guangdong Province, China (No. 2018A030313379) and Science, Technology and Innovation Commission of Shenzhen Municipality (No.GJHZ20180419190827179, No.KQJSCX20170322143848413). The work is also supported by grants from NIH GM36477 grant to K.S.Z.
Conflicts of interest The authors disclose no conflicts.
Public data access The data reported in this study is available in the CNGB Nucleotide Sequence Archive (CNSA: https://db.cngb.org/cnsa; accession number: CNP0000236).
Abbreviations used in this paper
- MET
- mesenchymal-epithelial transition
- EHT
- epithelial-hepatic transition
- E7.5
- Embryonic day 7.5
- MIRALCS
- microwell full-length mRNA amplification and library construction system
- RPKM
- Reads Per Kilobase per Million mapped reads
- t-SNE
- T-distributed Stochastic Neighbor Embedding
- PS
- primitive streak
- FG
- foregut endoderm
- VE
- visceral endoderm
- NT
- neural tube
- NC
- notochord
- IPA
- Ingenuity pathway analysis
- ECM
- extracellular matrix
- M score
- mesenchymal score
- E score
- epithelial score
- GT
- gut tube
- LP
- liver primordium
- LB
- liver bud
- GBP
- gallbladder primordium
- RaceID
- rare cell type identification
- L1
- gene group Liver 1
- L2
- gene group Liver 2
- L3
- gene group Liver 3
- G1
- gene group Gut tube 1
- G2
- gene group Gut tube 2
- G3
- gene group Gut tube 3
- LXR/RXR
- liver X receptors/retinoid X receptors
- TF
- transcription factor
- FACS
- Fluorescence-activated cell sorting