Laser Capture and Deep Sequencing Reveals the Transcriptomic Programmes Regulating the Onset of Pancreas and Liver Differentiation in Human Embryos.

Rachel E Jennings,James Strutt,Karen Piper Hanley,Ludovic Vallier,Andrew A Berry,Sarah Withey,Mariya Chhatriwala,Nicoletta Bobola,David T Gerrard,Stephen J Wearne,Neil A Hanley

doi:10.1016/j.stemcr.2017.09.018

Abstract

SummaryTo interrogate the alternative fates of pancreas and liver in the earliest stages of human organogenesis, we developed laser capture, RNA amplification, and computational analysis of deep sequencing. Pancreas-enriched gene expression was less conserved between human and mouse than for liver. The dorsal pancreatic bud was enriched for components of Notch, Wnt, BMP, and FGF signaling, almost all genes known to cause pancreatic agenesis or hypoplasia, and over 30 unexplored transcription factors. SOX9 and RORA were imputed as key regulators in pancreas compared with EP300, HNF4A, and FOXA family members in liver. Analyses implied that current in vitro human stem cell differentiation follows a dorsal rather than a ventral pancreatic program and pointed to additional factors for hepatic differentiation. In summary, we provide the transcriptional codes regulating the start of human liver and pancreas development to facilitate stem cell research and clinical interpretation without inter-species extrapolation.

Highlights

The liver bud is comprised of the first hepatic cords connected to a hepatobiliary primordium (HBP). We identified these structures in serial tissue sections in 13 human embryos from late CS12 to early CS14 and devised methodology for laser capture (LC), RNA isolation, amplification (A), and deep sequencing (LCA-RNA-seq; Figure 1A)
There were 655 transcription factor (TF) in the pancreatic bud, 574 in the HBP, and 637 in the hepatic cords. We identified those TFs imputed to regulate the most differentially expressed genes based on known binding events or motif discovery (Janky et al, 2014)
PLXNB3, the most characteristic HBP gene, is among seven genes on the X chromosome that can be deleted as part of the Contiguous ABCD1/DXS1375E Deletion Syndrome (CADDS; OMIM #300475), which includes severe cholestatic jaundice due to obstructed bile flow (Iwasa et al, 2013)

Summary

SUMMARY

To interrogate the alternative fates of pancreas and liver in the earliest stages of human organogenesis, we developed laser capture, RNA amplification, and computational analysis of deep sequencing. Pancreas-enriched gene expression was less conserved between human and mouse than for liver. The dorsal pancreatic bud was enriched for components of Notch, Wnt, BMP, and FGF signaling, almost all genes known to cause pancreatic agenesis or hypoplasia, and over 30 unexplored transcription factors. Analyses implied that current in vitro human stem cell differentiation follows a dorsal rather than a ventral pancreatic program and pointed to additional factors for hepatic differentiation. We provide the transcriptional codes regulating the start of human liver and pancreas development to facilitate stem cell research and clinical interpretation without inter-species extrapolation

RESULTS

Findings

DISCUSSION

EXPERIMENTAL PROCEDURES