Phenotypic chemical screening using a zebrafish neural crest EMT reporter identifies retinoic acid as an inhibitor of epithelial morphogenesis.

Laura Jimenez,Steven Warner,Jindong Wang,Katherine K Soh,Monique A Morrison,David Bearss,Cicely A Jette,Rodney A Stewart,Clifford Whatcott

doi:10.1242/dmm.021790

Abstract

ABSTRACTThe epithelial-to-mesenchymal transition (EMT) is a highly conserved morphogenetic program essential for embryogenesis, regeneration and cancer metastasis. In cancer cells, EMT also triggers cellular reprogramming and chemoresistance, which underlie disease relapse and decreased survival. Hence, identifying compounds that block EMT is essential to prevent or eradicate disseminated tumor cells. Here, we establish a whole-animal-based EMT reporter in zebrafish for rapid drug screening, called Tg(snai1b:GFP), which labels epithelial cells undergoing EMT to produce sox10-positive neural crest (NC) cells. Time-lapse and lineage analysis of Tg(snai1b:GFP) embryos reveal that cranial NC cells delaminate from two regions: an early population delaminates adjacent to the neural plate, whereas a later population delaminates from within the dorsal neural tube. Treating Tg(snai1b:GFP) embryos with candidate small-molecule EMT-inhibiting compounds identified TP-0903, a multi-kinase inhibitor that blocked cranial NC cell delamination in both the lateral and medial populations. RNA sequencing (RNA-Seq) analysis and chemical rescue experiments show that TP-0903 acts through stimulating retinoic acid (RA) biosynthesis and RA-dependent transcription. These studies identify TP-0903 as a new therapeutic for activating RA in vivo and raise the possibility that RA-dependent inhibition of EMT contributes to its prior success in eliminating disseminated cancer cells.

Highlights

The conversion of epithelial cells into migratory, invasive mesenchymal cells is a fundamental morphogenetic process during development and regeneration
This analysis revealed that GFP levels became higher as cells adopted mesenchymal morphologies, while at the same time snai1b mRNA levels decreased, consistent with snai1b mRNA being rapidly downregulated in migrating cranial neural crest (NC) cells
We found that GFP protein levels perdured in migratory NC cells until ∼36 hpf, which allows the Tg(snai1b: GFP) reporter to be used as a short-term lineage marker for the NC and visualize cells before obvious epithelial-tomesenchymal transition (EMT) morphologies are observed

Summary

Introduction

The conversion of epithelial cells into migratory, invasive mesenchymal cells is a fundamental morphogenetic process during development and regeneration. EMT can endow cancer cells with pro-invasive properties to allow dissemination from the primary tumor and promote the acquisition of stem-cell-like properties (Mani et al, 2008), therapeutic resistance (Kurrey et al, 2009; Li et al, 2009), increased survival and immunesuppression (Polyak and Weinberg, 2009); all of which contribute to poor patient prognosis. For these reasons, targeting EMT in cancer patients has gained substantial therapeutic interest

Methods

Results

Conclusion