GFRA2 Identifies Cardiac Progenitors and Mediates Cardiomyocyte Differentiation in a RET-Independent Signaling Pathway

Hidekazu Ishida,Rie Saba,Leo Dunkel,Hiroshi Sasaki,Kenta Yashiro,Anna-Katerina Hadjantonakis,Sonja Nowotschin,Ariel Poliandri,Yasushi Sakata,Yoshiki Sawa,Ken Suzuki,Prashant Ruchaya,Manabu Shiraishi,Osamu Yamaguchi,Keiichi Ozono,Shigetoyo Kogaki,Masakazu Hashimoto,Ioannis Kokkinopoulos,Andrew Tinker,Stephen C Harmer ,Duncan C Miller

doi:10.1016/j.celrep.2016.06.050

Abstract

SummaryA surface marker that distinctly identifies cardiac progenitors (CPs) is essential for the robust isolation of these cells, circumventing the necessity of genetic modification. Here, we demonstrate that a Glycosylphosphatidylinositol-anchor containing neurotrophic factor receptor, Glial cell line-derived neurotrophic factor receptor alpha 2 (Gfra2), specifically marks CPs. GFRA2 expression facilitates the isolation of CPs by fluorescence activated cell sorting from differentiating mouse and human pluripotent stem cells. Gfra2 mutants reveal an important role for GFRA2 in cardiomyocyte differentiation and development both in vitro and in vivo. Mechanistically, the cardiac GFRA2 signaling pathway is distinct from the canonical pathway dependent on the RET tyrosine kinase and its established ligands. Collectively, our findings establish a platform for investigating the biology of CPs as a foundation for future development of CP transplantation for treating heart failure.

Highlights

The heart is the first morphologically distinct developing organ in vertebrates
We found that Glial cell line-derived neurotrophic factor receptor alpha 2 (Gfra2) marks cardiac progenitors (CPs) of the FHF and second heart field (SHF) in mouse and human (Airaksinen and Saarma, 2002; Paratcha and Ledda, 2008)
Gfra2 Marks Both FHF and SHF CPs According to previous single-cell expression profiling of mouse embryonic CPs between days 7.5 and 8.0 post-conception (E7.5–E8.0), we observed that Gfra2, a specific receptor for a neurotrophic factor Neurturin (NRTN), was expressed in CPs but not in embryonic stem cells (ESCs) (42.31 ± 22.53 SEM of CPs versus 0.00 of ESCs in Reads Per Millions, respectively) (Brouilette et al, 2012; Kokkinopoulos et al, 2015)

Summary

Introduction

The heart is the first morphologically distinct developing organ in vertebrates. The primordial heart is derived from the anterior part of the lateral plate mesoderm as cardiac progenitors (CPs) being one of the earliest populations emerging from the primitive streak at gastrulation (Kinder et al, 1999; Rana et al, 2013). A major hurdle is the paucity of reliable and specific markers to identify CPs, especially for the robust isolation of living CPs using cell sorting, circumventing the requirement of genetic modification for tagging CPs. Previous reports have demonstrated that Kinase insert domain receptor (KDR, known as Flk-1), platelet-derived growth factor receptor alpha (PDGFRA), KIT, C-X-C chemokine receptor type 4 (CXCR4), and/or Prion protein (PrnP) can be used in defined combinations to identify and harvest CPs (Bondue et al, 2011; Hidaka et al, 2010; Kattman et al, 2006, 2011; Nelson et al, 2008; Yang et al, 2008). In vitro clonal-tracing studies have revealed that both KDR+/PDGFRA+ and KDRlow+/ KITneg cell populations contain highly enriched multipotent progenitors producing cardiomyocytes and endothelial and smooth muscle cells in mouse and human, respectively (Kattman et al, 2006, 2011; Yang et al, 2008). A cellsurface protein, hyperpolarization-activated cyclic nucleotidegated potassium channel 4 (HCN4), has been reported to be

Results

Discussion

Conclusion