Regulation of Sema3c and the Interaction between Cardiac Neural Crest and Second Heart Field during Outflow Tract Development

Kazuki Kodo,Sang-Ging Ong,Tsutomu Kume,Rumiko Matsuoka,Sachiko Miyagawa-Tomita,Hiroshi Takahashi,Hiroyuki Yamagishi,Shinsuke Shibata,Hideyuki Okano

doi:10.1038/s41598-017-06964-9

Abstract

The cardiac neural crest cells (cNCCs) and the second heart field (SHF) play key roles in development of the cardiac outflow tract (OFT) for establishment of completely separated pulmonary and systemic circulations in vertebrates. A neurovascular guiding factor, Semaphorin 3c (Sema3c), is required for the development of the OFT, however, its regulation of the interaction between cNCCs and SHF remains to be determined. Here, we show that a Sema3c is a candidate that mediates interaction between cNCCs and the SHF during development of the OFT. Foxc1/c2 directly activates the transcription of Sema3c in the OFT, whereas, a hypomorph of Tbx1, a key SHF transcription factor, resulted in the ectopic expression of Sema3c in the pharyngeal arch region. Fgf8, a downstream secreted factor of Tbx1, inhibited the expression of Sema3c in cNCCs via activation of ERK1/2 signaling. Blocking of FGF8 caused ectopic expression of SEMA3C and a migration defect of cNCCs, resulting in abnormal chick pharyngeal arch development. These results suggest that proper spatio-temporal expression of Sema3c, regulated positively by Foxc1/c2 and negatively by the Tbx1-Fgf8 cascade, respectively, is essential for the interaction between cNCCs and the SHF that correctly navigates cNCCs towards the OFT, composed of SHF-derived cells.

Highlights

In a recent study, it was found that Wnt1-positive neural crest cells form part of the intrapericardial aortic trunk, whereas lineages positive for the second heart field (SHF)-marker transcription factor Six[2] give rise to the intrapericardial pulmonary trunk
To test whether the abnormal upregulation of Semaphorin 3c (Sema3c) may disturb cardiac neural crest cells (cNCCs) migration, we utilized the primary culture of cNCCs and found that lentiviral-mediated Sema3c overexpression caused aggregation of cNCCs that could be rescued by Sema3c neutralizing antibodies (Supplementary Figure S5). These results suggest that Tbx[1] expression in the SHF is essential to inhibit the ectopic expression of Sema3c in cNCCs migrating in the pharyngeal arch region, and that loss of Tbx[1] function may lead to ectopic expression of Sema3c in cNCCs, which results in their abnormal migration and/or aggregation
We demonstrate a molecular mechanism for development of proper outflow tract (OFT) septation, where Sema3c is tightly regulated in a spatio-temporal fashion during the interaction between cNCCs and SHF

Summary

Introduction

It was found that Wnt1-positive neural crest cells form part of the intrapericardial aortic trunk, whereas lineages positive for the SHF-marker transcription factor Six[2] give rise to the intrapericardial pulmonary trunk. This suggests that the aortic and pulmonary trunks are derived from cNCCs and SHF progenitors, respectively[8]. Class 3 semaphorins are secreted proteins that act as axon repellents or attractants, controlling the formation of neuronal connections They are expressed in non-neuronal tissues and regulate cardiac morphogenesis and angiogenesis[10]. Our results suggest that Sema3c is a key signaling molecule that mediates interaction between cNCCs and the SHF, which is implicated in proper septation of the OFT and is critical to establishing separate systemic and pulmonary circulation systems

Methods

Results

Conclusion