A new transgenic reporter line reveals Wnt-dependent Snai2 re-expression and cranial neural crest differentiation in Xenopus

Jiejing Li,Kris Vleminckx,Mark Perfetto,Hong Thi Tran,Rebecca Li,Shuo Wei,Christopher Materna,Melinda K. Duncan

doi:10.1038/s41598-019-47665-9

Abstract

During vertebrate embryogenesis, the cranial neural crest (CNC) forms at the neural plate border and subsequently migrates and differentiates into many types of cells. The transcription factor Snai2, which is induced by canonical Wnt signaling to be expressed in the early CNC, is pivotal for CNC induction and migration in Xenopus. However, snai2 expression is silenced during CNC migration, and its roles at later developmental stages remain unclear. We generated a transgenic X. tropicalis line that expresses enhanced green fluorescent protein (eGFP) driven by the snai2 promoter/enhancer, and observed eGFP expression not only in the pre-migratory and migrating CNC, but also the differentiating CNC. This transgenic line can be used directly to detect deficiencies in CNC development at various stages, including subtle perturbation of CNC differentiation. In situ hybridization and immunohistochemistry confirm that Snai2 is re-expressed in the differentiating CNC. Using a separate transgenic Wnt reporter line, we show that canonical Wnt signaling is also active in the differentiating CNC. Blocking Wnt signaling shortly after CNC migration causes reduced snai2 expression and impaired differentiation of CNC-derived head cartilage structures. These results suggest that Wnt signaling is required for snai2 re-expression and CNC differentiation.

Highlights

The cranial neural crest (CNC) cells are a transient group of multipotent stem cells that exists during early vertebrate embryogenesis
Together with the in situ hybridization data, these results indicate that Snai[2] mRNA and protein are expressed in the post-migratory CNC, and that the snai2:enhanced green fluorescent protein (eGFP) transgenic line is suitable for tracing the CNC lineage at various developmental stages
Transgenic reporter animals provide powerful lineage-tracing tools for identifying CNC derivatives and understanding the mechanisms that control CNC differentiation, which are critical for the studies of CNC biology as well as the prevention and treatment of neurocristopathies

Summary

Introduction

The cranial neural crest (CNC) cells are a transient group of multipotent stem cells that exists during early vertebrate embryogenesis. The detection of CNC in Xenopus was almost solely dependent on in situ hybridization for CNC markers such as snai[2], sox[9] and twist, whereas Alcian blue staining was commonly used for visualization of the head cartilage structures that derive from the CNC. These procedures are time-consuming and labor-intensive, and require fixation that prevents further manipulations of the embryos. Studies published to date have been focused on the roles of Snai[2] in CNC induction and migration, and little is known about the expression or function of this important transcription factor at later stages of embryonic development. Blocking Wnt signaling shortly after the completion of CNC migration leads to reduction in snai[2] expression and under-differentiation of CNC-derived head cartilage structures, suggesting that Wnt is required for post-migratory CNC differentiation, probably by regulating snai[2] expression

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Results

Conclusion