Knockdown of Laminin gamma-3 (Lamc3) impairs motoneuron guidance in the zebrafish embryo.

Alexander M J Eve,James C Smith

doi:10.12688/wellcomeopenres.12394.1

Abstract

Background:Previous work in the zebrafish embryo has shown that laminin γ-3 ( lamc3) is enriched in endothelial cells marked by expression of fli1a, but the role of Lamc3 has been unknown. Methods: We use antisense morpholino oligonucleotides, and CRISPR/Cas9 mutagenesis of F0 embryos, to create zebrafish embryos in which lamc3 expression is compromised. Transgenic imaging, immunofluorescence, and in situ hybridisation reveal that Lamc3 loss-of-function affects the development of muscle pioneers, endothelial cells, and motoneurons. Results: Lamc3 is enriched in endothelial cells during zebrafish development, but it is also expressed by other tissues. Depletion of Lamc3 by use of antisense morpholino oligonucleotides perturbs formation of the parachordal chain and subsequently the thoracic duct, but Lamc3 is not required for sprouting of the cardinal vein. F0 embryos in which lamc3 expression is perturbed by a CRISPR/Cas9 approach also fail to form a parachordal chain, but we were unable to establish a stable lamc3 null line. Lamc3 is dispensable for muscle pioneer specification and for the expression of netrin-1a in these cells. Lamc3 knockdown causes netrin-1a up-regulation in the neural tube and there is increased Netrin-1 protein throughout the trunk of the embryo. Axonal guidance of rostral primary motoneurons is defective in Lamc3 knockdown embryos. Conclusions: We suggest that knockdown of Lamc3 perturbs migration of rostral primary motoneurons at the level of the horizontal myoseptum, indicating that laminin γ3 plays a role in motoneuron guidance.

Highlights

The zebrafish (Danio rerio) is a powerful model for studying the formation of the vascular system
We show that embryos lacking Lamc3 fail to form the parachordal chain (PAC) vasculature and that loss of the PAC is an indirect effect, caused by the failure of rostral primary motoneurons to extend at the level of the horizontal myoseptum (Lim et al, 2011)
To determine whether the Lamc3 detected in GFP+ cells derives from endothelial cells, we investigated the spatial expression of lamc3 by whole-mount in situ hybridisation

Summary

Introduction

The zebrafish (Danio rerio) is a powerful model for studying the formation of the vascular system. Zebrafish are amenable to genetic, cellular, and molecular studies, and many important features of vasculogenesis are conserved with other organisms, including mammals (Baldessari & Mione, 2008; Jing & Zon, 2011) For these reasons, zebrafish studies have already contributed to our understanding of blood and vascular development, often with relevance for human health and disease (Bertrand et al, 2010; Herbert et al, 2009; Wiley et al, 2011). High throughput RNA sequencing has revealed 388 novel transcripts enriched in zebrafish fli1a expressing cells between 26 and 28 hours post fertilisation (hpf), compared with the rest of the embryo (Cannon et al, 2013). Conclusions: We suggest that knockdown of Lamc perturbs migration of rostral primary motoneurons at the level of the horizontal myoseptum, indicating that laminin γ3 plays a role in motoneuron guidance

Methods

Results

Discussion

Conclusion