Abstract

A transient epithelial structure called the left-right organizer (LRO) establishes left-right asymmetry in vertebrate embryos. Developmental defects that alter LRO formation result in left-right patterning errors that often lead to congenital heart malformations. However, little is known about mechanisms that regulate individual cell behaviors during LRO formation. To address this, we developed a Cre-loxP based method to mosaically label precursor cells, called dorsal forerunner cells, that give rise to the zebrafish LRO known as Kupffer's vesicle. This methodology allows lineage tracing, 3-dimensional (3D) reconstruction and morphometric analysis of single LRO cells in living embryos. The ability to visualize and quantify individual LRO cell dynamics provides an opportunity to advance our understanding of LRO development, and in a broader sense, investigate the interplay between intrinsic biochemical mechanisms and extrinsic mechanical forces that drive morphogenesis of epithelial tissues.

Highlights

  • [Background] Recent efforts in the field of developmental biology have focused on understanding mechanisms underlying tissue and organ morphogenesis at the single-cell level

  • Taking advantage of the transparent zebrafish embryo–which is a useful system for conducting in vivo cell biology experiments–several approaches have been developed to analyze the dynamics of single cells in living embryos

  • Kupffer’s vesicle (KV) (Figure 1A) functions as a left-right organizer (LRO) in the zebrafish embryo (Essner et al, 2005; Kramer-Zucker et al, 2005) that is analogous to the ventral node in mouse and the gastrocoel roof plate in frog (Blum et al, 2014)

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Summary

Introduction

[Background] Recent efforts in the field of developmental biology have focused on understanding mechanisms underlying tissue and organ morphogenesis at the single-cell level. Expression of Cre recombinase in Zebrabow embryos generates differential fluorescent labeling of individual cells based on the stochastic recombination of the Zebrabow transgene. We describe a genetic mosaic labeling strategy to fluorescently label individual KV cells and provide a guide to analyze 3D data obtained from imaging live mosaic-labeled embryos using Imaris software. We determined a dose of 4-hydroxytamoxifen (4-OHT) that induces low levels of Cre activity in DFCs, and reliably results in mosaic labeling of DFC/KV cells in Tg(sox17:CreERT2); Tg(ubi:Zebrabow) embryos (Figure 3B).

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