Contemporary zebrafish transgenesis with Tol2 and application for Cre/lox recombination experiments.

Spatiotemporal transgene regulation by transgenic DNA recombinases is a central tool for reverse genetics in multicellular organisms, with excellent applications for misexpression and lineage tracing experiments. One of the most widespread technologies for this purpose is Cre recombinase-controlled lox site recombination that is attracting increasing interest in the zebrafish field. Tol2-mediated zebrafish transgenesis provides a stable platform to integrate lox cassette transgenes, while the amenability of the zebrafish embryo to drug treatments makes the model an ideal candidate for tamoxifen-inducible CreERT2 experiments. In addition, advanced transgenesis technologies such as phiC31 or CRISPR-Cas9-based knock-ins are even further promoting zebrafish transgenesis for Cre/lox applications. In this chapter, we will first introduce the basics of Cre/lox methodology, CreERT2 regulation by tamoxifen, as well as the utility of Tol2 and other contemporary transgenesis techniques for Cre/lox experiments. We will then outline in detail practical experimental steps for efficient transgenesis toward the creation of single-insertion transgenes and will introduce protocols for 4-hydroxytamoxifen-mediated CreERT2 induction to perform spatiotemporal lox transgene regulation experiments in zebrafish embryos. Last, we will discuss advanced experimental applications of Cre/lox beyond traditional lineage tracing approaches.

Generating and evaluating a ranked candidate gene list for potential vertebrate heart field regulators.

The vertebrate heart develops from two distinct lineages of cardiomyocytes that arise from the first and second heart fields (FHF and SHF, respectively). The FHF forms the primitive heart tube, while adding cells from the SHF allows elongation at both poles of the tube. Initially seen as an exclusive characteristic of higher vertebrates, recent work has demonstrated the presence of a distinct FHF and SHF in lower vertebrates, including zebrafish. We found that key transcription factors that regulate septation and chamber formation in higher vertebrates, including Tbx5 and Pitx2, influence relative FHF and SHF contributions to the zebrafish heart tube. To identify molecular modulators of heart field migration, we used microarray-based expression profiling following inhibition of tbx5a and pitx2ab in embryonic zebrafish (Mosimann & Panakova, et al, 2015; GSE70750). Here, we describe in more detail the procedure used to process, prioritize, and analyze the expression data for functional enrichment.