Lineage labeling with zebrafish hand2 Cre and CreERT2 recombinase CRISPR knock-ins.

BACKGROUND

The ability to generate endogenous Cre recombinase drivers using CRISPR-Cas9 knock-in technology allows lineage tracing, cell type-specific gene studies, and in vivo validation of inferred developmental trajectories from phenotypic and gene expression analyses. This report describes endogenous zebrafish hand2 Cre and CreERT2 drivers generated with GeneWeld CRISPR-Cas9 precision targeted integration.

RESULTS

hand2-2A-cre and hand2-2A-creERT2 knock-ins crossed with ubiquitous loxP-based Switch reporters led to broad labeling in expected mesodermal and neural crest-derived lineages in branchial arches, cardiac, fin, liver, intestine, and mesothelial tissues, as well as enteric neurons. Novel patterns of hand2 lineage tracing appeared in venous blood vessels. CreERT2 induction at 24 h reveals hand2-expressing cells in the 24- to 48-h embryo contribute to the venous and intestinal vasculature. Induction in 3 dpf larvae restricts hand2 lineage labeling to mesoderm-derived components of the branchial arches, heart, liver, and enteric neurons.

CONCLUSIONS

hand2 progenitors from the lateral plate mesoderm and ectoderm contribute to numerous lineages in the developing embryo. At later stages, hand2-expressing cells are restricted to a subset of lineages in the larva. The endogenous hand2 Cre and CreERT2 drivers establish critical new tools to investigate hand2 lineages in zebrafish embryogenesis and larval organogenesis.