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

Abstract

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 hours reveals late emerging hand2 progenitors in the 24–48 hour embryo contribute to the venous and intestinal vasculature. Induction in 3 dpf larva 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. Later emerging hand2 progenitors become restricted to a subset of lineages in the larva. The hand2 Cre and CreERT2 drivers establish critical new tools to investigate hand2 lineages in zebrafish embryogenesis and larval organogenesis.

Competing Interest Statement

MM and JJE have competing interests with Recombinetics Inc., Immusoft Inc., LifEngine and LifEngine Animal Health. KJC has competing interests with Recombinetics Inc., LifEngine and LifEngine Animal Health. SCE has competing interests with LifEngine and LifEngine Animal Health. ZM, FL, HRM, RLL, MA, NKR, PJ, SS, IF, CY, CM do not have competing interests.