How to Study Gene Expression and Gain of Function of Hoxb1 in Mouse Heart Development

Anterior Hox genes are required for genetic identity and anterior posterior patterning of the second heart field (SHF), which contributes to the formation of the embryonic heart in vertebrates. Defective contribution of SHF cells to the arterial or venous pole of the heart is often associated with severe congenital heart defects. The mouse Cre-lox system allows the activation of expression of any gene of interest in restricted tissues. We developed a gain of function approach that relies on the use of a CAG transgene to ectopically activate Hoxb1 expression in SHF cardiac progenitor cells through specific Cre activation. Therefore, we generated a floxed transgenic mouse line, CAG-Hoxb1-eGFP, which upon recombination by Cre recombinase conditionally induces robust Hoxb1 and eGFP expression. When induced within the anterior SHF lineage, we detected heart defects in mouse embryos such as right ventricular hypoplasia. Here, we describe the strategy for generating and genetically crossing this transgenic mouse line. We also provide detailed protocols for whole-mount embryo and paraffin section in situ RNAscope hybridization and X-gal staining allowing investigation of SHF contribution during heart development.