Genetic lineage tracing analysis of anterior hox expressing cells

Abstract

Cell lineage studies have been widely used in developmental biology to establish which cells, and how many cells, in the early embryo will give rise to a specifi c structure and its derivatives. Several methods have been developed to label progenitor cells in the early embryo. Here, we describe the genetic tracing approach that relies on the use of the recombinase to genetically and permanently label progenitor cells as well as their progeny through specifi c activation of a conditional reporter gene, the ROSA26 reporter mouse. Labeling of progenitor cells is spatially controlled by the use of a tissue-specifi c promoter driving Cre , the Hoxb1 IRES-Cre /+ and the Hoxa1-enhIII-cre . ROSA26R mice and Hoxb1 IRES-Cre /+ or Hoxa1 -enhIII-cre mice are crossed together to generate embryos at different stages of development. Embryos are collected and dissected at a specifi c stage of development and fi xed in paraformaldehyde. To follow Hoxb1 + and Hoxa1 + progeny, X-gal staining is performed to detect β-galactosidase activity in embryos or developing organ such as the heart. Finally, X-gal-positive cells are observed on whole-mount embryos or dissected organ to determine the lineage contribution of Hoxa1 and Hoxb1 during development.