Endogenous barcoding for in vivo fate mapping of lineage development in the blood and immune system

Objetivo

The immune system is a complex ensemble of diverse lineages. Studies on in-vivo-hematopoiesis have until
now largely rested on transplantation. More physiological experiments have been limited by the inability to
analyze hematopoietic stem (HSC) and progenitor cells in situ without cell isolation and other disruptive
manipulations. We have developed mouse mutants in which a fluorescent marker can be switched on in HSC
in situ (inducible fate mapping), and traced HSC lineage output under unperturbed conditions in vivo. These
experiments uncovered marked differences comparing in situ and post-transplantation hematopoiesis. These
new developments raise several important questions, notably on the developmental fates HSC realize in vivo
(as opposed to their experimental potential), and on the structure (routes and nodes) of hematopoiesis from
HSC to peripheral blood and immune lineages. Answers to these questions (and in fact the deconvolution of
any tissue) require the development of non-invasive, high resolution barcoding systems. We have now
designed, built and tested a DNA-based barcoding system, termed Polylox, that is based on an artificial
recombination locus in which Cre recombinase can generate several hundred thousand genetic tags in mice.
We chose the Cre-loxP system to link high resolution barcoding (i.e. the ability to barcode single cells and to
fate map their progeny) to the zoo of tissue- or stage-specific, inducible Cre-driver mice. Here, I will present
the principles of this endogenous barcoding system, demonstrate its experimental and analytical feasibilities
and its power to resolve complex lineages. The work program addresses in a comprehensive manner major
open questions on the structure of the hematopoietic system that builds and maintains the immune system.
This project ultimately aims at an in depth dissection of unique or common lineage pathways emerging from
HSC, and at resolving relationships within cell lineages of the immune system.