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HEMATOPOIETIC
STEM CELL GENERATION
We have performed a phenotypic characterization of cell populations
within the P-Sp/AGM and YS. We were able to isolate by functional
analysis i) the emerging hematopoietic precursors ; ii) a population
of primitive macrophages of unknown function ; iii) the endothelial
compartment ; and iv) the stromal environment. We will use this
characterization to study the signals that are expressed in these
hemogenic sits by a combined appraoch using real time RT-PCR and
in situ hybridization for the expression of signaling molecules
involved in different aspects of development. We will concentrate
on Notch and Notch ligands (such as Delta and Jagged), and Wingless
(Wnt) proteins. Both have been previously suggested to be involved
in different aspects of HSC maintenance. Once identified, the proteins
that are differentially expressed in YS and P-Sp/AGM will be transfected
on stromal lines and these will be used to induce hematopoiesis
in vitro from mesodermal precursors isolated from 7-8 day embryos.
The proteins capable to induce hematopoiesis from undifferentiated
mesodermal precursors will be used to construct knock-in mice under
the control of different promoters expressed in different mesodermal
tissues. These mice should then be capable to ectopic hematopoietic
cell generation. This approach will contribue to the understanding
of the environmental signals that play a role in the induction of
hematopoietic cells and will allow the dissection of stimuli responsible
for hematopoietic stem cell maintenance and/or self-renewal.
During our characterization of P-Sp/AGM and YS cell subsets, we
identified a population of hematopoietic precursors, at day 10.5
of gestation that expresses CD45 and is composed of macrophages
of undefined origin in mammals, but known to be generated in a third
hemogenic site (the heart region) in zebra fish (Herbomel P. Development
126 :3735, 1999). In order to elucidate the origin and functin of
these cells we established a collaboration with S.Jung (Rehovot,
Isreal) who has generated a mouse line where GFP is expressed under
the control of the gene encoding for the chemokine receptor CX3CR1.
This receptor binds the chemokine fractalkine and is expressed in
all macrophages. CX3CR1 has been shown to be involved in macrophage
migration to different tissues (Geissman et al. Immunity 19 :71,
2003). We will use embryos from these mice to analyze the migration
properties and eventually to elucidate the origin and function of
primitive macrophages in mammals, in collaboration with the «
Dynamic Imaging Platform» at the Pasteur Institute equipped
with confocal microscopes and a recently purchased two-photon confocal
microscope.
The murine embryonic dorsal aorta is also the site of origin of
hemagioblasts and mesangioblasts. The former cell type is a common
precursor for hematopoietic, endothelial and mural cells while the
latter is capable, after long-term culture, to generate most mesodermal
tissues in vitro and can functionally compensatge muscle dystrophic
conditions in vivo (Sampaolesi et al Science, 301 :487, 2003). In
collaboration with the group of M. Buckingham (Pasteur) we will
use GFP knock-in mice in loci of transcription factors important
in muscle cell determination (Pax-3) that will allow us to isolate
Pax-3 expressing embryonic precursors, or detect de novo Pax-3 expression
in cells in culture. We will attemp, in this set of experiments,
to establish the lineage relationship between the HSC, hemangioblasts
and mesangioblasts from undifferentiated mesodermal precursors.
Web site created by Marie-Christine Vougny (04/2004) |
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