ABSTRACT
The hematopoietic system is organized in a developmental hierarchy in which mature blood cells have a limited life-span and must be constantly replaced by the proliferation and differentiation of bone marrow progenitor cells. Hematopoietic stem cells, which are at the top of this hierarchy, have exstensive self-renewal, proliferative and differentiative capacities. They generate pluripotent progenitors with limited self-renewal potential which, in turn, give rise to unipotent progenitors committed to differentiate along a single hematopoietic lineage.
This developmental program is fundamentally a process of ordinate gene expression, governed by a myriad of transcriptional triggering events, many aspects of which are not clearly defined and represent one of the major themes of developmental biology.
There is growing evidence suggesting that common cellular and molecular mechanisms orchestrate differentiation in various tissues, including neurogenesis and hematopoiesis. Recent observations, obtained in our laboratory, have highlighted that Otx1, which plays a crucial role in brain morphogenesis, is also involved in the control of blood cell production.
Recently, we focused our attention on Sox2, an important neural gene. The Sry- type high-mobility-group box 2 gene (Sox2) encodes a transcription factor that is expressed in very early cells such as embryonic stem cells, primordial germ cells and neural stem cells.
This thesis is part of a research project carried out in our laboratory and aimed at investigating whether Sox2 plays a role also in blood cell production. Own results
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show that Sox2 is expressed at low levels in the bone marrow, the most important hematopoietic site during murine adult life, particularly, in primitive precursors.
The aim of my thesis has been to investigate the possible function of Sox2 in hematopoiesis. I used mutant mice in which one allele of Sox2 is fully inactivated, and the other has a shorter regulatory sequence. This kind of inactivation produces a significant decrease in the transcription of the gene, that results in brain abnormalities: these mutants are, however, viable, while the complete knock-out of Sox2 is lethal.
The effects of Sox2 downregulation will be investigated in: 1) mature cells, using blood cell counts; 2) precursors cells, using clonogenic assays; 3) hematopoietic stem cells, using in vivo competitive repopulation assays.
At the peripheral blood level, I did not detect relevant variations in the mutants, as compared to wild type animals.
However analysis of bone marrow precursors in clonogenic assays showed that Sox2 knock-down mice exhibited a significant increase in the number of
multipotent precursors, as compared to wild type animals.
In order to investigate the role of Sox2 in multipotent precursor cells mechanisms of self-replication, I analyzed the ability of primary CFU-Mix to give rise to secondary colonies. Replating assay of CFU-Mix showed that Sox2 knock-down mice exhibited a significant increase in the number of secondary CFU-Mix, as compared to wild type animals.
To study Sox2 knock-down stem cell proliferative and differentiative ability we used in vivo competitive repopulation assays, coinjecting Sox2 knock-down and wild type bone marrow cells in lethally irradiated recipients. Our data indicate that
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Sox2 knock-down bone marrow shows a reduced repopulating ability to wild type,
perhaps due to a decreased number of HSCs.
Taken together, these data support the view that Sox2 is involved in the regulation of blood cell production, likely at very early stages of differentiation.
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