Abstract
The hierarchy of transcription factors and signalling molecules involved in hematopoietic development has been dissected through transgenic and knock-out experiments, leading to the identification of several important genes. Less well known are the networks of transcription factors which regulate the activities of the main genes identified. Kit, encoding the membrane receptor of Stem Cell Factor (SCF), is a critical molecule for Hematopoietic Stem Cells (HSC) and some early progenitors, in which it is expressed. In a previous work (Cairns et al., Blood102, 3954;2003), we used mouse lines expressing transgenic Green Fluorescent Protein (GFP) under the control of Kit regulatory elements to investigate Kit regulation in different cell systems such as the hematopoietic and germ cell lineages. We generated a mouse Kit transgene capable of efficiently driving GFP expression both in PGC and in hematopoietic progenitors, such as CFU-Mix and BFU-Es.
In the present work, we evaluated the functional efficiency of the same transgene also in HSC residing in the Fetal Liver (FL) and adult Bone Marrow (BM). To test if the construct is expressed in HSC, we transplanted FL or BM cells, fractionated on the basis of Kit expression and the level of GFP fluorescence, into irradiated non-transgenic mice. At the same time, the proportion of hematopoietic progenitors in the various fractions was assessed by in vitro colony assays. Following long term hematological reconstitution, the contribution of transplanted GFP cells was evaluated by the proportion of fluorescent mixed colonies in colture as well as by the proportion of fluorescent bone marrow cells, as assessed by FACS analysis. Long term reconstitution was confirmed by secondary transplants.
Results show that the repopulating cells derived from fetal liver and adult bone marrow reside in a fraction of Kit+ cells with intermediate GFP fluorescence level, whereas CFU-Mix and BFU-E are in the highly GFP fluorescent fraction. Furthermore, flow cytometry of fetal liver shows that the intermediate fluorescence fraction is highly enriched in Kit+, Sca1+, CD11b+ cells (the expected HSC immunophenotype), whereas the high fluorescence fraction contains mainly Kit+, Sca1−, CD11b− cells. Similarly, the HSC-enriched tip of the Side Population (SP) of adult bone marrow is highly enriched in Kit+, Sca1+ cells of intermediate GFP fluorescence, whereas the upper part of the SP is enriched in Kit+, Sca1− cells of high GFP fluorescence. Our results indicate that the transgene (and possibly the endogenous Kit gene as well) might be transcribed at relatively low levels in HSC versus other progenitors. Noteworthy, the same transgene is also highly expressed in PGC and in Cardiac Stem Cells (CSC) (Messina et al., Circ. Res. 95,911;2004) and in blastocyst inner mass grown in vitro, indicating that the most 5′ part of the intron (4kb), added to the otherwise inactive promoter might include sites regulating Kit expression in multiple stem cell types.
Hemogenic endothelium during development and beyond
