- Top of page
- RESULTS AND DISCUSSION
- EXPERIMENTAL PROCEDURES
Hematopoiesis is the dynamic process whereby blood cells are continuously produced in an organism. Blood cell production is sustained by a population of self-renewing multipotent hematopoietic stem cells (HSCs) throughout the life of an organism. Cells with definitive HSC properties appear in the mid-gestation embryo as dense clusters of cells budding from the floor of the aorta, and that of the vitelline and umbilical arteries in the aorta-gonads-mesonephros region. Attempts to genetically modify the aortic floor from which these HSCs arise have been unsuccessful in the mouse, since the regulation of gene expression in the hemogenic endothelium is largely unknown. Here we report the implementation of gene transfer by electroporation into dorsal aortic endothelial cells in the chick embryo. This approach provides a quick and reproducible method of generating gain/loss-of-function models to investigate the function of genes involved in HSC birth. Developmental Dynamics 239:1748–1754, 2010. © 2010 Wiley-Liss, Inc.
- Top of page
- RESULTS AND DISCUSSION
- EXPERIMENTAL PROCEDURES
Hematopoiesis is the dynamic process whereby blood cells are continuously produced in an organism. Blood cell production is sustained by a population of self-renewing multipotent hematopoietic stem cells (HSCs) throughout the life of an organism. In early amniote embryos, the first functional definitive HSCs emerge from a splanchnopleura-derived region called AGM (Aorta-Gonads-Mesonephros), which includes the dorsal aorta floor, the mesonephros, mesentery, gonads, and the mesenchyme in between these structures (Cumano et al.,1996; Medvinsky and Dzierzak,1996; Muller et al.,1994). The first definitive HSCs produced in the embryo are thought to occur in dense clusters of hematopoietic cells budding from the floor of the aorta and also present in the vitelline and umbilical arteries. These intra-aortic clusters have been found in all vertebrate species studied so far (reviewed in Hartenstein,2006). Furthermore, transcription factors involved in HSC ontogeny are evolutionarily well conserved among vertebrates (for review, see Hsia and Zon,2005).
Transplantation experiments in birds have demonstrated that embryonic endothelial cells (ECs) originate from two different mesodermal lineages (Pardanaud et al., 1996). The aortic endothelium has a dual origin, roof and sides being contributed by somite-derived ECs and floor by splanchnopleura-derived ECs. As only splanchnopleura-born ECs display hemogenic capacities, intra-aortic clusters are restricted to the ventral aspect of the aorta. As hematopoiesis proceeds, the hemogenic endothelium also disappears from the aortic floor and is replaced by somitic ECs. Thus, the aortic floor appears as a transitory structure spent out in producing blood cells and replaced (Pouget et al.,2006). These experiments confirm that the dorsal aorta ventral endothelium has hemogenic ability, as proposed by the hemogenic endothelium hypothesis (Jordan,1916). This hypothesis has been supported as well by recent lineage tracing experiments in the mouse (Zovein et al.,2008). That close relationship of ECs and HCs led Murray to coin the term “hemangioblast” to refer to the components of the cell aggregates appearing in yolk sac and precursors of blood islands (Murray,1932). Today such hemangioblast is understood as a common precursor for both endothelial and hematopoietic lineages and its existence is still controversial.
Characterization of HSC is mainly done by bone marrow transplantation and relies mostly on the isolation of HSC populations from different sources according to specific surface markers (Sanchez et al.,1996). Actually, few attempts have been made to analyze HSC biology in their endogenous environment. The development of Cre transgenic lines in the mouse has approached the labeling of intra-aortic hematopoietic clusters, which contain functional HSCs based in transplantation experiments; however, up to now they failed to provide AGM-specific hemogenic labeling (reviewed in Yoshimoto et al.,2008). Further dissection of the regulatory sequences conferring HSC-specific expression would be required to achieve a more specific gene transfer strategy.
The accessibility of the chicken embryo has allowed the development of a number of manipulation techniques, including direct gene transfer, surgical manipulation, and time-lapse observation. Presently, gene transfer into chick cells is performed by three major systems: lipofection (which is based on the ability of liposomes to fuse with the cell membrane), electroporation (which uses electric pulses to make small holes in the cell membrane through which naked DNA molecules can enter the cells), and virus-mediated transfer (which allows integration of foreign genes into the chromosomes of host cells and, hence, the prolonged expression of the genes). Electroporation is the most widely used because it is highly efficient, fast, and requires only small amounts of DNA. Lipofection instead needs large amounts of DNA, whereas virus-mediated transfer requires a long period of time, to construct viral vectors, establish clones of producer cells, and assess viral titers. Also, the electroporation transfer has the advantage that naked plasmid DNA produces little antigenicity in the host (Gilkeson et al.,1991; Jiao et al.,1992). For these reasons, electroporation in ovo is today a routine tool for functional modification in the avian model (Muramatsu et al.,1997; reviewed in Itasaki et al.,1999).
This technique is efficient at introducing genes into adult vascular EC (Nishi et al.,1996) but efforts to electroporate DNA into the embryonic vascular EC have been unsuccessful, since labeling was not restricted to vascular EC but was also detected in tissues surrounding the vessel (Bollerot et al.,2006). Instead, lipoplex-mediated gene transfer was set up to modify embryonic vascular cells, with the drawback that modifications are driven to all the embryonic vasculature and not only to the hemogenic endothelium (Bollerot et al.,2006).
We present here a new protocol for gene transfer by electroporation into embryonic ECs and show that foreign DNA expression is local and constrained to the endothelial cells, not spreading initially to the surrounding tissues. By transferring expression constructs into the dorsal aorta ECs, we were able to direct gene expression to the hemogenic endothelium at the critical stages for HSC generation in the AGM. Moreover, gain-of-function using expression constructs for chick Lmo2 and Tal1 genes was achieved.
This method provides a fast and efficient means of generating gain- or loss-of-function models to investigate the function of genes involved in blood cell generation from hemogenic endothelium, including HSC birth. Furthermore, the simple experimental design and low cost make this an attractive tool for rapid large-scale screening of gene function.