During the search for novel surface molecules expressed on primitive hematopoietic cells, we noticed that cells in the hematopoietic and nervous systems share a number of expressed genes. For example, we have previously reported that mKirre, which is abundantly expressed in brain and BM stromal cells, plays a critical role in maintaining HSC functions [20
]. Ephrins and their receptors, Ephs, are expressed in both the nervous and the hematopoietic systems, and they play critical roles in various aspects of neurogenesis and hematopoiesis [23
]. Goolsby et al. also reported that hematopoietic progenitors express a set of neural genes [22
]. These observations prompted us to focus on Robo family proteins, cell surface receptors that play a critical role in the nervous system. Within this family, Robo4 drew our particular attention, as the expression of Robo4 was specific to endothelial cells (ECs), and many HSC markers, such as CD34 and Flk1, were shared with ECs [26
]. As expected, this study demonstrated that Robo4 is highly expressed in murine HSCs and immature hematopoietic progenitors. Moreover, transplantation experiments revealed that KSL cells with high Robo4 expression possessed higher long-term repopulating activity. These results demonstrate that the expression of Robo4 correlates with higher HSC capacities, such as long-term repopulation and differentiation to multiple lineages. During the course of our study, Forsberg et al. reported an extensive transcriptome analysis of LT-HSCs, ST-HSCs, and MPPs and showed that Robo4 is one of the differentially expressed genes among these three primitive hematopoietic cell populations [8
]. In the current study, however, we demonstrated that the expression of Robo4 is highest in LT-HSCs not only among primitive hematopoietic cells, but also among all hematopoietic cells, including various progenitors and lineage marker-positive cells. In addition, we confirmed this observation on both the mRNA and the protein levels. Furthermore, we demonstrated that Robo4 could be used for isolating HSCs with long-term repopulating potential when combined with KSL phenotype.
Robo/Slit signaling acts as a repulsive axon guidance cue and inhibits migration of neuronal cells. In addition, Slit2 inhibits migration of endothelial cells through Robo4 [7
]. On the basis of these published observations, it is reasonably speculated that Robo4 signaling inhibits HSC migration. It was also reported that Drosophila
Slit/Robo signaling inhibits N-cadherin function in mammalian cells [29
]. Since N-cadherin has been shown to play critical roles in the adhesion of HSCs and osteoblasts in the BM niche [9
], we also speculated that Robo4/Slit2 signaling might regulate HSC-osteoblast interaction upon myelosuppression. However, our preliminary analysis indicated that Slit2 did not inhibit migration of KSL cells toward SDF-1, and Robo4/Slit2 signaling did not inhibit N-cadherin-mediated adhesion in mammalian cells (unpublished observation). These data do not necessarily exclude the possibility that Robo4/Slit2 system is involved in migration or adhesion of HSCs in the physiological settings, and additional studies using gene-deficient animals will be required to unravel the precise roles of Robo4/Slit2 signaling in the HSC dynamics.
In a similar vein as the regulation of HSCs by the BM niche, it is intriguing that enhanced Slit2/Robo4 signaling resulted in the decreased residence of HSCs in the SP fraction. The SP phenotype is defined as a cell population with a high capacity of Hoechst 33342 dye efflux, and the efflux of Hoechst dye is accomplished by one of the ATP-binding cassette transporters, ABCG2/Bcrp-1 [30
]. A gene disruption study in mice revealed that Bcrp-1 is absolutely required for the SP phenotype [31
]; however, the expression of Bcrp-1 is equally observed in SP and non-SPs of CD34−
KSL cells, suggesting that the function of Bcrp-1 is regulated by other factors [32
]. Our finding that enhanced Slit2/Robo4 signaling led to decreased HSC residence in the SP fraction indicates that Slit2 could be a candidate factor regulating the Bcrp-1 function. In this regard, the induction of Slit2 in osteoblasts upon myelosuppression is of particular interest. It has been reported that HSCs are recruited from the SP to the non-SP fraction on days 4–6 after 5-FU treatment [13
]. The induction of Slit2 occurs on days 3–6 after 5-FU administration, which precedes or coincides with the SP to non-SP transition of HSCs. These data suggest that Slit2 induced in the BM niche upon myelosuppression acts on HSCs through Robo4 and may play a critical role in their transition from the SP to the non-SP fraction. Preliminary analysis of Robo4-deficient mice showed that there is only a slight difference, if any, in the proportion of KSL-SP cells between knockout and wild-type animals (data not shown). This suggests that other redundant pathways, in addition to Robo4, could regulate the SP to non-SP transition of HSCs, and further detailed analysis of Robo4-deficient mice would be required to unravel precise roles of Robo4 in vivo. Considering a well-recognized role of Robo/Slit signaling in cellular migration [1
], it is also tempting to speculate that Slit2 might be acting to induce HSC migration out of the niche in response to myelosuppression.