Evaluation of E13.5 fetal liver demonstrated significant chimerism with G_sα^−/− cells by β-gal (48% β-gal⁺; Fig.1d) and no difference in colony forming unit-cell (CFU-C) content between G_sα^+/+ and G_sα^−/− chimeric MNCs (Suppl. Fig.3). Therefore, HSPC form and have intact hematopoietic potential in the fetal liver even in the absence of G_sα, but without G_sα, there is a failure to engraft BM by E17.

To evaluate whether this developmental requirement was also present in adult HSPC, we crossed G_sα^fl/fl mice with Mx1-Cre mice, a strain that enables high efficiency polyI.polyC induced Cre expression in HSPC. Competitive repopulation was examined using BM mononuclear cells (BM MNCs) obtained from 6 week old G_sα^+/+Mx1-Cre⁺ or G_sα^fl/flMx-1-Cre⁺ mice treated with polyI.polyC for 5 days (hereafter referred to as Mx1-Cre WT and Mx1-Cre KO, respectively), and transplanted in competition (1:1) with wild-type cells. Deletion of the G_sα gene in primitive cells was confirmed by PCR (Suppl. Fig.4). Analysis of the number of HSPCs in the BM MNCs, phenotypically defined as lin⁻c-Kit⁺Sca-1⁺Flk-2⁻ was also identical between the Mx1-Cre WT and Mx1-Cre KO animals (WT: 0.031% ⁺/− 0.012; KO: 0.038% ⁺/− 0.002; p=0.545). Cells from the Mx1-Cre WT animals were able to efficiently engraft the BM, while BM MNCs from the Mx1-Cre KO mice were essentially absent from the BM at early (4 weeks) and late (16 weeks) time points following transplantation (Fig.2a). Furthermore, when animals were transplanted with BM cells from Mx1-Cre KO mice in the absence of competing wild type cells, spleen engraftment was significantly reduced as assessed by CFU-S₈ number (Suppl. Fig.5), and all animals died within two weeks indicating a failure of extramedullary engraftment. G_sα is therefore required for HSPC engraftment in adult as well as in embryonic settings.

A failure to successfully engraft may reflect multiple abnormalities that we sequentially examined. As others had previously found that pharmacologic modifiers of the adenylyl cyclase pathways influenced myeloid differentiation in vitro^8,9, BM MNCs were obtained from Mx1-Cre WT and KO mice and progenitor cell activity was analyzed using the CFU-C assay. No significant differences were observed between the genotypes in colony number (Fig.2b), morphology or size. Similarly, analysis of the more primitive cell fraction using the long-term culture initiating cell (LTC-IC) assay¹⁰, demonstrated no significant differences between the WT and KO cells (Fig.2c). Taken together, these data demonstrate that deletion of the G_sα gene does not alter the differentiation potential of primitive BM MNCs in vitro.

Migratory capacity was next assessed using the primitive lin⁻c-Kit⁺Sca-1⁺ (LKS⁺) or more mature lin⁻c-Kit⁺Sca-1⁻ (LKS⁻) BM MNCs of Mx1-Cre WT or Mx1-Cre KO mice. In vitro chemotaxis toward SDF-1α, the only known in vitro chemotactic agent for HSPCs¹¹, demonstrated comparable migratory capacity for each genotype of LKS⁺ cells for all but the lowest concentration of SDF-1α (Suppl. Fig.6a). In addition, there was no significant change in the chemotaxis of the more mature LKS⁻ subset of cells (Suppl. Fig.6b), or lymph node lymphocytes to SDF-1α (data not shown) indicating that there was no generalized impairment of in vitro cell motility related to the G_sα defect.

Primitive cells isolated from BM MNCs of Mx1-Cre WT or KO mice were then evaluated for in vivo capacity to home to the BM environment. Fluorescently-labelled LKS⁺ cells were injected into the peripheral circulation of mice and the numbers of cells present in the BM and spleen were measured using flow cytometry at 6 hours post-injection. Deletion of G_sα in LKS⁺ BM MNCs resulted in a marked impairment in their ability to home to both the BM and spleen (Fig.2d). To examine whether this was specific to primitive cell homing to the BM, or a more general defect, we examined the ability of the more mature LKS⁻ and lymph node lymphocytes to home to hematopoietic organs. While LKS⁻ cells demonstrated a similar impairment as LKS⁺ cells (Suppl. Fig.7a), no defect in G_sα^−/− lymphocytes (genotype confirmed by PCR) homing to lymph node, BM or spleen was observed (Suppl. Fig.7b). These data argue against a generalized defect in cell homing associated with a deficiency of G_sα and identify a specific BM homing defect of primitive hematopoietic cells. To assess the specific homing defect of primitive G_sα^−/− cells, we visualized HSPC homing in the calvarial BM endothelium using intravital confocal/two-photon videomicroscopy¹². We found that G_sα^−/− LKS⁺ cells were defective in their interaction with the BM endothelium with significantly fewer cells observed rolling on the endothelial cells, while the number of cells retained in the circulation was significantly increased (Fig.2e and Suppl. Movies 1 and 2). Therefore, G_sα is critical for the engagement of the BM vasculature by primitive hematopoietic cells thereby enabling subsequent steps in stem cell engraftment.

Deletion of the G_sα subunit resulted in significant reductions in the ability of primitive cells to establish hematopoiesis in the BM environment. Therefore, we examined whether the same deletion had any effects once engraftment of the cells in the BM had been achieved. To study this, BM MNCs from G_sα^+/+Mx1-Cre⁺ or G_sα^fl/flMx-1-Cre⁺ mice were transplanted into wild-type mice. Eight weeks following transplantation, the mice were treated with polyI.polyC to induce gene deletion (Fig.3a). Longitudinal analysis demonstrated no significant differences between mice with Mx1-Cre WT or KO cells in terms of BM CBC, CFU-C frequency (Suppl. Fig.8a–c) or HSPC frequency as assessed by immunophenotype (Fig.3b). Specific analysis of HSPC cell cycling status or number of apoptotic cells also indicated that there were no differences between the two different cell genotypes (Supp. Fig.9a,b). Further, there was no evidence of HSPC enriched cell movement from the BM to the peripheral blood by immunophenotypic analysis to indicate a retention defect. Rather, we did observe a striking defect in the ability of the Mx1-Cre KO cells to be mobilized using G-CSF (Fig.3c). These data demonstrate that G_sα signalling is not required for HSPC retention in the marrow, but under conditions of stress, mimicked here by G-CSF stimulation, trafficking out of the marrow space is impaired.

Genetic alteration of G_sα signalling demonstrated a central role in BM homing of HSPCs. However, to determine if these findings might be relevant for medicine, we tested whether pharmacologic modifiers of G_sα signalling could alter stem cell function in vivo. We treated BM MNCs from wild-type C57Bl/6 mice with cholera toxin, a compound known to constitutively activate G_sα by preventing GTP hydrolysis from the ADP-ribose-G_sα-GTP complex. Using a simple ex vivo exposure of cells for 1hour, we observed increased intracellular cAMP concentrations within hematopoietic cells by ELISA (data not shown). This resulted in an enhancement in the ability of treated cells to both home and engraft in the BM with approximately two-fold increased engraftment at 16 weeks (Fig.4a,b). To test whether the enhancement by cholera toxin was due to a non-specific action of the compound, homing of G_sα^−/− cells treated with cholera toxin was assessed; however no improvement in the homing defect was observed (Suppl. Fig.10). Since stimulation of G_sα-coupled receptors has also been noted to increase proliferation of HSPCs in developing zebrafish¹³, we tested whether murine LKS⁺ cells treated with cholera toxin demonstrated increased cell cycling following transplantation. Cell cycle evaluation of labelled cells two days following injection into lethally irradiated hosts did not detect any difference between untreated and cholera toxin treated cells (Suppl. Fig.11). Therefore, cholera toxin acting at least in part through the G_sα-coupled pathway, augments the homing of the cells and engraftment in the BM. This effect on engraftment was durable and without distortion of mature cell subsets, indicating that the cholera toxin induced improvement in HSPC engagement of the niche did not compromise cellular function.

Elucidating the mechanisms by which HSPCs home to the BM environment has biologic and medical implications that has made it an area of intense investigation. A number of molecules have been implicated, often they were studied because of their effects on other hematopoietic populations such as lymphocytes. Here we show that G_sα plays a key role in the specific homing of HSPCs to the BM, in a manner not shared by lymphocytes.

The inability of stem cells to migrate to the BM in development strongly resembles the phenotype observed with the deletion of CXCR4 or SDF-1^14,15. This, coupled with an impairment in SDF-1α responsive migration at specific concentrations in vitro, suggests that CXCR4 may be using G_sα as its predominant signalling pathway in that cell type. The data presented here do not rule out this possibility for fetal hematopoiesis. However, two important distinctions between the G_sα and CXCR4 null phenotypes exist for stem cell function in adult animals. First, pharmacological interruption of CXCR4 signalling results in prompt mobilization of stem cells into the circulation¹⁶. Genetic disruption of G_sα resulted in no such change in location. Indeed, G-CSF induced mobilization of cells from the BM was impaired in the absence of G_sα suggesting that egress from the marrow may be diminished. Therefore, while CXCR4 is a key component of HSPC retention in the BM¹⁷, it is not exerting its effect through G_sα. Second, CXCR4 null cells engraft in the BM, while G_sα null cells do not¹⁸. It is therefore unlikely that the role of G_sα is simply downstream of CXCR4 as this molecule is dispensable for HSPC engraftment in the transplant setting.

Our results demonstrate that G_sα is critical at the HSPC homing stage, enabling engagement of the niche, however continued G_sα signalling is not required to retain the cells there. There is then meaningful molecular distinction between the homing, engraftment and retention processes. It could be hypothesized that the deficiency in the ability of the G_sα^−/− cells to home to the BM could lead to a depletion of the HSPCs in the marrow space and an accumulation in the blood. However, the decreased egress from the marrow may balance the decreased ingress. Alternatively, there may be compensatory changes in resident cells that mask changes in the population.

Another unknown from these studies is the potential upstream receptor utilizing this pathway. The candidates are numerous and may include adrenergic and prostaglandin receptors. The report of prostaglandin E₂ (PGE₂) serving as a means of enhancing stem cell transplantation in mice¹³ indicates that this is likely at least one receptor capable of activating the pathway. Whether PGE₂ mediates an effect physiologically in mammals could not be discerned from the data of North and colleagues, however, our data indicate that the downstream pathway is critical for physiologic processes in development.

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