influenza a virus br Materials and methods br Results br Dis

Materials and methods
Results
Discussion In this study, we explored the expression pattern and functional significance of GPR56 in the regulation of hematopoietic stem and progenitor maintenance and function during steady-state and stress-induced hematopoiesis. We found that Gpr56 is predominantly expressed in primitive HSPCs during embryonic definitive and adult hematopoiesis and is regulated in adult BM by core HSC transcription factors. Yet, despite its predominant expression in HSCs, high level Gpr56 expression appears largely dispensable for HSC maintenance in the bone marrow of mice. While we were undertaking this study, Saito et al. (2013), reported that GPR56 signaling maintains HSC quiescence and retention in BM niches via regulation of apoptosis, cell cycle, adhesion, and migration through Rho-GTPase signaling, and in a Evi1-regulated manner (Saito et al., 2013). However, in our studies, we were unable to identify any differences in HSPC maintenance or differentiation capacity associated with the reduction of GPR56. Although the mice studied by Saito et al. (2013), and the Gpr56-deficient mice used in this study were generated by and obtained from the same source as that used in other studies of GPR56 function (Saito et al., 2013; Li et al., 2008; Koirala et al., 2009; Wu et al., 2013), all of the hematopoietic subsets we investigated, including phenotypic HSPCs and mature cell lineages in bone marrow, spleen, and blood were intact in the Gpr56-deficient mice, at least under the conditions tested here. There were, however, subtle phenotypes observed in our Gpr56-deficient mice, including an enlarged thymus, increased frequency of early thymic precursors and CD4+ T cells, and lower percentages of CD4+ and CD8+ T cells among lymphocytes in the spleen suggesting a possible role for GPR56 in the regulation of mature T lymphopoiesis in the thymus. Increased ETP and DN2 frequency despite normal proliferation and survival suggests that in the steady state GPR56 signaling may negatively regulate the entry of progenitors into the thymus. Similar observations were reported for mice deficient for the transcription factor Egr1 (Schnell et al., 2006), although further studies will be required to unravel the cellular and molecular mechanisms through which GPR56 regulates thymus size. Gpr56-deficient HSPCs displayed proliferation and apoptotic rates similar to that of WT HSPCs in vivo. Moreover, in contrast to previous studies (Saito et al., 2013), Gpr56-deficient HSPCs were able to regenerate the hematopoietic system normally in irradiated recipient mice in primary competitive BM transplantation settings. Gpr56-deficient HSPCs also displayed a normal pattern of hematopoietic recovery from myelosuppression after treatment with 5-FU in vivo, indicating that high levels of Gpr56 expression are not required for the repopulating activity of HSCs in vivo, although the mild impairment of reconstituting activity seen upon serial transplant of Gpr56-deficient HSPCs suggests that sustained GPR56 activity may be important for maintaining hematopoietic function during prolonged hematopoietic stress. Our observations that HSPC numbers and functions remain intact in Gpr56-deficient mice despite disruption of both Gpr56 influenza a virus and substantial reduction of GPR56 protein is consistent with a recent report focused on stem cell function in skeletal muscle, which likewise found no significant muscle phenotypes in Gpr56-deficient animals or patients (Wu et al., 2013). G-protein coupled receptors are highly conserved across species and show structural homology with the other members of this family, and their mechanisms of action can be context-dependent and tissue specific (Venkatakrishnan et al., 2013; Wu et al., 2013; Kinzer-Ursem and Linderman, 2007). GPR56 signaling has been implicated in the regulation of neuronal progenitor cell adhesion and migration in the brain; however, we failed to detect such functions for GPR56 in HSPCs in the BM microenvironment, illuminating the tissue-specific, and context-dependent regulation of GPR56 signaling. The underlying basis for the distinct functions of GPR56 in the brain versus other tissue remains unclear. It is possible that closely related GPCR proteins or other unknown factors can compensate for reduced GPR56 in the adult hematopoietic, but not neural, progenitors. Potentially confounding compensatory signals arising from hematopoietic and non-hematopoietic cells (such as, HSPC niche components) in response to germline disruption of Gpr56 and could also provide a selection advantage during homeostasis toward maintaining normal HSPC numbers in vivo. Finally, given our surprising observation that residual GPR56 protein can be detected using a mouse anti-human GPR56 monoclonal antibody in Gpr56-deficient mice, which previously have been reported to lack this protein entirely based on staining with a rabbit anti-human GPR56 antibody that was pre-cleared with mouse brain homogenates from GPR56 knockout mice, we cannot exclude the possibility that the residual protein expression we detect in these mice (Fig. 2B and Supplemental Figs. S2B–E) is sufficient to mediate the crucial functions of GPR56 in HSPCs, though perhaps not in other cell types. Furthermore, humans and mice harbor four splicing variants of GPR56, among which the S4 variant has its starting ATG in exon 4 of the gene (Kim et al., 2009). The targeting strategy used to generate the Gpr56-deficient mice used in this and prior studies (Luo et al., 2011a; Saito et al., 2013; Koirala et al., 2009; Wu et al., 2013) was designed to delete exons 2 and 3, and likely fails to delete the S4 variant. Thus, it is possible that the existing Gpr56 knockout allele is actually a hypomorphic allele and that the generation of new targeting constructs that delete all forms of GPR56 will reveal a role for this protein in physiological processes that can proceed relatively normally with even very low levels of receptor. It is also possible that different levels of residual protein expression or different ratios of GPR56 splice variants in mice housed in different animal facilities might ultimately provide an explanation for the different hematopoietic phenotypes observed in Gpr56-deficient mice in our studies and those of Saito et al. (2013), as a slightly higher level of residual GPR56 protein in our animals might be sufficient for GPR56 to perform its normal physiological functions. Such impacts of housing conditions on animal phenotype have been noted in other studies, which have further implicated differences in microbiome composition as a critical underlying variable (Kriegel et al., 2011). Further studies to identify GPR56 regulatory or compensatory signals and/or conditional deletion of Gpr56 in specific hematopoietic lineages will be very helpful for further dissecting the activities of Gpr56 in physiological and regenerative hematopoiesis. In any event, our data clearly argue that high level Gpr56 expression is largely dispensable for adult hematopoietic stem and progenitor cell maintenance in the BM niche and regenerative functions in mice.