Glucose-regulated protein 94 (GRP94; also referred to as gp96, CaBP4, endoplasmin, Tra-1) is a metazoan-restricted member of the HSP90 protein family. It is traditionally regarded as an endoplasmic reticulum (ER) localized chaperone assisting in protein folding, assembly and secretion [30]. Due to its client selectivity and interactions with late folding intermediates, GRP94 is postulated to perform unique chaperone functions in the ER, and control specific pathways critical for cell growth and differentiation [31]. In Drosophila, Gp93 (ortholog of GRP94) is required for gut epithelial homeostasis and nutrient assimilation- coupled growth control, suggesting an essential role in the functional expression of specific secretory/integral membrane proteins in tissue specialization [32]. Loss of GRP94 function in mouse models revealed that it is required for mouse embryonic development [33] and plays an important role in immune functions [34–36]. For example, GRP94 is required for the expression of a large number of integrins, as well as Toll-like receptors and selectively regulates innate immunity of macro- phages [37,38], and early T and B lymphopoiesis [39,40]. Furthermore, using a BM chimeric approach, an increase in HSCs and multipotent progenitors was observed in BM devoid of GRP94 [40], raising the interesting issue of how GRP94 deficiency could lead to HSC expansion. Recently, we described the creation of a floxed mouse model of GRP94 targeting the exon 2 of the Grp94 allele (Grp94F/F) for examining GRP94 function in specific tissues [41,42]. We report here that by breeding the Grp94F/F mice with the transgenic mice expressing Mx-1 promoted Cre recombinase [43], we generated Grp94F/F; Mx-1-Cre (referred to below as cKO) mice that allows inducible, acute homozygous knockout (KO) of GRP94 in the hematopoietic system through injection of polyinosinic-polycytidylic GRP94 Regulates Stem Cell-Niche Interactions acid (pI.pC). GRP94 deficiency results in a striking 2-fold expansion of the HSC-enriched Lin2 c-Kit+ Sca-1+ (LSK) cell population (p,0.001) with increases in both the long-term (LT) and short-term (ST) HSC populations. Cell cycle analysis showed loss of quiescence and lodgment assays revealed impaired HSC- niche interaction as a major contributing factor for this increase. Through transplantation assays we further determined that this is a HSC cell autonomous effect, which can be mediated by the loss of integrin a4 expression on the cell surface of Grp94 KO HSCs, leading to impaired HSC retention in the niche. Collectively, our studies establish GRP94 as a novel cell intrinsic factor required to maintain the interaction of HSCs with their niche, and thus regulate their physiology. Results Creation of a mouse model with inducible knockout of Grp94 in the hematopoietic system To study the in vivo function of GRP94, we created Grp94 mutant mice with the floxed or knockout allele (Figure 1A). Deletion of exon 2 flanked by the loxP-FRT sites led to an early frameshift mutation resulting in the inactivation of the Grp94 allele. To determine the role of GRP94 in the hematopoietic system, we bred Grp94F/F mice with transgenic mice bearing the Mx-1-Cre transgene that allows GRP94 to be acutely eliminated in the hematopoietic system in an inducible manner by administration of pI.pC. In these studies, Grp94 was deleted in 5.5 to 6.5 week old Grp94F/F; Mx-1-Cre (referred to below as cKO) mice. Littermates lacking the Cre transgene (Grp94F/F), which are phenotypically equivalent to animals with wild-type (WT) Grp94 alleles, were also injected with pI.pC and served as controls for any side effects of Figure 1. Conditional knockout of Grp94 in the bone marrow. A) Schematic drawings of the Grp94 wild-type (WT) allele, the floxed allele and the knockout (KO) allele. The exons are boxed and numbered. The loxP sites (closed triangle) and the FRT site (open triangle) and expected PCR products for genotyping is indicated. B) Representative BM PCR genotyping results of mice with indicated genotypes after pI.pC injection. C) Grp94 mRNA expression measured by quantitative real-time PCR from WT (n = 16) and cKO (n = 18) mouse BM after pI.pC injection. The level of Grp94 mRNA was normalized against the level of internal control 18S RNA. The data are presented as mean 6 s.e., ***p,0.001. doi:10.1371/journal.pone.0020364.g001 PLoS ONE | www.plosone.org 2 May 2011 | Volume 6 | Issue 5 | e20364