In this study we demonstrate that the Pbx1 proto-oncogene and global developmental regulator serves a critical role within the hematopoietic system to support the self-renewal of adult HSCs as one of few known factors involved in maintaining their quiescence. Transcriptional profiling suggests that multiple stem cell maintenance programs are perturbed in the absence of Pbx1, including genes associated with the response to Tgf-b signaling. This suggests potential mechanisms for facilitating loss of quiescence, inappropriate cell cycle entry, and initiation of a transcriptional program resembling that of more mature multipotent progenitors. As a consequence, LT-HSCs lose their major defining characteristic, i.e. self-renewal, and progressively exhaust themselves in the absence of Pbx1.
Among the multiple stem cell maintenance factors that are altered in Pbx1-deficient LT-HSCs, a significant proportion are linked with the Tgf-b pathway, which is increasingly implicated in regulation of HSC quiescence. Tgf-b has recently been suggested as a possible BM niche signal necessary to induce LT-HSC “hibernation” through inhibition of lipid raft clustering in response to cytokine stimulation (Yamazaki et al., 2007
). The CDKI p57 appears to be responsible for regulating this quiescent HSC state (Yamazaki et al., 2007
). Consistent with these observations, cell cycle arrest of human cord blood CD34+
cells in response to Tgf-b requires p57 (Scandura et al., 2004
). Moreover, p57 is responsible for cell cycle arrest of mouse KSL side population (SP) cells (Umemoto et al., 2005
), suggesting that it is one of the main contributors to maintenance of LT-HSC quiescence. We observed higher expression levels of Cdkn1c in LT-HSCs compared to ST-HSCs (although the difference did not meet our stringent statistical requirements for inclusion in Table S3
). This is consistent with previous studies performed with different methods and mouse strains showing that Cdkn1c is preferentially expressed in LT-HSCs compared to ST-HSCs and MPPs (Kiel et al., 2005
), and at higher baseline levels in mouse CD34−
KSL cells (highly enriched for LT-HSCs) than other CDKIs such as p21 or p27 (Yamazaki et al., 2006
). Although the latter are also implicated in HSC or progenitor self-renewal, we did not observe their expression levels to be altered in Pbx1-deficient LT-HSCs in contrast to the marked reduction in p57.
In support of a possible role for Pbx1 on a pathway that regulates the balance between stem cell quiescence/hibernation and cell cycle entry, the Cdkn1c
gene was not induced by Tgf-b stimulation of prospectively isolated Pbx1 mutant LT-HSCs. Although these data do not rule out a secondary role for Cdkn1c, it is one of very few in our Pbx1-dependent gene set to contain a Pbx1 heterodimer binding site in proximity to its promoter. Furthermore, Pbx1/Prep transcriptional complexes have been shown to interact with Smads (Bailey et al., 2004
), the nuclear effectors of Tgf-b signaling, and Pbx1 transcriptional complexes regulate expression of the p21 gene in a myelomonocytic cell line (Bromleigh and Freedman, 2000
). In epithelial cells, a FoxO/Smad complex is implicated in Tgf-b up regulation of p15ink4a, another CDKI (Gomis et al., 2006
). Thus, there are precedents for regulation of CDKI genes by Pbx and/or Smad proteins. However, perturbed expression of Smad7, a Tgf-b pathway regulator that has been implicated in HSC self-renewal (Blank et al., 2006
), was also observed in Pbx1-deficient HSCs in vitro and in vivo raising the possibility of a more global compromise of the response to Tgf-b. Since our data were obtained 4 weeks after Pbx1 deletion, it is theoretically possible that they reflect in part compensatory changes in the proliferation activity or differentiation state of the stem cell compartment. Further investigations are necessary to establish a causal relationship between the hematopoietic defects caused by Pbx1 loss and perturbed regulation of Tgf-b pathway-associated genes implicated in HSC self-renewal.
HSC reductions associated with the absence of Pbx1 were only apparent from approximately 3 weeks post-birth despite the fact that expression of the Tie2Cre transgene initiates in definitive HSCs with their emergence in the aorta-gonad-mesonephros and yolk sac during embryonic development. The postnatal onset of the Pbx1 phenotype likely reflects differences in the proliferation states of embryonic/fetal and adult BM HSCs. During development, HSCs undergo massive expansion while preserving their self-renewal capacity, whereas HSCs in the post-natal BM are mostly quiescent starting from 3–4 weeks of age (Bowie et al., 2006
), and their ability to maintain quiescence correlates with the maintenance of self-renewal and engraftment potential (Passegue et al., 2005
). Thus, onset of the Pbx1-deficient HSC phenotype in Tie2Cre.Pbx1 mice correlates with the post-natal requirement for HSC quiescence for maintenance of self-renewal.
A role for Pbx1 in maintaining stem cell quiescence significantly broadens its contributions beyond previous studies, which have consistently implicated it in promoting progenitor cell expansion during development of multiple organs (Brendolan et al., 2005
; Kim et al., 2002
; Manley et al., 2004
; Selleri et al., 2001
), including the hematopoietic system (DiMartino et al., 2001
). Thus, Pbx1 appears to have divergent but functionally complementary roles in the maintenance of HSC quiescence versus promotion of progenitor proliferation, and our current results raise the possibility that other Pbx1 deficient phenotypes may in part reflect its impact on tissue-specific stem cells. The potential complexity of its contributions in the hematopoietic progenitor compartment was suggested by earlier studies demonstrating that HoxB4-mediated HSC in vitro expansion is further enhanced by concurrent reduction of Pbx1 expression (Krosl et al., 2003
), which appeared inconsistent with its role in promoting progenitor expansion. However, a major function for Pbx1 in maintaining LT-HSC quiescence provides a likely explanation for its antagonistic effects since HoxB4 over-expression enhances HSC expansion without affecting long-term self-renewal potential. The divergent roles for Pbx1 in HSCs versus progenitors may reflect possible context-dependent contributions to the expression of CDKI and other cell cycle regulatory genes.
In addition to a stem cell defect, a marked reduction was observed in the BM pro-and pre-B cell compartments, as well as a striking reduction in CLPs, suggesting a role for Pbx1 at a critical stage of lymphoid development where acute leukemia likely originates, and confirming previous results from our laboratory obtained with a different experimental approach (Sanyal et al., 2007
). The extremely low number of CLPs in mutant mice prevented studies of their cell cycle status, although the cycling rates of pro- and pre-B cells were only mildly decreased (not shown), consistent with previous conclusions that Pbx1 may be dispensable from the pro-B stage onward (Sanyal et al., 2007
). The striking CLP hypoplasia may reflect a cell intrinsic requirement for Pbx1 in CLPs, or alternatively be the consequence of insufficient CLP generation due to an aberrant differentiation program initiated by Pbx1-null HSCs. In support of the latter, GSEA revealed a defect in lymphoid priming present at the LT-HSC stage. The expression of lymphoid-specific genes in LT-HSCs and the concept of lineage priming in stem cells has been previously reported (Mansson et al., 2007
; Rossi et al., 2005
). In Pbx1-deficient mice, the lymphoid priming defect does not completely compromise further differentiation capacity, since mature B cells are present in BM, spleen and PB of primary ko mice and transplant recipients. In future studies it will be of interest to further characterize the roles of Pbx1 in HSC lymphoid priming and generation of CLPs, and the potential relevance of these contributions for Pbx1 oncoproteins in B-cell precursor leukemias.