This study began with the question of what Ikaros’ role was in regulating developmental decisions during early hematopoiesis; we knew that mutant Ikaros HSC activity in vivo was impaired [11
], and that Ikaros null mutants lacked the cKit+Lin(-/lo)Sca1+(KLS) pool which contained the LT-HSC subfraction [14
]. Here, we have shown Ikaros is a molecular regulator of the HSC pool and that it clearly distinguishes LT-HSC self-renewal from progenitor proliferation. Ikaros is most highly expressed in self-renewing fractions of stem cells, and point mutant mice selectively lack the six-parameter cKit+Thy.1.1(lo)Lin(-/lo)Sca1+CD150+Flk2− phenotype of LT-HSCs which they fail to maintain and expand, while all non-self-renewing fractions of cells accumulate in number. An ectopic Notch signal could not rescue the Ikaros developmental defect in primitive hematopoietic cells, including self-renewing LT-HSCs, and these cells preferentially committed to the NK lineage in lieu of B and T lymphopoiesis.
A vast majority of HSC analyses focus upon the three-color KLS population. Our initial cursory observation of an accumulated KLS fraction in Plastic
homozygotes at E14.5-15.5 was surprising given earlier reports Ikaros was essential for maintenance of the LT-HSC pool. This data suggested that a point mutation in Ikaros had in fact caused an increase in HSCs even though previous experiments reported the lack of these cells. It is well-established that the KLS fraction represents a heterogeneous cellular population containing LT- and ST-HSCs as well as MPPs. A depleted/expanded KLS fraction may thus represent depleted/expanded progenitors rather than a HSC-specific effect. In order to analyze LT-HSCs, this necessitates the use of more than three flow cytometric parameters. Beyond the KLS phenotype, our experiments with Plastic
found the highly-characterized cKit+Thy1.1(lo)Lin(-/lo)Sca1+ (KTLS) fraction of cells is further heterogeneous for LT-HSCs since these mutants had a population of KTLS cells at E14.5-15.5 that did not express CD150. Before utilizing CD150 in conjunction with the Plastic
mutant, our observation of a population of cells characteristic as KTLS LT-HSCs was, as with the finding of an expanded KLS subfraction, irreconcilable with Ikaros’ status as a stem cell regulator. The point mutation in Ikaros
, along with wild-type in vivo assays, allowed us to positively identify the bona fide LT-HSC population. Our experiences with Plastic
shows the value of genetic mutants in helping clarify lineage relationships [32
] and underscores the importance of analyzing the complete stem cell phenotype.
Large-scale gene expression studies have varied in their success rate for identifying stem cell regulators. For example, despite strong indications that JamB/Jam2 is robustly expressed in multiple stem cell populations, functional studies of this gene failed to produce any stem cell phenotypes [37
]. Differences in input cells and bioinformatics protocols and the complexity of alternatively spliced transcripts in stem cells [38
] have been highlighted as underpinning these variable findings. When we analyzed the splice isoform distribution of Ikaros in stem and progenitor cells using earlier cell sorting protocols, the transition from LT-HSC to ST-HSC/MPP came with the addition of Ikaros isoforms, while the transition to lymphoid precursors and lymphocytes, as well as neutrophils, added another isoform (Ik-6) lacking DNA binding sequences [12
]. Based solely on transcriptional repertoire, Ikaros would not have emerged as a candidate stem cell gene: it was robustly expressed in purified LT-HSCs (defined as KLSCD34−Flk2−) in one [39
] of two [21
] microarray studies by our laboratory. A similar pattern was observed for the polycomb group member Bmi-1 which has a significant role in HSC self-renewal [40
]. Furthermore, although frequently overlooked, genes expressed at relatively low levels in stem cells may in fact be the most important regulators of regeneration by virtue of (a) a stem cell’s uncommittedness to any particular developmental lineage, and (b) their coordinated connectivity within mammalian protein networks and extensive combinatorial assembly to produce phenotypes [7
]. Appropriate mutations which selectively alter key protein domains, particularly in stem cells, are pivotal for dissecting links between genome sequence and phenomic diversity. This was recently shown for the non-homologous end-joining DNA Ligase IV enzyme which, through hypomorphic point mutation, was identified as a sensitive regulator of HSC stress over time when all previously generated null mutants were early embryonic lethal [9
We found Plstc/Plstc
FLs fail to undergo significant T cell differentiation in response to an ectopic Notch signal provided by the OP9-DL1 stromal line in vitro, and in vivo preferentially commit to the NK lineage. These findings indicate that Ikaros has potentially two distinct functional roles to play during T/NK cell lineage commitment from HSCs and progenitors. First, the expression of Ikaros in the BM niche confers to the thymus-seeding progenitor cell an ability to respond to a Notch1 signal that directs the commitment to the T/NK cell lineage, presumably through promoting the transcription of Notch target genes [42
]. Second, at the double-positive T cell stage, Ikaros is required to repress Notch1 signaling and prepare cells for positive and negative selection [43
] (Y.S. and G.F.H., unpublished data, June 26, 2009), and a failure to silence Notch signaling at this checkpoint can facilitate leukemogenesis.
harbors a point mutation in Ikaros exon 4 and the full-length DNA-binding isoform Ik-1 is generated and localizes to its normal cellular niche but fails to bind DNA [11
]. This isoform is normally expressed in a broad range of purified populations including LT- and ST-HSC, MPP, CLP, pro-B and -T cells, and neutrophils [12
]. However, since LT-HSCs specifically expressed only Ik-1 and -2, both of which contain exon 4, while more severely-truncated isoforms appeared in the differentiation subsets downstream of LT-HSCs [12
], our in vivo findings corroborate with the Plastic
allele having its greatest corruptive effect at the LT-HSC level. Ikaros and its family member Aiolos have been shown to operate along with other regulatory factors in a developmentally-stage-specific manner during discrete stages of B cell differentiation [44
]. In this instance, the abundance of each factor determined regulatory outcome, with, for example, Ikaros’ chromatin structure, and therefore its potential to be expressed at certain concentrations, posited as key to the activation/silencing of genes under its control. In line with reports of less compacted chromatin in undifferentiated stem cells compared with differentiated cells [45
], a transcriptionally-permissive environment featuring a interplay of multiple lineage-affiliated expression programs in HSCs [46
], and Ikaros’ role as a DNA binding moiety of several chromatin remodeling complexes and bivalent role as both an endogenous gene repressor/activator, it seems likely Ikaros operates via a similar mechanism in HSCs to antagonize other proteins (whether family members or other as yet unidentified regulators) and direct commitment to particular daughter progenitors. Based on expression trends and the extensive interaction between Ikaros and its family proteins through heterodimerization, family members such as Helios and Eos would be attractive candidates. A recent study by Georgopoulos and colleagues has shown that Ikaros operates via such a mechanism by on the one hand promoting lymphoid priming in HSC and in lymphoid-primed multipotent progenitors (LMPPs), and on the other preventing the expression of stem cell genes in LMPPs [48
]. Red cell formation is notable in this respect since KTLS(CD150+) LT-HSCs have a highly accessible erythroid-specific Gata1
]. Gata1 is essential for formation of the β-globin active chromatin hub [49
] and chromosome conformation capture assays have found Ikaros plays an essential role in the formation of this complex [29
]. The development of refined chromatin immuno precipitation (ChIP) assays on low cell numbers including miniChIP [50
] means it will be interesting to examine the epigenetic features of KTLS(CD150+) LT-HSCs and progenitors as they step-wise differentiate toward mature erythroid cells from both wild-type and Plastic
mutants. Similarly, more detailed CD150 promoter studies via ChIP will be valuable to determine whether Ikaros directly regulates the CD150 gene, specifically in only LT-HSCs or more generally within hematopoietic tissues. Given the critical role of Ikaros in HSC self-renewal we have shown here and the synchronous regulation of the erythroid lineage as revealed by anemia in Plastic
homozygotes, it will also be important to examine whether Ikaros exerts a concomitant bivalent regulatory mechanism within progenitor cells primed to erythro/megakaryopoiesis in addition to lymphopoiesis. As we previously noted [12
], alternate Ikaros isoforms in different hematopoietic subsets might identify and play a role in silencing loci, or possibly act as tags for chromatin opening complexes to identify regulatory regions, similar to bipotential chromatin marks, or both [48
]. Other mouse mutants with phenotypes resembling Plastic
may also be required to connect Ikaros target genes within molecular pathways in HSCs and progenitors.
The data presented here provides clear evidence of a critical and dynamic role for Ikaros throughout the hematopoietic hierarchy, most pertinently in the KTLS(CD150+) LT-HSC subfraction from which definitive blood cells begin developing during embryogenesis. Whilst dispensable for the initial embryonic development of blood, Ikaros is clearly needed for maintenance of this tissue. Elucidating precisely how this factor mechanistically controls self-renewal and cell fate choice remains a vital future area of research, in addition to the identification of new switches and components controlling the genetic circuitry of normal and cancer stem cells.