In this study, we describe an in vivo RNA interference functional genetics approach to evaluate the role of 20 different conserved polarity factors and fate determinants in mouse hematopoietic stem cell (HSC) activity. In total, this screen revealed three enhancers and one suppressor of HSC-derived reconstitution. Pard6a, Prkcz, and Msi2 shRNA-mediated depletion significantly impaired HSC repopulation. An in vitro promotion of differentiation was observed after the silencing of these genes, consistent with their function in regulating HSC self-renewal. Conversely, Prox1 knockdown led to in vivo accumulation of primitive and differentiated cells. HSC activity was also enhanced in vitro when Prox1 levels were experimentally reduced, identifying it as a potential antagonist of self-renewal. HSC engineered to overexpress Msi2 or Prox1 showed the reverse phenotype to those transduced with corresponding shRNA vectors. Gene expression profiling studies identified a number of known HSC and cell cycle regulators as potential downstream targets to Msi2 and Prox1.
Despite tremendous progress made toward the identification of the molecular circuitry that governs cell fate in embryonic stem cells, genes controlling this process in the adult hematopoietic stem cell have proven to be more difficult to unmask. We now report the results of a novel gain-of-function screening approach, which identified a series of 18 nuclear factors that affect hematopoietic stem cell activity. Overexpression of ten of these factors resulted in an increased repopulating activity compared to unmanipulated cells. Interestingly, at least four of the 18 factors, Fos, Tcfec, Hmgb1, and Sfpi1, show non-cell-autonomous functions. The utilization of this screening method together with the creation of a database enriched for potential determinants of hematopoietic stem cell self-renewal will serve as a resource to uncover regulatory networks in these cells.
The stem cell-intrinsic model of selfrenewal via asymmetric cell division (ACD) posits that fate determinants be partitioned unequally between daughter cells to either activate or suppress the stemness state. ACD is a purported mechanism by which hematopoietic stem cells (HSCs) self-renew, but definitive evidence for this cellular process remains open to conjecture. To address this issue, we chose 73 candidate genes that function within the cell polarity network to identify potential determinants that may concomitantly alter HSC fate while also exhibiting asymmetric segregation at cell division. Initial gene-expression profiles of polarity candidates showed high and differential expression in both HSCs and leukemia stem cells. Altered HSC fate was assessed by our established in vitro to in vivo screen on a subcohort of candidate polarity genes, which revealed 6 novel positive regulators of HSC function: Ap2a2, Gpsm2, Tmod1, Kif3a, Racgap1, IntroductionSelf-renewal is inextricably linked to stem cell division, and despite the premise that these processes in mammalian systems likely involve asymmetric cell division (ACD), the molecular details remain enigmatic. Our approach to addressing self-renewal via ACD in the hematopoietic stem cell (HSC) is based on increasing evidence that the mechanistic insights pertaining to polarity molecular networks, which are integral to ACD and cell fate in the invertebrate models of Drosophila melanogaster and Caenorhabditis elegans, are functionally conserved throughout evolution. [1][2][3] Studies from invertebrate models support both extrinsic (niche) and stem cell-intrinsic mechanisms of ACD. In relation to the cell intrinsic machinery, polarity is initiated by asymmetrically localizing protein complexes to the cell membrane. Subcomponents of these complexes act as cell fate determinants that are maintained asymmetrically during mitosis and subsequently segregated differentially into daughter cells. During this process, at the simplest level and without factoring in other potential organelle 4,5 or cell cycle component interactions, 6,7 these membrane complexes interact with centrosomes and the cytoskeletal network to, respectively, anchor and enable correct mitotic spindle orientation for an ACD. [8][9][10] The distinct advantages of these invertebrate models include the ability to follow the end fate of daughter cells during successive rounds of ACD together with real-time video tracking to observe the clear segregation of established cell-fate determinants during and after the ACD process. In contrast, within the hematopoietic system, these advantages are attenuated by the absence of definitive HSC markers or cell fate determinants that could allow for investigations of successive divisions of long-term repopulating HSCs (LT-HSCs). The added factor of HSC motility outside of its niche further hinders prospective daughter cell fate analysis.Despite these limitations, important aspects of HSC selfrenewal with indirect implications for ACD as a mechanism have been reported. F...
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