Commitment Point Research Articles

Temporal competition between differentiation programs determines cell fate choice

Multipotent differentiation, where cells adopt one of several possible fates, occurs in diverse systems ranging from bacteria to mammals. This decision-making process is driven by multiple differentiation programs that operate simultaneously in the cell. How these programs interact to govern cell fate choice is poorly understood. To investigate this issue, we simultaneously measured activities of the competing sporulation and competence programs in single Bacillus subtilis cells. This approach revealed that these competing differentiation programs progress independently without cross-regulation before the decision point. Cells seem to arrive at a fate choice through differences in the relative timing between the two programs. To test this proposed dynamic mechanism, we altered the relative timing by engineering artificial cross-regulation between the sporulation and competence circuits. Results suggest a simple model that does not require a checkpoint or intricate cross-regulation before cellular decision-making. Rather, cell fate choice appears to be the outcome of a 'molecular race' between differentiation programs that compete in time, providing a simple dynamic mechanism for decision-making.

Temporal anatomy of an epigenetic switch in cell programming: the white‐opaque transition of C. albicans

The human pathogen Candida albicans undergoes a well-defined switch between two distinct cell types, named 'white' and 'opaque'. White and opaque cells differ in metabolic preferences, mating behaviours, cellular morphologies and host interactions. Each cell type is stable through many generations; switching between them is rare, stochastic and occurs without any known changes in the primary sequence of the genome; thus the switch is epigenetic. The white-opaque switch is regulated by a transcriptional circuit, composed of four regulators arranged in a series of interlocking feedback loops. To understand how switching occurs, we investigated the order of regulatory changes that occur during the switch from the opaque to the white cell type. Surprisingly, changes in key transcriptional regulators occur gradually, extending over several cell divisions with little cell-to-cell variation. Additional experiments, including perturbations to regulator concentrations, refine the signature of the commitment point. Transcriptome analysis reveals that opaque cells begin to globally resemble white cells well before they irreversibly commit to switching. We propose that these characteristics of the switching process permit C. albicans to 'test the waters' before making an all-or-none decision.

Mixed Lineage Kinase-3 Stabilizes and Functionally Cooperates with TRIBBLES-3 to Compromise Mitochondrial Integrity in Cytokine-induced Death of Pancreatic Beta Cells

Mixed lineage kinases (MLKs) have been implicated in cytokine signaling as well as in cell death pathways. Our studies show that MLK3 is activated in leukocyte-infiltrated islets of non-obese diabetic mice and that MLK3 activation compromises mitochondrial integrity and induces apoptosis of beta cells. Using an ex vivo model of islet-splenocyte co-culture, we show that MLK3 mediates its effects via the pseudokinase TRB3, a mammalian homolog of Drosophila Tribbles. TRB3 expression strongly coincided with conformational change and mitochondrial translocation of BAX. Mechanistically, MLK3 directly interacted with and stabilized TRB3, resulting in inhibition of Akt, a strong suppressor of BAX translocation and mitochondrial membrane permeabilization. Accordingly, attenuation of MLK3 or TRB3 expression each prevented cytokine-induced BAX conformational change and attenuated the progression to apoptosis. We conclude that MLKs compromise mitochondrial integrity and suppress cellular survival mechanisms via TRB3-dependent inhibition of Akt.

C-Myb is crucial for B-lymphocyte development (138.30)

Abstract c-Myb is abundantly expressed in lymphocyte progenitors yet little is known about c-Myb function during lymphocyte development due to the embryonic lethality of Myb null mutations. We have targeted the murine Myb locus with loxP sites for conditional deletion and crossed Mybff mice to the Mb1Cre and CD19Cre strains to ablate c-Myb expression during different stages of B cell development. Mb1Cre mediated deletion begins in pre-pro-B cells and is complete by the pro-B cell stage. We detect a no decrease in the number pre-pro-B cells but <1% the normal number of CD19+ B-lineage cells at any subsequent stage of development, demonstrating that c-Myb is essential for the development past the point of commitment to the B cell lineage. In Mybf/f CD19Cre mice, B-lineage cells are reduced in number by 80% and blocked in transition from pro-B to pre-B cells, resulting in about 10% the normal number of IgM+ bone marrow B cells. c-Myb deficient B cells are hyporesponsive to IL-7 in methylcellulose cloning assays and in vitro proliferation assays, suggesting that at least part of the defect in pro-B to pre-B cell transition is due to a defect in the IL-7 signaling pathway.

Cyclic ADP-ribose links metabolism to multiple fission in the dinoflagellate Crypthecodinium cohnii

Cellular metabolism is required for cell proliferation. However, the way in which metabolic signals are conveyed to cell cycle decisions is unclear. Cyclic ADP-ribose (cADPR), the NAD + metabolite, mobilizes calcium from calcium stores in many cells. We found that dinoflagellate cells with higher metabolic rate underwent multiple fission (MF), a division mode in which cells can exceed twice their sizes at G1. A temperature shift-down experiment suggested that MF involves a commitment point at late G1. In fast-growing cells, cADPR level peaked in G 1 and increased with increasing concentrations of glucose in the medium. Addition of glycolytic poison iodoacetate inhibited cell growth, reduced cADPR levels as well as the commitment of cell cycles in fast-growing cells. Commitment of MF cell cycles was induced by a cell permeant cADPR agonist, but blocked by a specific antagonist of cADPR-induced Ca 2+ release. Our results establish cADPR as a link between cellular metabolism and cell cycle control.

Endophilin B1/Bif-1 Stimulates BAX Activation Independently from Its Capacity to Produce Large Scale Membrane Morphological Rearrangements

Endophilin B1/BAX-interacting factor 1 (Bif-1) is a protein that cooperates with dynamin-like protein 1 (DLP1/Drp1) to maintain normal mitochondrial outer membrane (MOM) dynamics in healthy cells and also contributes to BAX-driven MOM permeabilization (MOMP), the irreversible commitment point to cell death for the majority of apoptotic stimuli. However, despite its importance, exactly how Bif-1 fulfils its proapoptotic role is unknown. Here, we demonstrate that the stimulatory effect of Bif-1 on BAX-driven MOMP and on BAX conformational activation observed in intact cells during apoptosis can be recapitulated in a simplified system consisting of purified proteins and MOM-like liposomes. In this reconstituted model system the N-BAR domain of Bif-1 reproduced the stimulatory effect of Bif-1 on functional BAX activation. This process was dependent on physical interaction between Bif-1 N-BAR and BAX as well as on the presence of the mitochondrion-specific lipid cardiolipin. Despite that Bif-1 N-BAR produced large scale morphological rearrangements in MOM-like liposomes, this phenomenon could be separated from functional BAX activation. Furthermore, DLP1 also caused global morphological changes in MOM-like liposomes, but DLP1 did not stimulate BAX-permeabilizing function in the absence or presence of Bif-1. Taken together, our findings not only provide direct evidence for a functional interplay between Bif-1, BAX, and cardiolipin during MOMP but also add significantly to the growing body of evidence indicating that components of the mitochondrial morphogenesis machinery possess proapoptotic functions that are independent from their recognized roles in normal mitochondrial dynamics.

PRO: Anesthesia-Induced Developmental Neuroapoptosis: Status of the Evidence

PRO: Anesthesia-Induced Developmental Neuroapoptosis: Status of the Evidence

Candida albicans Tup1 Is Involved in Farnesol-Mediated Inhibition of Filamentous-Growth Induction

Candida albicans is a dimorphic fungus that can interconvert between yeast and filamentous forms. Its ability to regulate morphogenesis is strongly correlated with virulence. Tup1, a transcriptional repressor, and the signaling molecule farnesol are both capable of negatively regulating the yeast to filamentous conversion. Based on this overlap in function, we tested the hypothesis that the cellular response to farnesol involves, in part, the activation of Tup1. Tup1 functions with the DNA binding proteins Nrg1 and Rfg1 as a transcription regulator to repress the expression of hypha-specific genes. The tup1/tup1 and nrg1/nrg1 mutants, but not the rfg1/rfg1 mutant, failed to respond to farnesol. Treatment of C. albicans cells with farnesol caused a small but consistent increase in both TUP1 mRNA and protein levels. Importantly, this increase corresponds with the commitment point, beyond which added farnesol no longer blocks germ tube formation, and it correlates with a strong decrease in the expression of two Tup1-regulated hypha-specific genes, HWP1 and RBT1. Tup1 probably plays a direct role in the response to farnesol because farnesol suppresses the haploinsufficient phenotype of a TUP1/tup1 heterozygote. Farnesol did not affect EFG1 (a transcription regulator of filament development), NRG1, or RFG1 mRNA levels, demonstrating specific gene regulation in response to farnesol. Furthermore, the tup1/tup1 and nrg1/nrg1 mutants produced 17- and 19-fold more farnesol, respectively, than the parental strain. These levels of excess farnesol are sufficient to block filamentation in a wild-type strain. Our data are consistent with the role of Tup1 as a crucial component of the response to farnesol in C. albicans.

TGFβ-mediated apoptosis of Burkitt's lymphoma BL41 cells is associated with the relocation of mitochondrial BimEL

In this study, we showed that the transforming growth factor beta (TGFbeta)-mediated apoptosis of Burkitt's lymphoma BL41 cells is dependent on the BH3-only protein Bim. In contrast to what has been observed with other cell types, TGFbeta activation did not promote Bim upregulation in BL41 cells, but instead resulted in Bim release from the mitochondria. Indeed, Bim levels were high in healthy BL41 cells, in which they dimerized with the Bcl-2-like protein Mcl-1 at the mitochondrial surface. In healthy and TGFbeta-activated BL41 cells, unlike in epithelial cells or hepatocytes, Bim did not associate with Bcl-2 or Bcl-xL. TGFbeta activation of BL41 cells triggered the p38-dependent activation of caspase-8, causing the cleavage of Mcl-1 and the transfer of Bim from the mitochondria to the cytoskeleton. In addition to mitochondrial activation, this relocation of Bim may facilitate the complete demise of a cell death that is beyond the commitment point to apoptosis and may represent a hallmark of the TGFbeta-mediated apoptosis of human lymphoma B cells.

Systems Biology Analysis of Human Primary T Cells Identifies SAP145 as Rate Limiting for the G1→S Phase Transition.

We identified a G0→G1 commitment point in primary human T cells that controls entry into the cell cycle from quiescence. We demonstrated proof of principle that cellular pathways regulating cell cycle progression and effector functions that normally coincide during CD3/CD28 stimulation can be uncoupled experimentally. We have now used systems biology approaches to identify nuclear protein networks in primary human T cells that are regulated during the transition from quiescence into the cell cycle (G0→G1→S-phase). First we sequenced proteins that became bound to chromatin & nuclear matrix in G1 but were not bound in G0 and vice versa by mass spectrometry. Bioinformatic analysis identified 76 proteins specifically bound in G0 not G1 and 254 bound in G1 not G0. 179 of the 254 proteins bound in G1 not G0 (i.e. dynamic protein changes) were mapped to the 55,000 human protein interaction dataset. These are involved in numerous cellular functions, including epigenetics, transcription, RNA splicing & transport, and others. Cell cycle regulated chromatin/matrix binding of a subset was verified by western blotting (2/2 bound in G0 not G1 and 22/23 bound in G1 not G0). One of the proteins induced and bound in G1 was SAP145 (SF3B2). This is a component of the ubiquitous SF3b RNA splicing complex, involved in both major (U2-type) and minor (U12-type) spliceosomes. Since SAP145 is induced during G1 we investigated whether there was a role for SAP145 in regulating cell cycle progression. T cells depleted of SAP145 by siRNA enter G1 from G0 but progress poorly through S phase and die, probably by apoptosis. The same occurs if another component of the SF3B complex, SAP49 (SF3B4) is depleted with siRNA, indicating that the effect is due to depleting the complex rather than the individual SF3B protein. Proteins that are induced during G1 by CD3/CD28 stimulation e.g. cyclin D3, Cdc6 and cdc2 are produced normally when SAP145 is depleted, suggesting that their pre-mRNAs are spliced normally. In contrast, the expression of p107 and cyclin A2 are reduced markedly when SAP145 is depleted. Therefore, a systems biology approach to analysing cell cycle transitions identifies the splicing protein, SAP145 as rate-limiting for the G1 →S phase cell cycle transition but not for the transition from G0→G1.

Reducing MCM Loading Causes Chromosomal Aneuploidy.

Normal DNA replication must be accurate and occur only once per cell cycle. Sites of DNA replication are specified by binding the origin recognition complex, that includes minichromosome maintenance (MCM) proteins. Paradoxically, in higher eukaryotes MCM proteins are present in >20 fold excess of that required for DNA replication. They are also downregulated by elevated expression of proteins such as cyclin E that occurs in cancers, including AML and breast cancer. We investigated why human cells need “excess” MCM proteins and whether the reduction of MCM protein levels might contribute to a malignant phenotype. We determined the consequences of reducing the levels of MCM proteins in primary human T cells in which cell cycle controls and DNA damage responses are normal. Mass spectrometry sequencing of chromatin/nuclear matrix-bound proteins and western blotting identified that Mcm7 is not present in quiescent, normal primary human T cells. Mcm7 is induced in mid G1after the G0→G1 commitment point, the point beyond which T cells are committed to entering the cell cycle. Reduction of Mcm7 with siRNA to <5% of normal during G0→G1→S-phase reduces chromatin-binding of each of the MCM proteins that form the DNA helicase. However, these cells still enter S-phase and replicate DNA. Reducing MCM levels by titrating siRNA causes dose-dependent DNA-damage responses involving activation of ATR & ATM and Chk1 & Chk2. However, cells depleted of Mcm7 do not undergo apoptosis, rather reducing MCM levels even by 50% causes gross non-clonal chromosomal abnormalities normally found in genomic instability syndromes. M-FISH identified chromosome translocations, as well as loss and gain of individual chromosomes, which can occur individually or together in the same cell. Reducing MCM levels also causes misrepair by non-homologous end joining (NHEJ), and both NHEJ and homologous recombination (HR) are necessary for chromosomal abnormalities to occur. Therefore, “excess” MCM proteins that are present in a normal, proliferating cell are necessary for maintaining genome stability and reduction of MCM loading onto DNA that occurs in cancers is sufficient to cause genomic instability.

The ΔN Isoform of the p73 Gene Is Induced during the G0→G1 Transition in Primary Human T Cells.

p73 is a member of the p53-family of transcription factors (p73, p53 and p63). A truncated form of p73 (ΔNp73) also exists that lacks the transcriptional activation domain and inhibits both p53 & p73. However, ΔNp73 transcripts have not been reported in normal tissues or peripheral blood. We now show for the first time that ΔNp73 mRNA and protein are not present in quiescent (G0), primary human T cells but are induced post the G0→G1 cell cycle commitment point following stimulation with anti-CD3/CD28 or PMA/ionomycin. The ΔNp73 transcript could be produced either by activation of a promoter in intron 3 or from the 5′ P1 promoter by alternative splicing. Bisulfite sequencing shows that the intron 3 promoter is hypermethylated in T cells in G0 and throughout the cell cycle while the 5′ P1 promoter remains largely unmethylated. 5′-RACE results with quiescent and stimulated T cells verify that ΔNp73 transcripts originate from the P1 promoter. Additionally, RT-PCR analyses show that the transcript originating from the P1 promoter is present in G0 and mature ΔNp73 mRNA is produced by cell cycle-dependent splicing during the G0→G1 transition. We investigated the function of ΔNp73 during G0→G1 by inhibiting its induction with two different siRNAs. Microarray analyses show that expression of mRNA encoding a number of cell cycle regulators like E2F1, TCFL2 and CDT1 during G0→G1 are dependent on ΔNp73 and ΔNp73 represses the expression of the pro-apoptotic PUMA, a known p73 target. We conclude that ΔNp73 is produced in normal, human T cells during cell cycle entry and regulates normal gene expression. Moreover, the intron 3 promoter is hypermethylated, the ΔNp73 transcript originates at the P1 promoter and ΔNp73 mRNA is produced by cell cycle-dependent splicing.

GAPDH and Autophagy Preserve Survival after Apoptotic Cytochrome c Release in the Absence of Caspase Activation

In cells undergoing apoptosis, mitochondrial outer-membrane permeabilization (MOMP) is followed by caspase activation promoted by released cytochrome c. Although caspases mediate the apoptotic phenotype, caspase inhibition is generally not sufficient for survival following MOMP; instead cells undergo a "caspase-independent cell death" (CICD). Thus, MOMP may represent a point of commitment to cell death. Here, we identify glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a critical regulator of CICD. GAPDH-expressing cells preserved their clonogenic potential following MOMP, provided that caspase activation was blocked. GAPDH-mediated protection of cells from CICD involved an elevation in glycolysis and a nuclear function that correlated with and was replaced by an increase in Atg12 expression. Consistent with this, protection from CICD reflected an increase in and a dependence upon autophagy, associated with a transient decrease in mitochondrial mass. Therefore, GAPDH mediates an elevation in glycolysis and enhanced autophagy that cooperate to protect cells from CICD.

The cell cycle ofChlamydomonas reinhardtii: the role of the commitment point

Chlamydomonas reinhardtii cells can double their size several times during the light period before they enter the division phase. To explain the role of the commitment point (defined as the moment in the cell cycle after which cells can complete the cell cycle independently of light) and the moment of initiation of cell division we investigated whether the timing of commitment to cell division and cell division itself are dependent upon cell size or if they are under control of a timer mechanism that measures a period of constant duration. The time point at which cells attain commitment to cell division was dependent on the growth rate and coincided with the moment at which cells have approximately doubled in size. The timing of cell division was temperature-dependent and took place after a period of constant duration from the onset of the light period, irrespective of the light intensity and timing of the commitment point. We concluded that at the commitment point all the prerequisites are checked, which is required for progression through the cell cycle; the commitment point is not the moment at which cell division is initiated but it functions as a checkpoint, which ensures that cells have passed the minimum cell size required for the cell division.

Mitochondriotoxic compounds for cancer therapy

One of the hallmarks of cancer cells is their increased resistance to apoptosis induction. Alterations in many apoptosis regulators belonging to the intrinsic pathway confer emerging neoplastic cells with a selective growth advantage in the hostile tumor microenvironment. The realization that those same defects contribute to resistance to radiation and chemotherapeutic agents have prompted the unrelenting search for mitochondria-targeted compounds for the treatment of cancer. Mitochondria play a central role in the process of cell death. They serve as integrators of upstream effector mechanisms. Most importantly, mitochondrial outer membrane permeabilization becomes a commitment point during cell death. Thus, strategies aimed at directly triggering this event by either blocking the activity of antiapoptotic factors or by interfering with vital mitochondrial functions may help to overcome resistance to standard cancer therapy.

Blue- and red-light regulation of the cell cycle in Chlamydomonas reinhardtii (Chlorophyta)

When cultures of the unicellular green alga Chlamydomonas reinhardtii were subjected to blue light, cells continued growing for a longer period and attained larger sizes than under red light. This resulted in more division rounds per cell cycle. In blue light, the commitment point for cell division (after which cells can complete the cell cycle independent of light) was shifted later and consequently coincided with a larger cell size, which allowed for two division rounds. We found that exposure to blue light, when cells had approximately doubled in size and until the division phase, resulted in delayed cell division. Transfer into red light during this period triggered cell division. Furthermore, for cells that had doubled in size but whose growth was stopped by addition of the photosynthesis inhibitor DCMU, red-light illumination counteracted the inhibitory effect of blue light on the initiation of cell division, whereas transfer into darkness was ineffective. We confirm that the commitment point functions as a check-point, coordinating the cell cycle using internal and external factors, including cell size. In addition, we conclude that light quality is one of these factors. Spectral light conditions (blue and red light) affect the time at which cells attain the commitment point, and consequently the timing of cell division.

High growth rate and substrate exhaustion results in rapid cell death and lysis in the thermophilic bacterium Geobacillus thermoleovorans

Batch cultures of the thermophilic bacterium Geobacillus thermoleovorans T80 attained extremely high-specific glucose utilization rates leading to high specific growth rates, followed by extensive cell death and lysis with the onset of substrate exhaustion. The dramatic decrease in live cell numbers, as determined by flow cytometry, was accompanied by the release of soluble protein. Once the growth phase reached the point of commitment to lysis created by the impending exhaustion of substrate, the addition of extra carbon substrate did not halt the rapid death rate and lysis, although, towards the end of the exponential growth phase, the substrate was utilized producing only a small additional biomass concentration as a result of the net effect of cell growth and death. This lytic phenomenon was observed when a range of different carbon substrates (glucose, pyruvate, acetate, n-hexadecane, nutrient broth), as well as ammonium (the nitrogen source) in the presence of excess carbon source, reached near exhaustion. The rate and extent of cell death and the ensuing lysis depend on the culture growth rate. Cultures batch grown with a lower initial substrate concentration, or at a lower temperature, or at lower dilution rates for continuous-flow cultures, exhibited a lower rate and extent of cell death and lysis. Batch re-culture of the persister cells resulted in a behavior identical to that of the original culture indicating that these cells were not genetically modified. The glucose utilization, cell growth and death rates were mathematically described based on Monod kinetics and estimated values of pertinent biokinetic constants are reported.

Lipid Biosynthesis and its Coordination with Cell Cycle Progression

The activation of cell cycle regulators at the G1/S boundary has been linked to the cellular protein synthesis rate. It is conceivable that regulatory mechanisms are required to allow cells to coordinate the synthesis of other macromolecules with cell cycle progression. The availability of highly synchronized cells and flow cytometric methods facilitates investigation of the dynamics of lipid synthesis in the entire cell cycle of the heterotrophic dinoflagellate Crypthecodinium cohnii. Flow cytograms of Nile red-stained cells revealed a stepwise increase in the polar lipid content and a continuous increase in neutral lipid content in the dinoflagellate cell cycle. A cell cycle delay at early G1, but not G2/M, was observed upon inhibition of lipid synthesis. However, lipid synthesis continued during cell cycle arrest at the G1/S transition. A cell cycle delay was not observed when inhibitors of cellulose synthesis and fatty acid synthesis were added after the late G1 phase of the cell cycle. This implicates a commitment point that monitors the synthesis of fatty acids at the late G1 phase of the dinoflagellate cell cycle. Reduction of the glucose concentration in the medium down-regulated the G1 cell size with a concomitant forward shift of the commitment point. Inhibition of lipid synthesis up-regulated cellulose synthesis and resulted in an increase in cellulosic contents, while an inhibition of cellulose synthesis had no effects on lipid synthesis. Fatty acid synthesis and cellulose synthesis are apparently coupled to the cell cycle via independent pathways.

CaM-kinaseII-dependent commitment to microcystin-induced apoptosis is coupled to cell budding, but not to shrinkage or chromatin hypercondensation

The protein phosphatase inhibitor microcystin-LR (MC) induced hepatocyte apoptosis mediated by the calcium-calmodulin-dependent multifunctional protein kinase II (CaMKII). CaMKII antagonists were added at various times after MC to define for how long the cells depended on CaMKII activity to be committed to execute the various parameters of death. Shrinkage and nonpolarized budding were reversible and not coupled to commitment. A critical commitment step was observed 15-20 min after MC (0.5 microM) addition. After this, CaMKII inhibitors no longer protected against polarized budding, DNA fragmentation, lost protein synthesis capability, and cell disruption. Commitment to chromatin hypercondensation occurred 40 min after MC addition. In conclusion, irreversible death commitment was coupled to polarized budding, but not to shrinkage or chromatin condensation. Antioxidant prevented chromatin condensation when given after the CaMKII-dependent commitment point, suggesting that CaMKII had mediated the accumulation of a second messenger of reactive oxygen species nature.

Bcl-x Is Indispensable for the Development of Natural Killer Cells.

The size of the Natural Killer (NK) cell pool is maintained through production and subsequently export from the bone marrow, peripheral survival and proliferation, and ultimately cell death. While there exists considerable knowledge about developmental stages of lymphoid, myeloid and erythroid cells, comparably little is known about NK cell intermediates and the genes required for their development. Most of the models of NK cell differentiation have been based on in vitro culture systems where NK cells could be generated from multipotent HSC precursors. However, this approach suffers from the problems inherent to in vitro cell manipulations. We have utilized conditional gene targeting in adult mice to examine the role of the bcl-x gene in the development of NK cells in bone marrow and after their export to the spleen. Bcl-x is an important member of the anti-apoptotic member of the Bcl-2 gene family, and a critical role for this gene in the survival of hematopoietic cells was demonstrated in bcl-x-deficient mice causing embryonic death due to massive apoptosis of immature hematopoietic cells and of neurons. Conditional deletion in erythroid cells lead to hemolytic anemia and extensive splenomegaly. Furthermore, bcl-x is critical for the maturation of pre-B cells to the pro B cell stage, while it is not essential for the development of effector and memory T cells. We have conditionally deleted the gene in the stem cells of adult mice by cross breeding them with the Mxi-cre deleter strain, which allows for induced expression of cre recombinase by injection with pIpC.As early as 9 days after the first injection of pIpC, the number of NK cells in the bone marrow of mice started to decline as demonstrated by multi-color FACS analysis staining for IL2 Receptor beta (CD122) and NKG2D, among other markers. Cultures of bone marrow and spleen cells in the presence of cytokines to generate lymphokine activated killer cells failed, while no such effect was observed in cultures from Mxi-cre mice that were subjected to pIpC injections and carried along as controls. Analysis of animals after 3 weeks of pIpC administration revealed absence of NK cell precursors in the bone marrow as demonstrated by the lack of CD122+/Lin- negative cells. This phenomenon was accompanied by a reduction in the number of mature NK cells in the spleen.To date, six stages of NK cell maturation are described with the acquisition of IL2 receptor beta expression marking commitment to the NK-cell lineage. IL2 Receptor beta as well as NKG2D are expressed throughout NK cell development and at all stages. In order to characterize the specific stage at which expression of bcl-x is essential for NK cell maturation, we employed multi-color FACS analysis staining for CD122, NKG2D, CD49b, and the integrins CD43 and Mac-1 after depletion of lineage positive cells, followed by sorting for defined populations. Real Time PCR on sorted cells demonstrated that Bcl-x mRNA is highly expressed throughout all stages of NK cell development. Taken together, the gradual reduction in the number of NK cell precursors eventually leading to complete loss of this lineage in the bone marrow and peripheral sites suggests that bcl-x is indispensable for the development of NK cells presumably from the earliest time point of commitment to this lineage.