Progenitor Cell Cycle Research Articles

Cell cycle and lineage progression of neural progenitors in the ventricular-subventricular zones of adult mice

Proliferating neural stem cells and intermediate progenitors persist in the ventricular-subventricular zone (V-SVZ) of the adult mammalian brain. This extensive germinal layer in the walls of the lateral ventricles is the site of birth of different types of interneurons destined for the olfactory bulb. The cell cycle dynamics of stem cells (B1 cells), intermediate progenitors (C cells), and neuroblasts (A cells) in the V-SVZ and the number of times these cells divide remain unknown. Using whole mounts of the walls of the lateral ventricles of adult mice and three cell cycle analysis methods using thymidine analogs, we determined the proliferation dynamics of B1, C, and A cells in vivo. Achaete-scute complex homolog (Ascl)1(+) C cells were heterogeneous with a cell cycle length (T(C)) of 18-25 h and a long S phase length (T(S)) of 14-17 h. After C cells, Doublecortin(+) A cells were the second-most common dividing cell type in the V-SVZ and had a T(C) of 18 h and T(S) of 9 h. Human glial fibrillary acidic protein (hGFAP)::GFP(+) B1 cells had a surprisingly short Tc of 17-18 h and a T(S) of 4 h. Progenitor population analysis suggests that following the initial division of B1 cells, C cells divide three times and A cells once, possibly twice. These data provide essential information on the dynamics of adult progenitor cell proliferation in the V-SVZ and how large numbers of new neurons continue to be produced in the adult mammalian brain.

Mutations in the β-Tubulin Gene TUBB5 Cause Microcephaly with Structural Brain Abnormalities

SummaryThe formation of the mammalian cortex requires the generation, migration, and differentiation of neurons. The vital role that the microtubule cytoskeleton plays in these cellular processes is reflected by the discovery that mutations in various tubulin isotypes cause different neurodevelopmental diseases, including lissencephaly (TUBA1A), polymicrogyria (TUBA1A, TUBB2B, TUBB3), and an ocular motility disorder (TUBB3). Here, we show that Tubb5 is expressed in neurogenic progenitors in the mouse and that its depletion in vivo perturbs the cell cycle of progenitors and alters the position of migrating neurons. We report the occurrence of three microcephalic patients with structural brain abnormalities harboring de novo mutations in TUBB5 (M299V, V353I, and E401K). These mutant proteins, which affect the chaperone-dependent assembly of tubulin heterodimers in different ways, disrupt neurogenic division and/or migration in vivo. Our results provide insight into the functional repertoire of the tubulin gene family, specifically implicating TUBB5 in embryonic neurogenesis and microcephaly.

A Unilateral Negative Feedback Loop BetweenmiR-200microRNAs and Sox2/E2F3 Controls Neural Progenitor Cell-Cycle Exit and Differentiation

MicroRNAs have emerged as key posttranscriptional regulators of gene expression during vertebrate development. We show that the miR-200 family plays a crucial role for the proper generation and survival of ventral neuronal populations in the murine midbrain/hindbrain region, including midbrain dopaminergic neurons, by directly targeting the pluripotency factor Sox2 and the cell-cycle regulator E2F3 in neural stem/progenitor cells. The lack of a negative regulation of Sox2 and E2F3 by miR-200 in conditional Dicer1 mutants (En1(+/Cre); Dicer1(flox/flox) mice) and after miR-200 knockdown in vitro leads to a strongly reduced cell-cycle exit and neuronal differentiation of ventral midbrain/hindbrain (vMH) neural progenitors, whereas the opposite effect is seen after miR-200 overexpression in primary vMH cells. Expression of miR-200 is in turn directly regulated by Sox2 and E2F3, thereby establishing a unilateral negative feedback loop required for the cell-cycle exit and neuronal differentiation of neural stem/progenitor cells. Our findings suggest that the posttranscriptional regulation of Sox2 and E2F3 by miR-200 family members might be a general mechanism to control the transition from a pluripotent/multipotent stem/progenitor cell to a postmitotic and more differentiated cell.

Left column: Upper: ESC‐derived Purkinje cells with highly arborized dendrites (red and yellow) and ESC‐derived other GABAergic interneurons (green) in long‐term culture. See Muguruma and Sasai, pp 349‐357. Center: The CNS of the larvalascidian Ciona intestinalis visualized with Kaede green (top) and Kaede red (bottom) fl uorescence. The middle schematicaly illustrates the cell lineage of the CNS. See Sasakura et

Development, Growth & DifferentiationVolume 54, Issue 3 p. i-i Free Access Left column: Upper: ESC-derived Purkinje cells with highly arborized dendrites (red and yellow) and ESC-derived other GABAergic interneurons (green) in long-term culture. See Muguruma and Sasai, pp 349-357. Center: The CNS of the larvalascidian Ciona intestinalis visualized with Kaede green (top) and Kaede red (bottom) fl uorescence. The middle schematicaly illustrates the cell lineage of the CNS. See Sasakura et al., pp 420-437. Bottom: A cortical excitatory neuron extending an axon-like long process in the intermediate zone (magenta). Accumulation of Kif5C560-EGFP in the tip of this process suggests axonal nature of this process. See Hatanaka et al., pp 398-407. Right column: Upper: Most RAX (white) and OTX2 (green) double-expressing cells express only low levels of HES1 (red). In the last retinal progenitor cell cycle, the NOTCH-HES signal pathway becomes inactive, and RAX becomes active to activate Otx2 transcription. See Muranishi et al., pp 341-348. Second: Scanning electron micrograph of a Xenopus laevis embryo in neural tube closure. See Suzuki et al., pp 266-276. Third: Aberrant Venus::UNC-6 localization in muscle cells in unc-18 (e234) mutant C. elegans UNC-6 secretion may require an unknown UNC-18-mediated signal from neurons. See Ogura et al., pp 390-397. First published: 24 April 2012 https://doi.org/10.1111/j.1440-169X.2011.01353.xAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article. Volume54, Issue3Special Issue: Neural Development Edited by T. Miyata.April 2012Pages i-i RelatedInformation

Interfollicular epidermal homeostasis: dicing with differentiation

In the 1970s, studies of tissue architecture and cell proliferation were used to formulate a new model of epidermal homeostasis. This asserted that the tissue was maintained by long-lived, slow-cycling, self-renewing stem cells that generate a short-lived population of transit amplifying (TA) cells, which undergo terminal differentiation after a set number of cell divisions. It was further hypothesized that in the epidermis, the tissue was organized into clonal epidermal proliferative units (EPUs) comprising a central stem cell with surrounding TA cells, which maintain the overlying differentiated cell layers. The stem/TA and EPU hypotheses have been widely influential. Here, we first revaluate older literature, finding numerous studies that conflict with the EPU model. We then review recent large-scale lineage tracing studies in transgenic mice which exclude the stem/TA and EPU hypotheses, and reveal that the epidermis is maintained by a single population of functionally equivalent cycling progenitor cells. The outcome of individual progenitor cell divisions is random, but the probabilities of generating differentiated and progenitor cell daughters are equal, so that homeostasis is maintained across the progenitor population. We reconcile this model with the older literature and place the epidermis in the context of other tissues that are also maintained by continually cycling cells with stochastic fate.

Tip110 Maintains Expression of Pluripotent Factors in and Pluripotency of Human Embryonic Stem Cells

Abstract 2353Embryonic stem cells (ESCs) are pluripotent, self-renew and can be differentiated into cells of all three germ layers, and nanog, Oct4 and Sox2, form a core of the self-renewal transcription network. Nanog expression is restricted to pluripotent cells and is down regulated upon differentiation; little is known about its regulation. Expression of the OCT4 gene maintains cell pluripotency via a stringent dose-dependent regulation with OCT4 levels above or below required dosages producing cellular differentiation; thus maintenance of a critical amount of OCT4 is necessary to prevent ESC differentiation. Sox2, a high-mobility group domain containing transcription factor, binds to the consensus motif CATTGTT. We recently reported in Blood that Tip110 is an essential gene expressed in earliest cells of adult bone marrow hematopoietic development. Increased TIP110 expression enhanced hematopoietic progenitor cell (HPC) numbers, survival, and cell cycling; decreased Tip110 expression manifested the opposite effect, demonstrating a role for TIP110 in regulation of hematopoiesis. Herein, we investigated TIP110 expression and actions in human (h)ESCs. Quantitative RT-PCR showed that TIP110, as well as Nanog, Oct4 and Sox2 were expressed in a hESC line. hESCs were removed from feeder layers and b-FGF for 5 days, to allow ESC differentiation. TIP110 expression levels were dramatically reduced (by 77%); this was associated with large decreases in expression of NANOG (82%), OCT4 (80%), and SOX2 (85%). We then assessed whether TIP110 might regulate hESC pluripotency. We exogenously over-expressed TIP110 in hESC cells. Feeder layers and b-FGF were withdrawn upon introducing the TIP110 vector and cells cultured for 5 days to test whether sustained TIP110 expression rendered ESCs less sensitive to differentiation. Compared with controls, TIP110 over-expressing cells stained positive for OCT4, NANOG and were negative for Tuji, SMA and AFP, demonstrating that over-expression of TIP110 rendered ESCs less responsive to differentiation. Next, we reduced TIP110 expression by transfection of the hESCs with TIP110 siRNA. Cells were cultured in mTeSR medium on Matrigel-coated dishes for an additional 5 days in order to maintain cells under undifferentiation conditions. TIP 110 siRNA vector expressing cells were negative for OCT4, NANOG, and positive for Tuji, SMA and AFP expression compared with control cells, demonstrating that enforced reduction of TIP110 expression in hESCs causes hESC differentiation. This demonstrated the importance of TIP110 in maintenance of ESC pluripotency. We speculated that TIP110 maintenance of hESC pluripotency might be through regulation of NANOG, OCT4 and SOX2. We silenced TIP110 expression in hESCs by transfection with a TIP110 siRNA vector, previously shown to reduce TIP110 expression by 70%. Cells were cultured in complete 20% KSR hESC medium for an additional 5 days. Expression of these three transcription factors was dramatically decreased, demonstrating that TIP110 is required for maintaining NANOG, OCT4 and SOX2 levels in this hESC line. Reduction of TIP110 expression caused hESC differentiation directly or indirectly through down-regulation of NANOG, OCT4 and SOX2 expression. Thus, TIP110 is preferentially expressed in the undifferentiated state in hESCs and plays a key role in regulating OCT4, SOX2, and NANOG, factors required to maintain pluripotency. Together, our present and previous studies suggest TIP110 expression as a useful marker to distinguish early from more differentiated cells. Modulating TIP110 expression in a controlled fashion may be relevant for cellular engineering and regenerative medicine. Disclosures:No relevant conflicts of interest to declare.

Oxygen-Independent Stabilization of the Oxygen-Labile Transcription Factor HIF-1α with Dimethyloxalyl Glycine or FG-4497 Increases Hematopoietic Stem Cell Quiescence In Vivo and Mobilization in Response to G-CSF

Abstract 2334The endosteal region of the bone marrow (BM) is hypoxic in steady-state and most quiescent hematopoietic stem cells (HSC) reside in hypoxic, poorly perfused niches. Mobilizing doses of G-CSF renders most of the BM space hypoxic. Most cellular effects of hypoxia are mediated by O2-labile hypoxia-inducible transcription factors (HIF). At O2 concentration above 2%, HIF-α is rapidly hydroxylated on Pro residues by the prolyl hydroxylases PHD1, PHD2, and PHD3. HIF-α prolyl hydroxylation recruits the E3 ubiquitin ligase VHL, which targets HIF-α to the proteasome. In hypoxia (O2 < 2%), HIF-α proteins are stable, associate with the β subunit ARNT, and translocate to the nucleus to activate transcription. It has emerged that HIF-1α regulates HSC proliferation and is critical to maintain long-term HSC self-renewal in vivo.In this study, we investigated the effect of pharmacological stabilization of HIF-1α protein on HSC cycling and mobilization in mice using two different HIF prolyl hydroxylase (PHD) inhibitors, dimethyloxalyl glycine (DMOG) and FG-4497. We first assessed whether DMOG and FG-4497 stabilized HIF-1α protein in BM leukocytes in vivo by western-blot. Following a single injection of 400mg/kg DMOG, HIF-1α protein was stabilized for up to 6 hrs in BM leukocytes. With 20mg/kg FG-4497, HIF-1α protein persisted over 12 hours. HIF-1α protein was below detection in the BM from saline injected animals.C57BL/6 mice were injected daily with 400mg/kg DMOG to measure effect on HSC cycling and BrdU incorporation by flow cytometry. Using Hoecht33342 and FITC-conjugated anti-Ki67 antibody, a 18 day DMOG treatment increased the proportion of Lin-negative Kit+ Sca1+ CD48- (L-K+S+48-) HSC in phase G0 from 61±11% to 84±6% (p<0.001, 5 mice/group). Conversely, DMOG decreased the proportion of HSC in phase G1 from 31±5% to 14±5% (p<0.001). Similar enhancement of quiescence was observed in less primitive cells such as L-K+S+48+ cells and L-K+S- myeloid progenitors. In mice given BrdU in their drinking water for the last 3 days of the experiment, a 18 day DMOG treatment reduced 3-fold the proportion of L-K+S+48- HSC that incorporated BrdU and halved the proportion of BrdU+ L-K+S+48+ progenitors. Shorter DMOG treatments (6 days or 12 days) did not alter hematopoietic stem and progenitor cell (HSPC) cycling or BrdU incorporation. In contrast, mice treated with 20mg/kg/day FG-4497 had significantly increased proportion of HSPC in G0 phase after only 6 days of treatment, likely due to the more lasting effect of FG-4497 on HIF-1α stabilization. Proportion of HSPC that incorporated BrdU was also significantly decreased. As HIF-PHD inhibitors also increase erythropoietin (EPO) expression by stabilizing HIF in the kidney, we injected a parallel cohort of mice with EPO daily for 18 days. Despite a very strong increase in red cells and hemoglobin in the blood, EPO had no effect of HSPC cycling and BrdU incorporation. Therefore the effects of HIF-PHD inhibitors on HSPC cycling are not an indirect effect of increased EPO.Since HIF-PHD inhibitors slow HSC cycling in vivo, we tested whether they could protect HSC from sublethal irradiation. Mice were treated with 400mg/kg DMOG or saline for 22 days and then irradiated with 9.0Gy. Both cohorts were leukopenic betweens days 7 and 14 post-irradiation but DMOG treated mice had significantly higher blood leukocytes at days 22 and 30, and higher platelet numbers day 22 and all subsequent time-points suggesting that DMOG enhances HSC survival with higher blood recovery.Finally mice were injected with DMOG for 4 days and pegylated rhuG-CSF 3 and 1.5 days before harvest. DMOG doubled mobilization in response to G-CSF with 1,620±530 CFC / mL blood in response G-CSF alone, and 3,250±830 CFC / mL in the G-CSF + DMOG group (p<0.05). Without G-CSF, circulating CFC were less than 10 / mL blood. Similarly, FG-4497 pre-treatment increased the number of CFC mobilized into the spleen 4-fold compared to G-CSF alone (p<0.01).In conclusion, these data highlight the importance of the hypoxia pathway and HIF in the regulation of HPSC cycling and trafficking in vivo. Furthermore HIF-PHD inhibitors may provide therapeutic opportunities to protect HSC from deleterious effects of irradiation as well as increasing mobilization efficiency for transplantation. Disclosures:Walkinshaw:Fibrogen Inc.: Employment, Equity Ownership.

Cycling Toward Leukemia Stem Cell Elimination Wtih a Selective Sonic Hedgehog Antagonist,

Abstract 3776Cumulative evidence suggests that dormant self-renewing leukemia stem cells (LSC) contribute to relapse and blast crisis transformation by evading therapies that target cycling cells. Previously, sonic hedgehog (Shh) signaling was shown to modulate cell cycle regulation and self-renewal in normal mouse hematopoietic stem cells. However, its role in human LSC regeneration and quiescence had not been elucidated. Here we investigated the role of Shh signaling in maintenance of dormancy. We show that, compared to chronic phase CML and normal progenitors, human blast crisis LSC harbor enhanced expression of the Shh transcriptional activator, GLI2, and decreased expression of a transcriptional repressor, GLI3. Treatment of human blast crisis LSC engrafted RAG2−/−gc−/− mice with a selective Shh inhibitor, PF-04449913, reduced leukemic burden in a niche-dependent manner commensurate with GLI downregulation. Full transcriptome RNA sequencing performed on FACS-purified human progenitors from PF-04449913 treated blast crisis LSC engrafted mice demonstrated greater Shh gene splice isoform concordance with normal progenitors than vehicle treated controls. In addition, RNA sequencing revealed significantly decreased cell cycle regulatory genes expression and splice isoform analysis demonstrated reversion towards a normal splice isoform signature for many cell cycle regulatory genes. Moreover, cell cycle FACS analysis showed that selective Shh inhibition permitted dormant blast crisis LSC to enter the cell cycle while normal progenitor cell cycle status was unaffected. Finally, PF-04449913 synergized with BCR-ABL inhibition to reduce blast crisis LSC survival and self-renewal in concert with increased expression of Shh pathway regulators. Our findings suggest that selective Shh antagonism induces cycling of dormant human blast crisis LSC, rendering them susceptible to BCR-ABL inhibition, while sparing normal progenitors. Implementation of novel LSC splice isoform detection platforms to assess efficacy of Shh inhibitor-mediated sensitization to molecularly targeted therapy may inform dormant cancer stem cell elimination strategies that ultimately avert relapse. Disclosures:Levin:Pfizer Oncology: Employment; Pfizer Oncology: Equity Ownership.

Retinoic acid signaling controls the formation, proliferation and survival of the blastema during adult zebrafish fin regeneration

Adult teleosts rebuild amputated fins through a proliferation-dependent process called epimorphic regeneration, in which a blastema of cycling progenitor cells replaces the lost fin tissue. The genetic networks that control formation of blastema cells from formerly quiescent stump tissue and subsequent blastema function are still poorly understood. Here, we investigated the cellular and molecular consequences of genetically interfering with retinoic acid (RA) signaling for the formation of the zebrafish blastema. We show that RA signaling is upregulated within the first few hours after fin amputation in the stump mesenchyme, where it controls Fgf, Wnt/β-catenin and Igf signaling. Genetic inhibition of the RA pathway at this stage blocks blastema formation by inhibiting cell cycle entry of stump cells and impairs the formation of the basal epidermal layer, a signaling center in the wound epidermis. In the established blastema, RA signaling remains active to ensure the survival of the highly proliferative blastemal population by controlling expression of the anti-apoptotic factor bcl2. In addition, RA signaling maintains blastema proliferation through the activation of growth-stimulatory signals mediated by Fgf and Wnt/β-catenin signaling, as well as by reducing signaling through the growth-inhibitory non-canonical Wnt pathway. The endogenous roles of RA in adult vertebrate appendage regeneration are uncovered here for the first time. They provide a mechanistic framework to understand previous observations in salamanders that link endogenous sources of RA to the regeneration process itself and support the hypothesis that the RA signaling pathway is an essential component of vertebrate tissue regeneration.

An Essential Role for RAX Homeoprotein and NOTCH–HES Signaling inOtx2Expression in Embryonic Retinal Photoreceptor Cell Fate Determination

The molecular mechanisms underlying cell fate determination from common progenitors in the vertebrate CNS remain elusive. We previously reported that the OTX2 homeoprotein regulates retinal photoreceptor cell fate determination. While Otx2 transactivation is a pivotal process for photoreceptor cell fate determination, its transactivation mechanism in the retina is unknown. Here, we identified an evolutionarily conserved Otx2 enhancer of ∼500 bp, named embryonic enhancer locus for photoreceptor Otx2 transcription (EELPOT), which can recapitulate initial Otx2 expression in the embryonic mouse retina. We found that the RAX homeoprotein interacts with EELPOT to transactivate Otx2, mainly in the final cell cycle of retinal progenitors. Conditional inactivation of Rax results in downregulation of Otx2 expression in vivo. We also showed that NOTCH-HES signaling negatively regulates EELPOT to suppress Otx2 expression. These results suggest that the integrated activity of cell-intrinsic and -extrinsic factors on EELPOT underlies the molecular basis of photoreceptor cell fate determination in the embryonic retina.

Histone deacetylase inhibition promotes Caspase-independent cell death of ventral midbrain neurons

Inhibition of histone deacetylase (HDAC) activity results in dedifferentiation of various neural precursor cell populations, but is also known to promote neuronal differentiation. We sought to determine the effects of HDAC inhibition on differentiated and non-differentiated midbrain cells in order to examine more closely the consequences of HDAC inhibition on cell fate in a heterogeneous population. We demonstrate that HDAC inhibitor (HDACi) treatment causes a significant attenuation in the numbers of neurons, but not astrocytes, within 48h, with no evidence of neuronal dedifferentiation. The loss of neurons is due to an initial morphological destabilization, which is not recoverable upon inhibitor removal, and ultimately leads to cell death. HDACi treatment results in progenitor cell cycle arrest and Caspase-dependent apoptosis. In contrast, the loss of midbrain neurons does not correlate with activated Caspase-3 expression. Treating cultures transiently with Caspase inhibitors blocks overall HDACi-induced cell death in the cultures, but does not prevent the loss of neurons. These data suggest that HDACi treated midbrain neurons undergo Caspase-independent cell death. Finally, we demonstrate that cortical neurons do not undergo cell death in response to HDACi treatment, suggesting that there may be tissue-specific or microenvironmental factors that promote the susceptibility of midbrain neurons to the neurotoxic effects of HDAC inhibition.

The lymphoma-associated NPM-ALK oncogene elicits a p16INK4a/pRb-dependent tumor-suppressive pathway

AbstractOncogene-induced senescence (OIS) is a barrier for tumor development. Oncogene-dependent DNA damage and activation of the ARF/p53 pathway play a central role in OIS and, accordingly, ARF and p53 are frequently mutated in human cancer. A number of leukemia/lymphoma-initiating oncogenes, however, inhibit ARF/p53 and only infrequently select for ARF or p53 mutations, suggesting the involvement of other tumor-suppressive pathways. We report that NPM-ALK, the initiating oncogene of anaplastic large cell lymphomas (ALCLs), induces DNA damage and irreversibly arrests the cell cycle of primary fibroblasts and hematopoietic progenitors. This effect is associated with inhibition of p53 and is caused by activation of the p16INK4a/pRb tumor-suppressive pathway. Analysis of NPM-ALK lymphomagenesis in transgenic mice showed p16INK4a-dependent accumulation of senescent cells in premalignant lesions and decreased tumor latency in the absence of p16INK4a. Accordingly, human ALCLs showed no expression of either p16INK4a or pRb. Up-regulation of the histone-demethylase Jmjd3 and de-methylation at the p16INK4a promoter contributed to the effect of NPM-ALK on p16INK4a, which was transcriptionally regulated. These data demonstrate that p16INK4a/pRb may function as an alternative pathway of oncogene-induced senescence, and suggest that the reactivation of p16INK4a expression might be a novel strategy to restore the senescence program in some tumors.

STAT3-dependent IL-21 production from T helper cells regulates hematopoietic progenitor cell homeostasis

AbstractThe contribution of specific cell types to the production of cytokines that regulate hematopoiesis is still not well defined. We have previously identified T cell–dependent regulation of hematopoietic progenitor cell (HPC) numbers and cycling. In this report, we demonstrated that HPC activity is decreased in mice with STAT3-deficient T cells, a phenotype that is not because of decreased expression of IL-17 or RORγt. STAT3 expression in T cells was required for IL-21 production by multiple T helper subsets, and neutralization of IL-21 resulted in decreased HPC activity identical to that in mice with STAT3-deficient T cells. Importantly, injection of IL-21 rescued HPC activity in mice with STAT3-deficient T cells. Thus, STAT3-dependent IL-21 production in T cells is required for HPC homeostasis.

Analysis of Thyroid Response Element Activity during Retinal Development

Thyroid hormone (TH) signaling components are expressed during retinal development in dynamic spatial and temporal patterns. To probe the competence of retinal cells to mount a transcriptional response to TH, reporters that included thyroid response elements (TREs) were introduced into developing retinal tissue. The TREs were placed upstream of a minimal TATA-box and two reporter genes, green fluorescent protein (GFP) and human placental alkaline phosphatase (PLAP). Six of the seven tested TREs were first tested in vitro where they were shown to drive TH-dependent expression. However, when introduced into the developing retina, the TREs reported in different cell types in both a TH-dependent and TH-independent manner, as well as revealed specific spatial patterns in their expression. The role of the known thyroid receptors (TR), TRα and TRβ, was probed using shRNAs, which were co-electroporated into the retina with the TREs. Some TREs were positively activated by TR+TH in the developing outer nuclear layer (ONL), where photoreceptors reside, as well as in the outer neuroblastic layer (ONBL) where cycling progenitor cells are located. Other TREs were actively repressed by TR+TH in cells of the ONBL. These data demonstrate that non-TRs can activate some TREs in a spatially regulated manner, whereas other TREs respond only to the known TRs, which also read out activity in a spatially regulated manner. The transcriptional response to even simple TREs provides a starting point for understanding the regulation of genes by TH, and highlights the complexity of transcriptional regulation within developing tissue.

Duplicate dmbx1genes regulate progenitor cell cycle and differentiation during zebrafish midbrain and retinal development

BackgroundThe Dmbx1 gene is important for the development of the midbrain and hindbrain, and mouse gene targeting experiments reveal that this gene is required for mediating postnatal and adult feeding behaviours. A single Dmbx1 gene exists in terrestrial vertebrate genomes, while teleost genomes have at least two paralogs. We compared the loss of function of the zebrafish dmbx1a and dmbx1b genes in order to gain insight into the molecular mechanism by which dmbx1 regulates neurogenesis, and to begin to understand why these duplicate genes have been retained in the zebrafish genome.ResultsUsing gene knockdown experiments we examined the function of the dmbx1 gene paralogs in zebrafish, dmbx1a and dmbx1b in regulating neurogenesis in the developing retina and midbrain. Dose-dependent loss of dmbx1a and dmbx1b function causes a significant reduction in growth of the midbrain and retina that is evident between 48-72 hpf. We show that this phenotype is not due to patterning defects or persistent cell death, but rather a deficit in progenitor cell cycle exit and differentiation. Analyses of the morphant retina or anterior hindbrain indicate that paralogous function is partially diverged since loss of dmbx1a is more severe than loss of dmbx1b. Molecular evolutionary analyses of the Dmbx1 genes suggest that while this gene family is conservative in its evolution, there was a dramatic change in selective constraint after the duplication event that gave rise to the dmbx1a and dmbx1b gene families in teleost fish, suggestive of positive selection. Interestingly, in contrast to zebrafish dmbx1a, over expression of the mouse Dmbx1 gene does not functionally compensate for the zebrafish dmbx1a knockdown phenotype, while over expression of the dmbx1b gene only partially compensates for the dmbx1a knockdown phenotype.ConclusionOur data suggest that both zebrafish dmbx1a and dmbx1b genes are retained in the fish genome due to their requirement during midbrain and retinal neurogenesis, although their function is partially diverged. At the cellular level, Dmbx1 regulates cell cycle exit and differentiation of progenitor cells. The unexpected observation of putative post-duplication positive selection of teleost Dmbx1 genes, especially dmbx1a, and the differences in functionality between the mouse and zebrafish genes suggests that the teleost Dmbx1 genes may have evolved a diverged function in the regulation of neurogenesis.

Human Satellite Progenitor Cells for Use in Myofascial Repair

Current use of prosthetic meshes and implants for myofascial reconstruction has been associated with infectious complications, long-term failure, and dissatisfying cosmetic results. Our laboratory has developed a small animal model for ventral hernia repair, which uses progenitor cells isolated from a skeletal muscle biopsy. In the model, progenitor cells are expanded in vitro, seeded onto a nonimmunogenic, novel aligned scaffold of bovine collagen and placed into the defect as a living adjuvant to the innate repair mechanism. The purpose of the current investigation is to examine the feasibility of translating our current model to humans. As a necessary first step we present our study on the efficacy of isolating satellite cells from 9 human donor biopsies. We were able to successfully translate our progenitor cell isolation and culture protocols to a human model with some modifications. Specifically, we have isolated human satellite muscle cells, expanded them in culture, and manipulated these cells to differentiate into myotubes in vitro. Immunohistochemical analysis allowed the characterization of distinct progenitor cell cycle stages and quantification of approximate cell number. Furthermore, isolated cells were tracked via cytoplasmic nanocrystal labeling and observed using confocal microscopy.

Auto-SCT induces a phenotypic shift from CMP to GMP progenitors, reduces clonogenic potential and enhances in vitro and in vivo cycling activity defined by 18F-FLT PET scanning

Autologous SCT (auto-SCT) introduces a reduced tolerance to chemotherapy even in patients with adequate engraftment, suggesting long-term effects of the transplantation procedure on the BM capacity. To study the hematopoietic cell compartment after auto-SCT, CD34(+) BM cells (n = 16) from patients at 6-9 months after auto-SCT were studied with regard to the progenitor subsets, colony frequency and cell cycle status. The BM compartments were studied in vivo using PET tracer 3-fluoro-3-deoxy-L-thymidine (¹⁸F-FLT PET). BM CD34(+) cells after auto-SCT were compared with normal CD34(+) cells and showed a phenotypic shift from common myeloid progenitor (CMP mean percentage 3.7 vs 19.4%, P=0.001) to granulocyte-macrophage progenitor (GMP mean percentage 51.8 vs 27.6%, P=0.01). In addition, a reduced clonogenic potential and higher cycling activity especially of the GMP fraction (41% ± 4 in G2/S phase vs 19% ± 2, P = 0.03) were observed in BM after auto-SCT compared with normal. The enhanced cycling activity was confirmed in vivo by showing a significantly higher uptake of the ¹⁸F-FLT PET tracer by the BM compartment. This study shows that auto-SCT results in defects of the hematopoietic compartment at least 6 months after auto-SCT, characterized by changes in the composition of progenitor subsets and enhanced in vitro and in vivo cycling activity.

Timing of captopril administration determines radiation protection or radiation sensitization in a murine model of total body irradiation

Angiotensin II (Ang II), a potent vasoconstrictor, affects the growth and development of hematopoietic cells. Mixed findings have been reported for the effects of angiotensin-converting enzyme (ACE) inhibitors on radiation-induced injury to the hematopoietic system. We investigated the consequences of different regimens of the ACE inhibitor captopril on radiation-induced hematopoietic injury. C57BL/6 mice were either sham-irradiated or exposed to (60)Co total body irradiation (0.6 Gy/min). Captopril was provided in the water for different time periods relative to irradiation. In untreated mice, the survival rate from 7.5 Gy was 50% at 30 days postirradiation. Captopril treatment for 7 days prior to irradiation resulted in radiosensitization with 100% lethality and a rapid decline in mature blood cells. In contrast, captopril treatment beginning 1 hour postirradiation and continuing for 30 days resulted in 100% survival, with improved recovery of mature blood cells and multilineage hematopoietic progenitors. In nonirradiated control mice, captopril biphasically modulated Lin(-) marrow progenitor cell cycling. After 2 days, captopril suppressed G(0)-G(1) transition and a greater number of cells entered a quiescent state. However, after 7 days of captopril treatment Lin(-) progenitor cell cycling increased compared to untreated control mice. These findings suggest that ACE inhibition affects hematopoietic recovery following radiation by modulating the hematopoietic progenitor cell cycle. The timing of captopril treatment relative to radiation exposure differentially affects the viability and repopulation capacity of spared hematopoietic stem cells and, therefore, can result in either radiation protection or radiation sensitization.

Alternative splicing produces high levels of noncoding isoforms of bHLH transcription factors during development

During development, multiple cell types within a tissue often arise from a common pool of progenitor cells (PCs). PCs typically expand in number, while simultaneously producing post-mitotic cells (PMCs). This balance is partly regulated by transcription factors that are expressed within PCs, such as the basic helix-loop-helix (bHLH) gene mouse atonal homolog 7 (Math5), which is expressed in retinal PCs. Here we report that alternative splicing (AS) of Math5 serves as another layer of regulation of Math5 activity. Specifically, Math5, a single exon gene, is alternatively spliced such that the major isoform lacks the entire coding sequence. Similarly, neurogenin 3 (Ngn3), a Math5 paralog expressed in pancreatic PCs, is also alternatively spliced such that the major isoform fails to code for Ngn3 protein. The consequence of reducing the abundance of protein-coding isoforms is likely crucial, as we found that introduction of coding isoforms leads to a reduction in cycling PCs. Thus, AS can limit the number of PCs expressing key regulatory proteins that control PC expansion versus PMC production.

Cardiac Progenitor Cell Cycling Stimulated by Pim-1 Kinase

Cardioprotective effects of Pim-1 kinase have been previously reported but the underlying mechanistic basis may involve a combination of cellular and molecular mechanisms that remain unresolved. The elucidation of the mechanistic basis for Pim-1 mediated cardioprotection provides important insights for designing therapeutic interventional strategies to treat heart disease. Effects of cardiac-specific Pim-1 kinase expression on the cardiac progenitor cell (CPC) population were examined to determine whether Pim-1 mediates beneficial effects through augmenting CPC activity. Transgenic mice created with cardiac-specific Pim-1 overexpression (Pim-wt) exhibit enhanced Pim-1 expression in both cardiomyocytes and CPCs, both of which show increased proliferative activity assessed using 5-bromodeoxyuridine (BrdU), Ki-67, and c-Myc relative to nontransgenic controls. However, the total number of CPCs was not increased in the Pim-wt hearts during normal postnatal growth or after infarction challenge. These results suggest that Pim-1 overexpression leads to asymmetric division resulting in maintenance of the CPC population. Localization and quantitation of cell fate determinants Numb and alpha-adaptin by confocal microscopy were used to assess frequency of asymmetric division in the CPC population. Polarization of Numb in mitotic phospho-histone positive cells demonstrates asymmetric division in 65% of the CPC population in hearts of Pim-wt mice versus 26% in nontransgenic hearts after infarction challenge. Similarly, Pim-wt hearts had fewer cells with uniform alpha-adaptin staining indicative of symmetrically dividing CPCs, with 36% of the CPCs versus 73% in nontransgenic sections. These findings define a mechanistic basis for enhanced myocardial regeneration in transgenic mice overexpressing Pim-1 kinase.