Identification of positionally distinct astrocyte subtypes whose identities are specified by a homeodomain code.

Astrocytes constitute the most abundant cell type in the central nervous system (CNS) and play diverse functional roles, but the ontogenetic origins of this phenotypic diversity are poorly understood. We have investigated whether positional identity, a fundamental organizing principle governing the generation of neuronal subtype diversity, is also relevant to astrocyte diversification. We identified three positionally distinct subtypes of white-matter astrocytes (WMA) in the spinal cord, which can be distinguished by the combinatorial expression of Reelin and Slit1. These astrocyte subtypes derive from progenitor domains expressing the homeodomain transcription factors Pax6 and Nkx6.1, respectively. Loss- and gain-of-function experiments indicate that the positional identity of these astrocyte subtypes is controlled by Pax6 and Nkx6.1 in a combinatorial manner. Thus, positional identity is an organizing principle underlying astrocyte, as well as neuronal, subtype diversification and is controlled by a homeodomain transcriptional code whose elements are reutilized following the specification of neuronal identity earlier in development.

Cyclin D1 promotes neurogenesis in the developing spinal cord in a cell cycle-independent manner.

Neural stem and progenitor cells undergo an important transition from proliferation to differentiation in the G1 phase of the cell cycle. The mechanisms coordinating this transition are incompletely understood. Cyclin D proteins promote proliferation in G1 and typically are down-regulated before differentiation. Here we show that motoneuron progenitors in the embryonic spinal cord persistently express Cyclin D1 during the initial phase of differentiation, while down-regulating Cyclin D2. Loss-of-function and gain-of-function experiments indicate that Cyclin D1 (but not D2) promotes neurogenesis in vivo, a role that can be dissociated from its cell cycle function. Moreover, reexpression of Cyclin D1 can restore neurogenic capacity to D2-expressing glial-restricted progenitors. The neurogenic function of Cyclin D1 appears to be mediated, directly or indirectly, by Hes6, a proneurogenic basic helic-loop-helix transcription factor. These data identify a cell cycle-independent function for Cyclin D1 in promoting neuronal differentiation, along with a potential genetic pathway through which this function is exerted.

Observation of Unique Circulating miRNA Signatures in Non-Human Primates Exposed to Total-Body vs. Whole Thorax Lung Irradiation.

A radiological/nuclear (RAD-NUC) incident, especially in an urban setting, results in diverse radiation-induced injuries due to heterogeneities in dose, the extent of partial-body shielding, human biodiversity and pre-existing health conditions. For example, acute radiation syndrome (ARS) can result in death within days to weeks of exposure to 0.7-10 Gy doses and is associated with destruction of the bone marrow, known as hematopoietic ARS (H-ARS). However, partial-body shielding that spares a portion of the bone marrow from exposure can significantly reduce the occurrence of H-ARS, but delayed effects of acute radiation exposure (DEARE) can still occur within months or years after exposure depending on the individual. In a mass casualty event, ideal triage must be able to pre-symptomatically identify individuals likely to develop radiation-induced injuries and provide an appropriate treatment plan. Today, while there are FDA approved treatments for hematopoietic ARS, there are no approved diagnosis for radiation injury and no approved treatments for the broad spectra of injuries associated with radiation. This has resulted in a major capability gap in the nations preparedness to a potentially catastrophic RAD-NUC event. Circulating microRNA (miRNA) are a promising class of biomarkers for this application because the molecules are accessible via a routine blood draw and are excreted by various tissues throughout the body. To test if miRNA can be used to predict distinct tissue-specific radiation-induced injuries, we compared the changes to the circulating miRNA profiles after total-body irradiation (TBI) and whole thorax lung irradiation (WTLI) in non-human primates at doses designed to induce ARS (day 2 postirradiation; 2-6.5 Gy) and DEARE (day 15 postirradiation; 9.8 or 10.7 Gy), respectively. In both models, miRNA sequences were identified that correlated with the onset of severe neutropenia (counts <500 µL-1; TBI) or survival (WTLI). This method identified panels of eleven miRNA for both model and assigned functional roles for the panel members using gene ontology enrichment analysis. A common signature of radiation-induced injury was observed in both models: apoptosis, DNA damage repair, p53 signaling, pro-inflammatory response, and growth factor/cytokine signaling pathways were predicted to be disrupted. In addition, injury-specific pathways were identified. In TBI, pathways associated with ubiquitination, specifically of histone H2A, were enriched, suggesting more impact to DNA damage repair mechanisms and apoptosis. In WTLI, pro-fibrotic pathways including transforming growth factor (TGF-ß) and bone morphogenetic protein (BMP) signaling pathways were enriched, consistent with the onset of late lung injury. These results suggest that miRNA may indeed be able to predict the onset of distinct types of radiation-induced injuries.

Identification of miRNA Associated with Reduced Survival after Whole-Thorax Lung Irradiation in Non-Human Primates.

Thoracic exposure to ionizing radiation can lead to delayed injuries to the heart and lung that are serious and even life-threatening. These injuries are difficult to predict since they manifest over many weeks and months. To identify noninvasive, tissue-specific biomarkers for the early detection of late radiation injury, circulating microRNA (miRNA) levels were measured in non-human primates (NHP, Macaca mulatta) that received a single exposure of whole-thorax lung irradiation (WTLI) at a dose likely to result in 20% or 75% mortality within 180 days (9.8 or 10.7 Gy). Animals were observed for 270 days after WTLI. Approximately 58% of 9.8 Gy WTLI animals (7 of 12) and 94% of 10.7 Gy WTLI animals (15 out of 16) did not survive to the primary end point. Evidence of pulmonary fibrosis/pneumonitis was observed in all animals. Animals that received 10.7 Gy WTLI experienced more severe and early-onset pneumonitis, as indicated by reduced aerated lung volume, high non-sedated respiratory rate, earlier and more frequent dexamethasone treatments, and evidence of onset of heart disease. Radiation-induced changes in the circulating miRNA profile were most prominent within the first 30 days postirradiation, before the manifestation of symptoms, and included miRNA sequences known to regulate pathways associated with pulmonary fibrosis (TGF-ß/SMAD signaling) and pneumonitis/inflammation (p53 signaling). The abundance of several circulating miRNA differentially expressed at day 6 or 15, such as miR-199a-3p and miR-25-3p, correlated with statistically significant differences in survival. This study supports the hypothesis that it is feasible to use plasma miRNA profiles to identify individuals at high risk of organ-specific late radiation injury. These miRNA profiles could improve radiation oncology clinical practice and serve as biomarkers to predict who might develop late complications in the aftermath of a radiological or nuclear (RAD-NUC) incident.

The transcription factor NFIA controls the onset of gliogenesis in the developing spinal cord.

The mechanisms controlling the transition from neurogenesis to gliogenesis in the vertebrate CNS are incompletely understood. We identified a family of transcription factors, called NFI genes, which are induced throughout the spinal cord ventricular zone (VZ) concomitantly with the induction of GLAST, an early marker of gliogenesis. NFIA is both necessary and sufficient for GLAST induction in the VZ. Unexpectedly, NFIA is also essential for the continued inhibition of neurogenesis in VZ progenitors. This function is mediated by the requirement of NFIA for the expression of HES5, a Notch effector. However, Notch effectors are unable to promote glial-fate specification in the absence of NFIA. Thus, NFIA links the abrogation of neurogenesis to a generic program of gliogenesis, in both astrocyte and oligodendrocyte VZ progenitors. At later stages, NFIA promotes migration and differentiation of astrocyte precursors, a function that is antagonized in oligodendrocyte precursors by Olig2.

Identification of miRNA signatures associated with radiation-induced late lung injury in mice.

Acute radiation exposure of the thorax can lead to late serious, and even life-threatening, pulmonary and cardiac damage. Sporadic in nature, late complications tend to be difficult to predict, which prompted this investigation into identifying non-invasive, tissue-specific biomarkers for the early detection of late radiation injury. Levels of circulating microRNA (miRNA) were measured in C3H and C57Bl/6 mice after whole thorax irradiation at doses yielding approximately 70% mortality in 120 or 180 days, respectively (LD70/120 or 180). Within the first two weeks after exposure, weight gain slowed compared to sham treated mice along with a temporary drop in white blood cell counts. 52% of C3H (33 of 64) and 72% of C57Bl/6 (46 of 64) irradiated mice died due to late radiation injury. Lung and heart damage, as assessed by computed tomography (CT) and histology at 150 (C3H mice) and 180 (C57Bl/6 mice) days, correlated well with the appearance of a local, miRNA signature in the lung and heart tissue of irradiated animals, consistent with inherent differences in the C3H and C57Bl/6 strains in their propensity for developing radiation-induced pneumonitis or fibrosis, respectively. Radiation-induced changes in the circulating miRNA profile were most prominent within the first 30 days after exposure and included miRNA known to regulate inflammation and fibrosis. Importantly, early changes in plasma miRNA expression predicted survival with reasonable accuracy (88-92%). The miRNA signature that predicted survival in C3H mice, including miR-34a-5p, -100-5p, and -150-5p, were associated with pro-inflammatory NF-κB-mediated signaling pathways, whereas the signature identified in C57Bl/6 mice (miR-34b-3p, -96-5p, and -802-5p) was associated with TGF-ß/SMAD signaling. This study supports the hypothesis that plasma miRNA profiles could be used to identify individuals at high risk of organ-specific late radiation damage, with applications for radiation oncology clinical practice or in the context of a radiological incident.

G1 phase regulation, area-specific cell cycle control, and cytoarchitectonics in the primate cortex.

We have investigated the cell cycle-related mechanisms that lead to the emergence of primate areas 17 and 18. These areas are characterized by striking differences in cytoarchitectonics and neuron number. We show in vivo that (1) area 17 precursors of supragranular neurons exhibit a shorter cell cycle duration, a reduced G1 phase, and a higher rate of cell cycle reentry than area 18 precursors; (2) area 17 and area 18 precursors show contrasting and specific levels of expression of cyclin E (high in area 17, low in area 18) and p27Kip1 (low in area 17, high in area 18); (3) ex vivo up- and downmodulation of cyclin E and p27Kip1 show that both regulators influence cell cycle kinetics by modifying rates of cell cycle progression and cell cycle reentry; (4) modeling the areal differences in cell cycle parameters suggests that they contribute to areal differences in numbers of precursors and neuron production.

The Mode of Stem Cell Division Is Dependent on the Differential Interaction of ß-Catenin with the Kat3 Coactivators CBP or p300.

Normal long-term repopulating somatic stem cells (SSCs) preferentially divide asymmetrically, with one daughter cell remaining in the niche and the other going on to be a transient amplifying cell required for generating new tissue in homeostatic maintenance and repair processes, whereas cancer stem cells (CSCs) favor symmetric divisions. We have previously proposed that differential ß-catenin modulation of transcriptional activity via selective interaction with either the Kat3 coactivator CBP or its closely related paralog p300, regulates symmetric versus asymmetric division in SSCs and CSCs. We have previously demonstrated that SSCs that divide asymmetrically per force retain one of the dividing daughter cells in the stem cell niche, even when treated with specific CBP/ß-catenin antagonists, whereas CSCs can be removed from their niche via forced stochastic symmetric differentiative divisions. We now demonstrate that loss of p73 in early corticogenesis biases ß-catenin Kat3 coactivator usage and enhances ß-catenin/CBP transcription at the expense of ß-catenin/p300 transcription. Biased ß-catenin coactivator usage has dramatic consequences on the mode of division of neural stem cells (NSCs), but not neurogenic progenitors. The observed increase in symmetric divisions due to enhanced ß-catenin/CBP interaction and transcription leads to an immediate increase in NSC symmetric differentiative divisions. Moreover, we demonstrate for the first time that the complex phenotype caused by the loss of p73 can be rescued in utero by treatment with the small-molecule-specific CBP/ß-catenin antagonist ICG-001. Taken together, our results demonstrate the causal relationship between the choice of ß-catenin Kat3 coactivator and the mode of stem cell division.

Detection of Acute Radiation Sickness: A Feasibility Study in Non-Human Primates Circulating miRNAs for Triage in Radiological Events.

Development of biomarkers capable of estimating absorbed dose is critical for effective triage of affected individuals after radiological events. Levels of cell-free circulating miRNAs in plasma were compared for dose-response analysis in non-human primates (NHP) exposed to lethal (6.5 Gy) and sub-lethal (1 and 3 Gy) doses over a 7 day period. The doses and test time points were selected to mimic triage needs in the event of a mass casualty radiological event. Changes in miRNA abundance in irradiated animals were compared to a non-irradiated cohort and a cohort experiencing acute inflammation response from exposure to lipopolysaccharide (LPS). An amplification-free, hybridization-based direct digital counting method was used for evaluation of changes in microRNAs in plasma from all animals. Consistent with previous murine studies, circulating levels of miR-150-5p exhibited a dose- and time-dependent decrease in plasma. Furthermore, plasma miR-150-5p levels were found to correlate well with lymphocyte and neutrophil depletion kinetics. Additionally, plasma levels of several other evolutionarily and functionally conserved miRNAs were found altered as a function of dose and time. Interestingly, miR-574-5p exhibited a distinct, dose-dependent increase 24 h post irradiation in NHPs with lethal versus sub-lethal exposure before returning to the baseline level by day 3. This particular miRNA response was not detected in previous murine studies but was observed in animals exposed to LPS, indicating distinct molecular and inflammatory responses. Furthermore, an increase in low-abundant miR-126, miR-144, and miR-21 as well as high-abundant miR-1-3p and miR-206 was observed in irradiated animals on day 3 and/or day 7. The data from this study could be used to develop a multi-marker panel with known tissue-specific origin that could be used for developing rapid assays for dose assessment and evaluation of radiation injury on multiple organs. Furthermore this approach may be utilized to screen for tissue toxicity in patients who receive myeloablative and therapeutic radiation.

Contrasting effects of basic fibroblast growth factor and neurotrophin 3 on cell cycle kinetics of mouse cortical stem cells.

Basic fibroblast growth factor (bFGF) exerts a mitogenic effect on cortical neuroblasts, whereas neurotrophin 3 (NT3) promotes differentiation in these cells. Here we provide evidence that both the mitogenic effect of bFGF and the differentiation-promoting effect of NT3 are linked with modifications of cell cycle kinetics in mouse cortical precursor cells. We adapted an in vitro assay, which makes it possible to evaluate (1) the speed of progression of the cortical precursors through the cell cycle, (2) the duration of individual phases of the cell cycle, (3) the proportion of proliferative versus differentiative divisions, and (4) the influence on neuroglial differentiation. Contrary to what has been claimed previously, bFGF promotes proliferation via a change in cell cycle kinetics by simultaneously decreasing G1 duration and increasing the proportion of proliferative divisions. In contrast, NT3 lengthens G1 and promotes differentiative divisions. We investigated the molecular foundations of these effects and show that bFGF downregulates p27(kip1) and upregulates cyclin D2 expression. This contrasts with NT3, which upregulates p27(kip1) and downregulates cyclin D2 expression. Neither bFGF nor NT3 influences the proportion of glia or neurons in short to medium term cultures. The data point to links between the length of the G1 phase and the type of division of cortical precursors: differentiative divisions are correlated with long G1 durations, whereas proliferative divisions correlate with short G1 durations. The present results suggest that concerted mechanisms control the progressive increase in the cell cycle duration and proportion of differentiative divisions that is observed as corticogenesis proceeds.

The concerted modulation of proliferation and migration contributes to the specification of the cytoarchitecture and dimensions of cortical areas.

Regionalization of cell cycle kinetics of cortical precursors has been described in nonhuman primates and rodents indicating a fate map of areas as distinct proliferative programs in the germinal zones of the neocortex. It remains to be understood how proliferative gradients during corticogenesis are transcribed into a stepwise function to form adult areal borders. Here we have used the monkey areas 17 and 18, which show striking cytoarchitectonic differences, as a model system for studying how developmental events establish areal boundaries in the adult. We present data indicating that the events that are involved in the formation of a sharp border separating 2 areas involve an orchestration of diverse phenomena including differential rates of proliferation, migration, and tangential expansion.

Olig2+ neuroepithelial motoneuron progenitors are not multipotent stem cells in vivo.

Neurons and glia are thought to arise from multipotent and self-renewing stem cells, which comprise the majority of neuroepithelial cells in the ventricular zone (VZ) of the early embryonic CNS. However, this idea remains to be tested rigorously, because CNS stem cells have been identified only by using in vitro assays, from which their abundance in vivo cannot be directly inferred. In the hematopoietic system, stem cells are characterized by using prospective isolation and direct in vivo transplantation. Here we have used this approach to ask whether most VZ progenitors behave as stem cells in vivo. The best-studied region of the embryonic CNS for addressing this problem is, arguably, the ventral spinal cord, within which progenitors in the motoneuron progenitor (pMN) domain sequentially generate motoneurons (MNs) and oligodendrocyte precursors (OPs). Virtually all VZ cells in pMN express the transcription factor Olig2. If most of these cells are stem cells, then they should maintain neurogenic potential, even at later, gliogenic stages. To test this hypothesis, we have prospectively isolated Olig2(+) cells from murine embryonic day (E)9.5 and E13.5 spinal cord and directly transplanted them to E2 chick spinal cord. Transplanted E9.5 cells generate both neurons, including MNs and OPs, whereas E13.5 cells generate. The observation that most Olig2(+) progenitors do not maintain neurogenic potential into the period of gliogenesis argues that they do not self-renew. These results do not support the commonly held view that most neuroepithelial cells in the embryonic CNS VZ are stem cells in vivo.

Interaction of PRMT1 with BTG/TOB proteins in cell signalling: molecular analysis and functional aspects.

BACKGROUND: Several recent reports have connected protein methylation with differentiation. Furthermore, the BTG/TOB proteins have also been implicated in such control. BTG1 and 2 have been shown to interact with PRMT1 (predominant cellular arginine N-methyltransferase of type I). RESULTS: First, we have studied the interaction between PRMT1 and the proteins of the BTG/TOB family. We show that boxC, a sequence present only in BTG1 and BTG2, is essential for this association. Using boxC peptide, we have investigated the importance of PRMT1/BTG protein association during type I protein methylation reactions. Finally, we show that the addition of boxC fused to penetratin interferes with the neuronal differentiation of PC12 cells and ES cell-derived neurones. CONCLUSIONS: Taken together, these results indicate that PRMT1/BTG proteins could play a key role in the arginine methylation-mediated signalling pathway as well as in neuronal differentiation.