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The glycoprotein Prominin-1 and the carbohydrate Lewis X stage-specific embryonic antigen 1 (LeX-SSEA1) both have been extensively used as cell surface markers to purify neural stem cells (NSCs). While Prominin-1 labels a specialized membrane region in NSCs and ependymal cells, the specificity of LeX-SSEA1 expression and its biological significance are still unknown. To address these issues, we have here monitored the expression of the carbohydrate in neonatal and adult NSCs and in their progeny. Our results show that the percentage of immunopositive cells and the levels of LeX-SSEA1 immunoreactivity both increase with postnatal age across all stages of the neural lineage. This is associated with decreased proliferation in precursors including NSCs, which accumulate the carbohydrate at the cell surface while remaining quiescent. Exposure of precursors to bone morphogenetic protein (BMP) increases LEX-SSEA1 expression, which promotes cell cycle withdrawal by a mechanism involving LeX-SSEA1-mediated interaction at the cell surface. Conversely, interference with either BMP signaling or with LeX-SSEA1 promotes proliferation to a similar degree. Thus, in the postnatal germinal niche, the expression of LeX-SSEA1 increases with age and exposure to BMP signaling, thereby downregulating the proliferation of subependymal zone precursors including NSCs.

Niche homeostasis in the postnatal subependymal zone of the lateral ventricle (lSEZ) requires coordinated proliferation and differentiation of neural progenitor cells. The mechanisms regulating this balance are scarcely known. Recent observations indicate that the orphan nuclear receptor Tlx is an intrinsic factor essential in maintaining this balance. However, the effect of Tlx on gene expression depends on age and cell-type cues. Therefore, it is essential to establish its expression pattern at different developmental ages. Here, we show for the first time that in the neonatal lSEZ activated neural stem cells (NSCs) and especially transit-amplifying progenitors (TAPs) express Tlx and that its expression may be regulated at the posttranscriptional level. We also provide evidence that in both cell types Tlx affects gene expression in a positive and negative manner. In activated NSCs, but not in TAPs, absence of Tlx leads to overexpression of negative cell cycle regulators and impairment of proliferation. Moreover, in both cell types, the homeobox transcription factor Dlx2 is downregulated in the absence of Tlx. This is paralleled by increased expression of Olig2 in activated NSCs and glial fibrillary acidic protein in TAPs, indicating that in both populations Tlx decreases gliogenesis. Consistent with this, we found a higher proportion of cells expressing glial makers in the neonatal lSEZ of mutant mice than in the wild type counterpart. Thus, Tlx playing a dual role affects the expression of distinct genes in these two lSEZ cell types.

Fine-tuning of X chromosomal gene expression in Drosophila melanogaster involves the selective interaction of the Dosage Compensation Complex (DCC) with the male X chromosome, in order to increase the transcription of many genes. However, the X chromosomal DNA sequences determining DCC binding remain elusive. By adapting a 'one-hybrid' assay, we identified minimal DNA elements that direct the interaction of the key DCC subunit, MSL2, in cells. Strikingly, several such novel MSL2 recruitment modules have very different DNA sequences. The assay revealed a novel, 40 bp DNA element that is necessary for recruitment of DCC to an autosomal binding site in flies in the context of a longer sequence and sufficient by itself to direct recruitment if trimerized. Accordingly, recruitment of MSL2 to the single 40 bp element in cells was weak, but as a trimer approached the power of the strongest DCC recruitment site known to date, the roX1 DH site. This element is the shortest MSL2 recruitment sequence known to date. The results support a model for MSL2 recruitment according to which several different, degenerate sequence motifs of variable affinity cluster and synergise to form a high affinity site.

Background and Purpose — It has previously been shown that activation will increase cerebral blood flow (CBF) and cerebral glucose uptake (CMR glc ) in excess of cerebral oxygen uptake (CMRO 2 ). Our purpose was to investigate the influence of β-adrenergic blockade with propranolol on the activation-induced uncoupling of cerebral glucose and oxygen metabolism. Methods — Using awake rats, we determined the cerebral arteriovenous differences of oxygen [(a−v) O2 ], glucose [(a−v) glc ], and lactate [(a−v) lac ] both under baseline conditions and during activation. The molar ratio between CMRO 2 and CMR glc , the oxygen-glucose index (OGI), was calculated. Results — Without β-adrenergic blockade, activation decreased the (a−v) O2 but not the (a−v) glc , reducing the OGI from 6.1 during baseline conditions to 4.0 under activation ( P O2 decreased, indicating that the ratio CBF/CMRO 2 had increased. Under baseline conditions, a slight flux of lactate from the brain was observed. Activation increased the arterial lactate concentration, and during this condition, the lactate flux from the brain was reversed into a slight lactate uptake. Propranolol administration did not change the behavior of the animals during activation. After administration of propranolol, baseline values were unaffected, but β-adrenergic blockade totally abolished the activation-induced uncoupling of (a−v) O2 from (a−v) glc , because both remained constant with an unchanged OGI. The unchanged (a−v) O2 indicates that CBF remained unchanged compared with CMRO 2 . Conclusions — β-Adrenergic blockade by propranolol abolishes the activation-induced uncoupling of cerebral oxygen to glucose metabolism and the changes in (a−v) O2 . This may be of most significance to studies of cerebral activation by the blood oxygen level–dependent fMRI method.

The activation of epidermal growth factor receptor (EGFR) affects multiple aspects of neural precursor behaviour, including proliferation and migration. Telencephalic precursors acquire EGF responsiveness and upregulate EGFR expression at late stages of development. The events regulating this process and its significance are still unclear. We here show that in the developing and postnatal hippocampus (HP), growth/differentiation factor (GDF) 15 and EGFR are co-expressed in primitive precursors as well as in more differentiated cells. We also provide evidence that GDF15 promotes responsiveness to EGF and EGFR expression in hippocampal precursors through a mechanism that requires active CXC chemokine receptor (CXCR) 4. Besides EGFR expression, GDF15 ablation also leads to decreased proliferation and migration. In particular, lack of GDF15 impairs both processes in the cornu ammonis (CA) 1 and only proliferation in the dentate gyrus (DG). Importantly, migration and proliferation in the mutant HP were altered only perinatally, when EGFR expression was also affected. These data suggest that GDF15 regulates migration and proliferation by promoting EGFR signalling in the perinatal HP and represent a first description of a functional role for GDF15 in the developing telencephalon.

Controversy exists as to whether the metabolic activity of the brain as a whole increases during physical exercise. For example, there appears to be no change in brain O2 uptake during cycling (Zobl et al. 1965; Madsen et al. 1993), whereas during vigorous exercise on the treadmill an increase in brain O2 uptake and a tendency for glucose uptake to increase has been reported (Scheinberg et al. 1954). In the case of mental stress, there is no appreciable change in brain O2 uptake but there is an increase in cerebral glucose uptake (Madsen et al. 1995a). Thus, the rate of glucose uptake is enhanced compared to that of O2. Interestingly, the ‘uncoupling’ between the O2 and glucose uptake rates is sustained even after the cessation of brain activation (Madsen et al. 1995a,b) and may be associated with a decrease in the glycogen level in the brain (Madsen et al. 1995a). During maximal exercise, blood lactate increases to as much as 30 mmol l−1 (Nielsen, 1999) and the brain is known to take up lactate (Ahlborg & Wahren, 1972). Lactate transport across mammalian plasma membranes is mainly carrier mediated (Poole et al. 1993) and a monocarboxylate transporter is found in rat brain endothelium cells (Gerhart et al. 1997). Brain tissue, including neurons (Dringen et al. 1993) and astrocytes (Tildon et al. 1993), possesses the capacity to take up and utilise lactate as an energy source. In fact, lactate rather than glucose may be the primary energy source during neuronal activation (Larrabee, 1996) when lactate is supplied by the glial cells to the neurons (Poitry-Yamate et al. 1995). A positive arterial-jugular venous concentration difference (a–v difference) for lactate has been demonstrated in the dog (Nemoto et al. 1974; Avogaro et al. 1990) and in humans during cardiopulmonary resuscitation (Rivers et al. 1991). Taken together, these reports led us to hypothesise that the metabolic rate for the brain is increased during exercise and also in the recovery phase when lactate, in addition to glucose and O2, is taken into consideration as a metabolic substrate. Therefore, we investigated the a–v differences for glucose, lactate and O2 at rest, during exercise and in the immediate recovery period. Since arterial CO2 tension increases during moderate exercise and decreases during maximal exercise (Jorgensen et al. 1992), we applied an index of the metabolic rate of the brain that would be independent of flow, i.e. we calculated the O2/carbohydrate uptake ratio. In a separate experiment in the rat, we investigated whether an increase in blood lactate per se would influence brain uptake of lactate.

Neural stem cells (NSCs) generate new neurons in vivo and in vitro throughout adulthood and therefore are physiologically and clinically relevant. Unveiling the mechanisms regulating the lineage progression from NSCs to newborn neurons is critical for the transition from basic research to clinical application. However, the direct analysis of NSCs and their progeny is still elusive due to the problematic identification of the cells. We here describe the isolation of highly purified genetically unaltered NSCs and transit-amplifying precursors (TAPs) from the adult subependymal zone (SEZ). Using this approach we show that a primary cilium and high levels of epidermal growth factor receptor (EGFR) at the cell membrane characterize quiescent and cycling NSCs, respectively. However, we also observed non-ciliated quiescent NSCs and NSCs progressing into the cell cycle without up-regulating EGFR expression. Thus, the existence of NSCs displaying distinct molecular and structural conformations provides more flexibility to the regulation of quiescence and cell cycle progression.

The ubiquitous tandem kinase JIL-1 is essential for Drosophila development. Its role in defining decondensed domains of larval polytene chromosomes is well established, but its involvement in transcription regulation has remained controversial. For a first comprehensive molecular characterisation of JIL-1, we generated a high-resolution, chromosome-wide interaction profile of the kinase in Drosophila cells and determined its role in transcription. JIL-1 binds active genes along their entire length. The presence of the kinase is not proportional to average transcription levels or polymerase density. Comparison of JIL-1 association with elongating RNA polymerase and a variety of histone modifications suggests two distinct targeting principles. A basal level of JIL-1 binding can be defined that correlates best with the methylation of histone H3 at lysine 36, a mark that is placed co-transcriptionally. The additional acetylation of H4K16 defines a second state characterised by approximately twofold elevated JIL-1 levels, which is particularly prominent on the dosage-compensated male X chromosome. Phosphorylation of the histone H3 N-terminus by JIL-1 in vitro is compatible with other tail modifications. In vivo, phosphorylation of H3 at serine 10, together with acetylation at lysine 14, creates a composite histone mark that is enriched at JIL-1 binding regions. Its depletion by RNA interference leads to a modest, but significant, decrease of transcription from the male X chromosome. Collectively, the results suggest that JIL-1 participates in a complex histone modification network that characterises active, decondensed chromatin. We hypothesise that one specific role of JIL-1 may be to reinforce, rather than to establish, the status of active chromatin through the phosphorylation of histone H3 at serine 10.

We analysed the nuclear organization of the Polycomb/Trithorax group response element (PRE/TRE) Fab-7 and of other PRE/TREs in larval tissues of D. melanogaster. The results show that pairing/clustering of transgenic and endogenous Fab-7 elements and of other endogenous PRE/TREs occurs only to a limited degree in a highly locus-specific and tissue-specific manner. However, transgenic Fab-7 elements as well as the Fab-7-regulated Abd-B gene and other endogenous loci preferentially occupied defined nuclear regions. Preferred association with the nuclear periphery was observed in the inactive state. However, also in the active state, Fab-7 was often found associated with the nuclear periphery as well as with the boundary of heterochromatin in a fly line- and tissue-specific manner. The boundary between heterochromatin and euchromatin revealed a highly complex architecture in the three-dimensional nuclear space with a close juxtaposition of active and repressed domains. The results suggest that such complex architectures create nuclear microenvironments sustaining specific states of activity of defined PRE/TREs. However, the data also show that the positional behaviour of the transgenic Fab-7 element does not apply to PRE/TREs in general. Altogether, this finding and the highly locus-, tissue-, and fly line-specific behaviour with regard to nuclear positioning and pairing/clustering suggest that the relationships between nuclear organization and functional regulation of PRE/TREs are highly complex and that simple models making general predictions might not be appropriate.

Dosage compensation involves fine-tuning of gene expression at the level of entire chromosomes. The principles that assure selective targeting of the male X chromosome in Drosophila and the mechanism by which transcription levels are adjusted in a twofold range are still mysterious. We discuss the prevalent models in the context of recent experimental observations.

The Drosophila trithorax group gene kismet (kis)was identified in a screen for extragenic suppressors of Polycomb(Pc) and subsequently shown to play important roles in both segmentation and the determination of body segment identities. One of the two major proteins encoded by kis (KIS-L) is related to members of the SWI2/SNF2 and CHD families of ATP-dependent chromatin-remodeling factors. To clarify the role of KIS-L in gene expression, we examined its distribution on larval salivary gland polytene chromosomes. KIS-L is associated with virtually all sites of transcriptionally active chromatin in a pattern that largely overlaps that of RNA Polymerase II (Pol II). The levels of elongating Pol II and the elongation factors SPT6 and CHD1 are dramatically reduced on polytene chromosomes from kis mutant larvae. By contrast, the loss of KIS-L function does not affect the binding of PC to chromatin or the recruitment of Pol II to promoters. These data suggest that KIS-L facilitates an early step in transcriptional elongation by Pol II.

The dosage compensation complex (DCC) in Drosophila melanogaster is responsible for up-regulating transcription from the single male X chromosome to equal the transcription from the two X chromosomes in females. Visualization of the DCC, a large ribonucleoprotein complex, on male larval polytene chromosomes reveals that the complex binds selectively to many interbands on the X chromosome. The targeting of the DCC is thought to be in part determined by DNA sequences that are enriched on the X. So far, lack of knowledge about DCC binding sites has prevented the identification of sequence determinants. Only three binding sites have been identified to date, but analysis of their DNA sequence did not allow the prediction of further binding sites. We have used chromatin immunoprecipitation to identify a number of new DCC binding fragments and characterized them in vivo by visualizing DCC binding to autosomal insertions of these fragments, and we have demonstrated that they possess a wide range of potential to recruit the DCC. By varying the in vivo concentration of the DCC, we provide evidence that this range of recruitment potential is due to differences in affinity of the complex to these sites. We were also able to establish that DCC binding to ectopic high-affinity sites can allow nearby low-affinity sites to recruit the complex. Using the sequences of the newly identified and previously characterized binding fragments, we have uncovered a number of short sequence motifs, which in combination may contribute to DCC recruitment. Our findings suggest that the DCC is recruited to the X via a number of binding sites of decreasing affinities, and that the presence of high- and moderate-affinity sites on the X may ensure that lower-affinity sites are occupied in a context-dependent manner. Our bioinformatics analysis suggests that DCC binding sites may be composed of variable combinations of degenerate motifs., Synopsis In fruit flies, just like in humans, the two sexes are distinguished by different sex chromosomes. Females have two X chromosomes and hence a double dose of all X-linked genes when compared to males, which only have a single X chromosome. This different gene dosage needs to be compensated for by adjusting transcription levels such that male and female cells synthesize equal amounts of gene products. In Drosophila melanogaster, dosage compensation occurs by doubling the transcription of many genes on the single male X. This chromosome-wide control is achieved by a male-specific dosage compensation complex (DCC), which contains enzymes, structural proteins, and non-coding RNA. How is the DCC able to distinguish the X chromosome from the autosomes for selective interaction? In the following article, the authors identify and characterize several novel DNA sequences on the X chromosome that can recruit the DCC. Their results suggest that the X chromosome contains a large number of binding sites for the DCC, which are made up of combinations of degenerate sequence elements. These elements constitute binding sites with varying affinities for the complex. Collectively, their abundance on the X chromosome restricts the action of DCC to the X chromosomal territory.

Twofold differences in gene expression levels can be vital for an organism. This is beautifully illustrated by the process of `dosage compensation' in Drosophila, which doubles transcription from the single male X chromosome to equal the mRNA levels originating from the two X chromosomes in female cells. Failure of the process leads to male-specific lethality. A number of recent publications have furthered our understanding of the ribonucleoprotein complex, which mediates dosage compensation and how it targets the male X chromosome. Deciphering the principles of X chromosome recognition and the nature of the chromatin configuration, that allows fine-tuning of transcription, remain the most interesting challenges.