A novel Arabidopsis vacuolar glucose exporter is involved in cellular sugar homeostasis and affects the composition of seed storage compounds.

Subcellular sugar partitioning in plants is strongly regulated in response to developmental cues and changes in external conditions. Besides transitory starch, the vacuolar sugars represent a highly dynamic pool of instantly accessible metabolites that serve as energy source and osmoprotectant. Here, we present the molecular identification and functional characterization of the vacuolar glucose (Glc) exporter Arabidopsis (Arabidopsis thaliana) Early Responsive to Dehydration-Like6 (AtERDL6). We demonstrate tonoplast localization of AtERDL6 in plants. In Arabidopsis, AtERDL6 expression is induced in response to factors that activate vacuolar Glc pools, like darkness, heat stress, and wounding. On the other hand, AtERDL6 transcript levels drop during conditions that trigger Glc accumulation in the vacuole, like cold stress and external sugar supply. Accordingly, sugar analyses revealed that Aterdl6 mutants have elevated vacuolar Glc levels and that Glc flux across the tonoplast is impaired under stress conditions. Interestingly, overexpressor lines indicated a very similar function for the ERDL6 ortholog Integral Membrane Protein from sugar beet (Beta vulgaris). Aterdl6 mutant plants display increased sensitivity against external Glc, and mutant seeds exhibit a 10% increase in seed weight due to enhanced levels of seed sugars, proteins, and lipids. Our findings underline the importance of vacuolar Glc export during the regulation of cellular Glc homeostasis and the composition of seed reserves.

VEGFR-1 regulates adult olfactory bulb neurogenesis and migration of neural progenitors in the rostral migratory stream in vivo.

The generation of new neurons in the olfactory bulb (OB) persists into adulthood and is a multistep process that includes proliferation, fate choice, migration, survival, and differentiation. Neural precursor cells destined to form olfactory interneurons arise in the subventricular zone (SVZ) and migrate along the rostral migratory stream (RMS) to the OB. Recently, some factors classically known from their effects on the vascular system have been found to influence different steps of adult neurogenesis. In the present study, we report a modulatory function for the vascular endothelial growth factor receptor-1 (VEGFR-1) in adult olfactory neurogenesis. We identified expression of VEGFR-1 in GFAP-positive cells within regions involved in neurogenesis of the adult mouse brain. To determine functions for VEGFR-1 in adult neurogenesis, we compared neural progenitor cell proliferation, migration, and differentiation from wild-type and VEGFR-1 signaling-deficient mice (Flt-1TK(-/-) mice). Our data show that VEGFR-1 signaling is involved in the regulation of proliferation of neuronal progenitor cells within the SVZ, migration along the RMS, and in neuronal differentiation and anatomical composition of interneuron subtypes within the OB. RMS migration in Flt-1TK(-/-) mice was altered mainly as a result of increased levels of its ligand VEGF-A, which results in an increased phosphorylation of VEGFR-2 in neuronal progenitor cells within the SVZ and the RMS. This study reveals that proper RMS migration is dependent on endogenous VEGF-A protein.

Identification of a novel E3 ubiquitin ligase that is required for suppression of premature senescence in Arabidopsis.

During leaf senescence, resources are recycled by redistribution to younger leaves and reproductive organs. Candidate pathways for the regulation of onset and progression of leaf senescence include ubiquitin-dependent turnover of key proteins. Here, we identified a novel plant U-box E3 ubiquitin ligase that prevents premature senescence in Arabidopsis plants, and named it SENESCENCE-ASSOCIATED E3 UBIQUITIN LIGASE 1 (SAUL1). Using in vitro ubiquitination assays, we show that SAUL1 has E3 ubiquitin ligase activity. We isolated two alleles of saul1 mutants that show premature senescence under low light conditions. The visible yellowing of leaves is accompanied by reduced chlorophyll content, decreased photochemical efficiency of photosystem II and increased expression of senescence genes. In addition, saul1 mutants exhibit enhanced abscisic acid (ABA) biosynthesis. We show that application of ABA to Arabidopsis is sufficient to trigger leaf senescence, and that this response is abolished in the ABA-insensitive mutants abi1-1 and abi2-1, but enhanced in the ABA-hypersensitive mutant era1-3. We found that increased ABA levels coincide with enhanced activity of Arabidopsis aldehyde oxidase 3 (AAO3) and accumulation of AAO3 protein in saul1 mutants. Using label transfer experiments, we showed that interactions between SAUL1 and AAO3 occur. This suggests that SAUL1 participates in targeting AAO3 for ubiquitin-dependent degradation via the 26S proteasome to prevent premature senescence.

Impaired brain angiogenesis and neuronal apoptosis induced by conditional homozygous inactivation of vascular endothelial growth factor.

Vascular endothelial growth factor (VEGF) is essential for the differentiation of the primitive embryonic vascular system and has been implicated in the vascularization of organs. Recently, VEGF has also been proposed to play a role in neural development, neuroprotection, and adult neurogenesis. Here we have investigated the function of VEGF in the developing brain by cre-lox technology. We show that VEGF produced by the embryonic neuroectoderm is required for the vascularization and the development of the brain. Both the invasion and the directed growth of capillaries were severely impaired in the fore-, mid- and hindbrain of VEGF(lox/lox)/nestin-cre mouse embryos homozygous for a VEGF mutation in the neural tube. These observations demonstrate that VEGF, via local secretion by neural progenitors, induces brain angiogenesis and guides the growth of capillaries toward the ventricular zone. VEGF deficiency led to developmental retardation and progressive destruction of neural tissue in all brain regions. The defect was most pronounced in telencephalic structures, such as the hippocampus, and caused microcephaly. Taken together, the findings establish the critical importance of neuroectoderm-derived VEGF in the morphogenesis of the brain. VEGF acts as a key regulator of brain angiogenesis and provides instructive cues for the correct spatial organization of the vasculature.

Arabidopsis zinc-finger protein 2 is a negative regulator of ABA signaling during seed germination.

The hormone abscisic acid (ABA) mediates plant development and adaptation to environmental stresses. ABA-dependent transcription factors are central regulators of ABA signaling. Here, we report on the identification of the ABA-induced transcriptional repressor Arabidopsis zinc-finger protein 2 (AZF2) as ABA signaling component. We isolated azf2-1 mutants lacking AZF2 full-length transcripts that were hypersensitive to ABA during seed germination. In line with a function of AZF2 in seed germination and seedling development, AZF2-promoter activity was observed in radicles and young cotyledons of AZF2-promoter:GUS plants. Our results indicate that AZF2 is a negative regulator of ABA signaling in seeds.

VEGFR-1 signaling regulates the homing of bone marrow-derived cells in a mouse stroke model.

Vascular endothelial growth factor receptor 1 (VEGFR-1) is highly expressed in endothelial cells and regulates developmental angiogenesis by acting as a decoy receptor and trapping VEGF-A. Vascular endothelial growth factor receptor 1 is also expressed in monocytes and macrophages; mice lacking the VEGFR-1 tyrosine kinase (TK) domain (VEGFR-1 TK mice) display impaired macrophage function. Because macrophages are recruited to sites of cerebral ischemic infarcts, we hypothesized that lack of VEGFR-1 TK in bone marrow(BM) cells would affect the outcome in an experimental stroke model. We performed BM transplantation experiments in C57BL/6J mice using VEGFR-1 TK and VEGFR-1 TK mice as BM donors and analyzed cell infiltration after cerebral ischemia. There was reduced initial recruitment of VEGFR-1 TK myeloid cells into the infarcted tissue and reduced postischemic angiogenesis at 3days postischemia. By 10 days, the numbers of infiltrating cells and the densities of vessels in the infarct peri-infarct zone were similar for both groups. Neither infarct size at 3 and 10 days postischemia nor neurological performance at 24 hours was different between the experimental groups. These results support a role of VEGFR-1 signaling in the early regulation of BM infiltration and angiogenesis after brain ischemia.

Different networks, common growth factors: shared growth factors and receptors of the vascular and the nervous system.

Growth factors and their respective receptors are key regulators during development and for homeostasis of the nervous system. In addition, changes in growth factor function, availability or downstream signaling is involved in many neuropathological disorders like Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis, stroke and brain tumours. Research of the recent years revealed that some growth factors, initially discovered as neural growth factors are also affecting blood vessels [e.g. nerve growth factor (NGF) or brain-derived neurotrophic factor (BDNF)]. Likewise, vascular growth factors, such as vascular endothelial growth factor (VEGF), which was previously described as an endothelial cell specific mitogen, also affect neural cells. The discovery of shared growth factors affecting the vascular and the nervous system is of relevance for potential therapies of vascular and neurological diseases. This review aims to give an overview about the growing field of common growth factors and receptors within the two different networks.

ABA-responsive RNA-binding proteins are involved in chloroplast and stromule function in Arabidopsis seedlings.

The phytohormone abscisic acid (ABA) regulates essential growth and developmental processes in plants. Recently, RNA-binding proteins have been described as components of ABA signaling during germination. We have identified ten ABA-regulated RNA-binding proteins in Arabidopsis seedlings. Among those genes, AtCSP41B and cpRNP29 are highly expressed in seedlings. Using promoter:reporter gene analyses, we showed that both AtCSP41B and cpRNP29 were in particular expressed in photosynthetically active organs like green cotyledons, leaves, and petioles. The analysis of CFP-fusion proteins demonstrates that cpRNP29 localized to chloroplasts and AtCSP41B to chloroplasts and stromules. Whereas RNA-binding of cpRNP29 has previously been shown, we demonstrated through in vitro RNA-binding assays that recombinant AtCSP41B binds to RNA, and that chloroplast petD RNA can serve as a target of AtCSP41B. Developmental or environmental stimuli affected the expression of AtCSP41B and cpRNP29 in seedlings. Both genes were repressed during senescence, but only AtCSP41B was significantly repressed upon water stress. In addition, AtCSP41B and cpRNP29 exhibited low expression in etiolated seedlings compared to green seedlings, and cpRNP29 was regulated during the day photoperiod. Homozygous T-DNA insertion lines were isolated, characterized on the molecular level, and monitored for phenotypic changes. Taken together, the data show that both proteins are regulated during processes that are known to involve ABA signaling. Their localization in chloroplasts and RNA-binding activity suggest a role in chloroplast RNA metabolism in Arabidopsis seedlings.

Functional expression of the ecto-ATPase NTPDase2 and of nucleotide receptors by neuronal progenitor cells in the adult murine hippocampus.

An astrocyte-like cell population corresponding to residual radial glia represents the neuronal progenitors of the adult mammalian hippocampus. We show that radial glia-like cells of the dentate gyrus express surface-located ATP-hydrolyzing activity and are immunopositive for NTPDase2. This enzyme hydrolyzes extracellular nucleoside triphosphates such as ATP and UTP to their nucleoside diphosphates and is thus involved in the control of signaling via P2 receptors. NTPDase2 is expressed from embryonic day 17 onward. In the hippocampus, the embryonic pattern of NTPDase2 expression mirrors that of the dentate migration of neuroblasts. Double-immunolabeling revealed that NTPDase2 is associated with subpopulations of glial fibrillary acidic protein-, nestin- and doublecortin-positive radial cells. It is absent from mature granule cells or S100-positive astrocytes. NTPDase2-positive cells proliferate. Furthermore, after mitosis, progenitor cells preferentially reveal an NTPDase2-positive phenotype. Patch-clamp analysis demonstrates functional nucleotide receptors in progenitor cells expressing nestin promotor-driven green fluorescent protein. Our results identify the ecto-nucleotidase NTPDase2 and functional P2X receptors at hippocampal progenitor cells. We infer that signaling pathways via extracellular nucleotides may play a role in the control of hippocampal neurogenesis.

Circular vection during voluntary suppression of optokinetic reflex.

Optokinetic circular vection (CV) was investigated in 12 subjects using an optokinetic pattern rotating at 15 degrees /s, 30 degrees /s, or 60 degrees /s, and four viewing conditions: FOL, subjects attentively followed details of pattern; STA, subjects stared at the pattern; SUP, subjects suppressed their optokinetic reflex (OKR) voluntarily (this was facilitated by a white, featureless band at eye level which separated the pattern in an upper and lower half); FIX, subjects suppressed OKR by fixating at a stationary fixation point (FP). To quantify CV, subjects pressed a signal button each time they felt rotated by a further 90 degrees; OKR was recorded by electro-oculography. Voluntary suppression of OKR was achieved during 2-70% of stimulus duration. Total apparent self-displacement (cumulated 90 degrees indications) was smallest during FOL, increasing gradually in the order FOL < STA < SUP < FIX (all inequalities significant); CV latency decreased in the same order. Slow eye velocity was identical during FOL and STA, and was reduced by 70-30% during SUP. We conclude from these results: (1) the effect of eye movements on CV depends on whether these are intentional (FOL) or not (STA); (2) the increase in CV during voluntary OKR suppression without FP suggests that afferent motion cues (retinal slip) are processed with larger gain than efferent motion cues (eye movement); hence (3) the enhancement of CV during fixation of FP is not, or not solely, the result of the apparent motion of the FP counter to the direction of pattern movement (Duncker illusion).

Fusion of vestibular and podokinesthetic information during self-turning towards instructed targets.

When observers step about their vertical axis ("active turning") without vision they dispose of essentially two sources of information that can tell them by how much they have turned: the vestibular cue which reflects head rotation in space and the "podokinesthetic" cue, a compound of leg proprioceptive afferents and efference copy signals which reflects the observer's motion relative to his support. We ask how these two cues are fused in the process leading to the perception of self-displacement during active turning. To this end we compared the performance of observers in three angular navigation tasks which differed with regard to the number and type of available motion cues: (1) Passive rotation, vestibular cue ( ves) only; observers are standing on a platform which is being rotated. (2) Treadmill stepping, podokinesthetic cue ( pod) only; observers step counter to the rotating platform so as to remain stable in space. (3) Active turning, ves and pod available; observers step around on the stationary platform. In all three tasks, angular velocity varied from trial to trial (15, 30, 60 degrees /s) but was constant during trials. Perception was probed by having the observers signal when they thought to have reached a previously instructed angular displacement, either in space or relative to the platform ("target"; range 60-1080 degrees ). Performance was quantified in terms of the targeting gain (displacement reached by the observer divided by target angle) and of the random error ( E(r)), which records an observer's deviation during single trials from his average performance. Confirming previous observations, E(r) was found to be significantly smaller during active turning than during passive turning, and we now complement these observations by showing that it is also significantly smaller than during treadmill stepping. This behaviour of E(r) is compatible with the idea that ves and pod be averaged during active turning. On the other hand, the observed characteristics of the targeting gain ( G(T)) support this idea only for the case of fast rotations (60 degrees /s); at lower velocities, the gain found during active turning was clearly not the average of the G(T) values recorded in the passive and the treadmill modes. We therefore also discuss alternative scenarios as to how ves and pod could interact, among these one based on the concept of a vestibular eigenmodel. A common denominator of these scenarios is that ves assumes the role of a prerequisite for an optimal use of pod during turning on a stationary support, without itself entering the calculation of displacement perception; this perception would be based exclusively on pod. Finally, it was a consistent observation that during passive rotations cognitive mechanisms fill in for the decaying vestibular signal in the context of the present navigation task, enabling observers to achieve large displacements surprisingly well although the duration of these movements exceeds by far the conventionally cited value of the central vestibular time constant (=20 s).

Endothelium-specific Cre recombinase activity in flk-1-Cre transgenic mice.

The use of the Cre-loxP recombination system allows the conditional inactivation of genes in mice. The availability of transgenic mice in which the Cre recombinase expression is highly cell type specific is a prerequisite to successfully use this system. We previously have characterized regulatory regions of the mouse flk-1 gene sufficient for endothelial cell-specific expression of the LacZ reporter gene in transgenic mice. These regions were fused to the Cre recombinase gene, and transgenic mouse lines were generated. In the resulting flk-1-Cre transgenic mice, specificity of Cre activity was determined by cross-breeding with the reporter mouse lines Rosa26R or CAG-CAT-LacZ. We examined double-transgenic mice at different stages of embryonic development (E9.5-E16.5) and organs of adult animals by LacZ staining. Strong endothelium-specific staining of most vascular beds was observed in embryos older than E11.5 in one or E13.5 in a second line. In addition, the neovasculature of experimental BFS-1 tumors expressed the transgene. These lines will be valuable for the conditional inactivation of floxed target genes in endothelial cells of the embryonic vascular system.