Philippe Mourrain

Arabidopsis SGS2 and SGS3 Genes Are Required for Posttranscriptional Gene Silencing and Natural Virus Resistance

Posttranscriptional gene silencing (PTGS) in plants resuits from the degradation of mRNAs and shows phenomenological similarities with quelling in fungi and RNAi in animals. Here, we report the isolation of sgs2 and sgs3 Arabidopsis mutants impaired in PTGS. We establish a mechanistic link between PTGS, quelling, and RNAi since the Arabidopsis SGS2 protein is similar to an RNA-dependent RNA polymerase like N. crassa QDE-1, controlling quelling, and C. elegans EGO-1, controlling RNAi. In contrast, SGS3 shows no significant similarity with any known or putative protein, thus defining a specific step of PTGS in plants. Both sgs2 and sgs3 mutants show enhanced susceptibility to virus, definitively proving that PTGS is an antiviral defense mechanism that can also target transgene RNA for degradation.

Characterization of sleep in zebrafish and insomnia in hypocretin receptor mutants

Sleep is a fundamental biological process conserved across the animal kingdom. The study of how sleep regulatory networks are conserved is needed to better understand sleep across evolution. We present a detailed description of a sleep state in adult zebrafish characterized by reversible periods of immobility, increased arousal threshold, and place preference. Rest deprivation using gentle electrical stimulation is followed by a sleep rebound, indicating homeostatic regulation. In contrast to mammals and similarly to birds, light suppresses sleep in zebrafish, with no evidence for a sleep rebound. We also identify a null mutation in the sole receptor for the wake-promoting neuropeptide hypocretin (orexin) in zebrafish. Fish lacking this receptor demonstrate short and fragmented sleep in the dark, in striking contrast to the excessive sleepiness and cataplexy of narcolepsy in mammals. Consistent with this observation, we find that the hypocretin receptor does not colocalize with known major wake-promoting monoaminergic and cholinergic cell groups in the zebrafish. Instead, it colocalizes with large populations of GABAergic neurons, including a subpopulation of Adra2a-positive GABAergic cells in the anterior hypothalamic area, neurons that could assume a sleep modulatory role. Our study validates the use of zebrafish for the study of sleep and indicates molecular diversity in sleep regulatory networks across vertebrates.

Arabidopsis Mutants Impaired in Cosuppression

Post-transcriptional gene silencing (cosuppression) results in the degradation of RNA after transcription. A transgenic Arabidopsis line showing post-transcriptional silencing of a 35S–uidA transgene and uidA-specific methylation was mutagenized using ethyl methanesulfonate. Six independent plants were isolated in which uidA mRNA accumulation and β-glucuronidase activity were increased up to 3500-fold, whereas the transcription rate of the 35S–uidA transgene was increased only up to threefold. These plants each carried a recessive monogenic mutation that is responsible for the release of silencing. These mutations defined two genetic loci, called sgs1 and sgs2 (for suppressor of gene silencing). Transgene methylation was distinctly modified in sgs1 and sgs2 mutants. However, methylation of centromeric repeats was not affected, indicating that sgs mutants differ from ddm (for decrease in DNA methylation) and som (for somniferous) mutants. Indeed, unlike ddm and som mutations, sgs mutations were not able to release transcriptional silencing of a 35S–hpt transgene. Conversely, both sgs1 and sgs2 mutations were able to release cosuppression of host Nia genes and 35S–Nia2 transgenes. These results therefore indicate that sgs mutations act in trans to impede specifically transgene-induced post-transcriptional gene silencing.

Arabidopsis histone deacetylase HDA6 is required for maintenance of transcriptional gene silencing and determines nuclear organization of rDNA repeats

Histone acetylation and deacetylation are connected with transcriptional activation and silencing in many eukaryotic organisms. Gene families for enzymes that accomplish these modifications show a surprising multiplicity in sequence and expression levels, suggesting a high specificity for different targets. We show that mutations in Arabidopsis (Arabidopsis thaliana) HDA6, a putative class I histone deacetylase gene, result in loss of transcriptional silencing from several repetitive transgenic and endogenous templates. Surprisingly, total levels of histone H4 acetylation are only slightly affected, whereas significant hyperacetylation is restricted to the nucleolus organizer regions that contain the rDNA repeats. This switch coincides with an increase of histone 3 methylation at Lys residue 4, a modified DNA methylation pattern, and a concomitant decondensation of the chromatin. These results indicate that HDA6 might play a role in regulating activity of rRNA genes, and this control might be functionally linked to silencing of other repetitive templates and to its previously assigned role in RNA-directed DNA methylation.

DNA methylation and chromatin structure affect transcriptional and post-transcriptional transgene silencing in Arabidopsis

In plants, transgenes can be silenced at both the transcriptional [1] and post-transcriptional levels [2]. Methylation of the transgene promoter correlates with transcriptional gene silencing (TGS) [3] whereas methylation of the coding sequence is associated with post-transcriptional gene silencing (PTGS) [4]. In animals, TGS requires methylation and changes in chromatin conformation [5]. The involvement of methylation during PTGS in plants is unclear and organisms with non-methylated genomes such as Caenorhabditis elegans or Drosophila can display RNA interference (RNAi), a silencing process mechanistically related to PTGS [6]. Here, we crossed Arabidopsis mutants impaired in a SWI2/SNF2 chromatin component (ddm1 [7]) or in the major DNA methyltransferase (met1 [8] and E. Richards, personal communication) with transgenic lines in which a reporter consisting of the cauliflower mosaic virus 35S promoter fused to the beta-glucuronidase (GUS) gene (35S-GUS) was silenced by TGS or PTGS. We observed an efficient release of 35S-GUS TGS by both the ddm1 and met1 mutations and stochastic release of 35S-GUS PTGS by these two mutations during development. These results show that DNA methylation and chromatin structure are common regulators of TGS and PTGS.

Circadian and Homeostatic Regulation of Structural Synaptic Plasticity in Hypocretin Neurons

Neurons exhibit rhythmic activity that ultimately affects behavior such as sleep. In living zebrafish larvae, we used time-lapse two-photon imaging of the presynaptic marker synaptophysin in hypocretin/orexin (HCRT) neurons to determine the dynamics of synaptic modifications during the day and night. We observed circadian rhythmicity in synapse number in HCRT axons. This rhythm is regulated primarily by the circadian clock but is also affected by sleep deprivation. Furthermore, NPTX2, a protein implicated in AMPA receptor clustering, modulates circadian synaptic changes. In zebrafish, nptx2b is a rhythmic gene that is mostly expressed in hypothalamic and pineal gland cells. Arrhythmic transgenic nptx2b overexpression (hcrt:NPTX2b) increases synapse number and abolishes rhythmicity in HCRT axons. Finally, hcrt:NPTX2b fish are resistant to the sleep-promoting effects of melatonin. This behavioral effect is consistent with NPTX2b-mediated increased activity of HCRT circuitry. These data provide real-time in vivo evidence of circadian and homeostatic regulation of structural synaptic plasticity.

Sleep–wake regulation and hypocretin–melatonin interaction in zebrafish

In mammals, hypocretin/orexin (HCRT) neuropeptides are important sleep–wake regulators and HCRT deficiency causes narcolepsy. In addition to fragmented wakefulness, narcoleptic mammals also display sleep fragmentation, a less understood phenotype recapitulated in the zebrafish HCRT receptor mutant (hcrtr−/−). We therefore used zebrafish to study the potential mediators of HCRT-mediated sleep consolidation. Similar to mammals, zebrafish HCRT neurons express vesicular glutamate transporters indicating conservation of the excitatory phenotype. Visualization of the entire HCRT circuit in zebrafish stably expressing hcrt:EGFP revealed parallels with established mammalian HCRT neuroanatomy, including projections to the pineal gland, where hcrtr mRNA is expressed. As pineal-produced melatonin is a major sleep-inducing hormone in zebrafish, we further studied how the HCRT and melatonin systems interact functionally. mRNA level of arylalkylamine-N-acetyltransferase (AANAT2), a key enzyme of melatonin synthesis, is reduced in hcrtr−/− pineal gland during the night. Moreover, HCRT perfusion of cultured zebrafish pineal glands induces melatonin release. Together these data indicate that HCRT can modulate melatonin production at night. Furthermore, hcrtr−/− fish are hypersensitive to melatonin, but not other hypnotic compounds. Subthreshold doses of melatonin increased the amount of sleep and consolidated sleep in hcrtr−/− fish, but not in the wild-type siblings. These results demonstrate the existence of a functional HCRT neurons-pineal gland circuit able to modulate melatonin production and sleep consolidation.

Regulation of Hypocretin (Orexin) Expression in Embryonic Zebrafish

Hypocretins/orexins are neuropeptides involved in the regulation of sleep and energy balance in mammals. Conservation of gene sequence, hypothalamic localization of cell bodies, and projection patterns in adult zebrafish suggest that the architecture and function of the hypocretin system are conserved in fish. We report on the complete genomic structure of the zebrafish and Tetraodon hypocretin genes and the complete predicted hypocretin protein sequences from five teleosts. Using whole mount in situ hybridization, we have traced the development of hypocretin cells in zebrafish from onset of expression at 22 h post-fertilization through the first week of development. Promoter elements of similar size from zebrafish and Tetraodon were capable of driving efficient and specific expression of enhanced green fluorescent protein in developing zebrafish embryos, thus defining a minimal promoter region able to accurately mimic the native hypocretin pattern. This enhanced green fluorescent protein expression also revealed a complex pattern of projections within the hypothalamus, to the midbrain, and to the spinal cord. To further analyze the promoter, a series of deletion and substitution constructs were injected into embryos, and resulting promoter activity was monitored in the first week of development. A critical region of 250 base pairs was identified containing a core 13-base pair element essential for hypocretin expression.

Synaptic plasticity in sleep: learning, homeostasis and disease.

Sleep is a fundamental and evolutionarily conserved aspect of animal life. Recent studies have shed light on the role of sleep in synaptic plasticity. Demonstrations of memory replay and synapse homeostasis suggest that one essential role of sleep is in the consolidation and optimization of synaptic circuits to retain salient memory traces despite the noise of daily experience. Here, we review this recent evidence and suggest that sleep creates a heightened state of plasticity, which may be essential for this optimization. Furthermore, we discuss how sleep deficits seen in diseases such as Alzheimers disease and autism spectrum disorders might not just reflect underlying circuit malfunction, but could also play a direct role in the progression of those disorders.

Zebrafish: An integrative system for neurogenomics and neurosciences

Rapid technological advances over the past decade have moved us closer to a high throughput molecular approach to neurobiology, where we see the merging of neurogenetics, genomics, physiology, imaging and pharmacology. This is the case more in zebrafish than in any other model organism commonly used. Recent improvements in the generation of transgenic zebrafish now allow genetic manipulation and live imaging of neuronal development and function in early embryonic, larval, and adult animals. The sequenced zebrafish genome and comparative genomics give unprecedented insights into genome evolution and its relation to genome structure and function. There is now information on embryonic and larval expression of over 12,000 genes and just under 1000 mutant phenotypes. We review the remarkable similarity of the zebrafish genetic blueprint for the nervous system to that of mammals and assess recent technological advances that make the zebrafish a model of choice for elucidating the development and function of neuronal circuitry, transgene-based neuroanatomy, and small molecule neuropharmacology.