Unknown,Transcriptomics,Genomics,Proteomics

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A developmental atlas of transcription in the sea hare Aplysia californica

ABSTRACT: The monitoring of global transcription patterns during development is a useful first step to understand mechanisms underlying growth, differentiation and patterning in a given species. However such large scale developmental studies are so far only available from a few selected model organisms such as mouse, Drosophila and the nematode C. elegans. Genomic scale information from the lophotrochozoa is now emerging. The recently characterized neuronal transcriptome of the sea hare Aplysia californica (~200’000 ESTs and ~40,000 unique non-redundant sequences representing about 55-70% of all neuronal transcripts including splice forms and non-coding RNAs) provides new insights and opportunities into problems of molluscan development and specifically neurogenesis. Regulatory genes used in development are recruited for the functions of adult nervous systems such as synaptogenesis, specific wiring in networks and structural changes underlying synaptic plasticity. While this similarity reflects a basic concept in biology, namely the recruitment of the same genes for different functions, it has never been tested on a large scale genomic level. We used representative oligo-arrays constructed from transcripts obtained from the Aplysia nervous system to explore the following two questions: 1) What neuronal genes are differentially expressed during embryonic development of the sea hare Aplysia. 2) What gene expression changes can be observed in correlation with gastrulation, metamorphosis, and larval neurogenesis. For this purpose we hybridized RNA from 3 embryonic (64 cells, gastrula, trochophore) and 4 larval stages (pre-hatching, post-hatching, pre-metamorphosis, postmetamorphosis) against a reference sample consisting of equal contributions from all stages. Our analysis revealed that all embryonic and larval stages can be distinguished based on their transcription profiles. A comparison of the Aplysia atlas with similar studies in Drosophila and mouse reveals interesting differences. We identified several new transcription factors which are differentially expressed during early embryonic development. Various other transcripts involved in metabolism and differentiation were characterized. These genes can be candidate targets for understanding neuronal growth, synaptogenesis and memory mechanisms. We conclude that the microarray technology provides us with a powerful tool for efficient survey and functional annotation of the neuronal transcriptome in Aplysia. We used a reference design for this study where we hybridized each of the 8 developmental stages of Aplysia (cleavage, gastrula, trochophore, first veliger, hatching veliger, pre-metamorphosis (stage 6), post-metamorphosis (stage 7), post-metamorphosis 60 hours (pm60)

ORGANISM(S): Aplysia californica

SUBMITTER: Andreas Heyland

PROVIDER: E-GEOD-14941 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:The monitoring of global transcription patterns during development is a useful first step to understand mechanisms underlying growth, differentiation and patterning in a given species. However such large scale developmental studies are so far only available from a few selected model organisms such as mouse, Drosophila and the nematode C. elegans. Genomic scale information from the lophotrochozoa is now emerging. The recently characterized neuronal transcriptome of the sea hare Aplysia californica (~200’000 ESTs and ~40,000 unique non-redundant sequences representing about 55-70% of all neuronal transcripts including splice forms and non-coding RNAs) provides new insights and opportunities into problems of molluscan development and specifically neurogenesis. Regulatory genes used in development are recruited for the functions of adult nervous systems such as synaptogenesis, specific wiring in networks and structural changes underlying synaptic plasticity. While this similarity reflects a basic concept in biology, namely the recruitment of the same genes for different functions, it has never been tested on a large scale genomic level. We used representative oligo-arrays constructed from transcripts obtained from the Aplysia nervous system to explore the following two questions: 1) What neuronal genes are differentially expressed during embryonic development of the sea hare Aplysia. 2) What gene expression changes can be observed in correlation with gastrulation, metamorphosis, and larval neurogenesis. For this purpose we hybridized RNA from 3 embryonic (64 cells, gastrula, trochophore) and 4 larval stages (pre-hatching, post-hatching, pre-metamorphosis, postmetamorphosis) against a reference sample consisting of equal contributions from all stages. Our analysis revealed that all embryonic and larval stages can be distinguished based on their transcription profiles. A comparison of the Aplysia atlas with similar studies in Drosophila and mouse reveals interesting differences. We identified several new transcription factors which are differentially expressed during early embryonic development. Various other transcripts involved in metabolism and differentiation were characterized. These genes can be candidate targets for understanding neuronal growth, synaptogenesis and memory mechanisms. We conclude that the microarray technology provides us with a powerful tool for efficient survey and functional annotation of the neuronal transcriptome in Aplysia.

Project description:The complexity of events associated with age-related memory loss (ARML) cannot be overestimated. The problem is further complicated by the enormous diversity of neurons in the CNS and even synapses of one neuron within a neural circuit. Large-scale single-neuron analysis is not only challenging but mostly impractical for any model currently used in ARML. We simply do not know: do all neurons and synapses age differently or are some neurons (or synapses) more resistant to aging than others? What is happening in any given neuron while it undergoes “normal” aging? What are the genomic changes that make aging apparently irreversible? What would be the balance between neuron-specific vs global genome-wide changes in aging? In the proposed paper we address these questions and develop a new model to study the entire scope of genomic and epigenomic regulation in aging at the resolution of single functionally characterized cells and even cell compartments. In particular, the mollusc Aplysia californica has been implemented as a powerful paradigm in addressing fundamental questions of the neurobiology of aging. The proposed manuscript will consist of four parts. First, we will provide an introduction to Aplysia as a representative of the largest superclade of bilaterian animals (Lophotrochozoa). Aplysia has a short lifespan of 220-300 days with a well-characterized life cycle and characterized phenomenology of aging. Most importantly, Aplysia possess the largest nerve cells in the entire animal kingdom (only eggs are larger); these cells can be uniquely identified and mapped in terms of their well-defined interactions with other neurons forming relatively simpler neural circuits underlying several stereotypic and learned behaviors. Second, we have identified in Aplysia more that a hundred neurological- and age-related genes that were lost in other established invertebrate models (such as Drosophila and C. elegans). The proposed long-term regulatory age-related mechanisms include a high level of conservation among many epigenetic processes known to be lost in nematodes and flies with extremely short lifecycles and particularly derived genomes. We also identify and cloned more than 30 evolutionarily conserved homologs of genes involved in Alzheimer’s, Parkinson’s and Huntington’s diseases as well as age-related hormones. Third, we performed genome-wide analysis of expression patterns of more than 55,000 unique transcripts by comparing two different identified cholinergic neurons (R2 and LPl1) among young and aged animals. This direct single neuron genomic analysis indicates that there are significant cell-specific changes in gene-expression profiles as a function of aging. We estimated that only ~10-20% of genes that are differently expressed in the aging brain are common for all neuronal types - the remaining 80% are neuron-specific (i.e. found in aging neurons of one but not another type). The list of “common aging genes” includes components of insulin growth factor pathways, cell bioenergetics, telomerase-associated proteins, antioxidant enzymes, water channels and estrogen receptors. The rest were neuron-specific gene products (including apoptosis-related proteins, Alzheimer-related genes, growth factors and their receptors, ionic channels, transcription factors and more than 120 identified proteins known to be involved in neurodevelopment and synaptogenesis). Surprisingly, even two different identified cholinergic motoneurons age differently and each of them has a unique subset of genes differentially expressed in older animals. Fourth, we showed that the activity of the entire genome and associated epigenomic modifications (e.g. DNA methylation, histone dynamics) can be efficiently monitored within a single Aplysia neuron and can be modified as a function of aging in a neuron-specific manner including selective histones and histone-modifying enzymes and DNA methylation-related enzymes. This genome-wide analysis of aging allows us to propose novel mechanisms of active DNA demethylation and cell-specific methylation as well as regional relocation of RNAs as three key processes underlying age-related memory loss. These mechanisms tune the dynamics of long-term chromatin remodeling, control weakening and the loss of synaptic connections in aging. At the same time, our genomic tests revealed evolutionarily conserved gene clusters in the Aplysia genome associated with senescence and regeneration (e.g. apoptosis- and redox- dependent processes, insulin signaling, etc.). This is a reference design experiment with all samples being compared to one CNS from Aplysia. Two cholinergic neurons (R2 and LPl1), two ages (young and old), two arrays (AAA and DAA), three biological replicates each sample type. Two direct comparison experiments were also performed. One with young and old abdominal ganglion and the other with young and old R2.

Project description:Larval settlement and metamorphosis is a vital transition period for marine invertebrates and can have far-reaching effects on the ecology and evolution of a species. To explore the molecular mechanisms of this critical process in a non-model organism, the tropical abalone Haliotis asinina, we employed cDNA microarray methods. By comparing gene expression profiles through mid to late larval development and metamorphosis, we identified 144 genes as likely candidates for a role in competence and/or metamorphosis. Gene characterization showed that ~60% of these were significantly similar to previously described genes from other taxa, while ~40% had no significant similarities to any known genes. A high 49.3% of genes were gastropod- or abalone-specific, but none appear to be Lophotrochozoan-specific, despite the fact that metamorphosis is thought to have had a separate origin in this group. Based on temporal expression profiles, the differentially expressed larval and postlarval genes can be clustered into 5 categories that reveal there are strikingly different transcriptional patterns occurring during this phase of development. Some classes of gene activation are contingent upon exogenous cues and correlate with the initiation of settlement and metamorphosis. Importantly, there is also extensive gene activity associated with the endogenous attainment of competence, which occurs prior to, and independent of, the exogenous induction of settlement. Our results show that as the haliotid veliger larva matures, it requires the coordinated regulation of temporally different batteries of genes involved in a wide range of physiological and developmental processes associated with colonisation of the benthos. Although the signalling pathways operating at metamorphosis may be conserved across the animal kingdom, it appears they are regulating the expression of novel genes specific to abalone, gastropods and molluscs during H. asinina metamorphosis. Keywords: timecourse; metamorphosis; marine ecology Each microarray slide contained a different combination of 2 of the 9 developmental stages used in the experiment (66 hpf, 78 hpf, 90 hpf, 108 hpf, 120 hpf, 144 hpf, 12 hpi, 24 hpi, 48 hpi). Each developmental stage was subjected to 4 hybridisations â amounting to 4 technical replicates per stage - in a loop design (Churchill 2002; Oleksiak et al. 2002). This design led to raw data consisting of 72 measurements - 9 stages with 8 replicates (including 2 replicates per chip) - for each of 5541 spots.

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A developmental atlas of transcription in the sea hare Aplysia californica

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