Project description:To form operative neuronal networks neurons connect to their synaptic partners by axonal outgrowth guided by the movement of the axon tip, a specialized structure known as the growth cone (GC), which travels along stereotypical paths by sensing extracellular guidance cues. Interestingly, GCs contain complex transcriptomes and different guidance cues can rapidly regulate the translation of specific mRNAs which help to remodel the GC cytoskeleton thus steering axon navigation. However, the full landscape of proteomic changes elicited by different cues is not known and the precise protein synthesis (PS)-dependent molecular mechanisms underlying GC repulsive and attractive turning remain largely elusive. Here we have investigated the cue-induced changes in the nascent proteome by pulse Stable Isotope Labeling by Amino acids in Cell culture (pSILAC) in somaless developing retinal axons derived from Xenopus laevis primary cultures. The experimental approach presented major challenges due to the limited quantity of axonal material (~2 μg per condition) and to the desired rapid (5-15 min) time-scale (no previous work has accomplished SILAC with a pulse less than 20 min). To overcome these difficulties we used a superior protocol, termed Single-Pot Solid- Phase-enhanced Sample Preparation, based on ultrasensitive paramagnetic bead technology which allows high proteomic sample recovery from sub-microgram amounts of material. By employing this method we were able to identify up to 500 newly synthesized proteins after only 5 min of pSILAC. Subsequently, we successfully validated the purity and viability of the axonal samples and the reliability of the method. Finally, to characterize and quantify the global patterns of the axonal translational changes in response to specific guidance cues and to unravel the switch between repulsive and attractive responses, we carried out pSILAC in the presence of different repulsive cues and pharmacological reagents that convert repulsion into attraction. Our results uncovered a surprisingly high level of translational activity within the axonal compartment, even in basal conditions. Remarkably, comparative analysis revealed many changes in the nascent proteome in response to different cues, some distinct and some common. Interestingly, several proteins showed cue-specific downregulation. Taken together, our findings suggest a comprehensive GC PS-dependent chemotropic model and novel cue-specific molecular mechanisms possibly underlying the integration of chemotropic responses.

Project description:Genome-wide analysis of gene expression has documented differential gene expression between closely related species in plants or animals and nonadditive gene expression in hybrids and allopolyploids compared to the parents. In Arabidopsis, 15-43% of genes are expressed differently between the related species, A. thaliana and A. arenosa, and the majority of them are nonadditvely expressed (differently from mid-parent value) in allotetraploids relative to the parents. The gene expression variation is associated with transcriptional and posttranscriptional mechanisms. Although transcriptional regulation by epigenetic mechanisms plays a role in nonadditive gene expression, the effects of posttranscriptional regulation on nonadditive gene expression is largely unknown. Here we reported a comprehensive genome-wide analysis of mRNA decay in Arabidopsis allotetraploids using oligo gene microarrays. Among ~26,000 annotated genes on the arrays, over 1% of genes showed a rapid decay rate with an estimated half-life of less than 60 min, and these genes are known as genes with unstable transcripts (GUTs). Interestingly, 30% of GUTs matched the nonadditively expressed genes, and their expression levels were significantly negatively correlated. These nonadditively expressed GUTs are highly enriched with a 2-8-fold increase compared to the proportion of all genes in the Gene Ontology (GO) classifications in response to stress, abiotic and biotic stimulus, signal transduction, and transcription. The data are consistent with the enrichment of nonadditvely-expressed genes in those categories in allopolyploids of Arabidopsis, wheat and cotton. Interestingly, the GUTs include some key transcription factors including circadian clock regulators, suggesting a mechanism for rapid response to light signalling and clock regulation in allotetraploids. These data collectively suggest an important role of mRNA stability in nonadditive expression of homoeologous genes in allopolyploids.

Project description:Genome-wide analysis of gene expression has documented differential gene expression between closely related species in plants or animals and nonadditive gene expression in hybrids and allopolyploids compared to the parents. In Arabidopsis, 15-43% of genes are expressed differently between the related species, A. thaliana and A. arenosa, and the majority of them are nonadditvely expressed (differently from mid-parent value) in allotetraploids relative to the parents. The gene expression variation is associated with transcriptional and posttranscriptional mechanisms. Although transcriptional regulation by epigenetic mechanisms plays a role in nonadditive gene expression, the effects of posttranscriptional regulation on nonadditive gene expression is largely unknown. Here we reported a comprehensive genome-wide analysis of mRNA decay in Arabidopsis allotetraploids using oligo gene microarrays. Among ~26,000 annotated genes on the arrays, over 1% of genes showed a rapid decay rate with an estimated half-life of less than 60 min, and these genes are known as genes with unstable transcripts (GUTs). Interestingly, 30% of GUTs matched the nonadditively expressed genes, and their expression levels were significantly negatively correlated. These nonadditively expressed GUTs are highly enriched with a 2-8-fold increase compared to the proportion of all genes in the Gene Ontology (GO) classifications in response to stress, abiotic and biotic stimulus, signal transduction, and transcription. The data are consistent with the enrichment of nonadditvely-expressed genes in those categories in allopolyploids of Arabidopsis, wheat and cotton. Interestingly, the GUTs include some key transcription factors including circadian clock regulators, suggesting a mechanism for rapid response to light signalling and clock regulation in allotetraploids. These data collectively suggest an important role of mRNA stability in nonadditive expression of homoeologous genes in allopolyploids. A total of 8 slides were used for comparison (0 min vs. 120 min), including two biological and four technical replications. Each biological replication consists of two technical replications (four slides) that were hybridized with reverse-labeled probes.

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Personal and transgenerational cues are nonadditive at the phenotypic and molecular level

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