Neuronal transcriptome of Cancer borealis
Neuronal transcriptome of Cancer borealis
Total RNA was extrated for the CNS,stomatogastric ganglion, and the single neuron LP for cancer borealis. cDNA libraries were made from these individual tissues and sequenced using the 454 platform.
Despite the many advantages of the crab Cancer borealis as a model organism for cellular and system neuroscience; a major limitation has been the lack of genomic information.  First, we have sequenced ~400,000 ESTs/cDNAs representing >50,000 putative unique gene products including splice forms and non-coding RNAs, which likely correspond to ~50-60% of the total number of protein-coding genes expressed in the nervous system. Specifically, we have constructed three groups of cDNA libraries amenable for both standard EST collection/cloning applications and high-throughout pyrosequencing: (i) the entire CNS of Cancer, (ii) the stomatogastric ganglion (STG), and (iii) individual neurons  representing the pyloric central pattern generator. As a result, we have annotated >6,000 protein coding genes expressed in the Cancer CNS and the STG. A selected list of genes identified in this study represents major groups of transcripts relevant for control of neural excitability, synaptic functions, receptors and adhesion molecules, novel neuropeptides, etc. There are also >50 neuronal genes that are orthologues of defined markers for various neurodegenerative diseases. We have also identified candidates for regulatory molecules known to be involved in Ca-mediated signal transduction pathways linking electrical activity to gene expression as well as developmental genes.  Secondly, we developed a reliable in situ hybridization protocol for mapping studies. To this end, full-length cDNA sequences were obtained for three neuropeptide prohormones (Orcokinin, Red Pigment Concentrating Hormone, and Pigment Dispersing Hormone) that were not previously cloned before from Cancer but are important modulators of the rhythmic activity generated in the STG. We have mapped their expression in whole-mount preparations of the entire brain as well as in the STG and associated ganglia.  Each of the three neuropeptides has its own unique expression profile pattern and most neuropeptide releasing neurons are located outside of the STG supporting their role as mediators of descending modulatory control. Finally, using electrophysiological tests combined with double-labeling, we were able to unambiguously identify two orcokinin-containing neurons in the STG as lateral posterior gastric (LPG) neurons. In summary, the developed molecular resources and the ability to map gene expression should allow detailed study of the genomics of identified cells and circuits and provide a critical bridge between genes, circuits and behavior in Cancer and related decapod species.