Genetic Approaches for Synaptogenesis

Worms move in a stereotypical sinusoidal wave pattern. The excitatory cholinergic and inhibitory GABAergic NMJs coordinate the wave propagation. Disruption of NMJ development and function leads to a wide range of uncoordinated behaviors. Early screens for C. elegans uncoordinated behavior yielded a large number of genes important for nervous system development and function, known as the "unc" genes11. Subsequent decades of molecular analyses have revealed that many of these genes are important players at the NMJ, a few of which contribute to synapse development.

Direct visual inspection of fluorescently tagged synapse molecules has allowed for targeted identification of synapse genes. Genetic screens using SNB::GFP have played a pivotal role in the identification of new genes specific to synaptogenesis. Most screens have used neuron-type specific promoters to drive SNB::GFP expression in type D motor neurons6,12, mechanosensory neurons13, chemosensory neurons14, and hermaphrodite-specific neurons (HSNs)15. Mutations isolated using this approach generally have wide effects on many or all synapses, but often cause few behavioral abnormalities. Moreover, the particular synaptic phenotype differs in a synapse-type specific manner. Although these mutations were isolated based on abnormal patterns of the SV component SNB::GFP, the characterizations thus far have shown that most of these genes do not encode integral SV components, but rather encode a wide range of proteins involved in synapse development and function, validating the SNB::GFP screening approach. Recently, SYD-2::GFP has been used to screen for mutants defective in active zone assembly7. This screen yielded alleles of genes previously identified in SNB::GFP screens, and also identified new genes involved in active zone formation.

In addition to traditional forward genetic screens, RNA interference (RNAi) is a powerful reverse genetic approach useful for characterizing gene function. The reverse genetic approach of RNAi allows for the targeted analysis of specific genes or unbiased whole genome screens. However, poorly understood mechanisms reduce RNAi effectiveness in neurons. This problem was overcome by the identification of a number of C. elegans mutants that are more susceptible to RNAi. These include mutants in the 5'exonuclease, eri-1, the RNA-dependent RNA polymerase, rrf-3, and a subset of retinoblastoma mutants16,17. These mutants have enabled potent RNAi effects on neuronal targets. A large-scale screen using an RNAi-sensitized strain has led to the identification of 132 genes that were not previously implicated in synaptic development and transmission8.

4.2.2. Drosophila

Initial Drosophila screens for synapse target recognition defects used antibodies specific to a subset of NMJs. Many individual terminal branches and synapses are clearly visible in preparations stained by this method. These antibody screens identified a number of mutants with normal gross CNS morphology, but defective outgrowth or target recognition9.

The generation of a modular misexpression system in Drosophila allowed screening based on gain-of-function effects at specific times and in specific cells. The modular misexpression system is based on Gal4 transactivation of a mobile enhancer and promoter that "targets" random endogenous genes for expression18. This approach was used to assess high-level expression of genes in moto neurons19. Defects in axon guidance or synaptogenesis in Drosophila larvae were analyzed using a pan neuronal GFP reporter. This screen identified new genes in a wide range of classes, including kinases and phosphatases, GTPases and their regulatory proteins, RNA-binding proteins, and transcriptional regulators.

The reverse genetic RNAi approach is used in Drosophila to analyze gene function. RNAi was used to identify genes involved in stabilization of the Drosophila NMJ20. Synaptic sites develop the SSR postsynaptic specialization only in the presence of a presynaptic terminal. If the terminal retracts, the SSR is temporarily maintained leaving a "footprint" that contains SSR markers, but no presynaptic markers. By screening for synaptic footprints in RNAi-treated flies, this screen successfully identified necessary cytoskeletal components for synapse stabilization. A GAL4/UAS RNAi expression system has been developed to allow tissue specific knockdown of target genes21. This approach allows dissection of presynaptic versus postsynaptic effects at specific synapses.

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