Systematic Identification of Genes that Regulate Neuronal Wiring in the Drosophila Visual System

Forward genetic screens in model organisms are an attractive means to identify those genes involved in any complex biological process, including neural circuit assembly. Although mutagenesis screens are readily performed to saturation, gene identification rarely is, being limited by the considerable effort generally required for positional cloning. Here, we apply a systematic positional cloning strategy to identify many of the genes required for neuronal wiring in the Drosophila visual system. From a large-scale forward genetic screen selecting for visual system wiring defects with a normal retinal pattern, we recovered 122 mutations in 42 genetic loci. For 6 of these loci, the underlying genetic lesions were previously identified using traditional methods. Using SNP-based mapping approaches, we have now identified 30 additional genes. Neuronal phenotypes have not previously been reported for 20 of these genes, and no mutant phenotype has been previously described for 5 genes. The genes encode a variety of proteins implicated in cellular processes such as gene regulation, cytoskeletal dynamics, axonal transport, and cell signalling. We conducted a comprehensive phenotypic analysis of 35 genes, scoring wiring defects according to 33 criteria. This work demonstrates the feasibility of combining large-scale gene identification with large-scale mutagenesis in Drosophila, and provides a comprehensive overview of the molecular mechanisms that regulate visual system wiring

Epigenetic memory of an active gene state depends on histone H3.3 incorporation into chromatin in the absence of transcription

The remarkable stability of gene expression in somatic cells is exemplified by the way memory of an active gene state is retained when an endoderm cell nucleus is transplanted to an enucleated egg. Here we analyse the mechanism of a similar example of epigenetic memory. We find that memory can persist through 24 cell divisions in the absence of transcription and applies to the expression of the myogenic gene MyoD in non-muscle cell lineages of nuclear transplant embryos. We show that memory is not explained by the methylation of promoter DNA. However, we demonstrate that epigenetic memory correlates with the association of histone H3.3 with the MyoD promoter in embryos that display memory but not in those where memory has been lost. The association of a mutated histone H3.3 (H3.3 E4, which lacks the methylatable H3.3 lysine 4) with promoter DNA eliminates memory, indicating a requirement of H3.3 K4 for memory. We also show that overexpression of H3.3 can enhance memory in transplanted nuclei. We therefore conclude that the association of histone H3.3 with the MyoD promoter makes a necessary contribution to this example of memory. Hence, we suggest that epigenetic memory helps to stabilize gene expression in normal development; it might also help to account for the inefficient reprogramming in some transplanted nuclei.link_to_subscribed_fulltex