Multiple enhancers contribute to spatial but not temporal complexity in the expression of the proneural gene, amos

Background: The regulation of proneural gene expression is an important aspect of neurogenesis. In the study of the Drosophila proneural genes, scute and atonal, several themes have emerged that contribute to our understanding of the mechanism of neurogenesis. First, spatial complexity in proneural expression results from regulation by arrays of enhancer elements. Secondly, regulation of proneural gene expression occurs in distinct temporal phases, which tend to be under the control of separate enhancers. Thirdly, the later phase of proneural expression often relies on positive autoregulation. The control of these phases and the transition between them appear to be central to the mechanism of neurogenesis. We present the first investigation of the regulation of the proneural gene, amos. Results: Amos protein expression has a complex pattern and shows temporally distinct phases, in common with previously characterised proneural genes. GFP reporter gene constructs were used to demonstrate that amos has an array of enhancer elements up- and downstream of the gene, which are required for different locations of amos expression. However, unlike other proneural genes, there is no evidence for separable enhancers for the different temporal phases of amos expression. Using mutant analysis and site-directed mutagenesis of potential Amos binding sites, we find no evidence for positive autoregulation as an important part of amos control during neurogenesis. Conclusion: For amos, as for other proneural genes, a complex expression pattern results from the sum of a number of simpler sub- patterns driven by specific enhancers. There is, however, no apparent separation of enhancers for distinct temporal phases of expression, and this correlates with a lack of positive autoregulation. For scute and atonal, both these features are thought to be important in the mechanism of neurogenesis. Despite similarities in function and expression between the Drosophila proneural genes, amos is regulated in a fundamentally different way from scute and atonal.