In Drosophila, the retinal determination network comprises a set of nuclear factors whose loss-of-function phenotypes often include the complete or near total elimination of the developing eye. These genes also share the ability of being able to induce ectopic eye formation when forcibly expressed in nonretinal tissues such as the antennae, legs, halteres, wings, and genitals. However, it appears that the ability to redirect and transform tissue fates is limited; not all tissues and cell populations can be forced into adopting an eye fate. In this report, we demonstrate that ectopic eye formation by teashirt and its paralog tiptop, a potential new eye specification gene, is restricted to the developing antennae. Of interest, tiptop appears to be a more effective inducer of retinal formation than teashirt. A genetic screen for interacting proteins failed to identify paralog-specific relationships suggesting that the differences between these two genes may be attributed instead to structural differences between the duplicates. We also demonstrate that in addition to being expressed in coincident patterns within the developing eye, both paralogs are transcribed at very similar levels.

The ancient mechanisms that caused developmental gene regulatory networks to diversify among distantly related taxa are not well understood. Here we use ancestral protein reconstruction, biochemical experiments, and developmental assays of transgenic animals carrying reconstructed ancestral genes to investigate how the transcription factor Bicoid (Bcd) evolved its central role in anterior-posterior patterning in flies. We show that most of Bcd’s derived functions are attributable to evolutionary changes within its homeodomain (HD) during a phylogenetic interval >140 million years ago. A single substitution from this period (Q50K) accounts almost entirely for the evolution of Bcd’s derived DNA specificity in vitro. In transgenic embryos expressing the reconstructed ancestral HD, however, Q50K confers activation of only a few of Bcd’s transcriptional targets and yields a very partial rescue of anterior development. Adding a second historical substitution (M54R) confers regulation of additional Bcd targets and further rescues anterior development. These results indicate that two epistatically interacting mutations played a major role in the evolution of Bcd’s controlling regulatory role in early development. They also show how ancestral sequence reconstruction can be combined with in vivo characterization of transgenic animals to illuminate the historical mechanisms of developmental evolution.

The K50 (lysine at amino acid position 50) homeodomain (HD) protein Orthodenticle (Otd) is critical for anterior patterning and brain and eye development in most metazoans. In , another K50HD protein, Bicoid (Bcd), has evolved to replace Otd's ancestral function in embryo patterning. Bcd is distributed as a long-range maternal gradient and activates transcription of a large number of target genes, including Otd and Bcd bind similar DNA sequences in vitro, but how their transcriptional activities are integrated to pattern anterior regions of the embryo is unknown. Here we define three major classes of enhancers that are differentially sensitive to binding and transcriptional activation by Bcd and Otd. Class 1 enhancers are initially activated by Bcd, and activation is transferred to Otd via a feed-forward relay (FFR) that involves sequential binding of the two proteins to the same DNA motif. Class 2 enhancers are activated by Bcd and maintained by an Otd-independent mechanism. Class 3 enhancers are never bound by Bcd, but Otd binds and activates them in a second wave of zygotic transcription. The specific activities of enhancers in each class are mediated by DNA motif variants preferentially bound by Bcd or Otd and the presence or absence of sites for cofactors that interact with these proteins. Our results define specific patterning roles for Bcd and Otd and provide mechanisms for coordinating the precise timing of gene expression patterns during embryonic development.

In the Drosophila eye the retinal determination (RD) network controls both tissue specification and cell proliferation. Mutations in network members result in severe reductions in the size of the eye primordium and the transformation of the eye field into head cuticle. The zinc-finger transcription factor Teashirt (Tsh) plays a role in promoting cell proliferation in the anterior most portions of the eye field as well as in inducing ectopic eye formation in forced expression assays. Tiptop (Tio) is a recently discovered paralog of Tsh. It is distributed in an identical pattern to Tsh within the retina and can also promote ectopic eye development. In a previous study we demonstrated that Tio can induce ectopic eye formation in a broader range of cell populations than Tsh and is also a more potent inducer of cell proliferation. Here we have focused on understanding the molecular and biochemical basis that underlies these differences. The two paralogs are structurally similar but differ in one significant aspect: Tsh contains three zinc finger motifs while Tio has four such domains. We used a series of deletion and chimeric proteins to identify the zinc finger domains that are selectively used for either promoting cell proliferation or inducing eye formation. Our results indicate that for both proteins the second zinc finger is essential to the proper functioning of the protein while the remaining zinc finger domains appear to contribute but are not absolutely required. Interestingly, these domains antagonize each other to balance the overall activity of the protein. This appears to be a novel internal mechanism for regulating the activity of a transcription factor. We also demonstrate that both Tsh and Tio bind to C-terminal Binding Protein (CtBP) and that this interaction is important for promoting both cell proliferation and eye development. And finally we report that the physical interaction that has been described for Tsh and Homothorax (Hth) do not occur through the zinc finger domains.

The retinal determination (RD) network in Drosophila comprises fourteen known nuclear proteins that include DNA binding proteins, transcriptional co-activators, kinases and phosphatases. The composition of the network varies considerably throughout the animal kingdom, with the network in several basal insects having fewer members and with vertebrates having potentially significantly higher numbers of retinal determination genes. One important contributing factor for the variation in gene number within the network is gene duplication. For example, ten members of the RD network in Drosophila are derived from duplication events. Here we present an analysis of the coding regions of the five pairs of duplicate genes from within the retinal determination network of several different Drosophila species. We demonstrate that there is differential selection across the coding regions of all RD genes. Additionally, some of the most significant differences in ratios of non-silent to silent site substitutions (dN/dS) between paralog pairs are found within regions that have no ascribed function. Previous structure/function analyses of several duplicate genes have identified areas within one gene that contain novel activities when compared to its paralog. The evolutionary analysis presented here identifies these same areas in the paralogs as being under high levels of relaxed selection. We suggest that sequence divergence between paralogs and selection signatures can be used as a reasonable predictor of functional changes in rapidly evolving motifs.