Multi-level regulation of even-skipped stripes by the ubiquitous factor Zelda.

The zinc-finger protein Zelda (Zld) is a key activator of zygotic transcription in early Drosophila embryos. Here, we study Zld-dependent regulation of the seven-striped pattern of the pair-rule gene even-skipped (eve). Individual stripes are regulated by discrete enhancers that respond to broadly distributed activators; stripe boundaries are formed by localized repressors encoded by the gap genes.

In Situ Hybridization as a Method to Examine Gene Regulatory Activity In Vivo.

Transcription factor-enhancer binding events are among the most well-studied protein-DNA interactions, allowing researchers to determine mechanisms of transcriptional activation or repression during development. While large-scale ChIP-sequence datasets, together with computational predictions and chromatin accessibility data, yield information on potential transcription factor binding activities, reporter gene assays provide measurable information on whether these binding activities are functional in particular cell types during development. Here, we present a detailed protocol to examine enhancer activity in Drosophila embryos using cloning, transgenesis, and in situ hybridization.

The power of the (imperfect) palindrome: Sequence-specific roles of palindromic motifs in gene regulation.

In human languages, a palindrome reads the same forward as backward (e.g., 'madam').

Ancient mechanisms for the evolution of the bicoid homeodomain's function in fly development.

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 feed-forward relay integrates the regulatory activities of Bicoid and Orthodenticle via sequential binding to suboptimal sites.

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.

A dissection of the teashirt and tiptop genes reveals a novel mechanism for regulating transcription factor activity.

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.

Gene regulation: piecing together the puzzle of enhancer evolution.

The sequences of some gene regulatory elements diverge considerably, even between closely related species. A detailed analysis of the fast-evolving sparkling enhancer in Drosophila now identifies key compensatory mechanisms and 'grammar' elements that are critical for maintaining functional integrity.

Differential selection within the Drosophila retinal determination network and evidence for functional divergence between paralog pairs.

The retinal determination (RD) network in Drosophila comprises 14 known nuclear proteins that include DNA-binding proteins, transcriptional coactivators, 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 RD genes. One important contributing factor for the variation in gene number within the network is gene duplication.

Restriction of ectopic eye formation by Drosophila teashirt and tiptop to the developing antenna.

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.