A UAS site substitution approach to the in vivo dissection of promoters: interplay between the GATAb activator and the AEF-1 repressor at a Drosophila ecdysone response unit.

An ecdysone response unit (EcRU) directs the expression of the Fat body protein 1 (Fbp1) gene in the third instar larval Drosophila fat body. The tissue-specific activity of this regulatory element necessitates the binding of both the ligand-activated EcR/USP ecdysone receptor and GATAb. To analyze the role played by GATAb in the regulation of the Fbp1 EcRU activity, we have replaced the GATA-binding sites GBS1, GBS2 and GBS3 in the Fbp1 EcRU with UAS sites for the yeast GAL4 activator and tested the activity of the mutagenized Fbp1 EcRUs in transgenic lines, either in the presence or absence of ubiquitously expressed GAL4. Our results reveal that GATAb plays two distinguishable roles at the Fbp1 EcRU that contribute to the tissue-specific activity of this regulatory element. On the one hand, GATAb mediates a fat body-specific transcriptional activation. On the other hand, it antagonizes specifically in the fat body a ubiquitous repressor that maintains the Fbp1 EcRU in an inactive state, refractory to activation by GAL4. We identified this repressor as AEF-1, a factor previously shown to be involved in the regulation of the Drosophila Adh and yp1-yp2 genes. These results show that, for a functional dissection of complex promoter-dependent regulatory pathways, the replacement of specific regulatory target sites by UAS GAL4 binding sites is a powerful alternative to the widely used disruption approach.

Insect oenocytes: a model system for studying cell-fate specification by Hox genes.

During insect development, morphological differences between segments are controlled by the Hox gene family of transcription factors. Recent evidence also suggests that variation in the regulatory elements of these genes and their downstream targets underlies the evolution of several segment-specific morphological traits. This review introduces a new model system, the larval oenocyte, for studying the evolution of fate specification by Hox genes at single-cell resolution. Oenocytes are found in a wide range of insects, including species using both the short and the long germ modes of development. Recent progress in our understanding of the genetics and cell biology of oenocyte development in the fruitfly Drosophila melanogaster is discussed. In the D. melanogaster embryo, the formation of this cell type is restricted to the first 7 abdominal segments and is under Hox gene control. Oenocytes delaminate from the dorsal ectoderm of A1-A7 in response to an induction that involves the epidermal growth factor receptor (EGFR) signalling pathway. Although the receptor itself is required in the presumptive oenocytes, its ligand Spitz (Spi) is secreted by a neighbouring chordotonal organ precursor (COP). Thus, in dorsal regions, local signalling from this component of the developing peripheral nervous system induces the formation of oenocytes. In contrast, in lateral regions of the ectoderm, Spi signal from a different COP induces the formation of secondary COPs in a homeogenetic manner. This dorsoventral difference in the fate induced by Spi ligand is controlled by a prepattern in the responding ectoderm that requires the Spalt (Sal) transcription factor. Sal protein is expressed in the dorsal but not lateral ectoderm and acts as a competence modifier to bias the response to Spi ligand in favour of the oenocyte fate. We discuss a recently proposed model that integrates the roles of Sal and the EGFR pathway in oenocyte/chordotonal organ induction. This model should provide a useful starting point for future comparative studies of these ectodermal derivatives in other insects.

spalt-dependent switching between two cell fates that are induced by the Drosophila EGF receptor.

Signaling from the EGF receptor (EGFR) can trigger the differentiation of a wide variety of cell types in many animal species. We have explored the mechanisms that generate this diversity using the Drosophila peripheral nervous system. In this context, Spitz (SPI) ligand can induce two alternative cell fates from the dorsolateral ectoderm: chordotonal sensory organs and non-neural oenocytes. We show that the overall number of both cell types that are induced is controlled by the degree of EGFR signaling. In addition, the spalt (sal) gene is identified as a critical component of the oenocyte/chordotonal fate switch. Genetic and expression analyses indicate that the SAL zinc-finger protein promotes oenocyte formation and supresses chordotonal organ induction by acting both downstream and in parallel to the EGFR. To explain these findings, we propose a prime-and-respond model. Here, sal functions prior to signaling as a necessary but not sufficient component of the oenocyte prepattern that also serves to raise the apparent threshold for induction by SPI. Subsequently, sal-dependent SAL upregulation is triggered as part of the oenocyte-specific EGFR response. Thus, a combination of SAL in the responding nucleus and increased SPI ligand production sets the binary cell-fate switch in favour of oenocytes. Together, these studies help to explain how one generic signaling pathway can trigger the differentiation of two distinct cell types.

Dynamic expression of broad-complex isoforms mediates temporal control of an ecdysteroid target gene at the onset of Drosophila metamorphosis.

Metamorphosis in Drosophila melanogaster is orchestrated by the steroid hormone ecdysone, which triggers a cascade of primary-response transcriptional regulators and secondary effector genes during the third larval instar and prepupal periods of development. The early ecdysone-response Broad-Complex (BR-C) gene, a key regulator of this cascade, is defined by three complementing functions (rbp, br, and 2Bc) and encodes several distinct zinc-finger-containing isoforms (Z1 to Z4). Using isoform-specific polyclonal antibodies we observe in the fat body a switch in BR-C isoform expression from the Z2 to the other three isoforms during the third instar. We show that the 2Bc(+) function that corresponds presumably to the Z3 isoform is required for the larval fat body-specific expression of a transgenic construct (AE) in which the lacZ gene is under the control of the ecdysone-regulated enhancer and minimal promoter of the fat body protein 1 (Fbp1) gene. Using hs(BR-C) transgenes, we demonstrate that overexpression of Z1, Z3, or Z4, but not Z2, is able to rescue AE activity with faithful tissue specificity in a BR-C null (npr1) genetic context, demonstrating a partial functional redundancy between Z1, Z3, and Z4 isoforms. We also show that continuous overexpression of Z2 during the third instar represses AE, while conversely, expression of Z3 earlier than its normal onset induces precocious expression of the construct. This finding establishes a tight correlation between the dynamic pattern of expression of the BR-C isoforms and their individual repressive or inductive roles in AE regulation. Altogether our results demonstrate that the balance between BR-C protein isoforms in the fat body mediates, in part, the precise timing of the ecdysone activation of the AE construct but does not modulate its tissue specificity.

Dual requirement for the EcR/USP nuclear receptor and the dGATAb factor in an ecdysone response in Drosophila melanogaster.

The EcR/USP nuclear receptor controls Drosophila metamorphosis by activating complex cascades of gene transcription in response to pulses of the steroid hormone ecdysone at the end of larval development. Ecdysone release provides a ubiquitous signal for the activation of the receptor, but a number of its target genes are induced in a tissue- and stage-specific manner. Little is known about the molecular mechanisms involved in this developmental modulation of the EcR/USP-mediated pathway. Fbp1 is a good model of primary ecdysone response gene expressed in the fat body for addressing this question. We show here that the dGATAb factor binds to three target sites flanking an EcR/USP binding site in a 70-bp enhancer that controls the tissue and stage specificity of Fbp1 transcription. We demonstrate that one of these sites and proper expression of dGATAb are required for specific activation of the enhancer in the fat body. In addition, we provide further evidence that EcR/USP plays an essential role as a hormonal timer. Our study provides a striking example of the integration of molecular pathways at the level of a tissue-specific hormone response unit.