Identification of new Anopheles gambiae transcriptional enhancers using a cross-species prediction approach.

The success of transgenic mosquito vector control approaches relies on well-targeted gene expression, requiring the identification and characterization of a diverse set of mosquito promoters and transcriptional enhancers. However, few enhancers have been characterized in Anopheles gambiae to date. Here, we employ the SCRMshaw method we previously developed to predict enhancers in the A. gambiae genome, preferentially targeting vector-relevant tissues such as the salivary glands, midgut and nervous system. We demonstrate a high overall success rate, with at least 8 of 11 (73%) tested sequences validating as enhancers in an in vivo xenotransgenic assay. Four tested sequences drive expression in either the salivary gland or the midgut, making them directly useful for probing the biology of these infection-relevant tissues. The success of our study suggests that computational enhancer prediction should serve as an effective means for identifying A. gambiae enhancers with activity in tissues involved in malaria propagation and transmission.

Ras pathway specificity is determined by the integration of multiple signal-activated and tissue-restricted transcription factors.

Ras signaling elicits diverse outputs, yet how Ras specificity is generated remains incompletely understood. We demonstrate that Wingless (Wg) and Decapentaplegic (Dpp) confer competence for receptor tyrosine kinase-mediated induction of a subset of Drosophila muscle and cardiac progenitors by acting both upstream of and in parallel to Ras. In addition to regulating the expression of proximal Ras pathway components, Wg and Dpp coordinate the direct effects of three signal-activated (dTCF, Mad, and Pointed-functioning in the Wg, Dpp, and Ras/MAPK pathways, respectively) and two tissue-restricted (Twist and Tinman) transcription factors on a progenitor identity gene enhancer. The integration of Pointed with the combinatorial effects of dTCF, Mad, Twist, and Tinman determines inductive Ras signaling specificity in muscle and heart development.

The Toll pathway is required in the epidermis for muscle development in the Drosophila embryo.

The Toll signaling pathway functions in several Drosophila processes, including dorsal-ventral pattern formation and the immune response. Here, we demonstrate that this pathway is required in the epidermis for proper muscle development. Previously, we showed that the zygotic Toll protein is necessary for normal muscle development; in the absence of zygotic Toll, close to 50% of hemisegments have muscle patterning defects consisting of missing, duplicated and misinserted muscle fibers (Halfon, M.S., Hashimoto, C., and Keshishian, H., Dev. Biol. 169, 151-167, 1995). We have now also analyzed the requirements for easter, spätzle, tube, and pelle, all of which function in the Toll-mediated dorsal-ventral patterning pathway. We find that spätzle, tube, and pelle, but not easter, are necessary for muscle development. Mutations in these genes give a phenotype identical to that seen in Toll mutants, suggesting that elements of the same pathway used for Toll signaling in dorsal-ventral development are used during muscle development. By expressing the Toll cDNA under the control of distinct Toll enhancer elements in Toll mutant flies, we have examined the spatial requirements for Toll expression during muscle development. Expression of Toll in a subset of epidermal cells that includes the epidermal muscle attachment cells, but not Toll expression in the musculature, is necessary for proper muscle development. Our results suggest that signals received by the epidermis early during muscle development are an important part of the muscle patterning process.

Targeted gene expression without a tissue-specific promoter: creating mosaic embryos using laser-induced single-cell heat shock.

We have developed a method to target gene expression in the Drosophila embryo to a specific cell without having a promoter that directs expression in that particular cell. Using a digitally enhanced imaging system to identify single cells within the living embryo, we apply a heat shock to each cell individually by using a laser microbeam. A 1- to 2-min laser treatment is sufficient to induce a heat-shock response but is not lethal to the heat-shocked cells. Induction of heat shock was measured in a variety of cell types, including neurons and somatic muscles, by the expression of beta-galactosidase from an hsp26-lacZ reporter construct or by expression of a UAS target gene after induction of hsGAL4. We discuss the applicability of this technique to ectopic gene expression studies, lineage tracing, gene inactivation studies, and studies of cells in vitro. Laser heat shock is a versatile technique that can be adapted for use in a variety of research organisms and is useful for any studies in which it is desirable to express a given gene in only a distinct cell or clone of cells, either transiently or constitutively, at a time point of choice.

The Drosophila toll gene functions zygotically and is necessary for proper motoneuron and muscle development.

Toll is a maternally required Drosophila gene that encodes a transmembrane protein with an important function in embryonic dorsal-ventral patterning. The Toll protein is widely expressed zygotically, but its roles in late embryo-genesis have not been described in detail. We have examined the expression of Toll protein in the late embryonic central nervous system and somatic musculature. Toll is expressed in a dynamic pattern in teh musculature, initially in several muscle fibers in each hemisegment, with a later narrowing of expression to a single muscle fiber pair. Zygotic Toll mutants were used to investigate the development consequences of loss of Toll expression. We found that loss of one or both copies of the Toll gene leads to widespread defects in motoneuron number and muscle patterning. Loss of motoneurons prevents certain muscle fibers from receiving their wild-type innervation. Denervation in the mutants results in collateral sprouting from nearby nerve branches and leads to the appearance of ectopically placed motor endings. The limited expressivity observed suggests that Toll is only one of several genes required for proper motoneuron and muscle specification.

Elements controlling follicular expression of the s36 chorion gene during Drosophila oogenesis.

An 84-bp proximal regulatory protein (PRR) of the Drosophila melanogaster s36 chorion gene is sufficient for directing proper temporal and spatial expression of a reporter gene in three domains of the follicle: anterior, posterior, and main body. Here we show that the fidelity of PRR-directed s36 expression is dependent on the proper dorsal-ventral differentiation of the follicular epithelium, which requires the Drosophila epidermal growth factor receptor homolog. Transgenic analysis of site-directed mutants of the PRR suggests that s36 expression is regulated by the concerted action of multiple positive activators. Several cis-acting transcriptional elements have been identified: some appear to function in a quantitative manner, while others either are essential or appear to regulate expression in particular spatial domains. The approximate locations of these regulatory elements have been defined; some map within sequences that are strongly conserved in widely divergent dipteran species. In fact, the PRR analog of the medfly Ceratitis capitata Ccs36 gene directs expression in a manner similar to the D. melanogaster s36 PRR. We propose a model for transcriptional regulation of s36 based on the prechoriogenic polarization of the follicular epithelium that surrounds the developing egg chamber.

Cellular mechanisms governing synaptic development in Drosophila melanogaster.

The neuromuscular connections of Drosophila are ideally suited for studying synaptic function and development. Hypotheses about cell recognition can be tested in a simple array of pre- and postsynaptic elements. Drosophila muscle fibers are multiply innervated by individually identifiable motoneurons. The neurons express several synaptic cotransmitters, including glutamate, proctolin, and octopamine, and are specialized by their synaptic morphology, neurotransmitters, and connectivity. During larval development the initial motoneuron endings grow extensively over the surface of the muscle fibers, and differentiate synaptic boutons of characteristic morphology. While considerable growth occurs postembryonically, the initial wiring of motoneurons to muscle fibers is accomplished during mid-to-late embryogenesis (stages 15-17). Efferent growth cones sample multiple muscle fibers with rapidly moving filopodia. Upon reaching their target muscle fibers, the growth cones rapidly differentiate into synaptic contacts whose morphology prefigures that of the larval junction. Mismatch experiments show that growth cones recognize specific muscle fibers, and can do so when the surrounding musculature is radically altered. However, when denied their normal targets, motoneurons can establish functional synapses on alternate muscle fibers. Blocking synaptic activity with either injected toxins or ion channel mutants does not derange synaptogenesis, but may influence the number of motor ending processes. The molecular mechanisms governing cellular recognition during synaptogenesis remain to be identified. However, several cell surface glycoproteins known to mediate cellular adhesion events in vitro are expressed by the developing synapses. Furthermore, enhancer detector lines have identified genes with expression restricted to small subsets of muscle fibers and/or motoneurons during the period of synaptogenesis. These observations suggest that in Drosophila a mechanism of target chemoaffinity may be involved in the genesis of stereotypic synaptic wiring.