David Foronda

A simple and efficient method to identify replacements of P-lacZ by P-Gal4 lines allows obtaining Gal4 insertions in the bithorax complex of Drosophila

The functional replacement of one gene product by another one is a powerful method to study specificity in development and evolution. In Drosophila, the Gal4/UAS method has been used to analyze in vivo such functional substitutions. To this aim, Gal4 lines that inactivate a gene and reproduce its expression pattern are required, and they can be frequently obtained by replacing pre-existing P-lacZ lines with such characteristics. We have devised a new method to quickly identify replacements of P-lacZ lines by P-Gal4 lines, and applied it successfully to obtain Gal4 insertions in the Ultrabithorax and Abdominal-B Hox genes. We have used these lines to study the functional replacement of a Hox gene by another one. Our experiments confirm that the abdominal-A gene can replace Ultrabithorax in haltere development but that it cannot substitute for Abdominal-B in the formation of the genitalia.

Function and specificity of Hox genes

The Hox genes specify different structures along the anteroposterior axis of bilaterians. They code for transcription factors including a conserved domain, the homeodomain, that binds DNA. The specificity of Hox function is determined by each gene controlling the expression of different groups of downstream genes. These can be other transcription factors, elements in signaling pathways or realizator genes that carry out basic cellular functions. In regulating specific targets, the Hox genes interact with members of signaling pathways and with other proteins, thus forming part of gene networks that contribute to the modification of homologous structures or to the creation of new organs.

Requirement of abdominal-A and Abdominal-B in the developing genitalia of Drosophila breaks the posterior downregulation rule

The genitalia of Drosophila derive from the genital disc and require the activity of the Abdominal-B (Abd-B) Hox gene. This gene encodes two different proteins, Abd-B M and Abd-B R. We show here that the embryonic genital disc, like the larval genital disc, is formed by cells from the eighth (A8), ninth (A9) and tenth (A10) abdominal segments, which most likely express the Abd-B M, Abd-B R and Caudal products, respectively. Abd-B m is needed for the development of A8 derivatives such as the external and internal female genitalia, the latter also requiring abdominal-A (abd-A), whereas Abd-B r shapes male genitalia (A9 in males). Although Abd-B r represses Abd-B m in the embryo, in at least part of the male A9 such regulation does not occur. In the male A9, some Abd-B m–r– or Abd-B r– clones activate Distal-less and transform part of the genitalia into leg or antenna. In the female A8, many Abd-B m–r– mutant clones produce similar effects, and also downregulate or eliminate abdominal-A expression. By contrast, although Abd-B m is the main or only Abd-B transcript present in the female A8, Abd-B m– clones induced in this primordium do not alter Distal-less or abd-A expression, and transform the A8 segment into the A4. The relationship between Abd-B and abd-A in the female genital disc is opposite to that of the embryonic epidermis, and contravenes the rule that posteriorly expressed Hox genes downregulate more anterior ones.

Polycomb-dependent Ultrabithorax Hox gene silencing induced by high Ultrabithorax levels in Drosophila

The Ultrabithorax (Ubx) gene of Drosophila specifies the third thoracic and first abdominal segments. Ubx expression is controlled by several mechanisms, including negative regulation by its own product. We show here that if Ubx expression levels are inappropriately elevated, overriding the auto-regulatory control, a permanent repression of Ubx is established. This continuous repression becomes independent of the presence of exogenous Ubx and leads to the paradoxical result that an excess of Ubx results in a phenotype of Ubx loss. The mechanism of permanent repression depends on Polycomb-group genes. Absence of endogenous Ubx transcription when Ubx levels are highly elevated probably activates Polycomb complexes on a Polycomb response element located in the Ubx major intron. This, in turn, brings about permanent repression of Ubx transcription. Similar results are obtained with the gene engrailed, showing that this mechanism of permanent repression may be a general one for genes with negative auto-regulation when levels of expression are transitorily elevated.

Drosophila Hox and Sex-Determination Genes Control Segment Elimination through EGFR and extramacrochetae Activity

The formation or suppression of particular structures is a major change occurring in development and evolution. One example of such change is the absence of the seventh abdominal segment (A7) in Drosophila males. We show here that there is a down-regulation of EGFR activity and fewer histoblasts in the male A7 in early pupae. If this activity is elevated, cell number increases and a small segment develops in the adult. At later pupal stages, the remaining precursors of the A7 are extruded under the epithelium. This extrusion requires the up-regulation of the HLH protein Extramacrochetae and correlates with high levels of spaghetti-squash, the gene encoding the regulatory light chain of the non-muscle myosin II. The Hox gene Abdominal-B controls both the down-regulation of spitz, a ligand of the EGFR pathway, and the up-regulation of extramacrochetae, and also regulates the transcription of the sex-determining gene doublesex. The male Doublesex protein, in turn, controls extramacrochetae and spaghetti-squash expression. In females, the EGFR pathway is also down-regulated in the A7 but extramacrochetae and spaghetti-squash are not up-regulated and extrusion of precursor cells is almost absent. Our results show the complex orchestration of cellular and genetic events that lead to this important sexually dimorphic character change.

Dpp of posterior origin patterns the proximal region of the wing

The decapentaplegic (dpp) gene encodes a long-range morphogen that plays a key role in the patterning of the wing imaginal disc of Drosophila (Nellen, D., Burke, R., Struhl, G. and Basler, K. 1996. Direct and long-range action of a DPP morphogen gradient. Cell 85, 357-368.). The current view is that dpp is transcriptionally active in a narrow band of anterior compartment cells close to the anterio-posterior (A/P) compartment border. Once the Dpp protein is synthesised, it travels across the A/P border and diffuses forming concentration gradients in the two compartments (reviewed in Lawrence, P.A., Struhl, G. 1996. Morphogens, compartments, and pattern: lessons from drosophila? Cell 85, 951-961.). We have found a new site of dpp expression in the posterior wing compartment which appears during the third larval period. This source of Dpp signal generates a local gradient of Dpp pathway activity, which is independent of that originating in the anterior compartment. We show that this posterior tier of Dpp activity is functionally required for normal wing development: the elimination of dpp expression in the posterior compartment results in defective adult wings in which pattern elements such as the alula and much of the axillary cord are not formed. Moreover, these structures develop normally in the absence of anterior dpp expression. Thus the normal wing pattern requires distinct Dpp organizer activities in the anterior and posterior compartments. We further show that, unlike the anterior dpp expression domain, the posterior one is not dependent on Hedgehog activity but is dependant on the activity of the IRO complex gene mirror. Since there is a similar expression in the haltere disc, we suggest that this late appearing posterior Dpp activity may be an attribute of dorsal thoracic discs.

The Drosophila Hox gene Ultrabithorax controls appendage shape by regulating extracellular matrix dynamics

ABSTRACT Although the specific form of an organ is frequently important for its function, the mechanisms underlying organ shape are largely unknown. In Drosophila, the wings and halteres, homologous appendages of the second and third thoracic segments, respectively, bear different forms: wings are flat, whereas halteres are globular, and yet both characteristic shapes are essential for a normal flight. The Hox gene Ultrabithorax (Ubx) governs the difference between wing and haltere development, but how Ubx function in the appendages prevents or allows flat or globular shapes is unknown. Here, we show that Ubx downregulates Matrix metalloproteinase 1 (Mmp1) expression in the haltere pouch at early pupal stage, which in turn prevents the rapid clearance of Collagen IV compared with the wing disc. This difference is instrumental in determining cell shape changes, expansion of the disc and apposition of dorsal and ventral layers, all of these phenotypic traits being characteristic of wing pouch development. Our results suggest that Ubx regulates organ shape by controlling Mmp1 expression, and the extent and timing of extracellular matrix degradation. Summary: The Drosophila Hox gene Ultrabithorax, which distinguishes halteres from wings, downregulates Matrix metalloproteinase 1 expression to maintain ECM protein levels and so form the haltere globular shape instead of the flat wing.

The elimination of an adult segment by the Hox gene Abdominal-B.

Hox gene activity leads to morphological diversity of organs or structures in different species. One special case of Hox function is the elimination of a particular structure. The Abdominal-B Hox gene of Drosophila melanogaster provides an example of such activity, as this gene suppresses the formation of the seventh abdominal segment in the adult. This elimination occurs only in males, and is characteristic of more advanced Diptera. The elimination requires the differential expression or activity of genes that are downstream Abdominal-B, or that work together with it, and which regulate cell proliferation or cell extrusion. Here, we review the mechanisms responsible for such elimination and provide some new data on processes taking place within this segment.

A genetic strategy to obtain P-Gal4 elements in the Drosophila Hox genes.

The Drosophila Gal4/UAS system allows the expression of any gene of interest in restricted domains. We devised a genetic strategy, based on the P-element replacement and UAS-y (+) techniques, to generate Gal4 lines inserted in Hox genes of Drosophila that are, at the same time, mutant for the resident genes. This makes possible to express different wild-type or mutant Hox proteins in the precise domains of Hox gene expression, and thus to test the functional value of these proteins in mutant rescue experiments.

02-P010 Abdominal-B, homothorax and dsx control of growth in the Drosophila genitalia

multifunctional regulatory nuclear compartment that, besides hosting ribosome biogenesis, regulates cellular growth, cell death, and the cell cycle. The Myc transcription factor stimulates tissue growth, in part by activating the expression of genes required for ribosome biogenesis. By qRT-PCR we show that Myc is necessary and sufficient for transcriptional activation of vito expression. Our results raise the hypothesis that the integration of signalling and nutritional cues that control cell growth in Drosophila requires Vito to regulate nucleolar function and architecture.