Asymmetric localization of frizzled and the establishment of cell polarity in the Drosophila wing.

The frizzled gene of Drosophila encodes a transmembrane receptor molecule required for cell polarity decisions in the adult cuticle. In the wing, a single trichome is produced by each cell, which normally points distally. In the absence of frizzled function, the trichomes no longer point uniformly distalward. We report that during cell polarization, the Frizzled receptor is localized to the distal cell edge, probably resulting in asymmetric Frizzled activity across the axis of the cell. Furthermore, Frizzled localization correlates with subsequent trichome polarity, suggesting that it may be an instructive cue in the determination of cell polarity. This differential receptor distribution may represent a novel mechanism for amplifying small differences in signaling activity across the axis of a cell.

Multiple roles for four-jointed in planar polarity and limb patterning.

Insect cuticles have been a model system for the study of planar polarity for many years and a number of genes required for this process have been identified. These genes organise the polarised arrangement of hairs on the legs, wings, thorax, and abdomen of adult Drosophila. It has previously been shown that four-jointed is involved in planar polarity decisions in the eye as well as proximal distal leg and wing development. We now present evidence that four-jointed is expressed in a gradient through the developing wing and show that it is required for planar polarity determination in both the wing and the abdomen. Clones of cells either lacking or ectopically expressing four-jointed cause both autonomous and nonautonomous repolarisation of hairs in these tissues. We propose that the inferred four-jointed expression gradient is important for planar polarity establishment and that local inversions of the gradient by the clones are the probable cause of the observed polarity phenotypes. In addition we observe defects in wing vein development. The subtle phenotypes of mutant flies, and the diverse patterning processes in which it is involved, suggest that four-jointed may act as a modifier of the activity of multiple other signalling factors.

Nuclear signaling by Rac and Rho GTPases is required in the establishment of epithelial planar polarity in the Drosophila eye.

BACKGROUND: The small GTPases Rac and Rho act as cellular switches in many important biological processes. In the fruit fly Drosophila, RhoA participates in the establishment of planar polarity, a process mediated by the receptor Frizzled (Fz). Thus far, analysis of Rac in this process has not been possible because of the absence of mutant Rac alleles. Here, we have investigated the role of Rac and Rho in establishing the polarity of ommatidia in the Drosophila eye. RESULTS: By expressing a dominant negative or a constitutively activated form of Rac1, we interfered specifically with Rac signaling and disrupted ommatidial polarity. The resulting defects were similar to the loss/gain-of-function phenotypes typical of tissue-polarity genes. Through genetic interaction and rescue experiments involving a polarity-specific, loss-of-function dishevelled (dsh) allele, we found that Rac1 acts downstream of Dsh in the Fz signaling pathway, but upstream of, or in parallel to, RhoA. Rac signaled to the nucleus through the Jun N-terminal kinase (JNK) cascade in this process. By generating point mutations in the effector loop of RhoA, we found that RhoA also signals to the nucleus during the establishment of ommatidial polarity. Nevertheless, Rac and RhoA activated transcription of distinct target genes. CONCLUSIONS: Rac is specifically required downstream of Dsh in the Fz pathway. It functions upstream or in parallel to RhoA and both signal to the nucleus, through distinct effectors, to establish planar polarity in the Drosophila eye.

The four-jointed gene is required in the Drosophila eye for ommatidial polarity specification.

BACKGROUND: The Drosophila eye is composed of about 800 ommatidia, each of which becomes dorsoventrally polarised in a process requiring signalling through the Notch, JAK/STAT and Wingless pathways. These three pathways are thought to act by setting up a gradient of a signalling molecule (or molecules) often referred to as the 'second signal'. Thus far, no candidate for a second signal has been identified. RESULTS: The four-jointed locus encodes a type II transmembrane protein that is expressed in a dorsoventral gradient in the developing eye disc. We have analysed the function and regulation of four-jointed during eye patterning. Loss-of-function clones or ectopic expression of four-jointed resulted in strong non-autonomous defects in ommatidial polarity on the dorsoventral axis. Ectopic expression experiments indicated that localised four-jointed expression was required at the time during development when ommatidial polarity was being determined. In contrast, complete removal of four-jointed function resulted in only a mild ommatidial polarity defect. Finally, we found that four-jointed expression was regulated by the Notch, JAK/STAT and Wingless pathways, consistent with it mediating their effects on ommatidial polarity. CONCLUSIONS: The clonal phenotypes, time of requirement and regulation of four-jointed are consistent with it acting in ommatidial polarity determination as a second signal downstream of Notch, JAK/STAT and Wingless. Interestingly, it appears to act redundantly with unknown factors in this process, providing an explanation for the previous failure to identify a second signal.

Polarity determination in the Drosophila eye: a novel role for unpaired and JAK/STAT signaling.

The JAK/STAT signaling pathway is required for many processes including cytokine signaling, hematopoiesis, gliagenesis, and Drosophila segmentation. In this report we present evidence demonstrating that the JAK/STAT pathway is also central to the establishment of planar polarity during Drosophila eye development. We show that a localized source of the pathway ligand, Unpaired, is present at the midline of the developing eye, which is capable of activating the JAK/STAT pathway over long distances. A gradient of JAK/STAT activity across the DV axis of the eye regulates ommatidial polarity via an unidentified second signal. Additionally, localized Unpaired influences the position of the equator via repression of mirror.

Dishevelled activates JNK and discriminates between JNK pathways in planar polarity and wingless signaling.

Frizzled family proteins have been described as receptors of Wnt signaling molecules. In Drosophila, the two known Frizzled proteins are associated with distinct developmental processes. Genesis of epithelial planar polarity requires Frizzled, whereas Dfz2 affects morphogenesis by wingless-mediated signaling. Dishevelled is required in both signaling pathways. Here, we use genetic and overexpression assays to show that Dishevelled activates JNK cascades. Rescue analysis reveals different protein domain requirements in Dishevelled for the two pathways; the C-terminal DEP domain is essential to rescue planar polarity defects and induce JNK signaling. Furthermore, the planar polarity-specific dsh1 allele is mutated in the DEP domain. Our results indicate that different Wnt/Fz signals activate distinct intracellular pathways, and Dishevelled discriminates among them by distinct domain interactions.

Hedgehog is an indirect regulator of morphogenetic furrow progression in the Drosophila eye disc.

Pattern formation in the eye imaginal disc of Drosophila occurs in a wave that moves from posterior to anterior. The anterior edge of this wave is marked by a contracted band of cells known as the morphogenetic furrow, behind which photoreceptors differentiate. The movement of the furrow is dependent upon the secretion of the signalling protein Hedgehog (Hh) by more posterior cells, and it has been suggested that Hh acts as an inductive signal to induce cells to enter a furrow fate and begin differentiation. To further define the role of Hh in this process, we have analysed clones of cells lacking the function of the smoothened (smo) gene, which is required for transduction of the Hh signal and allows the investigation of the autonomous requirement for hh signalling. These experiments demonstrate that the function of hh in furrow progression is indirect. Cells that cannot receive/transduce the Hh signal are still capable of entering a furrow fate and differentiating normally. However, hh is required to promote furrow progression and regulate its rate of movement across the disc, since the furrow is significantly delayed in smo clones.

The role of RhoA in tissue polarity and Frizzled signalling.

The tissue polarity genes of Drosophila are required for correct establishment of planar polarity in epidermal structures, which in the eye is shown in the mirror-image symmetric arrangement of ommatidia relative to the dorsoventral midline. Mutations in the genes frizzled (fz), dishevelled (dsh) and prickle-spiny-legs (pk-sple) result in the loss of this mirror-image symmetry. fz encodes a serpentine receptor-like transmembrane protein required for reception and transmission of a polarity signal. Little else is known of the signalling pathway(s) involved other than that Dsh acts downstream of Fz. We have identified mutations in the Drosophila homologue of RhoA p21 GTPase, and by analysis of their phenotype show that RhoA is required for the generation of tissue polarity. Genetic interactions indicate a role for RhoA in signalling mediated by Fz and Dsh, and furthermore suggest that JNK/SAPK-like kinases are involved. These data are consistent with a Fz/RhoA signalling cascade analogous to the yeast pheromone signalling pathway and that proposed for activation of the serum response factor (SRF) in vertebrate cells.

The regulation of hedgehog and decapentaplegic during Drosophila eye imaginal disc development.

The hedgehog signalling pathway is a conserved mechanism which acts in inductive processes in both vertebrate and invertebrate development to direct growth and patterning. In Drosophila, the secreted Hedgehog protein acts as a signal to induce non-autonomous activation in adjacent cells of either the decapentaplegic or wingless genes (both of which encode growth factor-like molecules), via inactivation of patched activity. In the eye disc, this pathway drives progression of the morphogenetic furrow, while in the wing (and leg and antennal) discs it is required to set up an organising centre along the anteroposterior compartment boundary. We have compared the regulation and function of hedgehog pathway activity in the eye and wing discs, and find that there are significant differences. Whereas in the wing disc, engrailed function is required for hedgehog expression, in the eye disc activation and maintenance of hedgehog expression is achieved independently of engrailed. Regulation of decapentaplegic expression also differs: in the wing disc it is repressed in the anterior compartment by patched and in the posterior compartment by engrailed. In the eye disc, however, it is repressed posterior to the morphogenetic furrow in the absence of either patched or engrailed activity. We conclude that in the eye disc there are novel aspects to hedgehog pathway function. Moreover, engrailed does not play an essential conserved role.

Ommatidial polarity in the Drosophila eye is determined by the direction of furrow progression and local interactions.

The adult eye of Drosophila is a highly ordered structure. It is composed of about 800 ommatidia, each displaying precise polarity. The ommatidia are arranged about an axis of mirror image symmetry, the equator, which lies along the dorsoventral midline of the eye. We use hedgehog pathway mutants to induce ectopic morphogenetic furrows and use these as a tool to investigate the establishment of ommatidial polarity. Our results show that ommatidial clusters are self-organising units whose polarity in one axis is determined by the direction of furrow progression, and which can independently define the position of an equator without reference to the global coordinates of the eye disc.

Regulation of furrow progression in the Drosophila eye by cAMP-dependent protein kinase A.

The earliest physical sign of differentiation in the Drosophila retina is the passage of the morphogenetic furrow across the epithelium of the eye disc. Secreted factors encoded by hedgehog (hh) and decapentaplegic (dpp) have been implicated in propagation of the furrow and the subsequent initiation of photoreceptor differentiation. The morphogenetic furrow initiates at the posterior edge of the third larval instar eye imaginal disc. Its continued progression towards the anterior is believed to depend upon secretion of Hh protein by the differentiating clusters of photoreceptors that emerge posterior to the moving furrow. This progression is marked by the initiation of expression of the transforming growth factor-beta homologue Dpp in cells entering the furrow anteriorly, and loss of dpp expression in cells emerging posteriorly. Although the transmembrane protein encoded by the patched gene has been genetically implicated as the Hh receptor, the intercellular signalling pathways involved in these inductive processes remain uncharacterized. Here we show that the catalytic subunit of cyclic AMP-dependent protein kinase A (Pka-C1) is required for the correct spatial regulation of dpp expression during eye development. Loss of Pka-C1 function is sufficient to produce an ectopic morphogenetic wave marked by premature ectopic photoreceptor differentiation and non-autonomous propagation of dpp expression. Our results indicate that Pka-C1 lies in a signalling pathway that controls the orderly temporal progression of differentiation across the eye imaginal disc.

Characterisation of T48, a target of homeotic gene regulation in Drosophila embryogenesis.

In Drosophila, the homeotic genes encode transcription factors which specify segmental identities during embryogenesis by directly regulating subordinate target genes. A chromatin immunopurification approach has been used to identify targets of the homeotic gene Ultrabithorax (Ubx). We report here the characterisation of a candidate target gene which is positively regulated by the homeotic genes Ubx and abdominal-A in both the epidermis and the midgut visceral mesoderm and encodes a novel transmembrane protein. By mapping the cis-regulatory regions of this gene, we have identified elements responsible for mediating homeotic regulation, and show that they contain in vitro Ubx binding sites. Therefore, we suggest that this is an example of a direct target of regulation by homeotic genes in two germlayers.

Targets of homeotic gene regulation in Drosophila.

We have used a chromatin immunopurification approach to identify target genes regulated by the homeotic gene Ultrabithorax. A monoclonal antibody against the Ultrabithorax gene product is used to immunopurify in vivo Ultrabithorax protein binding sites in embryonic chromatin. The procedure gives an enrichment of sequences with matches to a consensus homeodomain binding site. In one case we have shown that an immunopurified sequence lies within a 4 kb fragment that acts in vivo as a homeotic response element. We anticipate that this approach will enable us to identify further targets, allowing the analysis of their regulation and function. The chromatin immunopurification strategy may be of general application for the identification of direct in vivo targets of DNA-binding proteins.

Targets of homeotic gene control in Drosophila.

The homeotic gene Ultrabithorax (Ubx) encodes homeodomain-containing transcription factors that determine segmental identity in Drosophila. Here, an immunopurification procedure is described that enriches for embryonic chromatin fragments containing binding sites for Ubx protein. In two cases these binding sites are located near embryonic transcription units regulated by the Ubx locus in vivo. Thus, these transcripts may correspond to Ubx target genes involved in elaborating segment-specific developmental pathways.