Roles of wingless in patterning the larval epidermis of Drosophila.

The larval epidermis of Drosophila shows a stereotyped segmentally repeating pattern of cuticular structures. Mutants deficient for the wingless gene product show highly disrupted patterning of the larval cuticle. We have manipulated expression of the wg gene product to assess its role in this patterning process. We present evidence for four distinct phases of wg function in epidermal cells: (1) an early requirement in engrailed-expressing cells to establish and maintain stable expression of en, (2) a discrete period when wg and en gene products act in concert to generate positional values in the anterior portion of the ventral segment and all values of the dorsal and lateral epidermis, (3) a progressive function (dependent on prior interaction with the en-expressing cells) in conferring positional values to cells within the posterior portion of the segment, and (4) a late continuous requirement for maintaining some ventral positional values.     

Secretion and movement of wingless protein in the epidermis of the Drosophila embryo.

The segment polarity gene wingless encodes a cysteine rich protein which is essential for pattern formation in Drosophila. Using polyclonal antibodies against the product of the wingless gene, we demonstrate that this protein is secreted in the embryo and that it is taken up by neighbouring cells. The protein can be found two or three cell diameters away from the cells in which it is synthesized. We discuss the possible mechanisms which are responsible for this spatial distribution and its regulation during embryogenesis.     

The consequences of ubiquitous expression of the wingless gene in the Drosophila embryo.

The segment polarity gene wingless has an essential function in cell-to-cell communication during various stages of Drosophila development. The wingless gene encodes a secreted protein that affects gene expression in surrounding cells but does not spread far from the cells where it is made. In larvae, wingless is necessary to generate naked cuticle in a restricted part of each segment. To test whether the local accumulation of wingless is essential for its function, we made transgenic flies that express wingless under the control of a hsp70 promoter (HS-wg flies). Uniform wingless expression results in a complete naked cuticle, uniform armadillo accumulation and broadening of the engrailed domain. The expression patterns of patched, cubitus interruptus Dominant and Ultrabithorax follow the change in engrailed. The phenotype of heatshocked HS-wg embryos resembles the segment polarity mutant naked, suggesting that embryos that overexpress wingless or lack the naked gene enter similar developmental pathways. The ubiquitous effects of ectopic wingless expression may indicate that most cells in the embryo can receive and interpret the wingless signal. For the development of the wild-type pattern, it is required that wingless is expressed in a subset of these cells.     

Domain mapping of tube, a protein essential for dorsoventral patterning of the Drosophila embryo.

The tube protein plays an essential role in the signal transduction pathway that establishes dorsoventral polarity in the Drosophila melanogaster embryo. Characterization of each of four tube mutants revealed a substitution or insertion in the amino-terminal half of the protein. This portion of the tube protein is also evolutionarily conserved, as demonstrated by isolation and sequencing of the Drosophila virilis tube gene. Moreover, RNA microinjection assays and germline transformation experiments demonstrated that the amino-terminal domain alone provides substantial levels of gene function: constructs encoding only the amino-terminal domain restore dorsoventral polarity to embryos lacking any maternal tube function. In the carboxyterminal domain, sequence conservation is concentrated in the five octapeptide repeats. Although the repeat-containing domain by itself provides no rescue of the tube maternal effect phenotype, it is necessary for wild-type levels of tube activity. This domain is thus likely to play an ancillary role in axis formation.     

DmMyD88 controls dorsoventral patterning of the Drosophila embryo

MyD88 is an adapter protein in the signal transduction pathway mediated by interleukin-1 (IL-1) and Toll-like receptors. A Drosophila homologue of MyD88 (DmMyD88) was recently shown to be required for the Toll-mediated immune response. In Drosophila, the Toll pathway was originally characterized for its role in the dorsoventral patterning of the embryo. We found that, like Toll, DmMyD88 messenger RNA is maternally supplied to the embryo. Here we report the identification of a new mutant allele of DmMyD88, which generates a protein lacking the carboxy-terminal extension, normally located downstream of the Toll/IL-1 receptor domain. Homozygous mutant female flies lay dorsalized embryos that are rescued by expression of a transgenic DmMyD88 complementary DNA. The DmMyD88 mutation blocks the ventralizing activity of a gain-of-function Toll mutation. These results show that DmMyD88 encodes an essential component of the Toll pathway in dorsoventral pattern formation.     

Secretory and inductive properties of Drosophila wingless protein in Xenopus oocytes and embryos.

Like its vertebrate homologues, Xenopus wnt-8 and murine wnt-1, we find that Drosophila wingless (wg) protein causes axis duplication when overexpressed in embryos of Xenopus laevis after mRNA injection. In many cases, the secondary axes contain eyes and cement glands, which reflect the induction of the most dorsoanterior mesodermal type, prechordal mesoderm. We show that the extent of axis duplication is dependent on the embryonic site of expression, with ventral expression leading to a more posterior point of axis bifurcation. The observed duplications are due to de novo generation of new axes as shown by rescue of UV-irradiated embryos. The true dorsal mesoderm-inducing properties of wg protein are indicated by its ability to generate extensive duplications after mRNA injection into D-tier cells of 32-cell embryos. As revealed by lineage mapping, the majority of these D cell progeny populate the endoderm; injections into animal blastomeres at this stage are far less effective in inducing secondary axes. However, when expressed in isolated animal cap explants, wg protein induces only ventral mesoderm, unless basic fibroblast growth factor is added, whereupon induction of muscle and occasionally notochord is seen. We conclude that in intact embryos, wg acts in concert with other factors to cause axis duplication. Immunolocalisation studies in embryos indicate that wg protein remains localised to the blastomeres synthesizing it and has a patchy, often perinuclear distribution within these cells, although some gets to the surface. In oocytes, the pool of wg protein is entirely intracellular and relatively unstable. When the polyanion suramin is added, most of the intracellular material is recovered in the external medium.     

Bristle patterning in Drosophila.

The 5000 bristles that protrude from the cuticle of a Drosophila adult function as either mechanosensors or chemosensors, and they are arranged in surprisingly intricate patterns. Development of the patterns appears to involve five stages: (1) establishment of a coordinate system of 'positional information'; (2) partitioning of the epidermis into areas where bristles either can or cannot originate; (3) selection of one or more bristle mother cells within each permissible area; (4) suppression of bristle development in the neighborhood of each mother cell; and (5) differentiation of the mother cell to produce four or more descendant cells, each of which forms part of the bristle apparatus. Some of the genes that control these events participate in more than one stage, and others play key roles in seemingly unrelated developmental pathways, including embryonic neurogenesis, body segmentation, and sex determination.     

Anteroposterior patterning of the epidermis by inductive influences from the vegetal hemisphere cells in the ascidian embryo.

Patterning along the anteroposterior axis is a critical step during animal embryogenesis. Although mechanisms of anteroposterior patterning in the neural tube have been studied in various chordates, little is known about those of the epidermis. To approach this issue, we investigated patterning mechanisms of the epidermis in the ascidian embryo. First we examined expression of homeobox genes (Hrdll-1, Hroth, HrHox-1 and Hrcad) in the epidermis. Hrdll-1 is expressed in the anterior tip of the epidermis that later forms the adhesive papillae, while Hroth is expressed in the anterior part of the trunk epidermis. HrHox-1 and Hrcad are expressed in middle and posterior parts of the epidermis, respectively. These data suggested that the epidermis of the ascidian embryo is patterned anteroposteriorly. In ascidian embryogenesis, the epidermis is exclusively derived from animal hemisphere cells. To investigate regulation of expression of the four homeobox genes in the epidermis by vegetal hemisphere cells, we next performed hemisphere isolation and cell ablation experiments. We showed that removal of the vegetal cells before the late 16-cell stage results in loss of expression of these homeobox genes in the animal hemisphere cells. Expression of Hrdll-1 and Hroth depends on contact with the anterior-vegetal (the A-line) cells, while expression of HrHox-1 and Hrcad requires contact with the posterior-vegetal (the B-line) cells. We also demonstrated that contact with the vegetal cells until the late 32-cell stage is sufficient for animal cells to express Hrdll-1, Hroth and Hrcad, while longer contact is necessary for HrHox-1 expression. Contact with the A-line cells until the late 32-cell stage is also sufficient for formation of the adhesive papillae. Our data indicate that the epidermis of the ascidian embryo is patterned along the anteroposterior axis by multiple inductive influences from the vegetal hemisphere cells and provide the first insight into mechanisms of epidermis patterning in the chordate embryos.     

Drosophila APC2 is a cytoskeletally-associated protein that regulates wingless signaling in the embryonic epidermis.

The tumor suppressor adenomatous polyposis coli (APC) negatively regulates Wingless (Wg)/Wnt signal transduction by helping target the Wnt effector beta-catenin or its Drosophila homologue Armadillo (Arm) for destruction. In cultured mammalian cells, APC localizes to the cell cortex near the ends of microtubules. Drosophila APC (dAPC) negatively regulates Arm signaling, but only in a limited set of tissues. We describe a second fly APC, dAPC2, which binds Arm and is expressed in a broad spectrum of tissues. dAPC2's subcellular localization revealed colocalization with actin in many but not all cellular contexts, and also suggested a possible interaction with astral microtubules. For example, dAPC2 has a striking asymmetric distribution in neuroblasts, and dAPC2 colocalizes with assembling actin filaments at the base of developing larval denticles. We identified a dAPC2 mutation, revealing that dAPC2 is a negative regulator of Wg signaling in the embryonic epidermis. This allele acts genetically downstream of wg, and upstream of arm, dTCF, and, surprisingly, dishevelled. We discuss the implications of our results for Wg signaling, and suggest a role for dAPC2 as a mediator of Wg effects on the cytoskeleton. We also speculate on more general roles that APCs may play in cytoskeletal dynamics.     

Wounded cells drive rapid epidermal repair in the early Drosophila embryo

Epithelial tissues are protective barriers that display a remarkable ability to repair wounds. Wound repair is often associated with an accumulation of actin and nonmuscle myosin II around the wound, forming a purse string. The role of actomyosin networks in generating mechanical force during wound repair is not well understood. Here we investigate the mechanisms of force generation during wound repair in the epidermis of early and late Drosophila embryos. We find that wound closure is faster in early embryos, where, in addition to a purse string around the wound, actomyosin networks at the medial cortex of the wounded cells contribute to rapid wound repair. Laser ablation demonstrates that both medial and purse-string actomyosin networks generate contractile force. Quantitative analysis of protein localization dynamics during wound closure indicates that the rapid contraction of medial actomyosin structures during wound repair in early embryos involves disassembly of the actomyosin network. By contrast, actomyosin purse strings in late embryos contract more slowly in a mechanism that involves network condensation. We propose that the combined action of two force-generating structures—a medial actomyosin network and an actomyosin purse string—contributes to the increased efficiency of wound repair in the early embryo.     

A role for wingless in an early pupal cell death event that contributes to patterning the Drosophila eye

Programmed cell death (PCD) is utilized in a wide variety of tissues to refine structure in developing tissues and organs. However, little is understood about the mechanisms that, within a developing epithelium, combine signals to selectively remove some cells while sparing essential neighbors. One popular system for studying this question is the developing Drosophila pupal retina, where excess interommatidial support cells are removed to refine the patterned ommatidial array. In this paper, we present data indicating that PCD occurs earlier within the pupal retina than previously demonstrated. As with later PCD, this death is dependent on Notch activity. Surprisingly, altering Drosophila Epidermal Growth Factor Receptor or Ras pathway activity had no effect on this death. Instead, our evidence indicates a role for Wingless signaling to provoke this cell death. Together, these signals regulate an intermediate step in the selective removal of unneeded interommatidial cells that is necessary for a precise retinal pattern.     

Drosophila APC2 and APC1 play overlapping roles in wingless signaling in the embryo and imaginal discs

The regulation of signal transduction plays a key role in cell fate choices, and its disregulation contributes to oncogenesis. This duality is exemplified by the tumor suppressor APC. Originally identified for its role in colon tumors, APC family members were subsequently shown to negatively regulate Wnt signaling in both development and disease. The analysis of the normal roles of APC proteins is complicated by the presence of two APC family members in flies and mice. Previous work demonstrated that, in some tissues, single mutations in each gene have no effect, raising the question of whether there is functional overlap between the two APCs or whether APC-independent mechanisms of Wnt regulation exist. We addressed this by eliminating the function of both Drosophila APC genes simultaneously. We find that APC1 and APC2 play overlapping roles in regulating Wingless signaling in the embryonic epidermis and the imaginal discs. Surprisingly, APC1 function in embryos occurs at levels of expression nearly too low to detect. Further, the overlapping functions exist despite striking differences in the intracellular localization of the two APC family members.