A Drosophila Axin homolog, Daxin, inhibits Wnt signaling.

The vertebrate Axin protein, the product of the mouse fused gene, binds to beta-catenin to inhibit Wnt signaling. We have identified a homolog of Axin in Drosophila, Daxin. Using double-stranded RNA interference, we generated loss-of-function phenotypes that are similar to overexpression of the Drosophila Wnt gene wingless (wg). Overexpression of Daxin produces phenotypes similar to loss of wg. In addition, we show that Daxin overexpression can modify phenotypes elicited by wg and another Drosophila Wnt gene, DWnt-2. Using immunoprecipitation of endogenous Daxin protein from embryos we show that Daxin interacts with Armadillo and Zeste-white 3. The loss-of-function and overexpression phenotypes show that Daxin, like its mammalian counterpart, acts as a negative regulator of wg/Wnt signaling.     

Cloning of segment polarity gene homologues from the unsegmented brachiopod Terebratulina retusa (Linnaeus).

We have used the polymerase chain reaction (PCR) to amplify, clone and sequence homologues of the Drosophila segment polarity genes engrailed (en), cubitus interruptus Dominant (ciD) and wingless (wg) from the genome of the brachiopod, Terebratulina retusa (Linnaeus). The deduced translation products of brachiopod en and ciD share high levels of sequence identity with their Drosophila homologues. The brachiopod wg-related clone is divergent from Drosophila wg, although clearly a member of the wg/Wnt gene family. These results indicate that structural diversity of Drosophila segment polarity genes has been evolutionarily conserved in a divergent, ancient and unsegmented animal phylum.     

Role of segment polarity genes in the definition and maintenance of cell states in the Drosophila embryo

Segment polarity genes are expressed and required in restricted domains within each metameric unit of the Drosophila embryo. We have used the expression of two segment polarity genes engrailed (en) and wingless (wg) to monitor the effects of segment polarity mutants on the basic metameric pattern. Absence of patched (ptc) or naked (nkd) functions triggers a novel sequence of en and wg patterns. In addition, although wg and en are not expressed on the same cells absence of either one has effects on the expression of the other. These observations, together with an analysis of mutant phenotypes during development, lead us to suggest that positional information is encoded in cell states defined and maintained by the activity of segment polarity gene products.     

Interactions between segment polarity genes and the generation of the segmental pattern in Drosophila.

Although mutations in the segment polarity genes wingless, engrailed, hedgehog, gooseberry and cubitus-interruptusD all affect the region of naked cuticle within each segment of the Drosophila larva, subtle phenotypic differences suggest that these genes play different roles in segmental patterning. In this paper, the regulative interactions between these genes are analysed. They have revealed that the products of most of these genes accomplish more than one function during embryogenesis. Whereas early on a positive feed-back loop involving wg, en and hh maintains the expression of wg and en in the extremes of each parasegment, later on wg and en become independent from each other. en appears to regulate the expression of hh and ptc, while wg depends on gsb and ciD.     

Evolutionary flexibility of pair-rule patterning revealed by functional analysis of secondary pair-rule genes, paired and sloppy-paired in the short-germ insect, Tribolium castaneum

In the Drosophila segmentation hierarchy, periodic expression of pair-rule genes translates gradients of regional information from maternal and gap genes into the segmental expression of segment polarity genes. In Tribolium, homologs of almost all the eight canonical Drosophila pair-rule genes are expressed in pair-rule domains, but only five have pair-rule functions. even-skipped, runt and odd-skipped act as primary pair-rule genes, while the functions of paired (prd) and sloppy-paired (slp) are secondary. Since secondary pair-rule genes directly regulate segment polarity genes in Drosophila, we analyzed Tc-prd and Tc-slp to determine the extent to which this paradigm is conserved in Tribolium. We found that the role of prd is conserved between Drosophila and Tribolium; it is required in both insects to activate engrailed in odd-numbered parasegments and wingless (wg) in even-numbered parasegments. Similarly, slp is required to activate wg in alternate parasegments and to maintain the remaining wg stripes in both insects. However, the parasegmental register for Tc-slp is opposite that of Drosophila slp1. Thus, while prd is functionally conserved, the fact that the register of slp function has evolved differently in the lineages leading to Drosophila and Tribolium reveals an unprecedented flexibility in pair-rule patterning.     

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.     

Analysis of the expression pattern of Mysidium columbiae wingless provides evidence for conserved mesodermal and retinal patterning processes among insects and crustaceans

The Wnt family includes a number of genes, such as wingless ( wg), which encode secreted glycoproteins that function in numerous developmental patterning processes. In order to gain a better understanding of crustacean pattern formation, a wg orthologue was cloned from the malacostracan crustacean Mysidium columbiae(mysid), and the expression pattern of this gene was compared with that of Drosophila wg. Although Drosophila wg is expressed in many developing tissues, such as the ventral neuroectoderm, M. columbiae wg (mcowg)expression is detected within only a subset of these tissues. mcowg is expressed in the dorsal part of each developing segment and within the developing eye, but not within the ventral neuroectoderm. Dorsal wg expression in Drosophila is required for heart and muscle development, and conservation of this dorsal wgexpression pattern suggests that mcowgmay function to pattern these tissues in mysids. Consistent with this, expression of Even-skipped (Eve) protein in heart precursor and muscle cells, which is dependent on Wg signaling in Drosophila, is also conserved in mysids. Within the developing mysid eye, mcowg is expressed in a pattern that is similar to the expression pattern of Drosophila wg in the fly eye disc. In Drosophila,Wg inhibits neural differentiation at the anterior margin of the eye disc and patterns the dorsal/ventral axis of the eye. These data indicate that mcowg may function similarly during mysid eye development. Analysis of mcowgexpression provides molecular evidence suggesting that the processes of heart, muscle, and eye patterning are likely to be conserved among insects and crustaceans.     

Secretion of Wnt ligands requires Evi, a conserved transmembrane protein

Wnt signaling pathways are important for multiple biological processes during development and disease. Wnt proteins are secreted factors that activate target-gene expression in both a short- and long-range manner. Currently, little is known about how Wnts are released from cells and which factors facilitate their secretion. Here, we identify a conserved multipass transmembrane protein, Evenness interrupted (Evi/Wls), through an RNAi survey for transmembrane proteins involved in Drosophila Wingless (Wg) signaling. During development, evi mutants have patterning defects that phenocopy wg loss-of-function alleles and fail to express Wg target genes. evi's function is evolutionarily conserved as depletion of its human homolog disrupts Wnt signaling in human cells. Epistasis experiments and clonal analysis place evi in the Wg-producing cell. Our results show that Wg is retained by evi mutant cells and suggest that evi is the founding member of a gene family specifically required for Wg/Wnt secretion.     

Multiple functions of a Drosophila homeotic gene, zeste-white 3, during segmentation and neurogenesis

Lack of both maternal and zygotic gene activity at the zeste-white 3 (zw3) locus causes severe developmental transformations. Embryos derived from germ cells that lack zw3+ gene activity die during embryogenesis and have a phenotype that is similar to that of embryos mutant for the segment polarity gene naked (nkd). In both nkd and germ line clone-derived zw3 embryos the pattern elements derived from the anterior-most part of each segment, the denticle belts, are deleted. Similar abnormal patterns of the zygotically expressed genes engrailed and Ultrabithorax are detected in both mutants, suggesting that the two genes are involved in the same developmental process. Additionally, the induction of clones of zw3 mutant cells in imaginal discs causes homeotic transformations of noninnervated hair cells into innervated sensory bristles. The multiple roles of zw3 during development and its possible interactions with the zygotic gene nkd are discussed.     

Refinement of wingless expression by a wingless- and notch-responsive homeodomain protein,defective proventriculus

Pattern formation during animal development is often induced by extracellular signaling molecules, known as morphogens, which are secreted from localized sources. During wing development in Drosophila, Wingless (Wg) is activated by Notch signaling along the dorsal-ventral boundary of the wing imaginal disc and acts as a morphogen to organize gene expression and cell growth. Expression of wg is restricted to a narrow stripe by Wg itself, repressing its own expression in adjacent cells. This refinement of wg expression is essential for specification of the wing margin. Here, we show that a homeodomain protein, Defective proventriculus (Dve), mediates the refinement of wg expression in both the wing disc and embryonic proventriculus, where dve expression requires Wg signaling. Our results provide evidence for a feedback mechanism that establishes the wg-expressing domain through the action of a Wg-induced gene product.     

The patterns of wingless,decapentaplegic, and tinman position the Drosophila heart

Two secreted signaling molecules, wingless (wg) and decapentaplegic (dpp), are required to specify the heart in Drosophila. wg and dpp are also required to specify other cell types within the mesoderm and in many other regions of the embryo. Because the spatial patterns of wg and dpp are dynamic, different populations of mesodermal cells are exposed to different combinations of wg and/or dpp at different times. To determine whether the patterns of wg and dpp expression provide unique positional information for the specification of heart precursors, we altered these patterns. Our data suggest that wg and dpp contribute progressively to the elaboration of the expression pattern of the mesoderm-specific homeobox-containing gene tinman (tin), and that the overlap of wg and dpp at an early stage (9) as well as at a later stage (11) in the presence of tin-expressing cells directs cardiac-specific differentiation. Furthermore, ectopic tin expression in the ectoderm at wg/dpp intersects (the primordia of the thoracic imaginal disks) also leads to cardiac-specific differentiation, suggesting that tin confers mesoderm-specificity to the wg/dpp response. We conclude that ectopic heart can be generated by altering the patterns of wg and dpp within the tin-expressing mesoderm, or by ectopic induction of tin within the wg- and dpp-expressing ectoderm.     

Conservation of the expression of Dll, en, and wg in the eye-antennal imaginal disc of stalk-eyed flies

SUMMARY We studied the developmental basis of exaggerated eye span in two species of stalk-eyed flies ( Cyrtodiopsis dalmanni and Sphyracephala beccarri ). These flies have eyes laterally displaced at the end of eyestalks, and males have greatly exaggerated eye span, which they use as a sexual display. To investigate eye span development we have compared eye-antennal disc morphology and the expression of three key regulator genes of Drosophila head development, Distal-less ( Dll ) , engrailed ( en ), and wingless ( wg ), in the stalk-eyed flies and Drosophila . We found great similarity in the basic division of the disc into anterior-antennal and posterior-eye portions and in the general patterning of Dll, en , and wg . Unexpectedly, our results showed that although the eye and antenna are adjacent in adult stalk-eyed flies, their primordia are physically separated by the presence of an intervening region between the anterior and posterior portions of the disc. This region is absent from Drosophila eye-antennal discs. We chose two stalk-eyed fly species that differed in the degree of eyestalk exaggeration but surprisingly we found no corresponding difference in the size of the en-wg expression domains that mark the boundaries of the dorsal head capsule primordia. In summary, our expression data establish the regional identity of the eye-antennal disc and provide a framework from which to address the developmental genetics of hypercephaly.     

Interactions between fused, a segment-polarity gene in Drosophila, and other segmentation genes.

Fused (fu) is a segment polarity gene whose product is maternally required in the posterior part of each segment. To define further the role of fused and determine how it interacts with other segmentation genes, we examined the phenotypes obtained by combining fused with mutations of pair rule, homeotic and other segment polarity loci. When it was possible, we also looked at the distribution of corresponding proteins in fused mutant embryos. We observed that fused-naked (fu;nkd) double mutant embryos display a phenotypic suppression of simple mutant phenotypes: both naked cuticle and denticle belts, which would normally have been deleted by one of the two mutants alone, were restored. In fused mutant embryos, engrailed (en) and wingless (wg) expression was normal until germ band extension, but partially and completely disappeared respectively during germ band retraction. In the fu;nkd double mutant embryo, en was expressed as in nkd mutant at germ band extension, but later this expression was restricted and became normal at germ band retraction. On the contrary, wg expression disappeared as in fu simple mutant embryos. We conclude that the requirements for fused, naked and wingless activities for normal segmental patterning are not absolute, and propose mechanisms by which these genes interact to specify anterior and posterior cell fates.