Autonomous requirements for the segment polarity gene armadillo during Drosophila embryogenesis

Embryos hemizygous for armadillo produce a "segment polarity" phenotype in which the naked posterior two-thirds of each segment is replaced by denticles with reversed polarity. Small patches of homozygous arm cells induced by mitotic recombination also form such denticles, indicating that the changes in cellular fate observed in homozygous arm embryos are autonomous at the level of single cells. Clonally derived arm patches do not, however, show the characteristic arm polarity reversals, arguing that this feature of the phenotype depends on cell interactions in fully mutant embryos. Few, if any, clones were found in the posterior-most regions of the naked cuticle, and none were found in the posterior compartments of the thorax.     

Insect epidermis: polarity patterns after grafting result from divergent cell adhesions between host and graft tissue

Insect epidermal cells display planar polarity (i.e. polarity in the plane of the cell sheet) by secreting oriented cuticular denticles and bristles before each moult. We investigate how cell polarities in an abdominal segment are uniformly oriented towards the posterior of the animal. Recently we have shown for the cotton bug Dysdercus that, in 180 degrees-rotated grafts pretreated with colchicine, graft cells tend to adopt the orientation prevailing in surrounding host cells via an intermediate stage with outward oriented denticles (Nübler-Jung and Grau, 1987). Here we show that, in untreated grafts that were transposed along the anteroposterior segment axis, the denticles also always tend to point outwards. This independence of the polarity pattern from the direction of transposition is compatible neither with a gradient model for polarity control, nor with the assumption that epidermal cells orient according to the local sequence of distinctly differentiated cells. Instead we found that outward orientation of graft denticles correlates with an elongation of epidermal cells along a host-graft border with divergent cell adhesiveness. We therefore propose that outward orientation in a graft results from a combination of two factors: epidermal cells stretch along an interface with divergent cell adhesiveness, and they form a denticle perpendicular to their long axis. By analogy, the normal anteroposterior orientation of denticles in a segment may result because epidermal cells tend to elongate parallel to the segment boundary and to form denticles perpendicular to this mediolateral cell elongation, i.e. along the anteroposterior segment axis.     

Insect epidermis: disturbance of supracellular tissue polarity does not prevent the expression of cell polarity

Summary The insect integument displays planar tissue polarity in the uniform orientation of polarized cuticular structures. In a body segment, for example, the denticles and bristles produced by the constituent epidermal cells point posteriorly. Colchicine can abolish this uniform orientation while still allowing individual cells to form orientated cuticular structures and thereby to express cell polarity. This suggests that an individual cell in a sheet can establish planar polarity without reference to some kind of covert supracellular cue (such as a morphogen gradient) in the epidermis as a whole. The results also indicate that colchicine interferes — directly or indirectly — with the mechanisms involved in aligning the polarity axes of individual cells into a common orientation, thereby generating supracellular or tissue polarity.     

Oroshigane, a New Segment Polarity Gene of Drosophila Melanogaster, Functions in Hedgehog Signal Transduction

Here we describe a new segment polarity gene of Drosophila melanogaster, oroshigane (oro). Identified as a dominant enhancer of Bar (B), oro is also recessive embryonic lethal, and homozygous oro embryos show variable substitution of naked cuticle with denticles. These patterns are distinctly similar to those of hedgehog (hh) and wingless (wg) embryos, which indicates that oro functions in determining embryonic segment polarity. Evidence that oro function is involved in Hh signal transduction during embryogenesis is provided by its genetic interactions with the segment polarity genes patched (ptc) and fused (fu). Furthermore, ptc(IN) is a dominant suppressor of the oro embryonic lethal phenotype, suggesting a close and dose-dependent relationship between oro and ptc in Hh signal transduction. oro function is also required in imaginal development. The oro(1) allele significantly reduces decapentaplegic (dpp), but not hh, expression in the eye imaginal disc. Furthermore, oro enhances the fu(1) wing phenotype in a dominant manner. Based upon the interactions of oro with hh, ptc, and fu, we propose that the oro gene plays important roles in Hh signal transduction.     

hunchback, a gene required for segmentation of an anterior and posterior region of the Drosophila embryo

The locus hunchback (hb) is a member of the gap class of segmentation genes of Drosophila. A number of X-ray-induced deletions locate the hb locus at the chromosomal site 85A3-B1, to the right of the pink locus, which maps in the same interval. A total of 14 EMS and 3 X-ray-induced hb alleles have been studied. Homozygous mutant embryos show deletions of segments in two separate regions. In the six strong alleles, the labium and all three thoracic segments are deleted anteriorly while posteriorly the 8th abdominal segment and adjacent parts of the 7th abdominal segment are lacking. The eight weak alleles show smaller deletions both in the thoracic and posterior abdominal region. In the weakest allele only part of the mesothorax is deleted. Three hb alleles produce a homoeotic transformation: superimposed on a strong or weak deletion phenotype, head or thoracic segments are transformed into abdominal segments, respectively. This suggests that hb might also be involved in the regulation of genes in the Bithorax complex (BX-C). Fate mapping of the normal-appearing segments in strong mutant embryos using the UV-laser beam ablation technique (Lohs-Schardin et al., 1979) shows that these segments arise from the normal blastoderm regions. The mutant phenotype can be recognized soon after the onset of gastrulation in a failure to fully extend the germ band. In 6-hr-old mutant embryos, two clusters of dead cells are observed in the thoracic and posterior abdominal region. These observations indicate region specific requirement of hb gene function. The analysis of germ line chimeras by transplantation of homozygous mutant pole cells shows that hb is already expressed during oogenesis. Homozygous mutant embryos derived from a homozygous mutant germ line have a novel phenotype. The anterior affected region is enlarged, including all three gnathal segments and the anterior three abdominal segments. In addition three abdominal segments with reversed polarity are formed between the remaining head structures and the posterior abdomen. Heterozygous mutant embryos derived from a homozygous mutant germ line develop normally, indicating that maternal gene expression is not required for normal development.     

Planar cell polarity in the larval epidermis of Drosophila and the role of microtubules

We investigate planar cell polarity (PCP) in the Drosophila larval epidermis. The intricate pattern of denticles depends on only one system of PCP, the Dachsous/Fat system. Dachsous molecules in one cell bind to Fat molecules in a neighbour cell to make intercellular bridges. The disposition and orientation of these Dachsous–Fat bridges allows each cell to compare two neighbours and point its denticles towards the neighbour with the most Dachsous. Measurements of the amount of Dachsous reveal a peak at the back of the anterior compartment of each segment. Localization of Dachs and orientation of ectopic denticles help reveal the polarity of every cell. We discuss whether these findings support our gradient model of Dachsous activity. Several groups have proposed that Dachsous and Fat fix the direction of PCP via oriented microtubules that transport PCP proteins to one side of the cell. We test this proposition in the larval cells and find that most microtubules grow perpendicularly to the axis of PCP. We find no meaningful bias in the polarity of microtubules aligned close to that axis. We also reexamine published data from the pupal abdomen and find no evidence supporting the hypothesis that microtubular orientation draws the arrow of PCP.     

Cell polarity during wound healing in an insect epidermis

The insect integument displays uniform posterior orientation of cuticular denticles or bristles formed by the epidermal cells. We want to understand how cell polarities become uniformly oriented in the plane of the epidermal sheet. Here we test whether directed cell migration disturbs the orientation of denticles. Burning a circular area of epidermal cells beneath the cuticle causes cells to migrate into the resulting wound and the cuticle pattern observed after the subsequent moult depends on the time interval between burning and ecdysis. After a short wound-healing period cuticular protrusions tend to point away from the wound. With increasing would healing periods they tend to point more and more towards the wound centre. These results suggest that the migrating cells tend to orient cuticular protrusions in the direction of cell movement while continued cell movement will bend nascent cuticular protrusions outwards. Cell shape may also determine denticle orientation. I propose that the asymmetric localization of cell components known to determine the orientation of cell migration may also determine denticle orientation in insect epidermal cells.     

Multiple functions of segment polarity genes in Drosophila

l(1)dishevelled (l(1)dsh) is a late zygotic lethal mutation that exhibits a rescuable maternal effect lethal phenotype. l(1)dsh/Y embryos, derived from females possessing a homozygous l(1)dsh germline clone, exhibit a segment polarity embryonic phenotype. Analysis of the development of these embryos indicates: (1) that segmental boundaries do not form although the correct number of tracheal pits is formed; (2) that pockets of cell death occur between the tracheal pits; and (3) that engrailed expression becomes abnormal during germ band shortening. We propose that, in the absence of both maternal and zygotic expression of l(1)dsh+, cells from each posterior compartment die. Subsequently, cells from the anterior compartment must rearrange their positional values to generate the segment polarity phenotype. We have compared the phenotype of five other segment polarity loci: four embryonic lethals [l(1)armadillo, l(2)gooseberry, l(2)wingless, and l(3)hedgehog]; and the late zygotic lethal, l(1)fused. Only l(2)wingless embryos exhibit early segmentation defects similar to those found in l(1)dsh/Y embryos derived from homozygous germline clones. In contrast, segmentation is essentially normal in l(1)armadillo, l(2)gooseberry, l(3)hedgehog, and l(1)fused embryos. The respective maternal and zygotic contribution and the roles of the segment polarity loci for the patterning of the embryo and the adult are discussed.     

Requirements for Hedgehog, a Segmental Polarity Gene, in Patterning Larval and Adult Cuticle of Drosophila

Mutations of the hedgehod gene are generally embryonic lethal, resulting in a lawn of denticles on the ventral surface. In strong alleles, no segmentation is obvious and the anteroposterior polarity of ventral denticles is lost. Temperature shift analysis of a temperature-sensitive allele indicates an embryonic activity period for hedgehod between 2.5 and 6 hr of embryonic development (at 25 degrees) and a larval/pupal period from 4 to 7 days of development (at 25 degrees). Mosaic analysis of hedgehod mutations in the adult cuticle indicates a series of defined defects associated with the failure of appropriate hedgehod expression. In particular, defects in the distal portions of the legs and antenna occur in association with homozygous hedgehog clones in the posterior compartment of those structures. Because the defects are associated with homozygous clones, but are not co-extensive, a type of "domineering" nonautonomy is proposed for the activity of the hedgehog gene.     

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.     

The Drosophila patched gene encodes a putative membrane protein required for segmental patterning

The patched (ptc) gene is one of several segment polarity genes required for correct patterning within every segment of Drosophila. The absence of ptc gene function causes a transformation of the fate of cells in the middle part of each segment so that they form pattern elements characteristic of cells positioned around the segment border. Analysis of the mutant phenotype demonstrates that both segment and parasegment borders are included in the duplicated pattern of ptc mutants. We have cloned the ptc gene and deduced that the product is a 1286 amino acid protein with at least seven putative transmembrane alpha helices. ptc RNA is expressed in embryos in broad stripes of segmental periodicity that later split into two stripes per segment primordium. The pattern of expression does not directly predict the transformation seen in ptc mutant embryos, suggesting that ptc participates in cell interactions that establish pattern within the segment.     

Secretion and localized transcription suggest a role in positional signaling for products of the segmentation gene hedgehog.

The segment polarity genes engrailed and wingless are expressed in neighboring stripes of cells on opposite sides of the Drosophila parasegment boundary. Each gene is mutually required for maintenance of the other's expression; continued expression of both also requires several other segment polarity genes. We show here that one such gene, hedgehog, encodes a protein targeted to the secretory pathway and is expressed coincidently with engrailed in embryos and in imaginal discs; maintenance of the hedgehog expression pattern is itself dependent upon other segment polarity genes including engrailed and wingless. Expression of hedgehog thus functions in, and is sensitive to, positional signaling. These properties are consistent with the non-cell autonomous requirement for hedgehog in cuticular patterning and in maintenance of wingless expression.     

Cubitus interruptus acts to specify naked cuticle in the trunk of Drosophila embryos.

One function of the Wingless signaling pathway is to determine the naked, cuticle cell fate choice in the trunk epidermis of Drosophila larvae. The zinc finger protein Teashirt binds to the transactivator domain of Armadillo to modulate Wingless signaling output in the embryonic trunk and contributes to the naked cell fate choice. The Hedgehog pathway is also necessary for the correct specification of larval epidermal cell fate, which signals via the zinc finger protein, Cubitus interruptus. Here, we show that Cubitus interruptus also has a Wingless-independent function, which is required for the specification of the naked cell fate; previously, it had been assumed that Ci induces naked cuticle exclusively by regulation of wg. Wg and Hh signaling pathways may be acting combinatorially in the same, or individually in different, cells for this process, by regulating common sets of target genes. First, the loss of the naked cuticular phenotype in embryos lacking cubitus interruptus activity is very similar to that induced by a late loss of Wingless function. Second, overexpression of Cubitus interruptus causes the suppression of denticles (as Wingless does) in absence of Wingless activity in the anterior trunk. Using epistasis experiments, we conclude that different combinations of the three proteins Teashirt, Cubitus interruptus, and Armadillo are employed for the specification of naked cuticle at distinct positions both along the antero-posterior axis and within individual trunk segments. Finally, biochemical approaches suggest the existence of protein complexes consisting of Teashirt, Cubitus interruptus, and Armadillo.     

Homoeosis in Drosophila: anterior and posterior transformations of Polycomb lethal embryos

Lethal embryos homozygous for Polycomb (Pc) mutations show transformations of segment-specific cuticular features to those of more anterior or posterior segments; the frequency and extent of such changes show differences which depend on the genotype and the region. The mesothorax of Polycomb lethal embryos often shows posterior transformations of the anterior- and posterior-most portions of the segment, and anterior transformations of the medial portion. A comparison of Polycomb embryos also bearing various genetic lesions of the bithorax gene complex (BX-C) shows that the penetrance of anterior transformation and the extent of posterior transformation in the appears independent of posterior transformation, even though cells undergoing each of these changes lie in close proximity in the developing embryo. It has been shown previously that in Polycomb lethal embryos posterior transformations require the normal function of the BX-C. We show here that anterior transformations of the mesothorax and other segments require the normal function of the Sex combs reduced (Scr) locus, also necessary for the normal development of the prothorax and some head segments. Similar observations are also presented for a Polycomblike mutation. We suggest that in Polycomb embryos there are errors in the clonal transmission of determined states resulting in expression of the BX-C and Scr+ loci at abnormal locations, and that such events are probabilistic in nature and show marked regional differences in frequency.     

Targeted expression of the signaling molecule decapentaplegic induces pattern duplications and growth alterations in Drosophila wings.

In the wing imaginal disc, the decapentaplegic (dpp) gene is expressed in a stripe of anterior cells near the anterior-posterior compartment boundary, and it is required solely in these cells for the entire disc to develop. In some viable segment polarity mutants, alterations in dpp expression have been demonstrated that correlate with changes in wing morphology. To test the hypothesis that the abnormal patterns of dpp expression are responsible directly for the mutant phenotypes, we have expressed dpp in ectopic places in wing imaginal discs, and we have found that dpp is able to cause overgrowth and pattern duplications in both anterior and posterior compartments of the wing disc. The alterations of the anterior compartment are strikingly similar to those observed in some viable segment polarity mutants. Thus, ectopic dpp alone can account for the phenotype of these mutants. We also show that ectopic expression of the segment polarity gene hedgehog (hh) gives similar morphological changes and activates dpp expression in the anterior compartment. This strongly suggests that the organizating activity of hh is mediated by dpp. We propose that the expression of dpp near the anterior-posterior compartment boundary is directed by the interaction between patched and hh, and that dpp itself could act as a general organizer of the patterning in the wing imaginal disc.     

A pupal lethal mutation with a paternally influenced maternal effect on embryonic development in Drosophila melanogaster

The maternal effect and zygotic phenotype of l(1)pole hole (l(1)ph) is described. l(1)ph is a zygotic lethal mutation which affects cell division of adult precursor cells in Drosophila larvae. The locus is located in 2F6 on the salivary gland chromosome map and four alleles have been characterized. Germ-line clonal analysis of amorphic alleles indicates that l(1)ph has a maternal effect lethal phenotype. Two lethal phenotypes are observed among embryos derived from female germ-line clones homozygous for amorphic alleles dependent upon the zygotic activity of l(1)ph⁺ introduced via the sperm. Class 1: If no wild-type dose of the gene is introduced, embryos form abnormal blastoderms in which nuclear migration and cell formation is disrupted leading to an ill-defined cuticular pattern. Class 2: If a wild-type copy of the gene is introduced, blastoderm cells do not form beneath the pole cells (the pole hole phenotype); subsequently such embryos are missing cuticular structures posterior to the seventh abdominal segment (the torso phenotype). When the zygotic activity l(1)ph⁺ is modulated using position effect variegation a new phenotype is observed among class 2 embryos in which torso embryos are twisted along their longitudinal axis.