The segment polarity phenotype of Drosophila involves differential tendencies toward transformation and cell death

The segment polarity genes of Drosophila are required for intrasegmental organization, as revealed by their abnormal cuticular morphology in mutant embryos. Lesions in most of these loci result in a similar cuticular phenotype, in which the normally naked, posterior region of the segment is covered to varying degrees by ectopic denticles. A temperature-sensitive allele of armadillo, which allows us to vary the level of arm+ activity, generates this entire range of phenotypes, suggesting that these genes affect a common pathway. Previous work with a strong allele of arm revealed the locus to be cell-autonomous, in that small homozygous epidermal clones secreted denticles. We have conducted a similar clonal analysis at all levels of arm+ activity. This shows a differential tendency toward cell transformation and cell death within the segment. Antibodies to segmentation gene-fusion products show that the cell death is primarily in the most posterior region of the segment. We suggest that differential cell respecification, resulting in transformation or death, is involved in generating the segment polarity phenotype.     

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.     

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.     

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.     

Directionality of wingless protein transport influences epidermal patterning in the Drosophila embryo.

Active endocytotic processes are required for the normal distribution of Wingless (Wg) protein across the epidermal cells of each embryonic segment. To assess the functional consequences of this broad Wg distribution, we have devised a means of perturbing endocytosis in spatially restricted domains within the embryo. We have constructed a transgene expressing a dominant negative form of shibire (shi), the fly dynamin homologue. When this transgene is expressed using the GAL4-UAS system, we find that Wg protein distribution within the domain of transgene expression is limited and that Wg-dependent epidermal patterning events surrounding the domain of expression are disrupted in a directional fashion. Our results indicate that Wg transport in an anterior direction generates the normal expanse of naked cuticle within the segment and that movement of Wg in a posterior direction specifies diverse denticle cell fates in the anterior portion of the adjacent segment. Furthermore, we have discovered that interfering with posterior movement of Wg rescues the excessive naked cuticle specification observed in naked (nkd) mutant embryos. We propose that the nkd segment polarity phenotype results from unregulated posterior transport of Wg protein and therefore that wild-type Nkd function may contribute to the control of Wg movement within the epidermal cells of the segment.     

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.     

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.     

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.     

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.     

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.     

Krüppel, a gene whose activity is required early in the zygotic genome for normal embryonic segmentation

Embryos homozygous for Krüppel die as late embryos with an altered segmentation pattern. In strong alleles the normal thoracic and anterior abdominal segments are replaced by a partial mirror image duplication of the posterior abdomen. Weak alleles cause smaller pattern deletions in the thorax and abdomen and are not associated with mirror image duplications. The altered segmentation pattern can be traced back to 12 min after the onset of gastrulation, when the shorter germ bands in homozygous Kr embryos provide a first indication of abnormal patterning. The mutant was mapped to position 107.6 at the tip of the right arm of the second chromosome, cytologically to bands 60F2-5. Analysis of homozygous deficiency embryos indicate that the phenotype produced by strong point mutations probably represents the amorphic condition. The requirement for Kr+ gene activity is strictly zygotic. Maternal dosage of Kr+ has no effect on the embryonic phenotype, nor does homozygosity for Kr prevent germ cells from making normal eggs capable of normal embryonic development when fertilized by wild-type sperm. The requirement for Kr+ seems restricted to embryogenesis. Homozygous clones induced in imaginal discs during larval development survive and develop normally and in vivo cultures established from homozygous embryos proliferate normally and metamorphose into adult structures of normal morphology.     

RacGap50C negatively regulates wingless pathway activity during Drosophila embryonic development

The Wingless (Wg)/Wnt signal transduction pathway directs a variety of cell fate decisions in developing animal embryos. Despite the identification of many Wg pathway components to date, it is still not clear how these elements work together to generate cellular identities. In the ventral epidermis of Drosophila embryos, Wg specifies cells to secrete a characteristic pattern of denticles and naked cuticle that decorate the larval cuticle at the end of embryonic development. We have used the Drosophila ventral epidermis as our assay system in a series of genetic screens to identify new components involved in Wg signaling. Two mutant lines that modify wg-mediated epidermal patterning represent the first loss-of-function mutations in the RacGap50C gene. These mutations on their own cause increased stabilization of Armadillo and cuticle pattern disruptions that include replacement of ventral denticles with naked cuticle, which suggests that the mutant embryos suffer from ectopic Wg pathway activation. In addition, RacGap50C mutations interact genetically with naked cuticle and Axin, known negative regulators of the Wg pathway. These phenotypes suggest that the RacGap50C gene product participates in the negative regulation of Wg pathway activity.     

Studies on the expression of intracellular and surface polarity in animal pole cells of Xenopus embryos cultured on various substrata

The expression of intracellular and surface polarity in animal pole cells of Xenopus embryos (stage 10) cultured on various substrata was studied by electron microscopy. When animal pole cells of Xenopus embryos were cultured on type I collagen- or gelatin-coated dishes until control embryos reached stage 23, the cells in confluent layers expressed an apical-basal polarity so that the apical surface membrane domain faced the culture medium. However, the cells in confluent layers cultured on naked plastic dishes were suppressed to express intracellular and surface polarity. In addition, single attached cells which were formed by being sparsely plated neither spread on any substrata nor displayed the apical-basal polarity perpendicular to the dishes. These results indicate that the expression of intracellular and surface polarity in cultured animal pole cells of Xenopus embryos requires not only cell-cell contact but also an adhesive substrata such as type I collagen or gelatin.     

The segment polarity gene armadillo encodes a functionally modular protein that is the Drosophila homolog of human plakoglobin

The Drosophila segment polarity gene armadillo is required for pattern formation within embryonic segments and imaginal discs. We have found that armadillo is highly conserved during evolution; it is 63% identical to human plakoglobin, a protein found in adhesive junctions joining epithelial and other cells. We have examined arm protein localization in a number of larval tissues and found that arm protein accumulation within cells shares many features with the accumulation of plakoglobin. We have compared the phenotype and molecular lesions responsible for the different arm mutations. Surprisingly, severely truncated proteins retain some function; the degree of function is strictly correlated with the length of the truncated protein, suggesting that the internally repetitive arm protein is modular in function. We present a possible model for the cellular role of arm.     

Cell lineage of the thoracic muscles of drosophila

The thorax of the adult Drosophila contains about 80 muscles, which develop from the mesoderm. A new genetic marker was used to map the cell lineage of the myoblasts that form these muscles. Clones of marked cells were produced by irradiation of embryos and larvae, and these were detected in the adult by histochemical staining. The principal findings are that the muscles of each segment have separate origins, and that each becomes compartmented precisely into a dorsal-lineage and a ventral-lineage set of muscles, each set probably being formed by the adepithelial cells found in one imaginal disc. In contrast with the epidermis, the muscles of each thoracic segment are not subdivided into anterior and posterior compartments, and clones of muscle cells that are homozygous for recessivelethal alleles of engralled develop normally.     

Generation of segment polarity in the paraxial mesoderm of the zebrafish through a T-box-dependent inductive event

The first morphological sign of vertebrate postcranial body segmentation is the sequential production from posterior paraxial mesoderm of blocks of cells termed somites. Each of these embryonic structures is polarized along the anterior/posterior axis, a subdivision first distinguished by marker gene expression restricted to rostral or caudal territories of forming somites. To better understand the generation of segment polarity in vertebrates, we have studied the zebrafish mutant fused somites (fss), because its paraxial mesoderm lacks segment polarity. Previously examined markers of caudal half-segment identity are widely expressed, whereas markers of rostral identity are either missing or dramatically down-regulated, suggesting that the paraxial mesoderm of the fss mutant embryo is profoundly caudalized. These findings gave rise to a model for the formation of segment polarity in the zebrafish in which caudal is the default identity for paraxial mesoderm, upon which is patterned rostral identity in an fss-dependent manner. In contrast to this scheme, the caudal marker gene ephrinA1 was recently shown to be down-regulated in fss embryos. We now show that notch5, another caudal identity marker and a component of the Delta/Notch signaling system, is not expressed in the paraxial mesoderm of early segmentation stage fss embryos. We use cell transplantation to create genetic mosaics between fss and wild-type embryos in order to assay the requirement for fss function in notch5 expression. In contrast to the expression of rostral markers, which have a cell-autonomous requirement for fss, expression of notch5 is induced in fss cells at short range by nearby wild-type cells, indicating a cell-non-autonomous requirement for fss function in this process. These new data suggest that segment polarity is created in a three-step process in which cells that have assumed a rostral identity must subsequently communicate with their partially caudalized neighbors in order to induce the fully caudalized state.     

Developmental analysis of the torso-like phenotype in Drosophila produced by a maternal-effect locus

The segmental plan of the Drosophila embryo is already established at the blastoderm stage through the action of maternal effect genes which determine the polarity of the embryo and zygotically active genes involved in segmentation. We have analyzed the first example of a group of maternally acting genes which are necessary for establishing the developmental potential of the posterior 25% of the blastoderm. Females, homozygous for the X-linked maternal-effect mutation female sterile(1)Nasrat211 [fs(1)N211], produce embryos, characterized as torso-like, which lack all posterior endodermal derivatives as well as structures characteristic of abdominal segments 8 to 10. In addition, anterior endodermal derivatives are deficient and the absence of pharyngeal musculature causes a collapse of the cephalopharyngeal apparatus. The columnar blastoderm cell layer is defective at the posterior tip below the pole cells in these embryos. This defect, however, is presumably secondary to some abnormal feature of pole cell formation since in double mutants of fs(1)Nasrat211; tudor3 the blastoderm is normal but the embryos still show the torso-like phenotype. In situ hybridization with RNA probes derived from the fushi tarazu gene establishes that the cellular determination of the posterior blastoderm of embryos produced by fs(1)N211 is changed. This represents the first direct demonstration that a maternal-effect mutation alters the spatial distribution of a zygotic gene product involved in the segmental patterning of the embryo.     

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.