By means of low-stringency cross-species hybridization to Southern DNA blots, human c-jun sequences were used to identify a unique Drosophila melanogaster locus (Djun). The predicted DJun protein is highly homologous to members of the mammalian Jun family in both the DNA binding and leucine zipper regions. Djun was mapped by in situ hybridization to position 46E of the second chromosome. It encodes a 1.7-kilobase transcript constitutively expressed at all developmental stages. Functionally, Djun in cooperation with mouse c-fos can trans-activate activator protein 1 DNA binding site when introduced into mammalian cells. Taken together, these data suggest that Djun, much like its mammalian homolog, may activate transcription of genes involved in regulation of cell growth, differentiation, and development. Furthermore, the identification of Djun allows one to exploit the genetics of Drosophila to identify genes in signal transduction pathways involving Djun and thus c-jun.
The Drosophila heat shock cognate gene 4 (hsc4), a member of the hsp70 gene family, encodes an abundant protein, hsc70, that is more similar to the constitutively expressed human protein than the Drosophila heat-inducible hsp70. Developmental expression revealed that hsc4 transcripts are enriched in cells active in endocytosis and those undergoing rapid growth and changes in shape.
We have conducted a genetic and developmental analysis of the 26 contiguous genetic complementation groups within the 19D3-20F2 interval of the base of the X chromosome, a region of 34 polytene bands delimited by the maroon-like and suppressor of forked loci. Within this region there are four loci which cause visible phenotypes but which have little or no effect on zygotic viability (maroon-like, little fly, small optic lobes and sluggish). There are 22 loci which, when mutated, are zygotic lethals and three of these, legless/runt, folded gastrulation and 13E3, have severe effects on embryonic development. In addition, three visible phenotypes have been defined only by overlapping deficiencies (melanized-like, tumorous head, and varied outspread). We have analyzed the lethal phases and maternal requirement of 58 mutations at 22 of the zygotic lethal loci by means of germline clone analysis using the dominant female sterile technique. Additionally, all lethal complementation groups, as well as a specific subset of deficiencies, have been studied histologically for defects in the development of the central and peripheral embryonic nervous systems.
By cross hybridization with the mammalian growth-related protein, GAP-43, we have isolated several Drosophila cDNAs and genomic sequences. These sequences correspond to a single copy gene that encodes two developmentally regulated transcripts 2.4 and 2.0 kb in length. The predicted protein sequence from the cDNAs contains a stretch of 20 amino acids closely related to the mammalian GAP-43 protein. These residues are also highly conserved in a cDNA isolated from the nematode C. elegans. Prior to dorsal closure, expression of the Drosophila gene is observed in non-neuronal tissues, especially in the mesectoderm and presumptive epidermis, both in a metameric pattern. After dorsal closure, expression becomes restricted to sets of cells that are segmentally reiterated along the periphery of the nervous system. These cells appear to include at least one specific set of glia that may establish scaffolding for the development of the longitudinal neuropile.
Maternal expression of the l(1)pole hole (l(1)ph) gene product is required for the development of the Drosophila embryo. When maternal l(1)ph+ activity is absent, alterations in the embryonic fate map occur as visualized by the expression of segmentation genes fushitarazu and engrailed. If both maternal and zygotic activity is absent, embryos degenerate around 7 h of development. If only maternal activity is missing, embryos complete embryogenesis and show deletions of both anterior and posterior structures. Anteriorly, structures originating from labral and acron head regions are missing. Posteriorly, abdominal segments A8, 9 and 10, the telson and the proctodeum are missing. Similar pattern deletions are observed in embryos derived from the terminal class of female sterile mutations. Thus, the maternal l(1)ph+ gene product is required for the establishment of cell identities at the anterior and posterior poles of the Drosophila embryo.
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.
In Drosophila the correct formation of the most anterior and posterior regions of the larva, acron and telson is dependent on the maternally expressed terminal class of genes. In their absence, the anterior head skeleton is truncated and all the structures posterior to the abdominal segment seven are not formed. The protein predicted to be encoded by one of these genes, torso (tor), seems to be a transmembrane protein with an extracytoplasmic domain acting as a receptor and a cytoplasmic domain containing tyrosine kinase activity. Here we report that another member of the terminal-genes class, l(1)polehole (l(1)ph), which is also zygotically expressed, is the Drosophila homologue of the v-raf oncogene and encodes a potential serine-and-threonine kinase. We also show that functional l(1)ph gene product is required for the expression of a gain-of-function tor mutant phenotype, indicating that l(1)ph acts downstream of tor. Together, these results support the idea that the induction of terminal development occurs through a signal transduction system, involving the local activation of the tor-encoded tyrosine kinase at the anterior and posterior egg poles, resulting in the phosphorylation of the l(1)ph gene product. In turn, downstream target proteins may be phosphorylated, ultimately leading to the regionalized expression of zygotic target genes. Such a process is in agreement with the finding that both tor and l(1)ph messenger RNAs are evenly distributed.
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.
In order to identify all X-linked zygotic lethal loci that exhibit a specific maternal effect on embryonic development, germline clonal analyses of X-linked zygotic lethal mutations have been performed. Two strategies were employed. In Screen A germline clonal analysis of 441 mutations at 211 previously mapped X-linked loci within defined regions was performed. In Screen B germline clonal analysis of 581 larval and pupal mutations distributed throughout the entire length of the X chromosome was performed. These approaches provide an 86% level of saturation for X-linked late zygotic lethals (larval and pupal) with specific maternal effect embryonic lethal phenotypes. The maternal effect phenotypes of these mutations are described.
Hypomorphic alleles of the locus polyhomeotic (ph) produce multiple, homeotic-like transformations in adult flies that mimic dominant mutations in the Antennapedia and Bithorax complexes. Analysis of null alleles of ph has revealed a complex, embryonically lethal phenotype that includes cell death of the ventral epidermis and abnormalities in the patterns of expression of homeotic and segmentation genes. There is also a dramatic alteration in the pattern of axon pathways in the central nervous system, such that the wild-type array of segmentally repeated commissures and connectives is replaced by bundles of axons confined to the hemiganglia of origin. It is possible that this axonal phenotype is the result of loss of neuronal identity caused by abnormal homeotic and segmentation gene expression.
Females homozygous for sans fille1621 (= fs(1)1621) have an abnormal germ line. Instead of producing eggs, the germ-line cells proliferate forming ovarian tumors or excessive numbers of nurse cells. The Sex-lethal gene product(s) regulate the branch point of the dosage compensation and sex determination pathways in the soma. The role of Sex-lethal in the germ line is not clear but the germ line of females homozygous for female sterile Sex-lethal alleles or germ-line clones of loss-of-function alleles are characterized by ovarian tumors. Females heterozygous for sans fille1621 or Sex-lethal are phenotypically wild type with respect to viability and fertility but females trans-heterozygous for sans fille1621 and Sex-lethal show ovarian tumors, somatic sexual transformations, and greatly reduced viability.
The maternal effect phenotypes of recessive mutations at the Drosophila zygotic lethal gene l(1)discs-large-1 (l(1)dlg-1) are described. L(1)dlg-1 is located in 10B7-8 on the salivary gland chromosome map. A complex complementation pattern is observed among the nine characterized alleles. Larvae missing zygotic l(1)dlg-1+ gene activity die due to aberrant growth of imaginal cells at the larval-pupal transition. Embryos lacking both maternal and zygotic activity of l(1)dlg-1+, i.e., embryos derived from homozygous l(1)dlg-1 germ line clones for null alleles, show neurogenesis and morphogenesis defects that result in very abnormal embryos. Although differentiated, most tissues are morphologically misshapen. This maternal effect is rescuable to some extent. One allele, l(1)dlg-1HF321, is a temperature-sensitive mutation for the zygotic lethality. Embryos derived from homozygous l(1)dlg-1HF321 females at 18 degrees C exhibit defects associated with dorsal closure and head involution. More extreme phenotypes are observed when females are shifted to higher temperatures and include defective dorsal closure, collapse of the somatic musculature, and an oversized central nervous system. The possible involvement of the recessive oncogene l(1)dlg-1 in cell adhesion is discussed.
A murine v-raf probe, representing the kinase domain, was used to identify two unique loci in Drosophila melanogaster DNA. The most closely related to v-raf was mapped by in situ hybridization to position 2F5-6 (Draf-1) on the X chromosome, whereas the other raf-related gene (Draf-2) was found at position 43A2-5 on chromosome 2. The nucleotide and amino acid homologies of Draf-1 to the kinase domain of v-raf are 61 and 65%, respectively. The large amount of a 3.2-kilobase Draf-1 transcript detected in eggs as a maternal message decreases during embryonic development, and significant steady-state levels are observed throughout the remainder of morphogenesis. We speculate that the Draf-1 locus plays an important role in early embryogenesis.
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.
Mutations at the ovo locus result in a defective female germ line. The male germ line is not affected. Adult females homozygous for loss-of-function alleles have no germ line stem cells. The sex-specific phenotype is evident at late blastoderm and early gastrula stages when the pole cells of embryos homozygous for a loss-of-function allele begin to die. This is the only zygotically acting gene known that is required specifically for embryonic germ line survival. Females heterozygous for dominant alleles or homozygous for alleles reducing gene activity exhibit a range of defects in oogenesis. We have mapped the ovo locus to position 4E1-2 of the salivary gland X chromosome by using a set of cytologically visible deletions.
Lack of zygotic expression of the l(1)giant locus (l(1)gt;3A1), produces embryos with defects in abdominal A5, 6, and 7 and within the head. Scanning electron microscopy at the time of segment formation reveals two regions of defects in the segmentation pattern: anteriorly the labial lobe and thoracic segments T1 and T2 are fused; posteriorly, abdominal segments A5-7 are disrupted. The mature embryo shows incomplete head involution and defects within A5-7; fusion of T1 and T2 is no longer observed. Localized cell death within neural and mesodermal tissues is observed at 7 hr of development; later ventral ganglia, A5-7, are missing. Double-mutant analyses of l(1)gt with maternal effect lethal mutations and mutations that generate homeotic, segment number, gap, or segment polarity phenotypes indicate that normal activity of l(1)gt is required for differentiation of two embryonic domains: one corresponding to labial, T1 and T2 segments, and the second corresponding to abdominal segments 5, 6, and 7.
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.
The maternal and zygotic effect phenotypes of mutations at the l(1)hopscotch (l(1)hop) locus are described. l(1)hop is located in 10B6-8 on the salivary gland chromosome map and 17 alleles have been characterized. A complex complementation pattern is observed among the 17 alleles. The lethal phase of null alleles of l(1)hop occurs at the larval-pupal interface associated with a small disc phenotype. Embryos produced from homozygous l(1)hop germline clones show segment specific defects. The extent of these defects depends upon both the strength of the allele and the paternal contribution. In the most extreme case embryos exhibit defects associated with five segments T2, T3, A4, A5, and A8. In the less extreme phenotype defects are only associated with A5. Thus, activity of l(1)hop+ is required both for the maintenance and continued cell division of diploid imaginal precursors and for the establishment of the full array of segments.
We have examined the number of X-linked loci specifically required only during oogenesis. Complementation analyses among female-sterile (fs) mutations obtained in two mutagenesis screens--GANS' and MOHLER's--indicate that any fs locus represented by two or more mutant alleles in GANS' collection are usually present in MOHLER's collection. However, when a locus is represented by a single allele in one collection, it is generally not present in the other collection. We propose that this discrepancy is due to the fact that most "fs loci" represented by less than two mutant alleles are, in fact, vital (zygotic lethal) genes, and that the fs alleles are hypomorphic mutations of such genes. In support of this hypothesis we have identified lethal alleles at 12 of these "fs loci." The present analysis has possibly identified all maternal-effect lethal loci detectable by mutations on the X chromosome and has allowed us to reevaluate the number of "ovary-specific fs" loci in the Drosophila genome. Finally, germline clone analysis of a large number of fs mutations was performed in order to estimate the relative contribution of germline and somatic cell derivatives to oogenesis and to embryonic development. All the maternal-effect lethal loci tested are germline-dependent.
Two mutations in the gene, RpII215, were analyzed to determine their effects on cell differentiation and proliferation. The mutations differ in that one, RpII215ts (ts), only displays a conditional recessive lethality, while the other, RpII215Ubl (Ubl), is a recessive lethal mutation that also displays a dominant mutant phenotype similar to that caused by the mutation Ultrabithorax (Ubx). Ubl causes a partial transformation of the haltere into a wing; however, this transformation is more complete in flies carrying both Ubl and Ubx. The present study shows that patches of Ubl/-tissue in gynandromorphs are morphologically normal. cuticle that has lost the wild-type copy of the RpII215 locus fails to show a haltere to wing transformation, nor does it show the synergistic enhancement of Ubx by Ubl. We conclude that an interaction between the two RpII215 alleles, Ubl and RpII215+, is responsible for the mutant phenotype. Gynandromorphs carrying the ts allele, when raised at permissive temperature, display larger patches of ts/-cuticle than expected, possibly indicating that the proliferation of ts/+ cells is reduced. This might result from an antagonistic interaction between different RpII215 alleles. Classical negative complementation does not appear to be the cause of the antagonistic interactions described above, as only one RpII215 subunit is thought to be present in an active multimeric polymerase enzyme. We have therefore coined the term 'negative heterosis' to describe the aforementioned interactions. We also observed that the effects of mutationally altered RNA polymerase II on somatic cells are different from its effects on germ cells.(ABSTRACT TRUNCATED AT 250 WORDS)