The study of serine/threonine kinases in Drosophila is coming of age. Recently several kinases have been identified and their role in cell determination has been established. This review discusses these recent findings and describes the potential for genetic analyses of kinase activity and signal transduction.
Null mutations at the polyhomeotic locus of Drosophila produce a complex phenotype during embryogenesis, which includes death of the ventral epidermis, misregulation of homeotic and segmentation gene expression, and global misrouting of CNS axons. It is shown here, through the use of mosaic analyses, double mutant combinations, and in vitro culture experiments, that all aspects of the phenotype with the exception of the axonal phenotype are cell autonomous. The changes in homeotic and segmentation gene expression in the CNS are not caused by death of the ventral epidermis, but are cell autonomous effects which most likely cause changes in neuronal cell identity. The axonal phenotype associated with ph mutations is also independent of epidermal cell death, but may be due to the nonautonomous effects of altered neuronal identities or to death or transformation of some as yet unidentified cell type. Despite the apparent autonomy of the ph mutation, mutant neurons can influence the development of adjacent wild-type neurons, presumably by depriving them of their normal fasciculation partners.
The orthodenticle (otd) locus of Drosophila is required for embryonic development, and null mutations of otd cause defects in head development and segmental patterning. We show here that otd is necessary for the formation of the embryonic central nervous system (CNS). otd mutations result in the formation of an abnormal neuropil and in the disappearance of identified neurons associated with the midline of the CNS. In addition, otd is allelic to ocelliless (oc), a mutation that causes the deletion of the ocelli of the adult fly. We have identified a transcription unit corresponding to the otd locus and find that it is expressed early in a stripe near the anterior pole of the cellular blastoderm and later in the region of the CNS from which these neurons normally arise. The predicted otd protein contains a well-conserved homeo domain and is therefore likely to be a transcriptional regulator involved in specifying cell fate both in the embryonic CNS and in the ocelli.
In the Drosophila embryo, cell fate along the anterior-posterior axis is determined by maternally expressed genes. The activity of the bicoid (bcd) gene is required for the development of larval head and thoracic structures, and that of maternal torso (tor) for the development of the unsegmented region of the head (acron). In contrast to the case of thoracic and abdominal segmentation, the hierarchy of zygotically expressed genes controlling head development has not been clearly defined. The bcd protein, which is expressed in a gradient, activates zygotic expression of the gap gene hunchback (hb), but hb alone is not sufficient to specify head development. Driever et al. proposed that at least one other bcd-activated gene controls the development of head regions anterior to the hb domain. We report here that the homeobox gene orthodenticle (otd), which is involved in head development, could be such a gene. We also show that otd expression responds to the activity of the maternal tor gene at the anterior pole of the embryo.
The metameric pattern of the Drosophila embryo is regulated by a combination of maternal and zygotic genes. The segment-polarity class of genes are required for the correct patterning within each segmental unit. Mutations in any one of these genes results in deletions and duplications of parts of each segment. The segment-polarity genes act coordinately by means of local cellular interactions to assign and maintain an identity for each cell in the segment, and to establish segment boundaries. Here we describe the molecular characterization of a novel segment-polarity gene, zeste-white3 (zw3). Embryos derived from germ lines that are homozygous for zw3 mutations (zw3 embryos) have phenotypes similar to embryos that are mutant for the segment-polarity gene naked (nkd). These embryos lack most of the ventral denticles, which are differentiated structures derived from the most anterior region of each segment. We have isolated the zw3 gene and compared the structure of one maternal and one zygotic transcript encoded by the gene. The zw3 gene is unique among the segment-polarity genes so far characterized, in that it encodes proteins that have homology to serine-threonine protein kinases. This indicates that zw3 may play a part in a signal transduction pathway involved in the establishment of cell identity within each embryonic segment.
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.