Homeotic gene function in the muscles of Drosophila larvae

The segmental musculature of Drosophila melanogaster larvae consists of 24-30 muscles per segment. Unique patterns of muscles are found in the three thoracic segments and the first and last abdominal segments; the remaining abdominal segments share the same pattern. Mutations in Ultrabithorax (Ubx) cause partial transformation of the muscle pattern of larval abdominal segments towards metathorax. The muscles of the thorax are not affected. In the first two abdominal segments the changes include the loss of at least 11 `abdominal' muscles and the gain of 11 `thoracic' muscles. Less extensive transformations are seen in more posterior abdominal segments. Anterobithorax, bithorax, postbithorax and bithoraxoid mutations also induce transformations of the larval musculature. Each allelic group affects a domain that is a subset of the entire Ubx domain but these domains are not restricted to compartments or segments and may extend through as many as five segments. In the muscles the segmental distribution of Ubx antigen correlates with the segments affected by Ubx mutations. The different domains of Ubx in mesoderm and ectoderm argue that the segmental diversity of the muscle pattern is not simply induced by the overlying epidermis and that Ubx function in the mesoderm is required for the correct development of abdominal segments.     

Genetic dissection of meiotic cytokinesis in Drosophila males

We have used Drosophila male meiosis as a model system for genetic dissection of the cytokinesis mechanism. Drosophila mutants defective in meiotic cytokinesis can be easily identified by their multinucleate spermatids. Moreover, the large size of meiotic spindles allows characterization of mutant phenotypes with exquisite cytological resolution. We have screened a collection of 1955 homozygous mutant male sterile lines for those with multinucleate spermatids, and thereby identified mutations in 19 genes required for cytokinesis. These include 16 novel loci and three genes, diaphanous, four wheel drive, and pebble, already known to be involved in Drosophila cytokinesis. To define the primary defects leading to failure of cytokinesis, we analyzed meiotic divisions in male mutants for each of these 19 genes. Examination of preparations stained for tubulin, anillin, KLP3A, and F-actin revealed discrete defects in the components of the cytokinetic apparatus, suggesting that these genes act at four major points in a stepwise pathway for cytokinesis. Our results also indicated that the central spindle and the contractile ring are interdependent structures that interact throughout cytokinesis. Moreover, our genetic and cytological analyses provide further evidence for a cell type-specific control of Drosophila cytokinesis, suggesting that several genes required for meiotic cytokinesis in males are not required for mitotic cytokinesis.     

Genetic dissection of the Drosophila circadian system

Genetic experiments involving selected strains as well as single gene mutations have provided information concerning the organization of the Drosophila circadian system. The phase of the emergence rhythm of D. pseudoobscura can be altered by genetic selection without significantly affecting the phase and period of the light-sensitive pacemaker. The period of the D. melanogaster pacemaker, over the range 19 hours to 29 hours, can be encoded in the DNA sequence of a single genetic locus. The short-period and long-period mutations do not eliminate the pacemaker's temperature compensation mechanism. The short-period mutation alters the resetting behavior of the pacemaker from weak (type 1) in wild-type to strong (type 0) in the mutant. Five aperiodic mutations isolated in D. pseudoobscura belong to two complementation groups. In complements bearing one mutation from each group, the periodicity of the pacemaker is wild-type, but the phase of the emergence rhythm is 5 hours later than wild-type. Thus mutations in particular genetic loci have dramatic effects on the basic properties of circadian pacemakers and rhythms.     

Genetic dissection of a complex neurological mutant, polyhomeotic, in Drosophila

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.     

Furin cleavage is not a requirement for Drosophila Notch function

Notch (N) is a large transmembrane protein that acts as a receptor in an evolutionarily conserved intercellular signalling pathway. Because of this conservation, it has been assumed that biochemical events mediating N function are identical in all species. For instance, intracellular maturation by furin protease and subunit assembly leading to the formation of a heterodimeric cell surface N receptor are thought to be central to its function in both mammals and flies. However, in Drosophila the majority of N appears to be full-length. It has not been determined whether this full-length N protein is on the cell surface. We describe experiments which indicate that unlike mammalian N, the majority of Drosophila N on the cell surface is full-length and that in Drosophila, in vivo, furin cleavage is not required for biological activity. We further show that the behaviour of fly and mouse N can be interchanged simply by swapping the regions in which the mammalian furin-like cleavage site is located.     

Genetic and phenotypic analysis of alleles of the Drosophila chromosomal JIL-1 kinase reveals a functional requirement at multiple developmental stages

In this study we provide a cytological and genetic characterization of the JIL-1 locus in Drosophila. JIL-1 is an essential chromosomal tandem kinase and in JIL-1 null animals chromatin structure is severely perturbed. Using a range of JIL-1 hypomorphic mutations, we show that they form an allelic series. JIL-1 has a strong maternal effect and JIL-1 activity is required at all stages of development, including embryonic, larval, and pupal stages. Furthermore, we identified a new allele of JIL-1, JIL-1(h9), that encodes a truncated protein missing COOH-terminal sequences. Remarkably, the truncated JIL-1 protein can partially restore viability without rescuing the defects in polytene chromosome organization. This suggests that sequences within this region of JIL-1 play an important role in establishing and/or maintaining normal chromatin structure. By analyzing the effects of JIL-1 mutations we provide evidence that JIL-1 function is necessary for the normal progression of several developmental processes at different developmental stages such as oogenesis and segment specification. We propose that JIL-1 may exert such effects by a general regulation of chromatin structure affecting gene expression.     

Genetic dissection of centromere function.

A system to detect a minimal function of Saccharomyces cerevisiae centromeres in vivo has been developed. Centromere DNA mutants have been examined and found to be active in a plasmid copy number control assay in the absence of segregation. The experiments allow the identification of a minimal centromere unit, CDE III, independently of its ability to mediate chromosome segregation. Centromere-mediated plasmid copy number control correlates with the ability of CDE III to assemble a DNA-protein complex. Cells forced to maintain excess copies of CDE III exhibit increased loss of a nonessential artificial chromosome. Thus, segregationally impaired centromeres can have negative effects in trans on chromosome segregation. The use of a plasmid copy number control assay has allowed assembly steps preceding chromosome segregation to be defined.     

Genetic dissection of cyclophilin function. Saturation mutagenesis of the Drosophila cyclophilin homolog ninaA.

Cyclophilins, the intracellular receptors for the widely used immunosuppressant cyclosporin A have been found to be peptidyl-prolyl cis/trans isomerases and have been implicated in intracellular protein folding and trafficking. The Drosophila ninaA gene encodes a photoreceptor-specific cyclophilin homolog involved in rhodopsin biogenesis. ninaA mutants have a 90% reduction in the levels of Rh1 rhodopsin. To gain insight into the role of cyclophilins in vivo, we carried out a genetic screen designed to identify functionally important regions in the ninaA protein. Over 700,000 mutagenized flies were screened for a visible ninaA phenotype and 70 independent mutations in ninaA were isolated and characterized. These mutations provide a detailed dissection of the structure/function relationships in cyclophilin. We also show that mammalian cyclophilins engineered to contain missense mutations found in two temperature-sensitive ninaA alleles display temperature-sensitive prolyl cis/trans isomerase activity.     

Genetic dissection of Nucleoporin 160 (Nup160), a gene involved in multiple phenotypes of reproductive isolation in Drosophila

Previous reports have suggested that the Nucleoporin 160 (Nup160) gene of Drosophila simulans (Nup160(sim)) causes the hybrid inviability, female sterility, and morphological anomalies that are observed in crosses with D. melanogaster. Here we have confirmed this observation by transposon excision from the P{EP}Nup160(EP372) insertion mutation of D. melanogaster. Null mutations of the Nup160 gene resulted in the three phenotypes caused by Nup160(sim), but revertants of the gene did not. Interestingly, several mutations produced by excision partially complemented hybrid inviability, female sterility, or morphological anomalies. In the future, these mutations will be useful to further our understanding of the developmental mechanisms of reproductive isolation. Based on our analyses with the Nup160(sim) introgression line, the lethal phase of hybrid inviability was determined to be during the early pupal stage. Our analysis also suggested that homozygous Nup160(sim) in D. melanogaster leads to slow development. Thus, Nup160(sim) is involved in multiple aspects of reproductive isolation between these two species.     

Genetic analysis of yeast Yip1p function reveals a requirement for Golgi-localized rab proteins and rab-Guanine nucleotide dissociation inhibitor

Yip1p is the first identified Rab-interacting membrane protein and the founder member of the YIP1 family, with both orthologs and paralogs found in all eukaryotic genomes. The exact role of Yip1p is unclear; YIP1 is an essential gene and defective alleles severely disrupt membrane transport and inhibit ER vesicle budding. Yip1p has the ability to physically interact with Rab proteins and the nature of this interaction has led to suggestions that Yip1p may function in the process by which Rab proteins translocate between cytosol and membranes. In this study we have investigated the physiological requirements for Yip1p action. Yip1p function requires Rab-GDI and Rab proteins, and several mutations that abrogate Yip1p function lack Rab-interacting capability. We have previously shown that Yip1p in detergent extracts has the capability to physically interact with Rab proteins in a promiscuous manner; however, a genetic analysis that covers every yeast Rab reveals that the Rab requirement in vivo is exclusively confined to a subset of Rab proteins that are localized to the Golgi apparatus.     

Genetic mosaic analysis of decapentaplegic and wingless gene function in the Drosophila leg

 Genetically mosaic flies were constructed which lack a functional decapentaplegic (dpp) or wingless (wg) gene in portions of their leg epidermis, and the leg cuticle was examined for defects. Although dpp has previously been shown to be transcribed both ventrally and dorsally, virtually the only dpp-null clones that affect leg anatomy are those which reside dorsally. Conversely, wg-null clones only cause leg defects when they reside ventrally – a result that was expected, given that wg is only expressed ventrally. Both findings are consistent with models of leg development in which the future tip of the leg is specified by an interaction between dpp and wg at the center of the leg disc. Null clones can cause mirror-image cuticular duplications confined to individual leg segments. Double-ventral, mirror-image patterns are observed with dpp-null clones, and double-dorsal patterns with wg-null clones. Clones that are doubly mutant (null for both dpp and wg) manifest reduced frequencies for both types of duplications. Duplications can include cells from surrounding non-mutant territory. Such nonautonomy implies that both dpp and wg are involved in positional signaling, not merely in the maintenance of cellular identities. However, neither gene product appears to function as a morphogen for the entire leg disc, since the effects of each gene’s null clones are restricted to a discrete part of the circumference. Interestingly, the circumferential domains where dpp and wg are needed are complementary to one another. Received: 25 March 1996 / Accepted: 13 June 1996     

Genetic dissection of cadherin function during nephrogenesis

The distinct expression of R-cadherin in the induced aggregating metanephric mesenchyme suggests that it may regulate the mesenchymal-epithelial transition during kidney development. To address whether R-cadherin is required for kidney ontogeny, R-cadherin-deficient mice were generated. These mice appeared to be healthy and were fertile, demonstrating that R-cadherin is not essential for embryogenesis. The only kidney phenotype of adult mutant animals was the appearance of dilated proximal tubules, which was associated with an accumulation of large intracellular vacuoles. Morphological analysis of nephrogenesis in R-cadherin(-/-) mice in vivo and in vitro revealed defects in the development of both ureteric bud-derived cells and metanephric mesenchyme-derived cells. First, the morphology and organization of the proximal parts of the ureteric bud epithelium were altered. Interestingly, these morphological changes correlated with an increased rate of apoptosis and were further supported by perturbed branching and patterning of the ureteric bud epithelium during in vitro differentiation. Second, during in vitro studies of mesenchymal-epithelial conversion, significantly fewer epithelial structures developed from R-cadherin(-/-) kidneys than from wild-type kidneys. These data suggest that R-cadherin is functionally involved in the differentiation of both mesenchymal and epithelial components during metanephric kidney development. Finally, to investigate whether the redundant expression of other classic cadherins expressed in the kidney could explain the rather mild kidney defects in R-cadherin-deficient mice, we intercrossed R-cadherin(-/-) mice with cadherin-6(-/-), P-cadherin(-/-), and N-cadherin(+/-) mice. Surprisingly, however, in none of the compound knockout strains was kidney development affected to a greater extent than within the individual cadherin knockout strains.     

Genetic dissection of the anterior optic tract of Drosophila melanogaster

Electron microscopy shows that in wild-type Drosophila melanogaster the anterior optic tract (AOT) is formed by about 1260 fibers in males and slightly fewer in females. Golgi staining suggests that most AOT fibers connect the lobula with different regions of the central brain. In the sine oculis (so) and small optic lobes (sol) mutants the number of axons is drastically reduced, by 58% in sol and by 35% in so. In the double mutant sol:so there is a loss of up to 83% of the fibers in the AOT. Approximately half of the remaining 220 fibers form a well defined subbundle of very thin axons which is identifiable in wild type as well as in both single mutants. The fibers of this subbundle neither originate nor terminate in the visual ganglia: instead, they connect two different central brain regions. It is concluded that the combined action of the sol and so mutations abolishes more than 90% of the fibers of visual origin or destination in the AOT. Quantitative analysis of electron micrographs shows that the so and sol mutations act independently on nearly exclusive subsets of axons in the AOT.     

Genetic analysis of viable Hsp90 alleles reveals a critical role in Drosophila spermatogenesis

The Hsp90 chaperone protein maintains the activities of a remarkable variety of signal transducers, but its most critical functions in the context of the whole organism are unknown. Point mutations of Hsp83 (the Drosophila Hsp90 gene) obtained in two different screens are lethal as homozygotes. We report that eight transheterozygous mutant combinations produce viable adults. All exhibit the same developmental defects: sterile males and sterile or weakly fertile females. We also report that scratch, a previously identified male-sterile mutation, is an allele of Hsp82 with a P-element insertion in the intron that reduces expression. Thus, it is a simple reduction in Hsp90 function, rather than possible altered functions in the point mutants, that leads to male sterility. As shown by light and electron microscopy, all stages of spermatogenesis involving microtubule function are affected, from early mitotic divisions to later stages of sperm maturation, individualization, and motility. Aberrant microtubules are prominent in yeast cells carrying mutations in HSP82 (the yeast Hsp90 gene), confirming that Hsp90 function is connected to microtubule dynamics and that this connection is highly conserved. A small fraction of Hsp90 copurifies with taxol-stabilized microtubule proteins in Drosophila embryo extracts, but Hsp90 does not remain associated with microtubules through repeated temperature-induced assembly and disassembly reactions. If the spermatogenesis phenotypes are due to defects in microtubule dynamics, we suggest these are indirect, reflecting a role for Hsp90 in maintaining critical signal transduction pathways and microtubule effectors, rather than a direct role in the assembly and disassembly of microtubules themselves.     

Molecular dissection of zyxin function reveals its involvement in cell motility

Spatially controlled actin filament assembly is critical for numerous processes, including the vectorial cell migration required for wound healing, cell- mediated immunity, and embryogenesis. One protein implicated in the regulation of actin assembly is zyxin, a protein concentrated at sites where the fast growing ends of actin filaments are enriched. To evaluate the role of zyxin in vivo, we developed a specific peptide inhibitor of zyxin function that blocks its interaction with alpha-actinin and displaces it from its normal subcellular location. Mislocalization of zyxin perturbs cell migration and spreading, and affects the behavior of the cell edge, a structure maintained by assembly of actin at sites proximal to the plasma membrane. These results support a role for zyxin in cell motility, and demonstrate that the correct positioning of zyxin within the cell is critical for its physiological function. Interestingly, the mislocalization of zyxin in the peptide-injected cells is accompanied by disturbances in the distribution of Ena/VASP family members, proteins that have a well-established role in promoting actin assembly. In concert with previous work, our findings suggest that zyxin promotes the spatially restricted assembly of protein complexes necessary for cell motility.