Membrane fusion proteins are required for oskar mRNA localization in the Drosophila egg chamber

We used a genetic screen in Drosophila to identify mutations which disrupt the localization of oskar mRNA during oogenesis. Based on the hypothesis that some cytoskeletal components which are required during the mitotic divisions will also be required for oskar mRNA localization during oogenesis, we designed the following genetic screen. We screened for P-element insertions in genes which slow down the blastoderm mitotic divisions. A secondary genetic screen was to generate female germ-line clones of these potential cell division cycle genes and to identify those which cause the mislocalization of oskar mRNA. We identified mutations in ter94 which disrupt the localization of oskar mRNA to the posterior pole of the oocyte. Ter94 is a member of the CDC48p/VCP subfamily of AAA proteins which are involved in homotypic fusion of the endoplasmic reticulum during mitosis. Consistent with the function of the yeast ortholog, ter94-mutant egg chambers are defective in the assembly of the endoplasmic reticulum. We tested whether other membrane biosynthesis genes are required for localizing oskar mRNA during oogenesis. We found that ovaries that are mutant for syntaxin-1a, rop, and synaptotagmin are also defective in oskar mRNA localization during oogenesis. We suggest a pathway for the role of membrane assembly proteins on oskar mRNA localization.     

A screen for dynein synthetic lethals in Aspergillus nidulans identifies spindle assembly checkpoint genes and other genes involved in mitosis.

Cytoplasmic dynein is a ubiquitously expressed microtubule motor involved in vesicle transport, mitosis, nuclear migration, and spindle orientation. In the filamentous fungus Aspergillus nidulans, inactivation of cytoplasmic dynein, although not lethal, severely impairs nuclear migration. The role of dynein in mitosis and vesicle transport in this organism is unclear. To investigate the complete range of dynein function in A. nidulans, we searched for synthetic lethal mutations that significantly reduced growth in the absence of dynein but had little effect on their own. We isolated 19 sld (synthetic lethality without dynein) mutations in nine different genes. Mutations in two genes exacerbate the nuclear migration defect seen in the absence of dynein. Mutations in six other genes, including sldA and sldB, show a strong synthetic lethal interaction with a mutation in the mitotic kinesin bimC and, thus, are likely to play a role in mitosis. Mutations in sldA and sldB also confer hypersensitivity to the microtubule-destabilizing drug benomyl. sldA and sldB were cloned by complementation of their mutant phenotypes using an A. nidulans autonomously replicating vector. Sequencing revealed homology to the spindle assembly checkpoint genes BUB1 and BUB3 from Saccharomyces cerevisiae. Genetic interaction between dynein and spindle assembly checkpoint genes, as well as other mitotic genes, indicates that A. nidulans dynein plays a role in mitosis. We suggest a model for dynein motor action in A. nidulans that can explain dynein involvement in both mitosis and nuclear distribution.     

Identification of mutations that cause cell migration defects in mosaic clones

Cell movement is an important feature of animal development, wound healing and tumor metastasis; however, the mechanisms underlying cell motility remain to be elucidated. To further our understanding, it would be useful to identify all of the proteins that are essential for a cell to migrate, yet such information is not currently available for any cell type. We have carried out a screen for mutations affecting border cell migration in Drosophila. Mutations that cause defects in mosaic clones were identified, so that genes that are also required for viability could be detected. From 6000 mutagenized lines, 20 mutations on chromosome 2R were isolated that cause defects in border cell position. One of the mutations was dominant while all of the recessive mutations appeared to be homozygous lethal. This lethality was used to place the mutations into 16 complementation groups. Many of the mutations failed to complement cytologically characterized deficiencies, allowing their rapid mapping. Mutations in three loci altered expression of a marker gene in the border cells, whereas the remaining mutations did not. One mutation, which caused production of supernumerary border cells, was found to disrupt the costal-2 locus, indicating a role for Hedgehog signaling in border cell development. This screen identified many new loci required for border cell migration and our results suggest that this is a useful approach for elucidating the mechanisms involved in cell motility.     

poirot,a new regulatory gene of Drosophila oskar acts at the level of the short Oskar protein isoform

Embryonic germ cell formation and abdomen development in Drosophila requires localisation and site specific translation of oskar mRNA in the posterior part of the oocyte. Targeting of oskar function to the posterior pole of the oocyte needs a large set of proteins and RNAs, encoded by posterior group genes. Consequently, mutations in the posterior group genes can result in embryos without abdomens and/or germ cells. During a systematic hobo-mediated mutant isolation screen, we identified poirot, a novel posterior group gene, owing to its germ cell-less phenotype. We show that the lack of poirot activity dramatically decreases OSK protein levels, without affecting the oskar mRNA distribution. In poirot mutant oocytes, delocalised OSK protein is observed, indicating that wild-type poirot has a role in the anchoring process of the OSK protein at the posterior pole. Furthermore, we demonstrate that poirot acts in an isoform-specific manner, only the short OSK isoform is affected, while the long OSK isoform remains at wild-type levels in poirot mutants.     

A new genetic method for isolating functionally interacting genes: high plo1(+)-dependent mutants and their suppressors define genes in mitotic and septation pathways in fission yeast

We describe a general genetic method to identify genes encoding proteins that functionally interact with and/or are good candidates for downstream targets of a particular gene product. The screen identifies mutants whose growth depends on high levels of expression of that gene. We apply this to the plo1(+) gene that encodes a fission yeast homologue of the polo-like kinases. plo1(+) regulates both spindle formation and septation. We have isolated 17 high plo1(+)-dependent (pld) mutants that show defects in mitosis or septation. Three mutants show a mitotic arrest phenotype. Among the 14 pld mutants with septation defects, 12 mapped to known loci: cdc7, cdc15, cdc11 spg1, and sid2. One of the pld mutants, cdc7-PD1, was selected for suppressor analysis. As multicopy suppressors, we isolated four known genes involved in septation in fission yeast: spg1(+), sce3(+), cdc8(+), and rho1(+), and two previously uncharacterized genes, mpd1(+) and mpd2(+). mpd1(+) exhibits high homology to phosphatidylinositol 4-phosphate 5-kinase, while mpd2(+) resembles Saccharomyces cerevisiae SMY2; both proteins are involved in the regulation of actin-mediated processes. As chromosomal suppressors of cdc7-PD1, we isolated mutations of cdc16 that resulted in multiseptation without nuclear division. cdc16(+), dma1(+), byr3(+), byr4(+) and a truncated form of the cdc7 gene were isolated by complementation of one of these cdc16 mutations. These results demonstrate that screening for high dose-dependent mutants and their suppressors is an effective approach to identify functionally interacting genes.     

Mutations affecting liver development and function in Medaka, Oryzias latipes, screened by multiple criteria

We report here mutations affecting various aspects of liver development and function identified by multiple assays in a systematic mutagenesis screen in Medaka. The 22 identified recessive mutations assigned to 19 complementation groups fell into five phenotypic groups. Group 1, showing defective liver morphogenesis, comprises mutations in four genes, which may be involved in the regulation of growth or patterning of the gut endoderm. Group 2 comprises mutations in three genes that affect the laterality of the liver; in kendama mutants of this group, the laterality of the heart and liver is uncoupled and randomized. Group 3 includes mutations in three genes altering bile color, indicative of defects in hemoglobin-bilirubin metabolism and globin synthesis. Group 4 consists of mutations in three genes, characterized by a decrease in the accumulation of fluorescent metabolite of a phospholipase A(2) substrate, PED6, in the gall bladder. Lipid metabolism or the transport of lipid metabolites may be affected by these mutations. Mutations in Groups 3 and 4 may provide animal models for relevant human diseases. Group 5 mutations in six genes affect the formation of endoderm, endodermal rods and hepatic bud from which the liver develops. These Medaka mutations, identified by morphological and metabolite marker screens, should provide clues to understanding molecular mechanisms underlying formation of a functional liver.     

The Drosophila hnRNPA/B homolog, Hrp48, is specifically required for a distinct step in osk mRNA localization

The Staufen-dependent localization of oskar mRNA to the posterior of the Drosophila oocyte induces the formation of the pole plasm, which contains the abdominal and germline determinants. In a germline clone screen for mutations that disrupt the posterior localization of GFP-Staufen, we isolated three missense alleles in the hnRNPA/B homolog, Hrp48. These mutants specifically abolish osk mRNA localization, without affecting its translational control or splicing, or the localization of bicoid and gurken mRNAs and the organization of the microtubule cytoskeleton. Hrp48 colocalizes with osk mRNA throughout oogenesis, and interacts with its 5' and 3' regulatory regions, suggesting that it binds directly to oskar mRNA to mediate its posterior transport. The hrp48 alleles cause a different oskar mRNA localization defect from other mutants, and disrupt the formation of GFP-Staufen particles. This suggests a new step in the localization pathway, which may correspond to the assembly of Staufen/oskar mRNA transport particles.     

A systematic genome-wide screen for mutations affecting organogenesis in Medaka, Oryzias latipes

A large-scale mutagenesis screen was performed in Medaka to identify genes acting in diverse developmental processes. Mutations were identified in homozygous F3 progeny derived from ENU-treated founder males. In addition to the morphological inspection of live embryos, other approaches were used to detect abnormalities in organogenesis and in specific cellular processes, including germ cell migration, nerve tract formation, sensory organ differentiation and DNA repair. Among 2031 embryonic lethal mutations identified, 312 causing defects in organogenesis were selected for further analyses. From these, 126 mutations were characterized genetically and assigned to 105 genes. The similarity of the development of Medaka and zebrafish facilitated the comparison of mutant phenotypes, which indicated that many mutations in Medaka cause unique phenotypes so far unrecorded in zebrafish. Even when mutations of the two fish species cause a similar phenotype such as one-eyed-pinhead or parachute, more genes were found in Medaka than in zebrafish that produced the same phenotype when mutated. These observations suggest that many Medaka mutants represent new genes and, therefore, are important complements to the collection of zebrafish mutants that have proven so valuable for exploring genomic function in development.     

In vivo commitment to splicing in yeast involves the nucleotide upstream from the branch site conserved sequence and the Mud2 protein.

Pre-mRNA splicing is a stepwise nuclear process involving intron recognition and the assembly of the spliceosome followed by intron excision. We previously developed a pre-mRNA export assay that allows the discrimination between early steps of spliceosome formation and splicing per se. Here we present evidence that these two assays detect different biochemical defects for point mutations. Mutations at the 5' splice site lead to pre-mRNA export, whereas 3' splice site mutations do not. A genetic screen applied to mutants in the branch site region shows that all positions in the conserved TACTAAC sequence are important for intron recognition. An exhaustive analysis of pre-mRNA export and splicing defects of these mutants shows that the in vivo recognition of the branch site region does not involve the base pairing of U2 snRNA with the pre-mRNA. In addition, the nucleotide preceding the conserved TACTAAC sequence contributes to the recognition process. We show that a T residue at this position allows for optimal intron recognition and that in natural introns, this nucleotide is also used preferentially. Moreover, the Mud2 protein is involved in the recognition of this nucleotide, thus establishing a role for this factor in the in vivo splicing pathway.     

Identification of chromosome inheritance modifiers in Drosophila melanogaster

Faithful chromosome inheritance is a fundamental biological activity and errors contribute to birth defects and cancer progression. We have performed a P-element screen in Drosophila melanogaster with the aim of identifying novel candidate genes involved in inheritance. We used a "sensitized" minichromosome substrate (J21A) to screen approximately 3,000 new P-element lines for dominant effects on chromosome inheritance and recovered 78 Sensitized chromosome inheritance modifiers (Scim). Of these, 69 decreased minichromosome inheritance while 9 increased minichromosome inheritance. Fourteen mutations are lethal or semilethal when homozygous and all exhibit dramatic mitotic defects. Inverse PCR combined with genomic analyses identified P insertions within or close to genes with previously described inheritance functions, including wings apart-like (wapl), centrosomin (cnn), and pavarotti (pav). Further, lethal insertions in replication factor complex 4 (rfc4) and GTPase-activating protein 1 (Gap1) exhibit specific mitotic chromosome defects, discovering previously unknown roles for these proteins in chromosome inheritance. The majority of the lines represent mutations in previously uncharacterized loci, many of which have human homologs, and we anticipate that this collection will provide a rich source of mutations in new genes required for chromosome inheritance in metazoans.     

Mutations in Polycombeotic, a Drosophila Polycomb-Group Gene, Cause a Wide Range of Maternal and Zygotic Phenotypes

The polycomb-group genes, a set of genes characterized by mutations that cause similar phenotypes and dosage-dependent interactions, are required for the normal expression of segment-specific homeotic loci. Here we report that polycombeotic (formerly 1(3)1902), originally identified by a lethal mutation that causes a small-disc phenotype, is also a member of this group of essential genes. Adults homozygous for temperature-sensitive pco alleles that were exposed to the restrictive temperature during larval life display the second and third leg to first leg transformation characteristic of polycomb-group mutants. Adult females homozygous for temperature-sensitive alleles exposed to the restrictive temperature during oogenesis produce embryos that show anterior segments with structures normally unique to the eighth abdominal segment, another transformation characteristic of polycomb-group mutants. Mutations in the polycombeotic gene also cause defects not reported for mutations in other polycomb-group genes. Females homozygous for the most extreme temperature-sensitive allele are sterile, and larvae homozygous for null alleles have small imaginal discs and reduced frequencies of mitotic figures in the brain. Dominant mutations originally identified as enhancers or suppressors of zeste are gain-of-function alleles of polycombeotic. The type and variety of defects displayed by different mutations in this gene indicate that the product might be involved in chromosome structure and/or function.     

Drosophila development: RNA interference ab ovo

A novel protein required for RNA interference in Drosophila, Armitage, was identified in a screen for genes involved in embryonic axis formation. In armitage mutants, oocyte polarity and the regulation of oskar mRNA translation are impaired, suggesting that RNA silencing regulates the first steps of Drosophila development.     

Molecular genetic and clinical aspects of mitochondrial disorders in childhood

Mitochondrial OXPHOS disorders are caused by mutations in mitochondrial or nuclear genes, which directly or indirectly affect mitochondrial oxidative phosphorylation (OXPHOS). Primary mtDNA abnormalities in children are due to rearrangements (deletions or duplications) and point mutations or insertions. Mutations in the nuclear-encoded polypeptide subunits of OXPHOS result in complex I and II deficiency, whereas mutations in the nuclear proteins involved in the assembly of OXPHOS subunits cause defects in complexes I, III, IV, and V. Here, we review recent progress in the identification of mitochondrial and nuclear gene defects and the associated clinical manifestations of these disorders in childhood.     

Relationships between a Hyper-Rec Mutation (rem1 ) and Other Recombination and Repair Genes in Yeast

Mutations in the REM1 gene of Saccharomyces cerevisiae confer a semidominant hyper-recombination and hypermutable phenotype upon mitotic cells ( GOLIN and ESPOSITO 1977). These effects have not been observed in meiosis. We have examined the interactions of rem1 mutations with rad6-1, rad50 -1, rad52-1 or spo11 -1 mutations in order to understand the basis of the rem1 hyper-rec phenotype. The rad mutations have pleiotropic phenotypes; spo11 is only defective in sporulation and meiosis. The RAD6, RAD50 and SPO11 genes are not required for spontaneous mitotic recombination; mutations in the RAD52 gene cause a general spontaneous mitotic Rec- phenotype. Mutations in RAD50 , RAD52 or SPO11 eliminate meiotic recombination, and mutations in RAD6 prevent spore formation. Evidence for the involvement of RAD6 in meiotic recombination is less clear. Mutations in all three RAD genes confer sensitivity to X rays; the RAD6 gene is also required for UV damage repair. To test whether any of these functions might be involved in the hyper-rec phenotype conferred by rem1 mutations, double mutants were constructed. Double mutants of rem1 spo11 were viable and demonstrated rem1 levels of mitotic recombination, suggesting that the normal meiotic recombination system is not involved in producing the rem1 phenotype. The rem1 rad6 double mutant was also viable and had rem1 levels of mitotic recombination. Neither rem1 rad50 nor rem1 rad52 double mutants were viable. This suggests that rem1 causes its hyper-rec phenotype because it creates lesions in the DNA that are repaired using a recombination-repair system involving RAD50 and RAD52.     

A clonal genetic screen for mutants causing defects in larval tracheal morphogenesis in Drosophila

The initial establishment of the tracheal network in the Drosophila embryo is beginning to be understood in great detail, both in its genetic control cascades and in its cell biological events. By contrast, the vast expansion of the system during larval growth, with its extensive ramification of preexisting tracheal branches, has been analyzed less well. The mutant phenotypes of many genes involved in this process are probably not easy to reveal, as these genes may be required for other functions at earlier developmental stages. We therefore conducted a screen for defects in individual clonal homozygous mutant cells in the tracheal network of heterozygous larvae using the mosaic analysis with a repressible cell marker (MARCM) system to generate marked, recombinant mitotic clones. We describe the identification of a set of mutants with distinct phenotypic effects. In particular we found a range of defects in terminal cells, including failure in lumen formation and reduced or extensive branching. Other mutations affect cell growth, cell shape, and cell migration.     

A genetic screen for temperature-sensitive cell-division mutants of Caenorhabditis elegans.

A novel screen to isolate conditional cell-division mutants in Caenorhabditis elegans has been developed. The screen is based on the phenotypes associated with existing cell-division mutations: some disrupt postembryonic divisions and affect formation of the gonad and ventral nerve cord-resulting in sterile, uncoordinated animals-while others affect embryonic divisions and result in lethality. We obtained 19 conditional mutants that displayed these phenotypes when shifted to the restrictive temperature at the appropriate developmental stage. Eighteen of these mutations have been mapped; 17 proved to be single alleles of newly identified genes, while 1 proved to be an allele of a previously identified gene. Genetic tests on the embryonic lethal phenotypes indicated that for 13 genes, embryogenesis required maternal expression, while for 6, zygotic expression could suffice. In all cases, maternal expression of wild-type activity was found to be largely sufficient for embryogenesis. Cytological analysis revealed that 10 mutants possessed embryonic cell-division defects, including failure to properly segregate DNA, failure to assemble a mitotic spindle, late cytokinesis defects, prolonged cell cycles, and improperly oriented mitotic spindles. We conclude that this approach can be used to identify mutations that affect various aspects of the cell-division cycle.     

Potential mechanisms of mutations that affect neuronal migration in man and mouse

Mutations in the genes that encode filamin-1, Lis1 and doublecortin are responsible for X-linked lissencephaly in man, whereas mutations in the genes that encode Cdk5, its activator p35 and the reelin-signaling pathway disturb migration and architectonic development in mice. To understand the action of genes that control neuronal migration and the phenotype of corresponding defects, it might be as important to consider the positioning of the nucleus as it is to consider the guidance of the leading process.     

A targeted RNAi screen for genes involved in chromosome morphogenesis and nuclear organization in the Caenorhabditis elegans germline

We have implemented a functional genomics strategy to identify genes involved in chromosome morphogenesis and nuclear organization during meiotic prophase in the Caenorhabditis elegans germline. This approach took advantage of a gene-expression survey that used DNA microarray technology to identify genes preferentially expressed in the germline. We defined a subset of 192 germline-enriched genes whose expression profiles were similar to those of previously identified meiosis genes and designed a screen to identify genes for which inhibition by RNA interference (RNAi) elicited defects in function or development of the germline. We obtained strong germline phenotypes for 27% of the genes tested, indicating that this targeted approach greatly enriched for genes that function in the germline. In addition to genes involved in key meiotic prophase events, we identified genes involved in meiotic progression, germline proliferation, and chromosome organization and/or segregation during mitotic growth.     

An F1 genetic screen for maternal-effect mutations affecting embryonic pattern formation in Drosophila melanogaster

Large-scale screens for female-sterile mutations have revealed genes required maternally for establishment of the body axes in the Drosophila embryo. Although it is likely that the majority of components involved in axis formation have been identified by this approach, certain genes have escaped detection. This may be due to (1) incomplete saturation of the screens for female-sterile mutations and (2) genes with essential functions in zygotic development that mutate to lethality, precluding their identification as female-sterile mutations. To overcome these limitations, we performed a genetic mosaic screen aimed at identifying new maternal genes required for early embryonic patterning, including zygotically required ones. Using the Flp-FRT technique and a visible germline clone marker, we developed a system that allows efficient screening for maternal-effect phenotypes after only one generation of breeding, rather than after the three generations required for classic female-sterile screens. We identified 232 mutants showing various defects in embryonic pattern or morphogenesis. The mutants were ordered into 10 different phenotypic classes. A total of 174 mutants were assigned to 86 complementation groups with two alleles on average. Mutations in 45 complementation groups represent most previously known maternal genes, while 41 complementation groups represent new loci, including several involved in dorsoventral, anterior-posterior, and terminal patterning.