A screen for dominant modifiers of the irreC-rst cell death phenotype in the developing Drosophila retina

Programmed cell death (PCD) in the Drosophila retina requires activity of the irregular chiasmC-roughest (irreC-rst) gene. Loss-of-function mutations in irreC-rst block PCD during retinal development and lead to a rough eye phenotype in the adult. To identify genes that interact with irreC-rst and may be involved in PCD, we conducted a genetic screen for dominant enhancers and suppressors of the adult rough eye phenotype. We screened 150,000 mutagenized flies and recovered 170 dominant modifiers that localized primarily to the second and third chromosomes. At least two allelic groups correspond to previously identified death regulators, Delta and dRas1. Examination of retinae from homozygous viable mutants indicated two major phenotypic classes. One class exhibited pleiotropic defects while the other class exhibited defects specific to the cell population that normally undergoes PCD.     

HID-1 is a peripheral membrane protein primarily associated with the medial- and trans- Golgi apparatus

Caenorhabditis elegans hid-1 gene was first identified in a screen for mutants with a high-temperature-induced dauer formation (Hid) phenotype. Despite the fact that the hid-1 gene encodes a novel protein (HID-1) which is highly conserved from Caenorhabditis elegans to mammals, the domain structure, subcellular localization, and exact function of HID-1 remain unknown. Previous studies and various bioinformatic softwares predicted that HID-1 contained many transmembrane domains but no known functional domain. In this study, we revealed that mammalian HID-1 localized to the medial- and trans-Golgi apparatus as well as the cytosol, and the localization was sensitive to brefeldin A treatment. Next, we demonstrated that HID-1 was a peripheral membrane protein and dynamically shuttled between the Golgi apparatus and the cytosol. Finally, we verified that a conserved N-terminal myristoylation site was required for HID-1 binding to the Golgi apparatus. We propose that HID-1 is probably involved in the intracellular trafficking within the Golgi region.     

拟南芥温度诱导脂质运载蛋白TIL1参与雌配子体发育

雌配子体的正常发育是种子形成的前提条件之一,拟南芥温度诱导的脂质运载蛋白编码基因TIL1突变使胚珠败育,结实率下降明显。基因表达分析表明T-DNA插入使得TIL1基因敲除,突变体TIL1基因功能缺失;互交实验、Alexander染色、花粉离体培养和胚珠透明实验结果表明till-1突变体雄配子体发育正常、雌配子体胚囊发育有缺陷;通过遗传互补实验证明外源克隆的TIL1基因能恢复突变体的败育表型,并确定了TIL1基因主要在胚珠的胚囊中表达。实验结果表明TIL1基因参与了植物雌配子体发育这一重要的生理过程。     

The Transformer Genes of the Fern Ceratopteris Simultaneously Promote Meristem and Archegonia Development and Repress Antheridia Development in the Developing Gametophyte

The sex of the haploid gametophyte of the fern Ceratopteris is determined by the presence or absence of the pheromone antheridiogen, which, when present, promotes male development and represses female development of the gametophyte. Several genes involved in sex determination in Ceratopteris have been identified by mutation. In this study, the epistatic interactions among new and previously described sex-determining mutants have been characterized. These results show that sex expression is regulated by two sets of genes defined by the FEM1 and TRA loci. Each promotes the expression of either male or female traits and simultaneously represses the expression of the other. A model describing how antheridiogen regulates the expression of these genes and the sex of the gametophyte is described. The observation that some gametophytic sex-determining mutants have phenotypic effects on the sporophyte plant indicates that sex determination in the Ceratopteris gametophyte is regulated by a mechanism that also regulates sporophyte development.     

Characterization of the two maize embryo-lethal defective kernel mutants rgh*-1210 and fl*-1253b: effects on embryo and gametophyte development

We have examined the effects on embryonic and gametophytic development of two nonallelic defective-kernel mutants of maize. Earlier studies indicated that both mutants are abnormal in embryonic morphogenesis as well as in the formation of their endosperm. Mutant rgh*-1210 embryos depart from the normal embryogenic pathway at the proembryo and transition stage, by developing meristematic lobes and losing bilateral symmetry. They continue growth as irregular cell masses that enlarge and become necrotic. Somatic embryos arising in rgh*-1210 callus cultures display the rgh*-1210 mutant phenotype. Mutant fl*-1253B embryos are variably blocked from the coleoptilar stage through stage 2. Following formation of the shoot apex in the mutant embryos the leaf primordia and tissues surrounding the embryonic axis continue growth and cell division, while the scutellum ceases development and becomes hypertrophied. Mutant fl*-1253B embryos are unable to germinate, either in mutant kernels or as immature embryos in culture, and the mutant scutellar tissue does not produce regenerable callus. Expression of the fl*-1253B locus during male gametophytic development is revealed by a marked reduction in pollen transmission as a result of mutant expression during the interval between meiosis and the initiation of pollen tube growth. In both mutants, there is considerable proliferation of the aleurone cells of the endosperm. Mutant expression of rgh*-1210 in the female gametophyte is revealed by the abnormal antipodal cells of the embryo sac. These results show that these two gene loci play unique and crucial roles in normal morphogenesis of the embryo. In addition, it is evident that both mutants are pleiotropic in affecting the development of the endosperm and gametophyte as well as the embryo. These pleiotropisms suggest some commonality in the gene regulation of development in these three tissues.     

Disruption of apyrases inhibits pollen germination in Arabidopsis

In Arabidopsis, we previously identified two highly similar apyrases, AtAPY1 and AtAPY2. Here, T-DNA knockout (KO) mutations of each gene were isolated in a reverse genetic approach. The single KO mutants lacked a discernible phenotype. The double KO mutants, however, exhibited a complete inhibition of pollen germination, and this correlated with positive beta-glucuronidase staining in the pollen of apyrase promoter:beta-glucuronidase fusion transgenic lines. The vast majority of the pollen grains of these mutants were identical to wild type in size, shape, and nuclear state and were viable as assayed by metabolic activity and plasma membrane integrity. Complementation with either AtAPY1 or AtAPY2 cDNA rescued pollen germination, confirming that the phenotype was apyrase specific. Despite the redundancy of the two apyrases in rescue potential, transmission analyses suggested a greater role for AtAPY2 in male gamete success. The effect of mutant apyrase on the transmission through the female gametophyte was only marginal, and embryo development appeared normal in the absence of apyrases. The male-specific double KO mutation is fully penetrant and shows that apyrases play a crucial role in pollen germination.     

The role of salicylic acid in the induction of cell death in Arabidopsis acd11

Salicylic acid (SA) is implicated in the induction of programmed cell death (PCD) associated with pathogen defense responses because SA levels increase in response to PCD-inducing infections, and PCD development can be inhibited by expression of salicylate hydroxylase encoded by the bacterial nahG gene. The acd11 mutant of Arabidopsis (Arabidopsis thaliana L. Heynh.) activates PCD and defense responses that are fully suppressed by nahG. To further study the role of SA in PCD induction, we compared phenotypes of acd11/nahG with those of acd11/eds5-1 and acd11/sid2-2 mutants deficient in a putative transporter and isochorismate synthase required for SA biosynthesis. We show that sid2-2 fully suppresses SA accumulation and cell death in acd11, although growth inhibition and premature leaf chlorosis still occur. In addition, application of exogenous SA to acd11/sid2-2 is insufficient to restore cell death. This indicates that isochorismate-derived compounds other than SA are required for induction of PCD in acd11 and that some acd11 phenotypes require NahG-degradable compounds not synthesized via isochorismate.     

Drosophila RecQ5 is required for efficient SSA repair and suppression of LOH in vivo

RecQ5 in mammalian cells has been suggested to suppress inappropriate homologous recombination. However, the specific pathway(s) in which it is involved and the underlining mechanism(s) remain poorly understood. We took advantage of genetic tools in Drosophila to investigate how Drosophila RecQ5 (dRecQ5) functions in vivo in homologous recombination-mediated double strand break (DSB) repair. We generated null alleles of dRecQ5 using the targeted recombination technique. The mutant animals are homozygous viable, but with growth retardation during development. The mutants are sensitive to both exogenous DSB-inducing treatment, such as gamma-irradiation, and endogenously induced double strand breaks (DSBs) by I-Sce I endonuclease. In the absence of dRecQ5, single strand annealing (SSA) -mediated DSB repair is compromised with compensatory increases in either inter-homologous gene conversion, or non-homologous end joining (NHEJ) when inter-chromosomal homologous sequence is unavailable. Loss of function of dRecQ5 also leads to genome instability in loss of heterozygosity (LOH) assays. Together, our data demonstrate that dRecQ5 functions in SSA-mediated DSB repair to achieve its full efficiency and in suppression of LOH in Drosophila.     

NUCLEAR FUSION DEFECTIVE1 encodes the Arabidopsis RPL21M protein and is required for karyogamy during female gametophyte development and fertilization

Karyogamy, or nuclear fusion, is essential for sexual reproduction. In angiosperms, karyogamy occurs three times: twice during double fertilization of the egg cell and the central cell and once during female gametophyte development when the two polar nuclei fuse to form the diploid central cell nucleus. The molecular mechanisms controlling karyogamy are poorly understood. We have identified nine female gametophyte mutants in Arabidopsis (Arabidopsis thaliana), nuclear fusion defective1 (nfd1) to nfd9, that are defective in fusion of the polar nuclei. In the nfd1 to nfd6 mutants, failure of fusion of the polar nuclei is the only defect detected during megagametogenesis. nfd1 is also affected in karyogamy during double fertilization. Using transmission electron microscopy, we showed that nfd1 nuclei fail to undergo fusion of the outer nuclear membranes. nfd1 contains a T-DNA insertion in RPL21M that is predicted to encode the mitochondrial 50S ribosomal subunit L21, and a wild-type copy of this gene rescues the mutant phenotype. Consistent with the predicted function of this gene, an NFD1-green fluorescent protein fusion protein localizes to mitochondria and the NFD1/RPL21M gene is expressed throughout the plant. The nfd3, nfd4, nfd5, and nfd6 mutants also contain T-DNA insertions in genes predicted to encode proteins that localize to mitochondria, suggesting a role for this organelle in nuclear fusion.     

Arabidopsis hapless mutations define essential gametophytic functions

In flowering plants, the egg develops within a haploid embryo sac (female gametophyte) that is encased within the pistil. The haploid pollen grain (male gametophyte) extends a pollen tube that carries two sperm cells within its cytoplasm to the embryo sac. This feat requires rapid, precisely guided, and highly polarized growth through, between, and on the surface of the cells of the stigma, style, and ovary. Pollen tube migration depends on a series of long-range signals from diploid female cells as well as a short-range attractant emitted by the embryo sac that guides the final stage of tube growth. We developed a genetic screen in Arabidopsis thaliana that tags mutant pollen with a cell-autonomous marker carried on an insertion element. We found 32 haploid-disrupting (hapless) mutations that define genes required for pollen grain development, pollen tube growth in the stigma and style, or pollen tube growth and guidance in the ovary. We also identified genomic DNA flanking the insertion element for eleven hap mutants and showed that hap1 disrupts AtMago, a gene whose ortholog is important for Drosophila cell polarity.     

Characterization of mutations that feminize gametophytes of the fern Ceratopteris.

Gametophytes of the fern Ceratopteris are either male or hermaphroditic. Their sex is epigenetically determined by the pheromone antheridiogen, which is secreted by the hermaphrodite and induces male and represses female development in other young, sexually undetermined gametophytes. To understand how antheridiogen represses the development of female traits at the genetic level, 16 new mutations that feminize the gametophyte in the presence of antheridiogen were identified and characterized. Seven are very tightly linked to the FEM1 locus previously described. Nine others define another locus named NOTCHLESS1 (NOT1), as several of the not1 mutants lack a meristem notch. Some not1 mutations also affect sporophyte development only when homozygous, indicating that the not1 mutations are recessive and that NOT1 is also required for normal sporophyte development. The epistatic interactions among FEM1, NOT1, and other sex-determining genes are described. This information was used to expand the genetic model of the sex-determining pathway in Ceratopteris. On the basis of this model, we can say that the presence of antheridiogen leads to the activation of the FEM1 gene, which not only promotes the differentiation of male traits, but also represses female development by activating the NOT1 gene. NOT1 represses the TRA genes necessary for the development of female traits in the gametophyte.     

Loss-of-function mutations in a human gene related to Chlamydomonas reinhardtii dynein IC78 result in primary ciliary dyskinesia

Primary ciliary dyskinesia (PCD) is a group of heterogeneous disorders of unknown origin, usually inherited as an autosomal recessive trait. Its phenotype is characterized by axonemal abnormalities of respiratory cilia and sperm tails leading to bronchiectasis and sinusitis, which are sometimes associated with situs inversus (Kartagener syndrome) and male sterility. The main ciliary defect in PCD is an absence of dynein arms. We have isolated the first gene involved in PCD, using a candidate-gene approach developed on the basis of documented abnormalities of immotile strains of Chlamydomonas reinhardtii, which carry axonemal ultrastructural defects reminiscent of PCD. Taking advantage of the evolutionary conservation of genes encoding axonemal proteins, we have isolated a human sequence (DNAI1) related to IC78, a C. reinhardtii gene encoding a dynein intermediate chain in which mutations are associated with the absence of outer dynein arms. DNAI1 is highly expressed in trachea and testis and is composed of 20 exons located at 9p13-p21. Two loss-of-function mutations of DNAI1 have been identified in a patient with PCD characterized by immotile respiratory cilia lacking outer dynein arms. In addition, we excluded linkage between this gene and similar PCD phenotypes in five other affected families, providing a clear demonstration of locus heterogeneity. These data reveal the critical role of DNAI1 in the development of human axonemal structures and open up new means for identification of additional genes involved in related developmental defects.     

The PET1-CMS mitochondrial mutation in sunflower is associated with premature programmed cell death and cytochrome c release

In mammals, mitochondria have been shown to play a key intermediary role in apoptosis, a morphologically distinct form of programmed cell death (PCD), for example, through the release of cytochrome c, which activates a proteolytic enzyme cascade, resulting in specific nuclear DNA degradation and cell death. In plants, PCD is a feature of normal development, including the penultimate stage of anther development, leading to dehiscence and pollen release. However, there is little evidence that plant mitochondria are involved in PCD. In a wide range of plant species, anther and/or pollen development is disrupted in a class of mutants termed CMS (for cytoplasmic male sterility), which is associated with mutations in the mitochondrial genome. On the basis of the manifestation of a number of morphological and biochemical markers of apoptosis, we have shown that the PET1-CMS cytoplasm in sunflower causes premature PCD of the tapetal cells, which then extends to other anther tissues. These features included cell condensation, oligonucleosomal cleavage of nuclear DNA, separation of chromatin into delineated masses, and initial persistence of mitochondria. In addition, immunocytochemical analysis revealed that cytochrome c was released partially from the mitochondria into the cytosol of tapetal cells before the gross morphological changes associated with PCD. The decrease in cytochrome c content in mitochondria isolated from male sterile florets preceded a decrease in the integrity of the outer mitochondrial membrane and respiratory control ratio. Our data suggest that plant mitochondria, like mammalian mitochondria, play a key role in the induction of PCD. The tissue-specific nature of the CMS phenotype is discussed with regard to cellular respiratory demand and PCD during normal anther development.     

BIN2, a new brassinosteroid-insensitive locus in Arabidopsis

Brassinosteroids (BRs) play important roles throughout plant development. Although many genes have been identified that are involved in BR biosynthesis, genetic approaches in Arabidopsis have led to the identification of only one gene, BRI1, that encodes a membrane receptor for BRs. To expand our knowledge of the molecular mechanism(s) of plant steroid signaling, we analyzed many dwarf and semidwarf mutants collected from our previous genetic screens and identified a semidwarf mutant that showed little response to exogenous BR treatments. Genetic analysis of the bin2 (BR-INSENSITIVE 2) mutant indicated that the BR-insensitive dwarf phenotype was due to a semidominant mutation in the BIN2 gene that mapped to the middle of chromosome IV between the markers CH42 and AG. A direct screening for similar semidwarf mutants resulted in the identification of a second allele of the BIN2 gene. Despite some novel phenotypes observed with the bin2/+ mutants, the homozygous bin2 mutants were almost identical to the well-characterized bri1 mutants that are defective in BR perception. In addition to the BR-insensitive dwarf phenotype, bin2 mutants exhibited BR insensitivity when assayed for root growth inhibition and feedback inhibition of CPD gene expression. Furthermore, bin2 mutants displayed an abscisic acid-hypersensitive phenotype that is shared by the bri1 and BR-deficient mutants. A gene dosage experiment using triploid plants suggested that the bin2 phenotypes were likely caused by either neomorphic or hypermorphic gain-of-function mutations in the BIN2 gene. Thus, the two bin2 mutations define a novel genetic locus whose gene product might play a role in BR signaling.     

mummy/cystic encodes an enzyme required for chitin and glycan synthesis, involved in trachea, embryonic cuticle and CNS development--analysis of its role in Drosophila tracheal morphogenesis

Tracheal and nervous system development are two model systems for the study of organogenesis in Drosophila. In two independent screens, we identified three alleles of a gene involved in tracheal, cuticle and CNS development. Here, we show that these alleles, and the previously identified cystic and mummy, all belong to the same complementation group. These are mutants of a gene encoding the UDP-N-acetylglucosamine diphosphorylase, an enzyme responsible for the production of UDP-N-acetylglucosamine, an important intermediate in chitin and glycan biosynthesis. cyst was originally singled out as a gene required for the regulation of tracheal tube diameter. We characterized the cyst/mmy tracheal phenotype and upon histological examination concluded that mmy mutant embryos lack chitin-containing structures, such as the procuticle at the epidermis and the taenidial folds in the tracheal lumen. While most of their tracheal morphogenesis defects can be attributed to the lack of chitin, when compared to krotzkopf verkehrt (kkv) chitin-synthase mutants, mmy mutants showed a stronger phenotype, suggesting that some of the mmy phenotypes, like the axon guidance defects, are chitin-independent. We discuss the implications of these new data in the mechanism of size control in the Drosophila trachea.