![A synthetic miRNA directed against white is functional in vivo and a useful screening tool for miRNA biogenesis factors. (A) Predicted secondary structure of w-miR and its predicted interaction with the target sequence in the white 39 UTR. The predicted mature strand is indicated in red. (B) S2 cells were transfected with the indicated plasmids, together with ub-gal4. Values represent fold repression of luciferase activity relative to empty psiCHECK vector. (C) Wing disc sensor assay. w-miR expression driven by ptc-gal4 (C) results in repression of tubGFP-w-39UTR sensor (C9 and merge, C 00 ). (D) A white eye. (E) A wild-type (Canton S) eye. (F) Eye of an animal with pGUS-DsRed-w-miR transgene. (G) Clones of dcr-1[Q1147X] in the background of GMR-w-miR result in a mosaic eye with darker patches. (H) Example of presumed enhancer of miR-mediated silencing from our screen (gene unknown). (I) Example of suppressor of miR-mediated silencing from our screen (pasha[23D2], which is a newly isolated null mutant of pasha that results in the premature truncation of Pasha at R308).](https://www.researchgate.net/profile/Dong-Wang-109/publication/51674276/figure/fig6/AS:668669784899608@1536434708833/A-synthetic-miRNA-directed-against-white-is-functional-in-vivo-and-a-useful-screening_Q320.jpg)
![Identification and characterization of novel Microprocessor alleles. (A) Molecular lesions of pasha alleles. pasha[KO] is a complete locus deletion that has been described previously (Martin et al. 2009). (B) Molecular lesions of drosha alleles. (C–K) Phenotypic characterization of pasha alleles: (C–E) Adult eyes with clones of the indicated genotype in the background of GMR-w-miR. Note that the pasha[KO] allele itself has a w[+] transgene and is included only for comparison of morphology, not pigmentation. (F–H) Third instar disc clusters of indicated alleles in trans with pasha[KO] stained with Elav (green) and a tissue marker (Phalloidin, [KO] and [6B3]; or Hoechst, 33352-[14D4], purple). (I–K) Mitotic clones of indicated alleles marked by the absence of b-gal (red) and stained for GFP-ban sensor (green, I9–K9) and Mei-P26 (grayscale, I99–K99). (L–T) Phenotypic characterization of drosha alleles: (L,M) Adult eyes with clones of the indicated genotype in the background of GMR-w-miR. (O–Q) Third instar disc clusters of indicated alleles in trans with Df(2R)exel6055 stained with Elav (green) and a Hoechst 33352 (purple). (R–T) Mitotic clones of indicated alleles marked by the absence of b-gal (red) and stained for GFP-ban sensor (green, R9–T9) and Mei-P26 (grayscale, R99–T99).](https://www.researchgate.net/profile/Dong-Wang-109/publication/51674276/figure/fig1/AS:340515808661510@1458196703721/Identification-and-characterization-of-novel-Microprocessor-alleles-A-Molecular_Q320.jpg)
![Effects of Microprocessor mutants on small RNA maturation. (A) Accumulation of pri-miRNA transcripts in null Microprocessor mutants. Total RNA was extracted from Canton S, homozygous pasha[KO], or trans-heterozygous drosha[21K11]/ Df(2R)exel6055 animals and assayed by qRT-PCR. The y-axis represents the fold up-regulation of the indicated miRNA relative to the Canton S sample and standard deviation. (B) Mature miRNA levels in Microprocessor mutants. Total RNA was extracted from third instar larvae of the indicated genotypes. For the pasha alleles, indicated alleles were in trans with pasha[KO]. For the drosha alleles, indicated alleles were in trans with Df(2R)exel6055. The small RNA blot was sequentially probed as indicated. 2S rRNA was used as a loading and transferring control. (C) Ribosomal RNA maturation is unaffected in Microprocessor mutants. RNA from the same samples was run simultaneously on two gels, transferred, and probed for 5S and 5.8S rRNA.](https://www.researchgate.net/profile/Dong-Wang-109/publication/51674276/figure/fig2/AS:340515808661512@1458196703814/Effects-of-Microprocessor-mutants-on-small-RNA-maturation-A-Accumulation-of-pri-miRNA_Q320.jpg)
![In vivo cross-regulation of Microprocessor components in Drosophila. In all cases, mutant clones are marked by the absence of b-gal (stained in red). (A) A clone for drosha[21K11] has strongly reduced Drosha staining (A9). (B) A pasha[KO] clone also has strongly reduced Drosha staining (B9). (C) Schematic representation of pasha locus demonstrating position of 59-UTR hairpins. (D–G) Clones of pasha[KO] (D), dcr- 1[Q1147X] (E), and drosha[21K11], either in a wild-type background (F) or in a background homozygous for pasha[KO] rescued by two copies of a pasha genomic rescue fragment lacking the two hairpins in the 59 UTR (G) exhibit derepression of Mei-P26 (D9–G9), indicating loss of miRNA pathway activity. (H–K) Clones of the same genotypes as D–G have different effects on Pasha levels (H9–K9). (H) Clones of pasha[KO] have no detectable Pasha staining. (I) Pasha levels are unaffected in dcr-1[Q1147X] clones. (J) Pasha protein is elevated in clones of drosha[21K11]. (K) In a genetic background homozygous for the pasha[KO] allele but rescued with two copies of a pasha genomic rescue transgene lacking the two hairpins in the 59 UTR, clones for drosha[21K11] do not have altered Pasha levels.](https://www.researchgate.net/profile/Dong-Wang-109/publication/51674276/figure/fig3/AS:340515812855809@1458196704273/In-vivo-cross-regulation-of-Microprocessor-components-in-Drosophila-In-all-cases-mutant_Q320.jpg)
![Hypomorphic miRNA pathway mutants exhibit normal eye development. (A) Schematic representation of Dcr-1 protein with positions of missense mutations dcr-1[18E6] and dcr-1[19E2] and nonsense mutation dcr-1[Q1147X] (Lee et al. 2004) indicated. (B) Control w-miR eye. Eyes bearing clones of dcr-1[18E6] (C) or dcr- 1[19E2] (D) in the background of w-miR exhibit derepression of the white reporter, seen as darker red pigmentation, but otherwise lack roughness that might indicate aberrant development. (E–H) In comparison to wild type, hypomorphic mutants of dcr-1, drosha, and pasha all exhibit normal patterns of photoreceptor projections to the central brain.](https://www.researchgate.net/profile/Dong-Wang-109/publication/51674276/figure/fig4/AS:340515812855812@1458196704366/Hypomorphic-miRNA-pathway-mutants-exhibit-normal-eye-development-A-Schematic_Q320.jpg)
Content uploaded by Dong Wang
Author content
All content in this area was uploaded by Dong Wang on Jan 27, 2014
Content may be subject to copyright.
A preview of the PDF is not available
A Drosophila genetic screen yields allelic series of core microRNA biogenesis factors and reveals post-developmental roles for microRNAs
Abstract and Figures
Canonical animal microRNAs (miRNAs) are ∼22-nt regulatory RNAs generated by stepwise cleavage of primary hairpin transcripts by the Drosha and Dicer RNase III enzymes. We performed a genetic screen using an miRNA-repressed reporter in the Drosophila eye and recovered the first reported alleles of fly drosha, an allelic series of its dsRBD partner pasha, and novel alleles of dicer-1. Analysis of drosha mutants provided direct confirmation that mirtrons are independent of this nuclease, as inferred earlier from pasha knockouts. We further used these mutants to demonstrate in vivo cross-regulation of Drosha and Pasha in the intact animal, confirming remarkable conservation of a homeostatic mechanism that aligns their respective levels. Although the loss of core miRNA pathway components is universally lethal in animals, we unexpectedly recovered hypomorphic alleles that gave adult escapers with overtly normal development. However, the mutant photoreceptor neurons exhibited reduced synaptic transmission, without accompanying defects in neuronal development or maintenance. These findings indicate that synaptic function is especially sensitive to optimal miRNA pathway function. These allelic series of miRNA pathway mutants should find broad usage in studies of miRNA biogenesis and biology in the Drosophila system.
Figures - uploaded by Dong Wang
Author content
All figure content in this area was uploaded by Dong Wang
Content may be subject to copyright.
... It has been previously reported that homozygous drosha null mutants die during the course of post-embryonic development with 100% lethality before reaching adulthood (10). Death is marked at the end of the third instar larval stage and the beginning of pupariation due to the lack of imaginal discs (18). This is a severe and relatively rare developmental phenotype that prevents pupation (67). ...
... viable hypomorphic alleles exhibit synaptic transmission defects (18), indicating its role in retinal/neural development and function (68). ...
... To assess the phenotypes associated with the loss of Drosha, we examined four available null alleles; drosha Q884X , drosha Q938X , drosha R662X , and drosha W1123X ( Figure 2C). Homozygous mutant flies for each allele die at the end of the 3 rd instar larval stage or the beginning of pupariation (Supplemental Figure 2A), consistent with previously published data (18). We next looked for the presence of imaginal discs that surround the larval brain in the four drosha mutants ( Figure 3A). ...
DROSHA encodes a ribonuclease that is a subunit of the Microprocessor complex and is involved in the first step of microRNA (miRNA) biogenesis. To date, DROSHA has not yet been associated with a Mendelian disease. Here we describe two individuals with profound intellectual disability, epilepsy, white matter atrophy, microcephaly, and dysmorphic features, who carry damaging de novo heterozygous variants in DROSHA. DROSHA is constrained for missense variants and moderately intolerant to loss of function (o/e = 0.24). The loss of the fruit fly ortholog drosha causes developmental arrest and death in third instar larvae, a severe reduction in brain size, and loss of imaginal discs in the larva. Loss of drosha in eye clones causes small and rough eyes in adult flies. One of the identified DROSHA variants (p.Asp1219Gly) behaves as a strong loss-of-function allele in flies, while another variant (p.Arg1342Trp) is less damaging in our assays. In worms, a knock-in that mimics the p.Asp1219Gly variant at a worm equivalent residue causes loss of miRNA expression and heterochronicity, a phenotype characteristic of the loss of miRNA. Together, our data show that the DROSHA variants found in the individuals presented here are damaging based on functional studies in model organisms and likely underlie the severe phenotype involving the nervous system.
... However, with the completion of numerous genome sequences and the identification of many miRNAs in uni-and multi-cellular plants and animals that did not exhibit temporal expression concomitant with development it became evident that miRNAs perform more complex biological functions in regulating gene expression [6,7]. It is now recognised that most mRNAs are regulated by one or more miRNAs [8] and that they play central roles in co-ordinating gene expression during growth, development, differentiation, metabolism, reproduction, and pathogenesis [7,[9][10][11][12]. ...
... The increase in the expression of these genes reflects a significant developmental transition for the parasite as it acquires the ability to feed on host tissue and blood, migrate and undergo rapid periods of growth. These same biological processes are also evident in the adult parasites as they continue to feed and grow (clusters [8][9][10][11]. However, specific to the adult stage is the presence of zinc ion binding, which is consistent with the employment of metallo-peptidases in the process of egg development [59]. ...
Background
MiRNAs are small non-coding RNAs that post-transcriptionally regulate gene expression in organisms ranging from viruses to mammals. There is great relevance in understanding how miRNAs regulate genes involved in the growth, development, and maturation of the many parasitic worms (helminths) that together afflict more than 2 billion people.
Results
Here, we describe the miRNAs expressed by each of the predominant intra-mammalian development stages of Fasciola hepatica , a foodborne flatworm that infects a wide range of mammals worldwide, most importantly humans and their livestock. A total of 124 miRNAs were profiled, 72 of which had been previously reported and three of which were conserved miRNA sequences described here for the first time. The remaining 49 miRNAs were novel sequences of which, 31 were conserved with F. gigantica and the remaining 18 were specific to F. hepatica. The newly excysted juveniles express 22 unique miRNAs while the immature liver and mature bile duct stages each express 16 unique miRNAs. We discovered several sequence variant miRNAs (IsomiRs) as well as miRNA clusters that exhibit strict temporal expression paralleling parasite development. Target analysis revealed the close association between miRNA expression and stage-specific changes in the transcriptome; for example, we identified specific miRNAs that target parasite proteases known to be essential for intestinal wall penetration (cathepsin L3). Moreover, we demonstrate that miRNAs fine-tune the expression of genes involved in the metabolic pathways that allow the parasites to move from an aerobic external environment to the anerobic environment of the host.
Conclusions
These results provide novel insight into the regulation of helminth parasite development and identifies new genes and miRNAs for therapeutic development to limit the virulence and pathogenesis caused by F. hepatica .
... The behavior of pasha 5 ′ UTR hairpins was instructive. A feedback loop in which Drosha cleaves 5 ′ UTR foldbacks in pasha/DGCR8 is conserved from mammals to fruit flies (Han et al. 2009;Smibert et al. 2011). Although mammalian DGCR8 5 ′ UTR hairpin products are nuclearly retained (Han et al. 2009), sufficient mature reads of DGCR8 hairpins exist to justify annotation of mir-3618 and mir-1306. ...
... Although their resultant small RNAs are not very abundant, the coupling with our prior evidence of in vivo cleavage of these hairpins by Drosha (Smibert et al. 2011) indicates the depth of our small RNA profiling. ...
To assess miRNA evolution across the Drosophila genus, we analyzed several billion small RNA reads across 12 fruit fly species. These data permit comprehensive curation of species- and clade-specific variation in miRNA identity, abundance, and processing. Among well-conserved miRNAs, we observed unexpected cases of clade-specific variation in 5' end precision, occasional antisense loci, and putatively noncanonical loci. We also used strict criteria to identify a large set (649) of novel, evolutionarily restricted miRNAs. Within the bulk collection of species-restricted miRNAs, two notable subpopulations are splicing-derived mirtrons and testes-restricted, recently evolved, clustered (TRC) canonical miRNAs. We quantified miRNA birth and death using our annotation and a phylogenetic model for estimating rates of miRNA turnover. We observed striking differences in birth and death rates across miRNA classes defined by biogenesis pathway, genomic clustering, and tissue restriction, and even identified flux heterogeneity among Drosophila clades. In particular, distinct molecular rationales underlie the distinct evolutionary behavior of different miRNA classes. Mirtrons are associated with high rates of 3' untemplated addition, a mechanism that impedes their biogenesis, whereas TRC miRNAs appear to evolve under positive selection. Altogether, these data reveal miRNA diversity among Drosophila species and principles underlying their emergence and evolution.
... By contrast, DGCR8 stabilizes and solubilizes Drosha through protein-protein interactions. This cross-regulation between Drosha and DGCR8 enables homeostatic control of Microprocessor activity and is conserved in different animal species [137][138][139] . ...
Ever since microRNAs (miRNAs) were rst recognized as an extensive gene family >20 years ago, a broad community of researchers was drawn to investigate the universe of small regulatory RNAs. Although core features of miRNA biogenesis and function were revealed early on, recent years continue to uncover fundamental information on the structural and molecular dynamics of core miRNA machinery, how miRNA substrates and targets are selected from the transcriptome, new avenues for multilevel regulation of miRNA biogenesis and mechanisms for miRNA turnover. Many of these latest insights were enabled by recent technological advances, including massively parallel assays, cryogenic electron microscopy, single-molecule imaging and CRISPR-Cas9 screening. Here, we summarize the current understanding of miRNA biogenesis, function and regulation, and outline challenges to address in the future. Sections
... If such a factor did exist, the ability to reconstitute RNAi in S. cerevisiae by adding only DCR1 and AGO1 indicated that the additional factor either was not essential for RNAi or had other functions in budding yeast, leading to its retention in S. cerevisiae after loss of the RNAi pathway. Based on the success of genetic screens and selections in other systems, including Arabidopsis, C. elegans and Drosophila, which provided early insight into the core components of the RNAi pathway and identified additional factors that influence RNAi efficacy (36)(37)(38)(39)(40)(41)(42)(43)(44)(45)(46), we implemented a genetic selection in N. castellii to identify mutants with reduced RNAi activity. This selection identified mutants in the gene encoding Xrn1p. ...
RNA interference (RNAi) is a gene-silencing pathway that can play roles in viral defense, transposon silencing, heterochromatin formation, and post-transcriptional gene silencing. Although absent from Saccharomyces cerevisiae , RNAi is present in other budding-yeast species, including Naumovozyma castellii , which have an unusual Dicer and a conventional Argonaute that are both required for gene silencing. To identify other factors that act in the budding-yeast pathway, we performed an unbiased genetic selection. This selection identified Xrn1p, the cytoplasmic 5′-to-3′ exoribonuclease, as a cofactor of RNAi in budding yeast. Deletion of XRN1 impaired gene silencing in N. castellii , and this impaired silencing was attributable to multiple functions of Xrn1p, including affecting the composition of siRNA species in the cell, influencing the efficiency of siRNA loading into Argonaute, degradation of cleaved passenger strand, and degradation of sliced target RNA.
... Suppression of Pasha in Drosophila interferes with pri-miRNA processing, leading to an accumulation of pri-miRNAs and a reduction in mature miRNAs (Denli et al., 2004;Landthaler et al., 2004). Like in other animals, Pasha is essential for processing of canonical miRNAs but is dispensable for mirtrons (Flynt et al., 2010;Martin et al., 2009;Smibert et al., 2011). Drosophila Pasha is possibly phosphorylated by ERK/MAPK, as suggested by phosphorylation of human DGCR8 in insect cells; the phosphorylation appears to increase protein stability without altering miRNA processing activity (Herbert et al., 2013). ...
RNA silencing denotes sequence-specific repression mediated by small RNAs. In vertebrates, there are two closely related pathways, which share several protein factors: RNA interference (RNAi) and microRNA (miRNA) pathway. The miRNA pathway regulates endogenous protein-coding gene expression and has been implicated in many biological processes. RNAi generally serves as a form of innate immunity targeting viruses and mobile elements. While Arthropoda are an extremely large and diverse phylum, research on microRNA (miRNA) and RNA interference (RNAi) pathway in this phylum primarily used the Drosophila melanogaster model system and related species. Notably, both pathways are genetically separated; they utilize dedicated Dicer proteins to produce miRNAs and small interfering RNAs (siRNAs), which are sorted onto different Argonaute effector proteins. This review focuses on the miRNA pathway and pathways initiated by long dsRNA in arthropods. The first part introduces the key molecular players of RNA silencing. The second discussed biological roles of miRNA and dsRNA-induced pathways in Arthropods.
NOTE: This is a chapter from an open source book. The full text is available directly through the DOI link above.
... If such a factor did exist, the ability to reconstitute RNAi in S. cerevisiae by adding only DCR1 and AGO1 indicated that the additional factor either was not essential for RNAi or had other functions in budding yeast, leading to its retention in S. cerevisiae after loss of the RNAi pathway. Based on the success of genetic screens and selections in other systems, including Arabidopsis, C. elegans and Drosophila, which provided early insight into the core components of the RNAi pathway and identified additional factors that influence RNAi efficacy (36)(37)(38)(39)(40)(41)(42)(43)(44)(45)(46), we implemented a genetic selection in N. castellii to identify mutants with reduced RNAi activity. This selection identified mutants in the gene encoding Xrn1p. ...
RNA interference (RNAi) is a gene-silencing pathway that can play roles in viral defense, transposon silencing, heterochromatin formation and post-transcriptional gene silencing. Although absent from Saccharomyces cerevisiae, RNAi is present in other budding-yeast species, including Naumovozyma castellii, which have an unusual Dicer and a conventional Argonaute that are both required for gene silencing. To identify other factors that act in the budding-yeast pathway, we performed an unbiased genetic selection. This selection identified Xrn1p, the cytoplasmic 5'-to-3' exoribonuclease, as a cofactor of RNAi in budding yeast. Deletion of XRN1 impaired gene silencing in N. castellii, and this impaired silencing was attributable to multiple functions of Xrn1p, including affecting the composition of siRNA species in the cell, influencing the efficiency of siRNA loading into Argonaute, degradation of cleaved passenger strand and degradation of sliced target RNA.
... Although it remains challenging to reconcile the evidence for such broad miRNA regulatory networks with the often nominal defects observed in individual miRNA knockouts 8 , documented miRNA mutants exhibit developmental, physiological, metabolic, and/or behavioral defects 9 . Moreover, null mutants in core miRNA biogenesis factors are lethal in all animals [10][11][12][13] , and yield severe tissue-specific defects when inactivated conditionally 10,[14][15][16] . Strikingly, recent studies reveal that mutation of human DICER1 is cell lethal in human embryonic stem cells 17 . ...
Somatic mutations in the RNase IIIb domain of DICER1 arise in cancer and disrupt the cleavage of 5' pre-miRNA arms. Here, we characterize an unstudied, recurrent, mutation (S1344L) in the DICER1 RNase IIIa domain in tumors from The Cancer Genome Atlas (TCGA) project and MSK-IMPACT profiling. RNase IIIa/b hotspots are absent from most cancers, but are notably enriched in uterine cancers. Systematic analysis of TCGA small RNA datasets show that DICER1 RNase IIIa-S1344L tumors deplete 5p-miRNAs, analogous to RNase IIIb hotspot samples. Structural and evolutionary coupling analyses reveal constrained proximity of RNase IIIa-S1344 to the RNase IIIb catalytic site, rationalizing why mutation of this site phenocopies known hotspot alterations. Finally, examination of DICER1 hotspot endometrial tumors reveals derepression of specific miRNA target signatures. In summary, comprehensive analyses of DICER1 somatic mutations and small RNA data reveal a mechanistic aspect of pre-miRNA processing that manifests in specific cancer settings.
... The existence of extensive regulatory networks mediated by microRNAs (miRNAs) suggests broad possibilities for their requirement during development or physiology (Flynt and Lai, 2008;Sun and Lai, 2013). As is true for most tissues, loss of core miRNA biogenesis factors such as Dicer-1 or Pasha causes substantial defects in the developing Drosophila eye (Lee et al., 2004;Smibert et al., 2011). Beyond the general requirement for miRNA biogenesis in this tissue, some individual miRNAs and miRNA sites influence eye development. ...
Photoreceptors in the crystallineDrosophilaeye are recruited by receptor tyrosine kinase (RTK)/Ras signaling, mediated by the Epidermal Growth Factor receptor (EGFR) and Sevenless receptor. Analyses of an allelic deletion series of themir-279/996locus, along with a panel of modified genomic rescue transgenes, show that normalDrosophilaeye patterning depends on both miRNAs. Transcriptional reporter and activity sensor transgenes reveal expression and function of miR-279/996 in non-neural cells of the developing eye. Moreover,mir-279/996mutants exhibit substantial numbers of ectopic photoreceptors, particularly of R7, and cone cell loss. These miRNAs restrict RTK signaling in the eye, sincemir-279/996nulls are dominantly suppressed by positive components of the EGFR pathway and enhanced by heterozygosity for an EGFR repressor. miR-279/996 limit photoreceptor recruitment by targeting multiple positive RTK/Ras signaling components that promote photoreceptor/R7 specification. Strikingly, deletion ofmir-279/996sufficiently de-represses RTK/Ras signaling so as to rescue a population of R7 cells in R7-specific RTK null mutantsbossandsev, which otherwise completely lack this cell fate. Altogether, we reveal a rare setting of developmental cell specification that substantially requires miRNA control.
... In contrast, the mammalian Rnt1p ortholog Drosha processes double-stranded microRNA precursors in the nucleus (Lee et al., 2003). Knockdown of Drosha in human cells and genetic knockouts in mice and D. melanogaster showed unaltered mature and 47S pre-rRNA levels (Chong et al., 2008;Smibert et al., 2011;Wu et al., 2000). ...
Non-coding RNA biogenesis in higher eukaryotes has not been fully characterized. Here, we studied the Drosophila melanogaster Rexo5 (CG8368) protein, a metazoan-specific member of the DEDDh 3′-5′ single-stranded RNA exonucleases, by genetic, biochemical, and RNA-sequencing approaches. Rexo5 is required for small nucleolar RNA (snoRNA) and rRNA biogenesis and is essential in D. melanogaster. Loss-of-function mutants accumulate improperly 3′ end-trimmed 28S rRNA, 5S rRNA, and snoRNA precursors in vivo. Rexo5 is ubiquitously expressed at low levels in somatic metazoan cells but extremely elevated in male and female germ cells. Loss of Rexo5 leads to increased nucleolar size, genomic instability, defective ribosome subunit export, and larval death. Loss of germline expression compromises gonadal growth and meiotic entry during germline development.
exposed daily per ear followed a monomolecular model. Pollen shed and silk exsertion curves were translated to potential kernel set using observed also that modern hybrids tended to produce a published relationship. Excellent agreement was observed between kernels on a second (subapical) ear at low plant popula- predicted and measured number of florets per hectare available for tion densities. Therefore, they proposed a double-hyper- pollination. The model overpredicted kernel number per hectare at bola function to describe the relationship between ker- high pollen shed densities. Predicted kernels per hectare were within nel number per plant and plant growth rate. Otegui 5% of actual kernel numbers at low pollen shed densities when delayed (1995), however, reported that among Argentinean hy- development of subapical ears and asynchronous pollination within brids, only prolific types tended to produce kernels on ears were considered. These results indicate kernel set in maize can subapical ears at commercial plant densities. be predicted from simple measures of the flowering process. They Because canopy photosynthesis is controlled primar- confirm that kernel set is limited primarily by factors other than pollen ily by solar radiation (Christy et al., 1986), a similar
The genetic analysis of a gene at a late developmental stage can be impeded if the gene is required at an earlier developmental stage. The construction of mosaic animals, particularly in Drosophila, has been a means to overcome this obstacle. However, the phenotypic analysis of mitotic clones is often complicated because standard methods for generating mitotic clones render mosaic tissues that are a composite of both mutant and phenotypically normal cells. We describe here a genetic method (called EGUF/hid) that uses both the GAL4/UAS and FLP/FRT systems to overcome this limitation for the Drosophila eye by producing genetically mosaic flies that are otherwise heterozygous but in which the eye is composed exclusively of cells homozygous for one of the five major chromosome arms. These eyes are nearly wild type in size, morphology, and physiology. Applications of this genetic method include phenotypic analysis of existing mutations and F1 genetic screens to identify as yet unknown genes involved in the biology of the fly eye. We illustrate the utility of the method by applying it to lethal mutations in the synaptic transmission genes synaptotagmin and syntaxin.
One proposed strategy for controlling the transmission of insect-borne pathogens uses a drive mechanism to ensure the rapid spread of transgenes conferring disease refractoriness throughout wild populations. Here, we report the creation of maternal-effect selfish genetic elements in Drosophila that drive population replacement and are resistant to recombination-mediated dissociation of drive and disease refractoriness functions. These selfish elements use microRNA-mediated silencing of a maternally expressed gene essential for embryogenesis, which is coupled with early zygotic expression of a rescuing transgene.
neuralized (neu) represents one of the strong neurogenic mutants in Drosophila. Mutants of this class display, among other phenotypes, a strong overcommitment to neural fates at the expense of epidermal fates. We analyzed the role of neu during adult development by using mutant clonal analysis, misexpression of wild-type and truncated forms of Neu, and examination of genetic interactions with N-pathway mutations. We find that neu is required cell-autonomously for lateral inhibition during peripheral neurogenesis and for multiple asymmetric cell divisions in the sensory lineage. In contrast, neu is apparently dispensable for other N-mediated processes, including lateral inhibition during wing vein development and wing margin induction. Misexpression of wild-type Neu causes defects in both peripheral neurogenesis and wing vein development, while a truncated form lacking the RING finger is further capable of inhibiting formation of the wing margin. In addition, the phenotypes produced by misexpression of wild-type and truncated Neu proteins are sensitive to the dosage of several N-pathway components. Finally, using epitope-tagged Neu proteins, we localize Neu to the plasma membrane and reveal a novel morphology to the sensory organ precursor cells of wing imaginal discs. Collectively, these data indicate a key role for neu in the reception of the lateral inhibitory signal during peripheral neurogenesis.
Cell proliferation, cell death, and pattern formation are coordinated in animal development. Although many proteins that control cell proliferation and apoptosis have been identified, the means by which these effectors are linked to the patterning machinery remain poorly understood. Here, we report that the bantam gene of Drosophila encodes a 21 nucleotide microRNA that promotes tissue growth. bantam expression is temporally and spatially regulated in response to patterning cues. bantam microRNA simultaneously stimulates cell proliferation and prevents apoptosis. We identify the pro-apoptotic gene hid as a target for regulation by bantam miRNA, providing an explanation for bantam's anti-apoptotic activity.
Since the establishment of a canonical animal microRNA biogenesis pathway driven by the RNase III enzymes Drosha and Dicer, an unexpected variety of alternative mechanisms that generate functional microRNAs have emerged. We review here the many Drosha-independent and Dicer-independent microRNA biogenesis strategies characterized over the past few years. Beyond reflecting the flexibility of small RNA machineries, the existence of noncanonical pathways has consequences for interpreting mutants in the core microRNA machinery. Such mutants are commonly used to assess the consequences of "total" microRNA loss, and indeed, they exhibit many overall phenotypic similarities. Nevertheless, ongoing studies reveal a growing number of settings in which alternative microRNA pathways contribute to distinct phenotypes among core microRNA biogenesis mutants.