RESUMO
BACKGROUND: During Drosophila embryogenesis, Jun kinase (JNK) signaling has been shown to play a key role in regulating the morphogenetic process of dorsal closure, which also serves as a model for epithelial sheet fusion during wound repair. During dorsal closure the JNK signaling cascade in the dorsal-most (leading edge) cells of the epidermis activates the AP-1 transcription factor comprised of DJUN and DFOS that, in turn, upregulates the expression of the dpp gene. DPP is a secreted morphogen that signals lateral epidermal cells to elongate along the dorsoventral axis. The leading edge cells contact the peripheral cells of a monolayer extraembryonic epithelium, the amnioserosa, which lies on the dorsal side of the embryo. Focal complexes are present at the dorsal-most membrane of the leading edge cells, where they contact the amnioserosa. RESULTS: We show that the JNK signaling cascade is initially active in both the amnioserosa and the leading edge of the epidermis. JNK signaling is downregulated in the amnioserosa, but not in the leading edge, prior to dorsal closure. The subcellular localization of DFOS and DJUN is responsive to JNK signaling in the amnioserosa: JNK activation results in nuclear localization of DFOS and DJUN; the downregulation of JNK signaling results in the relocalization of DFOS and DJUN to the cytoplasm. The HINDSIGHT (HNT) Zn-finger protein and the PUCKERED (PUC) JNK phosphatase are essential for downregulation of the JNK cascade in the amnioserosa. Persistent JNK activity in the amnioserosa leads to defective focal complexes in the adjacent leading edge cells and to the failure of dorsal closure. CONCLUSIONS: Focal complexes are assembled at the boundary between high and low JNK activity. In the absence of focal complexes, miscommunication between the amnioserosa and the leading edge may lead to a premature "stop" signal that halts dorsalward migration of the leading edge. Spatial and temporal regulation of the JNK signaling cascade may be a general mechanism that controls tissue remodeling during morphogenesis and wound healing.
Assuntos
Proteínas de Drosophila , Drosophila/embriologia , Drosophila/enzimologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Âmnio/embriologia , Âmnio/enzimologia , Animais , Regulação para Baixo , Drosophila/genética , Epiderme/embriologia , Epiderme/enzimologia , Epitélio/embriologia , Epitélio/enzimologia , Proteínas Quinases JNK Ativadas por Mitógeno , Proteínas Quinases Ativadas por Mitógeno/genética , Mutação , Proteínas Nucleares/genética , Proteínas Proto-Oncogênicas c-fos/metabolismo , Proteínas Proto-Oncogênicas c-jun/metabolismo , Transdução de Sinais , Frações Subcelulares/metabolismo , Fator de Transcrição AP-1/metabolismo , Fatores de Transcrição/genéticaRESUMO
Maternally encoded RNAs and proteins program the early development of all animals. A subset of the maternal transcripts is eliminated from the embryo before the midblastula transition. In certain cases, transcripts are protected from degradation in a subregion of the embryonic cytoplasm, thus resulting in transcript localization. Maternal factors are sufficient for both the degradation and protection components of transcript localization. Cis-acting elements in the RNAs convert transcripts progressively (i) from inherently stable to unstable and (ii) from uniformly degraded to locally protected. Similar mechanisms are likely to act later in development to restrict certain classes of transcripts to particular cell types within somatic cell lineages. Functions of transcript degradation and protection are discussed.
Assuntos
RNA/genética , Transcrição Gênica , Animais , Sequência de Bases , Evolução Biológica , Blastocisto/fisiologia , Sequência Conservada , Drosophila/embriologia , Drosophila/genética , Feminino , Impressão Genômica , Masculino , RNA/químicaRESUMO
The major axes of the oocyte-antero-posterior and dorso-ventral-are established over a one-day period during mid-oogenesis in Drosophila. The same molecule, GURKEN (GRK), functions to initiate signaling between the oocyte and the surrounding, somatically derived follicle cells. This results first in specification of the antero-posterior axis and, later, the dorso-ventral axis of the oocyte and surrounding follicle cells. Central to specification of both axes is a combination of cytoplasmic localization and translational regulation of the grk RNA. Here we discuss the mechanisms by which the grk RNA is localized within the oocyte and the role of translational regulation in spatially restricting the production of GRK protein. We then discuss the generality of these mechanisms during oogenesis by focusing on a second transcript, oskar, whose function is also regulated through a combination of transcript localization and translational control.
Assuntos
Padronização Corporal/genética , Proteínas de Drosophila , Drosophila/embriologia , Proteínas de Insetos/genética , Oócitos/crescimento & desenvolvimento , Biossíntese de Proteínas/genética , RNA/genética , RNA/metabolismo , Fator de Crescimento Transformador alfa , Fatores de Crescimento Transformadores/genética , Animais , Drosophila/genética , Drosophila/metabolismo , Proteínas de Insetos/metabolismo , Oócitos/citologia , Oócitos/metabolismo , Fatores de Crescimento Transformadores/metabolismoRESUMO
Retrovirus vectors are de novo methylated and transcriptionally silent in mammalian stem cells. Here, we identify epigenetic modifications that mark retrovirus-silenced transgenes. We show that murine stem cell virus (MSCV) and human immunodeficiency virus type 1 (HIV-1) vectors dominantly silence a linked locus control region (LCR) beta-globin reporter gene in transgenic mice. MSCV silencing blocks LCR hypersensitive site formation, and silent transgene chromatin is marked differentially by a histone code composed of abundant linker histone H1, deacetylated H3 and acetylated H4. Retrovirus-transduced embryonic stem (ES) cells are silenced predominantly 3 days post-infection, with a small subset expressing enhanced green fluorescent protein to low levels, and silencing is not relieved in de novo methylase-null [dnmt3a-/-;dnmt3b-/-] ES cells. MSCV and HIV-1 sequences also repress reporter transgene expression in Drosophila, demonstrating establishment of silencing in the absence of de novo and maintenance methylases. These findings provide mechanistic insight into a conserved gene silencing mechanism that is de novo methylase independent and that epigenetically marks retrovirus chromatin with a repressive histone code.
Assuntos
Inativação Gênica , Vetores Genéticos , Retroviridae/genética , Animais , Animais Geneticamente Modificados , Sequência de Bases , Evolução Biológica , Cromatina/genética , Metilases de Modificação do DNA/metabolismo , Primers do DNA/genética , Drosophila/genética , Genes Reporter , Globinas/genética , HIV-1/genética , Histonas/genética , Humanos , Lentivirus/genética , Camundongos , Camundongos TransgênicosRESUMO
Transcript localization and translational regulation are two post-transcriptional mechanisms for the spatial and temporal regulation of protein production. During the past year, two transcript localization mechanisms have been elaborated in some detail. Where localization involves directional transport on cytoskeletal tracks, links between the transcripts and the cytoskeletal molecular motors have been elaborated. In the case of localization by generalized transcript degradation combined with localized protection, trans-acting pathways and cis-acting elements for degradation and protection have been identified. A third transcript localization mechanism, vectorial transport out of the nucleus into a particular cytoplasmic domain, was initially thought to localize pair-rule transcripts in Drosophila. However, these have now been shown to be localized by directional transport in the cytoplasm. Transcript localization and translational regulation can be intimately linked in that, for certain messenger RNAs, only the localized fraction of transcripts is translated whereas unlocalized transcripts are translationally repressed. Cis-acting sequences and trans-acting factors that function in translational repression have been identified along with factors involved in relief of translational repression at the site of localization.
Assuntos
Regulação da Expressão Gênica , Biossíntese de Proteínas/genética , RNA Mensageiro/metabolismo , Animais , Transporte Biológico , Citoesqueleto/metabolismo , Drosophila/genética , Drosophila/metabolismo , RNA Mensageiro/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMO
During animal development, morphogenesis of tissues and organs requires dynamic cell shape changes and movements that are accomplished without loss of epithelial integrity. Data from vertebrate and invertebrate systems have implicated several cell surface and cytoskeleton-associated molecules in the establishment and maintenance of epithelial architecture, but there has been little analysis of the genetic regulatory hierarchies that control epithelial morphogenesis in specific tissues. Here we show that the Drosophila Hindsight nuclear zinc-finger protein is required during tracheal morphogenesis for the maintenance of epithelial integrity and assembly of apical extracellular structures known as taenidia. In hindsight (hnt) mutants tracheal placodes form, invaginate, and undergo primary branching as well as early fusion events. Starting at midembryogenesis, however, the tracheal epithelium collapses or expands to give rise to sacs of tissue. While a subset of hnt mutant tracheal cells enters the apoptotic pathway, genetic suppression of apoptosis indicates that this is not the cause of the epithelial defects. Surviving hnt mutant tracheal cells retain cell-cell junctions and a normal subcellular distribution of apical markers such as Crumbs and DE-Cadherin. However, taenidia do not form on the lumenal surface of tracheal cells. While loss of epithelial integrity is a common feature of crumbs, stardust, and hnt mutants, defective assembly of taenidia is unique to hnt mutants. These data suggest that HNT is a tissue-specific factor that regulates maintenance of the tracheal epithelium as well as differentiation of taenidia.
Assuntos
Proteínas de Drosophila , Drosophila/embriologia , Drosophila/genética , Genes de Insetos , Traqueia/embriologia , Animais , Apoptose/genética , Drosophila/metabolismo , Epitélio/embriologia , Epitélio/metabolismo , Feminino , Proteínas de Insetos/genética , Masculino , Microscopia Eletrônica , Modelos Biológicos , Mutação , Proteínas Nucleares/genética , Traqueia/citologia , Traqueia/metabolismo , Fatores de Transcrição/genética , Dedos de Zinco/genéticaRESUMO
Cytoplasmic mRNA localization is one method by which protein production is restricted to a particular intracellular site. We report here a novel mechanism for localization of transcripts encoding distinct protein isoforms to different destinations. Alternative processing of transcripts produced in the Drosophila ovary by the hu-li tai shao (hts) locus introduces distinct 3' untranslated regions (3'UTRs) that differentially localize the mRNAs. Three classes of hts mRNA (R2, N32 and N4) are synthesized in the germ line nurse cells and encode proteins with adducin-homologous amino-terminal regions but divergent carboxy-terminal domains. The R2 and N32 classes of mRNA remain in the nurse cells and are not transported into the oocyte. In contrast, the N4 class of transcripts is transported from the nurse cells into the oocyte starting at stage 1, is subsequently localized to the oocyte cortex at stage 8 and then to the anterior pole from stage 9 on. All aspects of N4 transcript transport and localization are directed by the 345-nucleotide(nt)-long 3' untranslated region (3'UTR). The organization of localization elements in the N4 3'UTR is modular: a 150 nt core is sufficient to direct transport and localization throughout oogenesis. Additional 3'UTR elements function additively together with this core region at later stages of oogenesis to maintain or enhance anterior transcript anchoring. The swallow locus is required to maintain hts transcripts at the anterior pole of the oocyte and functions through the N4 3'UTR. In addition to the three classes of germ line-expressed hts transcripts, a fourth class (R1) is expressed in the somatic follicle cells that surround the germ line cells. This transcript class encodes the Drosophila orthologue of mammalian adducin.
Assuntos
Regiões 3' não Traduzidas/genética , Proteínas de Ligação a Calmodulina/genética , Proteínas de Ligação a Calmodulina/metabolismo , Proteínas de Drosophila , Oogênese/genética , Transcrição Gênica/fisiologia , Animais , Citoplasma/metabolismo , Citoesqueleto/metabolismo , Drosophila , Feminino , Regulação da Expressão Gênica/fisiologia , Mutação em Linhagem Germinativa , Mamíferos , Dados de Sequência Molecular , Oócitos/química , Oócitos/metabolismo , Ovário/citologia , Ovário/fisiologia , Splicing de RNA/fisiologia , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Ratos , Homologia de Sequência de AminoácidosRESUMO
As the germ band shortens in Drosophila melanogaster embryos, cell shape changes cause segments to narrow anteroposteriorly and to lengthen dorsoventrally. One of the genes required for this retraction process is the hindsight (hnt) gene. hnt encodes a nuclear Zinc-finger protein that is expressed in the extraembryonic amnioserosa and the endodermal midgut prior to and during germ band retraction (M. L. R. Yip, M. L. Lamka, and H. D. Lipshitz, 1997, Development 124, 2129-2141). Here we show, through analysis of hnt genetic mosaic embryos, that hnt activity in the amnioserosa-particularly in those cells that are adjacent to the epidermis-is necessary for germ band retraction. In hnt mutant embryos the amnioserosa undergoes premature cell death (L. C. Frank and C. Rushlow, 1996, Development 122, 1343-1352). We demonstrate that prevention of premature apoptosis in hnt mutants does not rescue retraction. Thus, failure of this process is not an indirect consequence of premature amnioserosal apoptosis; instead, hnt must function in a pathway that controls germ band retraction. We show that the Krüppel gene is activated by hnt in the amnioserosa while the Drosophila insulin receptor (INR) functions downstream of hnt in the germ band. We present evidence against a physical model in which the amnioserosa "pushes" the germ band during retraction. Rather, it is likely that the amnioserosa functions in production, activation, or presentation of a diffusible signal required for retraction.
Assuntos
Proteínas de Drosophila , Drosophila melanogaster/embriologia , Drosophila melanogaster/genética , Embrião não Mamífero/fisiologia , Animais , Apoptose , Tamanho Celular , Embrião não Mamífero/citologia , Células Epidérmicas , Epiderme/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Nucleares/genética , Fenótipo , Fatores de Transcrição/genética , Dedos de ZincoRESUMO
Neuronal cell fate decisions are directed in Drosophila by NUMB, a signaling adapter protein with two protein-protein interaction domains: a phosphotyrosine-binding domain and a proline-rich region (PRR) that functions as an SH3-binding domain. Here we show that there are at least four human NUMB isoforms and that these serve two distinct developmental functions in the neuronal lineage: differentiation (but not proliferation) is promoted by human NUMB protein isoforms with a type I (short) PRR. In contrast, proliferation (but not differentiation) is directed by isoforms that have a type II (long) PRR. The two types of PRR may promote distinct intracellular signaling pathways downstream of the NOTCH receptor during mammalian neurogenesis.
Assuntos
Drosophila/genética , Hormônios Juvenis/genética , Hormônios Juvenis/fisiologia , Neurônios/citologia , Processamento Alternativo , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados , Diferenciação Celular , Divisão Celular , Linhagem Celular , Proteínas de Drosophila , Humanos , Hormônios Juvenis/química , Dados de Sequência Molecular , Família Multigênica , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/fisiologia , RNA Mensageiro/genética , Transcrição Gênica , Transfecção , Asas de Animais/anatomia & histologiaRESUMO
Maternally synthesized RNAs program early embryonic development in many animals. These RNAs are degraded rapidly by the midblastula transition (MBT), allowing genetic control of development to pass to zygotically synthesized transcripts. Here we show that in the early embryo of Drosophila melanogaster, there are two independent RNA degradation pathways, either of which is sufficient for transcript elimination. However, only the concerted action of both pathways leads to elimination of transcripts with the correct timing, at the MBT. The first pathway is maternally encoded, is targeted to specific classes of mRNAs through cis-acting elements in the 3'-untranslated region and is conserved in Xenopus laevis. The second pathway is activated 2 h after fertilization and functions together with the maternal pathway to ensure that transcripts are degraded by the MBT.
Assuntos
Blastocisto/metabolismo , Proteínas de Drosophila , Drosophila melanogaster/metabolismo , RNA Mensageiro/metabolismo , Regiões 3' não Traduzidas , Animais , Sequência de Bases , Drosophila melanogaster/embriologia , Evolução Molecular , Feminino , Proteínas de Choque Térmico/biossíntese , Proteínas de Choque Térmico/genética , Dados de Sequência Molecular , Mutação , Óvulo/metabolismo , Fatores Sexuais , Fatores de Tempo , Xenopus , Zigoto/metabolismoRESUMO
Cytoplasmic RNA localization is an evolutionarily ancient mechanism for producing cellular asymmetries. This review considers RNA localization in the context of animal development. Both mRNAs and non-protein-coding RNAs are localized in Drosophila, Xenopus, ascidian, zebrafish, and echinoderm oocytes and embryos, as well as in a variety of developing and differentiated polarized cells from yeast to mammals. Mechanisms used to transport and anchor RNAs in the cytoplasm include vectorial transport out of the nucleus, directed cytoplasmic transport in association with the cytoskeleton, and local entrapment at particular cytoplasmic sites. The majority of localized RNAs are targeted to particular cytoplasmic regions by cis-acting RNA elements; in mRNAs these are almost always in the 3'-untranslated region (UTR). A variety of trans-acting factors--many of them RNA-binding proteins--function in localization. Developmental functions of RNA localization have been defined in Xenopus, Drosophila, and Saccharomyces cerevisiae. In Drosophila, localized RNAs program the antero-posterior and dorso-ventral axes of the oocyte and embryo. In Xenopus, localized RNAs may function in mesoderm induction as well as in dorso-ventral axis specification. Localized RNAs also program asymmetric cell fates during Drosophila neurogenesis and yeast budding.
Assuntos
Padronização Corporal , Compartimento Celular , RNA Mensageiro , Animais , Transporte Biológico , Indução Embrionária , Evolução Molecular , RNA Mensageiro/genéticaRESUMO
In both vertebrate and invertebrate development, cells are often programmed to adopt fates distinct from their neighbors. Genetic analyses in Drosophila melanogaster have highlighted the importance of cell surface and secreted proteins in these cell fate decisions. Homologues of these proteins have been identified and shown to play similar roles in vertebrate development. Fringe, a novel signalling protein, has been shown to induce wing margin formation in Drosophila. Fringe shares significant sequence homology and predicted secondary structure similarity with bacterial glycosyltransferases. Thus fringe may control wing development by altering glycosylation of cell surface and/or secreted molecules. Recently, two fringe genes were isolated from Xenopus laevis. We report here the cloning and characterization of three murine fringe genes (lunatic fringe, manic fringe and radical fringe). We find in several tissues that fringe expression boundaries coincide with Notch-dependent patterning centres and with Notch-ligand expression boundaries. Ectopic expression of murine manic fringe or radical fringe in Drosophila results in phenotypes that resemble those seen in Notch mutants.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento/genética , Glicosiltransferases , Proteínas de Membrana/genética , Proteínas/genética , Proteínas de Xenopus , Sequência de Aminoácidos , Animais , Padronização Corporal/genética , Diferenciação Celular , Sondas de DNA , Proteínas de Drosophila , Drosophila melanogaster/genética , Olho/citologia , Glucosiltransferases , Hibridização In Situ , Peptídeos e Proteínas de Sinalização Intercelular , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Dados de Sequência Molecular , Mutação/genética , Fenótipo , Receptores Notch , Asas de Animais/citologia , Xenopus/genéticaRESUMO
Drosophila embryos lacking hindsight gene function have a normal body plan and undergo normal germ-band extension. However, they fail to retract their germ bands. hindsight encodes a large nuclear protein of 1920 amino acids that contains fourteen C2H2-type zinc fingers, and glutamine-rich and proline-rich domains, suggesting that it functions as a transcription factor. Initial embryonic expression of hindsight RNA and protein occurs in the endoderm (midgut) and extraembryonic membrane (amnioserosa) prior to germ-band extension and continues in these tissues beyond the completion of germ-band retraction. Expression also occurs in the developing tracheal system, central and peripheral nervous systems, and the ureter of the Malpighian tubules. Strikingly, hindsight is not expressed in the epidermal ectoderm which is the tissue that undergoes the cell shape changes and movements during germ-band retraction. The embryonic midgut can be eliminated without affecting germ-band retraction. However, elimination of the amnioserosa results in the failure of germ-band retraction, implicating amnioserosal expression of hindsight as crucial for this process. Ubiquitous expression of hindsight in the early embryo rescues germ-band retraction without producing dominant gain-of-function defects, suggesting that hindsight's role in germ-band retraction is permissive rather than instructive. Previous analyses have shown that hindsight is required for maintenance of the differentiated amnioserosa (Frank, L. C. and Rushlow, C. (1996) Development 122, 1343-1352). Two classes of models are consistent with the present data. First, hindsight's function in germ-band retraction may be limited to maintenance of the amnioserosa which then plays a physical role in the retraction process through contact with cells of the epidermal ectoderm. Second, hindsight might function both to maintain the amnioserosa and to regulate chemical signaling from the amnioserosa to the epidermal ectoderm, thus coordinating the cell shape changes and movements that drive germ-band retraction.
Assuntos
Padronização Corporal , Proteínas de Drosophila , Drosophila/embriologia , Proteínas Nucleares/genética , Proteínas Repressoras , Fatores de Transcrição/genética , Dedos de Zinco , Sequência de Aminoácidos , Âmnio/química , Animais , Blastoderma/química , Núcleo Celular/química , DNA Complementar/genética , Proteínas de Ligação a DNA/análise , Sistema Digestório/química , Sistema Digestório/embriologia , Drosophila/genética , Regulação da Expressão Gênica no Desenvolvimento , Fatores de Transcrição Kruppel-Like , Dados de Sequência Molecular , Morfogênese , Proteínas Nucleares/análise , Proteínas Nucleares/fisiologia , Alinhamento de Sequência , Análise de Sequência de DNA , Membrana Serosa/química , Fatores de Transcrição/análise , Fatores de Transcrição/fisiologiaRESUMO
BACKGROUND: Drosophila numb was originally described as a mutation affecting binary divisions in the sensory organ precursor (SOP) lineage. The numb gene was subsequently shown to encode an asymmetrically localized protein which is required for binary cell-fate decisions during peripheral nervous system development. Part of the Drosophila NUMB protein exhibits homology to the SHC phosphotyrosine-binding (PTB) domain, suggesting a potential link to tyrosine-kinase signal transduction. RESULTS: A widely expressed mammalian homologue of Drosophila numb (dnumb) has been cloned from rat and is referred to here as mammalian Numb (mNumb). The mNUMB protein has a similar overall structure to dNUMB and 67 sequence similarity. Misexpression of mNumb in Drosophila during sensory nervous system precursor cell division causes identical cell fate transformations to those produced by ectopic dNUMB expression. In vitro, the mNUMB PTB domain binds phosphotyrosine-containing proteins, and SH3 domains of SRC-family tyrosine kinases bind to mNUMB presumably through interactions with proline-rich regions in the carboxyl terminus. Overexpression of full-length mNUMB in the multipotential neural crest stem cell line MONC-1 dramatically biases its differentiation towards neurons, whereas overexpression of the mNUMB PTB domain biases its differentiation away from neuronal fates. CONCLUSIONS: Our results demonstrate that mNUMB is an evolutionarily conserved functional homologue of dNUMB, and establish a link to tyrosine-kinase-mediated signal transduction pathways. Furthermore, our results suggest that mNUMB and dNUMB are new members of a family of signaling adapter molecules that mediate conserved cell-fate decisions during development.
Assuntos
Linhagem da Célula/genética , Drosophila/genética , Evolução Molecular , Hormônios Juvenis/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Sequência Conservada , DNA Complementar , Drosophila/embriologia , Proteínas de Drosophila , Regulação da Expressão Gênica no Desenvolvimento , Hormônios Juvenis/metabolismo , Dados de Sequência Molecular , Fosfotirosina/metabolismo , Ligação Proteica , Homologia de Sequência de Aminoácidos , Domínios de Homologia de srcRESUMO
Adducin is a cytoskeletal protein that can function in vitro to bundle F-actin and to control the assembly of the F-actin/spectrin cytoskeletal network. The Drosophila Adducin-like (Add) locus (also referred to as hu-li tai shao (hts)) encodes a family of proteins of which several are homologous to mammalian adducin (Ding et al., Proc. Natl. Acad. Sci. USA 90, 2512-16, 1993; Yue & Spradling, Genes Dev. 6, 2443-54, 1992). We report the identification of two novel adducin isoforms: a 95 x 10(3) Mr form (ADD-95) and an 87 x 10(3) Mr form (ADD-87). We present a detailed analysis of the distribution patterns of ADD-95 and ADD-87 during oogenesis and embryogenesis. The isoforms are co-expressed in several cell- and tissue-types; however, only ADD-87 is present in mid- to late-stage oocytes. ADD-87 is present throughout the oocyte cortex at stages 9 and 10 of oogenesis but is detectable only at the anterior pole from stage 11 onward, correlated with localisation of Add-hts mRNA first to the cortex and then to the anterior pole of the oocyte. ADD-87 co-localises with F-actin and spectrin in the cortex of the oocyte through stage 10 of oogenesis, consistent with a possible role in cytoskeletal assembly or function predicted by mammalian studies.
Assuntos
Proteínas de Ligação a Calmodulina/metabolismo , Drosophila/embriologia , Embrião não Mamífero/metabolismo , Oócitos/metabolismo , Actinas/metabolismo , Animais , Western Blotting , Proteínas de Ligação a Calmodulina/genética , Proteínas de Ligação a Calmodulina/imunologia , Feminino , Sistema Nervoso/embriologia , Sistema Nervoso/metabolismo , Oócitos/citologia , Oócitos/crescimento & desenvolvimento , Oogênese , Folículo Ovariano/metabolismo , Folículo Ovariano/fisiologia , Espectrina/metabolismoRESUMO
Adducin is a cytoskeletal protein that can function in vitro to bundle F-actin and to control the assembly of the F-actin/spectrin cytoskeletal network. We previously reported cloning of the Drosophila Adducin-like (Add) locus [Ding et al., 1993] also referred to as hu-li tai shao (hts) [Yue and Spradling, 1992], and identification of two adducin-related protein isoforms: a 95 x 10(3) Mr form (ADD-95) and an 87 x 10(3) Mr form (ADD-87) [Zaccai and Lipshitz, 1996]. ADD-87 protein is present throughout the oocyte cortex at stages 9 and 10 of oogenesis but is restricted to its anterior pole from stage 11 onward. This ADD-87 protein localization is preceded by localization of Add-hts mRNA first to the cortex and then to the anterior pole of the oocyte. Mutation of the swallow gene results in delocalization of Add-hts mRNA and ADD-87 protein from the cortex of stage 9 and 10 oocytes, and from the anterior pole of later stage oocytes. Early embryos produced by swallow or Add-hts mutant females have severe defects in the distribution of F-actin and spectrin as well as abnormalities in nuclear division, nuclear migration, and cellularization. In addition to their cytoskeletal defects, embryos produced by swallow females have an abnormal anterior pattern because bicoid mRNA is delocalized from the anterior pole. In contrast, bicoid mRNA is still found at the anterior of embryos produced by Add-hts mothers. Thus swallow functions to restrict bicoid mRNA and Add-hts mRNA to the cortex of the oocyte. Cortical restriction of Add-hts mRNA and protein is required for the normal structure and function of the early embryonic F-actin/spectrin cytoskeleton. A defective embryonic cytoskeleton can be induced in either of two ways: (1) by delocalization of functional ADD from the oocyte cortex (as in swallow mutants), or (2) by reduction of ADD function while retaining its normal cortical localization during oogenesis (as in Add-hts mutants).
Assuntos
Proteínas de Ligação a Calmodulina/genética , Proteínas do Citoesqueleto/genética , Proteínas de Drosophila , Drosophila/metabolismo , Proteínas de Homeodomínio , RNA Mensageiro/metabolismo , Transativadores , Actinas/metabolismo , Animais , Proteínas do Citoesqueleto/metabolismo , Drosophila/embriologia , Drosophila/genética , Feminino , Hormônios de Inseto/genética , Hormônios de Inseto/metabolismo , Masculino , Mutação , Oogênese , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Espectrina/metabolismoRESUMO
Over twenty distinct families of long terminal direct repeat (LTR)-containing retrotransposons have been identified in Drosophila melanogaster. While there have been extensive analyses of retrotransposon transcription in cultured cells, there have been few studies of the spatial expression of retrotransposons during normal development. Here we report a detailed analysis of the spatial expression patterns of fifteen families of retrotransposons during Drosophila melanogaster embryogenesis (17.6, 297, 412, 1731, 3S18, blood, copia, gypsy, HMS Beagle, Kermit/flea, mdg1, mdg3, opus, roo/B104 and springer). In each case, analyses were carried out in from two to four wild-type strains. Since the chromosomal insertion sites of any particular family of retrotransposons vary widely among wild-type strains, a spatial expression pattern that is conserved among strains is likely to have been generated through interaction of host transcription factors with cis-regulatory elements resident in the retrotransposons themselves. All fifteen families of retrotransposons showed conserved patterns of spatially and temporally regulated expression during embryogenesis. These results suggest that all families of retrotransposons carry cis-acting elements that control their spatial and temporal expression patterns. Thus, transposition of a retrotransposon into or near a particular host gene-possibly followed by an excision event leaving behind the retrotransposon's cis-regulatory sequences-might impose novel developmental control on such a host gene. Such a mechanism would serve to confer evolutionarily significant alterations in the spatio-temporal control of gene expression.