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1.
Andrology ; 2(5): 655-65, 2014 Sep.
Article in English | MEDLINE | ID: mdl-25079683

ABSTRACT

In both mammalian and Drosophila spermatids, the completely histone-based chromatin structure is reorganized to a largely protamine-based structure. During this histone-to-protamine switch, transition proteins are expressed, for example TNP1 and TNP2 in mammals and Tpl94D in Drosophila. Recently, we demonstrated that in Drosophila spermatids, H3K79 methylation accompanies histone H4 hyperacetylation during chromatin reorganization. Preceding the histone-to-protamine transition, the H3K79 methyltransferase Grappa is expressed, and the predominant isoform bears a C-terminal extension. Here, we show that isoforms of the Grappa-equivalent protein in humans, rats and mice, that is DOT1L, have a C-terminal extension. In mice, the transcript of this isoform was enriched in the post-meiotic stages of spermatogenesis. In human and mice spermatids, di- and tri-methylated H3K79 temporally overlapped with hyperacetylated H4 and thus accompanied chromatin reorganization. In rat spermatids, trimethylated H3K79 directly preceded transition protein loading on chromatin. We analysed the impact of bacterial infections on spermatid chromatin using a uropathogenic Escherichia coli-elicited epididymo-orchitis rat model and showed that these infections caused aberrant spermatid chromatin. Bacterial infections led to premature emergence of trimethylated H3K79 and hyperacetylated H4. Trimethylated H3K79 and hyperacetylated H4 simultaneously occurred with transition protein TNP1, which was never observed in spermatids of mock-infected rats. Upon bacterial infection, only histone-based spermatid chromatin showed abnormalities, whereas protamine-compacted chromatin seemed to be unaffected. Our results indicated that H3K79 methylation is a histone modification conserved in Drosophila, mouse, rat and human spermatids and may be a prerequisite for proper chromatin reorganization.


Subject(s)
Chromatin Assembly and Disassembly/physiology , Histones/metabolism , Spermatids/cytology , Spermatids/microbiology , Spermatogenesis/physiology , Acetylation , Animals , Cell Differentiation , Chromosomal Proteins, Non-Histone/metabolism , Drosophila melanogaster , Epididymitis/microbiology , Escherichia coli/pathogenicity , Escherichia coli Infections , Histone-Lysine N-Methyltransferase , Humans , Male , Methylation , Methyltransferases/genetics , Mice , Mice, Inbred C57BL , Orchitis/microbiology , Protamines/metabolism , Protein Isoforms/genetics , Rats , Testis/metabolism
2.
Development ; 128(24): 5061-73, 2001 Dec.
Article in English | MEDLINE | ID: mdl-11748142

ABSTRACT

Mutations in the rolling pebbles (rols) gene result in severe defects in myoblast fusion. Muscle precursor cells are correctly determined, but myogenesis does not progress significantly beyond this point because recognition and/or cell adhesion between muscle precursor cells and fusion-competent myoblasts is disturbed. Molecular analysis of the rols genomic region reveals two variant transcripts of rols due to different transcription initiation sites, rols6 and rols7. rols6 mRNA is detectable mainly in the endoderm during differentiation as well as in malpighian tubules and in the epidermis. By contrast, rols7 expression is restricted to the mesoderm and later to progenitor descendants during somatic and pharyngeal muscle development. Transcription starts at the extended germ band stage when progenitor/founder cells are determined and persists until stage 13. The proteins encoded by the rols gene are 1670 (Rols6) and 1900 (Rols7) amino acids in length. Both forms contain an N-terminal RING-finger motif, nine ankyrin repeats and a TPR repeat eventually overlaid by a coiled-coil domain. The longer protein, Rols7, is characterized by 309 unique N-terminal amino acids, while Rols6 is distinguishable by 79 N-terminal amino acids. Expression of rols7 in muscle founder cells indicates a function of Rols7 in these cells. Transplantation assays of rols mutant mesodermal cells into wild-type embryos show that Rols is required in muscle precursor cells and is essential to recruit fusion-competent myoblasts for myotube formation.


Subject(s)
Drosophila Proteins , Drosophila/embryology , Membrane Proteins/metabolism , Muscle Development , Muscle Proteins/metabolism , Muscles/embryology , Stem Cells/cytology , Amino Acid Sequence , Animals , Ankyrin Repeat , Cell Fusion , Chromosome Walking , Embryo, Nonmammalian/ultrastructure , Genes, Insect , Giant Cells , Membrane Proteins/genetics , Molecular Sequence Data , Muscle Fibers, Skeletal/cytology , Muscle Proteins/genetics , Mutation , Nuclear Pore Complex Proteins , Pharyngeal Muscles , Proto-Oncogene Proteins , Zinc Fingers
4.
Nucleic Acids Res ; 28(6): 1439-46, 2000 Mar 15.
Article in English | MEDLINE | ID: mdl-10684940

ABSTRACT

The tissue-specific expression of the Drosophila beta 2 tubulin gene ( B2t ) is accomplished by the action of a 14-bp activator element (beta2UE1) in combination with certain regulatory elements of the TATA-less, Inr-containing B2t core promoter. We performed an in vivo analysis of the Inr element function in the B2t core promoter using a transgenic approach. Our experiments demonstrate that the Inr element acts as a functional cis -regulatory element in vivo and quantitatively regulates tissue-specific reporter expression in transgenic animals. However, our mutational analysis of the Inr element demonstrates no essential role of the Inr in mediating tissue specificity of the B2t promoter. In addition, a downstream element seems to affect promoter activity in combination with the Inr. In summary, our data show for the first time the functionality of the Inr element in an in vivo background situation in Drosophila.


Subject(s)
Drosophila melanogaster/genetics , Gene Expression Regulation/genetics , Genes, Insect/genetics , Promoter Regions, Genetic/genetics , Response Elements/genetics , Spermatozoa/metabolism , Tubulin/genetics , Animals , Animals, Genetically Modified , Base Sequence , Conserved Sequence/genetics , DNA/genetics , DNA/metabolism , DNA-Binding Proteins/metabolism , Genes, Reporter/genetics , HSP70 Heat-Shock Proteins/genetics , Insect Proteins/metabolism , Male , Organ Specificity , Sequence Deletion/genetics , TATA Box/genetics , Testis/cytology , Testis/metabolism , Transcription, Genetic/genetics , Transgenes/genetics
5.
Dev Biol ; 216(1): 327-39, 1999 Dec 01.
Article in English | MEDLINE | ID: mdl-10588882

ABSTRACT

During Drosophila embryogenesis, the beta3 tubulin gene is expressed in the visceral and somatic mesoderm as well as in the dorsal vessel. Transcription of the gene is limited to four pairs of cardioblasts per segment. Here we show that its expression in the dorsal vessel (dv) is mediated by a 333-bp enhancer located upstream of the gene. The homeodomain protein Tinman is also expressed in these cardioblasts, implying that Tinman might be a key regulator of the beta3 tubulin gene. Gel retardation and footprint assays indeed revealed two Tinman binding sites within the dv-specific enhancer. We analyzed the relevance of the Tinman binding sites in a transgenic fly assay and observed distinct functions for both sites. The BS(Tin-1460) site is absolutely required for expression in cardioblasts, while BS(Tin-1425) is needed for high-level expression. Thus, these two Tinman binding sites act in concert to drive beta3 tubulin gene expression during heart development. Tinman initially functions in the specification of visceral mesoderm and heart progenitors, but remains expressed in cardioblasts until dorsal closure. Overall, our data demonstrate a late function for Tinman in the regulation of beta3 tubulin gene expression in the forming heart of Drosophila.


Subject(s)
Drosophila Proteins , Drosophila/embryology , Repressor Proteins/metabolism , Trans-Activators/metabolism , Tubulin/genetics , Animals , Animals, Genetically Modified , Base Sequence , Binding Sites , DNA-Binding Proteins/analysis , Enhancer Elements, Genetic , Gene Expression Regulation, Developmental , Genes, Reporter , Heart/embryology , Homeodomain Proteins/metabolism , Immunohistochemistry , Molecular Sequence Data , Mutation , Recombinant Fusion Proteins , Repressor Proteins/genetics , Trans-Activators/genetics
6.
Mech Dev ; 83(1-2): 17-26, 1999 May.
Article in English | MEDLINE | ID: mdl-10507836

ABSTRACT

In Drosophila, as in vertebrates, each muscle is a syncytium and arises from mesodermal cells by successive fusion. This requires cell-cell recognition, alignment, formation of prefusion complexes, followed by electron-dense plaques and membrane breakdown. Because muscle development in Drosophila is rapid and well-documented, it has been possible to identify several genes essential for fusion. Molecular analysis of two of these genes revealed the importance of cytoplasmic components. One of these, Myoblast city, is expressed in several tissues and is homologous to the mammalian protein DOCK180. Myoblast city is presumably involved in cell recognition and cell adhesion. Blown fuse, the second cytoplasmic component, is selectively expressed in the mesoderm and essential in order to proceed from the prefusion complex to electron-dense plaques at opposed membranes between adjacent myoblasts. The rolling stone gene is transiently expressed during myoblast fusion. The Rost protein is located in the membrane and thus might be a key component for cell recognition. The molecular characterization of further genes relevant for fusion such as singles bar and sticks and stones will help to elucidate the mechanism of myoblast fusion in Drosophila.


Subject(s)
Cytoskeletal Proteins , Drosophila Proteins , Drosophila/embryology , Muscles/embryology , rac GTP-Binding Proteins , Animals , Body Patterning/genetics , Cell Adhesion , DNA-Binding Proteins/physiology , Female , GTP-Binding Proteins/physiology , Insect Proteins/analysis , Insect Proteins/physiology , Intracellular Membranes/metabolism , MEF2 Transcription Factors , Male , Models, Biological , Muscle Proteins/analysis , Muscle Proteins/physiology , Mutagenesis , Myogenic Regulatory Factors , Spermatogenesis , Transcription Factors/physiology
7.
Cell Tissue Res ; 296(1): 151-60, 1999 Apr.
Article in English | MEDLINE | ID: mdl-10199975

ABSTRACT

This review describes briefly what is known about the early steps of mesoderm differentiation in the fruitfly Drosophila melanogaster. After a summary of general aspects including mesoderm differentiation, mesoderm cell migration and subdivision of the mesoderm, more detail is given about the specification of muscle progenitor cells, due to their role as the earliest obvious landmarks in muscle fiber development in Drosophila. Particular focus is given to recent results on the role of asymmetric cell division in muscle differentiation. Furthermore a short summary of myoblast fusion is provided.


Subject(s)
Drosophila melanogaster/embryology , Drosophila melanogaster/genetics , Mesoderm/physiology , Muscle, Skeletal/embryology , Animals , Body Patterning , Cell Division , Embryo, Nonmammalian/physiology , Larva , Mesoderm/cytology , Muscle Fibers, Skeletal/cytology , Muscle Fibers, Skeletal/physiology , Muscle, Skeletal/cytology
8.
Mol Gen Genet ; 262(4-5): 643-58, 1999 Dec.
Article in English | MEDLINE | ID: mdl-10628847

ABSTRACT

The beta3 tubulin gene of Drosophila is expressed in the major mesodermal derivatives during their differentiation. The gene is subject to complex stage- and tissue-specific transcriptional control by upstream as well as downstream regions. Analysis of the vm1 enhancer, which is responsible for tissue-specific expression in the visceral mesoderm and is localized in the intron, revealed a complex modular arrangement of regulatory elements. In vitro and in vivo experiments uncovered two binding sites, termed UBX1 and UBX2, for the product of the homeotic gene Ultrabithorax (Ubx), which are required for high-level expression in pPS6 and PS7. Further analysis of the vm1 enhancer revealed that deletion of a specific element, termed element 7 (e7), abolishes transcription of the lacZ reporter gene in all parasegments except pPS6/PS7. Gel-retardation and footprint analysis identified a binding site for the homeodomain protein Tinman, which is essential for the specification of the dorsal mesoderm, within e7. Simultaneous deletion of two further sequence blocks in the vml enhancer, named elements 3 (e3), and 6 (e6), results in a reduction analogous to that caused by removal of e7. The e6 sequence contains conserved motifs also found in the visceral enhancer of the Ubx gene. Therefore we conclude that these elements act in concert with the Tinman binding site to achieve high expression levels. Thus the vm1 enhancer of the beta3 tubulin gene contains a complex array of elements that are involved in transactivation by a combination of tissue- and position-specific factors including Tinman and UBX.


Subject(s)
DNA-Binding Proteins/metabolism , Drosophila Proteins , Drosophila/genetics , Enhancer Elements, Genetic , Homeodomain Proteins/metabolism , Nuclear Proteins , Repressor Proteins/metabolism , Trans-Activators/metabolism , Transcription Factors , Tubulin/genetics , Animals , Antennapedia Homeodomain Protein , Base Sequence , DNA, Complementary , DNA-Binding Proteins/genetics , Gene Expression Regulation/genetics , Homeodomain Proteins/genetics , Insect Proteins/genetics , Insect Proteins/metabolism , Mesoderm/metabolism , Molecular Sequence Data , Protein Binding , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Repressor Proteins/genetics , Sequence Deletion , Sequence Homology, Nucleic Acid , Species Specificity , Trans-Activators/genetics
9.
J Cell Sci ; 111 ( Pt 22): 3299-309, 1998 Nov.
Article in English | MEDLINE | ID: mdl-9788872

ABSTRACT

The Drosophila don juan gene encodes a basic protein (Don Juan protein), which is solely expressed postmeiotically during spermiogenesis in elongated spermatids and in mature sperm. Transgenic expression of a GFP-tagged Don Juan protein (DJ-GFP) in the male germ line showed an association of the fusion protein with the sperm tail. Detailed examination of DJ-GFP localization revealed novel insights into its distinct temporal and spatial distribution along the sperm tail during the last phase of spermatid maturation. Co-localization of DJ-GFP with actin-labeled cysts demonstrated its emergence in elongated spermatids during individualization. Additionally, the endogenous Don Juan protein was detected with epitope-specific antibodies in finally elongated nuclei of spermatids. After completion of nuclear shaping Don Juan is no longer detectable in the sperm heads with the onset of individualization. Mislocalization of the DJ-GFP protein in flagella of a mutant with defective mitochondrial differentiation provides evidence of mitochondrial association of the fusion protein with flagellar mitochondrial arrays. Ectopically expressed DJ-GFP in premeiotic germ cells as well as salivary gland cells confirmed the capability of the fusion protein to associate with mitochondria. Therefore we suppose that Don Juan is a nuclear-encoded, germ-cell specifically expressed mitochondrial protein, which might be involved in the final steps of mitochondrial differentiation within the flagellum.


Subject(s)
Drosophila Proteins , Drosophila melanogaster/genetics , Insect Proteins/genetics , Mitochondria/chemistry , Sperm Tail/chemistry , Spermatids/chemistry , Amino Acid Sequence , Animals , Cell Differentiation/physiology , Cell Division/physiology , Fluorescent Antibody Technique , GTP Phosphohydrolases/analysis , Gene Expression/physiology , Germ-Line Mutation , Green Fluorescent Proteins , Indicators and Reagents , Insect Proteins/analysis , Luminescent Proteins/genetics , Male , Meiosis/physiology , Membrane Proteins/analysis , Molecular Sequence Data , Recombinant Fusion Proteins/analysis , Recombinant Fusion Proteins/genetics , Salivary Glands/chemistry , Spermatids/cytology
10.
Dev Biol ; 199(1): 138-49, 1998 Jul 01.
Article in English | MEDLINE | ID: mdl-9676198

ABSTRACT

The beta 3 tubulin gene (beta Tub60D) is a structural gene expressed during mesoderm development from the extended germ band stage onward. Expression within the individual mesodermal derivatives is guided by different control elements. The upstream regions allow expression in the dorsal vessel and the somatic mesoderm while enhancers localized in the first intron guide expression in the visceral mesoderm. Deletion analysis carried out in transgenic flies revealed independent regulatory elements for the dorsal vessel and the somatic mesoderm. For expression in the somatic mesoderm, a 279-bp region is absolutely essential. This region contains a binding site for the Drosophila myocyte-specific enhancer binding factor 2 (D-MEF2), a MADS-box transcription factor known to be essential for mesoderm development. Deletion or mutation of this D-MEF2 binding site strongly reduces transcription. This pattern is consistent with the strongly reduced expression of beta 3 tubulin in D-mef2 mutant embryos. This analysis furthermore reveals that the D-MEF2 binding site acts in concert with nearby cis regulatory elements. These data show that the upstream control region of the beta 3 tubulin gene is an early target of the D-MEF2 transcriptional activator.


Subject(s)
Blood Vessels/embryology , Genes, Insect , Muscles/embryology , Regulatory Sequences, Nucleic Acid , Tubulin/genetics , Animals , Animals, Genetically Modified , Binding Sites , DNA-Binding Proteins/metabolism , Drosophila/embryology , Drosophila/genetics , Drosophila Proteins , Gene Expression Regulation, Developmental , MEF2 Transcription Factors , Mesoderm , Myogenic Regulatory Factors/metabolism , Recombinant Fusion Proteins/biosynthesis , Transcription Factors/metabolism , Transcription, Genetic , Tubulin/biosynthesis
11.
Mech Dev ; 67(1): 69-81, 1997 Sep.
Article in English | MEDLINE | ID: mdl-9347916

ABSTRACT

Not enough muscles (nem) mutants of Drosophila reveal defects in the development of embryonic muscles, a subset of pericardial cells, the CNS and derivatives of the PNS (Burchard, S., Paululat, A., Hinz, U. and Renkawitz-Pohl, R. (1995) The mutant not enough muscles (nem) reveals reduction of the Drosophila embryonic muscle pattern. J. Cell. Sci. 108, 1443-1454). The molecular analysis of the nem locus shows a complex genomic structure. One transcription unit was identified as inscuteable (insc). Within the first intron of insc we find another independent gene, skittles (sktl), which is not affected in nem mutants. insc transcripts are localised apically in neuroblasts and may prefigure the localisation of the protein. The skittles mRNA is ubiquitously distributed during early embryogenesis due to maternal contribution. Later, some enrichment of sktl is observed in the nervous system and the mesoderm. The muscle phenotype shows deletions as well as duplication of specific muscles which is reflected in a change of even-skipped (eve) and Krüppel (Kr) expressing cells. Our data suggest a role for insc in the specification process of a subset of muscle progenitors/founders. Furthermore, in insc mutants the eve expressing pericardial cells of the developing heart are significantly reduced in numbers.


Subject(s)
Cytoskeletal Proteins , Drosophila/embryology , Drosophila/genetics , Mesoderm/physiology , Phosphotransferases (Alcohol Group Acceptor)/genetics , Animals , Central Nervous System/embryology , Central Nervous System/physiology , DNA Transposable Elements , Drosophila Proteins , Embryo, Nonmammalian/physiology , Gene Expression Regulation, Developmental , Genes, Insect , Heart/embryology , Molecular Sequence Data , Muscles/embryology , Mutation , Myocardium/pathology , Neurons/metabolism , Neuropeptides , Peripheral Nervous System/embryology , Peripheral Nervous System/physiology , Phosphotransferases (Alcohol Group Acceptor)/metabolism
12.
J Cell Biol ; 138(2): 337-48, 1997 Jul 28.
Article in English | MEDLINE | ID: mdl-9230076

ABSTRACT

In homozygous rolling stone embryos, the fusion of myoblasts to syncytial myotubes is diminished. Nevertheless, the visceral mesoderm, the heart mesoderm, and few somatic muscles are properly formed. Thus, we postulate a central role of rolling stone for the fusion process within the somatic mesoderm. We have cloned the rolling stone gene, and the deduced protein sequence is in accordance with a transmembrane protein, which agrees with the enrichment of Rost in the membrane fraction of Drosophila embryos. No homologous genes have been described so far. rolling stone is expressed in the embryonic nervous system and cells of the somatic mesoderm, most notable in muscle founder cells. To elucidate the function of rolling stone for myoblast fusion, we applied a knock-out strategy. The expression of an antisense rolling stone transcript specifically within the mesoderm of wild-type embryos results in fusion defects of myoblasts, proving that the rolling stone expression in the mesoderm is responsible for the rolling stone phenotype. We suggest that rolling stone is a member of a group of genes that are necessary for the fusion process during myogenesis.


Subject(s)
Cell Fusion/genetics , Drosophila Proteins , Drosophila/genetics , Gene Expression Regulation, Developmental , Membrane Proteins/genetics , Muscle Proteins , Amino Acid Sequence , Animals , Base Sequence , Cloning, Molecular , Drosophila/embryology , Ectoderm/chemistry , Genes, Insect/genetics , Membrane Proteins/physiology , Mesoderm/chemistry , Molecular Sequence Data , Muscle Fibers, Skeletal/cytology , Muscles/cytology , Muscles/embryology , Mutation , Nervous System/embryology , Phenotype , RNA, Antisense , RNA, Messenger/analysis , Restriction Mapping , Sequence Analysis, DNA
13.
Mech Dev ; 64(1-2): 19-30, 1997 Jun.
Article in English | MEDLINE | ID: mdl-9232593

ABSTRACT

We identified and characterized the don juan gene (dj) of Drosophila melanogaster. The don juan gene codes for a sperm specific protein component with an unusual repetitive six amino acid motif (DPCKKK) in the carboxy-terminal part of the protein. The expression of Don Juan is limited to male germ cells where transcription of the dj gene is initiated during meiotic prophase. But Western blot experiments indicate that DJ protein occurs just postmeiotically. Examination of transgenic flies bearing a dj-promoter-lacZ reporter construct revealed lacZ mRNA distribution resembling the expression pattern of the endogenous dj mRNA in the adult testes, whereas beta-galactosidase expression is exclusively present in postmeiotic germ cells. Thus, these observations strongly suggest that dj transcripts are under translational repression until in spermiogenesis. To study the function and subcellular distribution of DJ in spermiogenesis we expressed a chimaeric dj-GFP fusion gene in the male germline exhibiting strong GFP fluorescence in the liver testes, where only elongated spermatids are decorated. With regard to the characteristic expression pattern of DJ protein and its conspicuous repeat units possible functional roles are discussed.


Subject(s)
Drosophila Proteins , Drosophila melanogaster/genetics , Genes, Insect , Insect Proteins/genetics , Spermatozoa/metabolism , Amino Acid Sequence , Animals , Animals, Genetically Modified , Base Sequence , DNA, Complementary/genetics , Drosophila melanogaster/growth & development , Drosophila melanogaster/metabolism , Gene Expression Regulation, Developmental , Green Fluorescent Proteins , Insect Proteins/metabolism , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Male , Molecular Sequence Data , Oligonucleotide Probes/genetics , Protein Biosynthesis , RNA, Messenger/genetics , RNA, Messenger/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Repetitive Sequences, Nucleic Acid , Spermatids/metabolism , Spermatogenesis/genetics
14.
Dev Genes Evol ; 207(2): 127-130, 1997 Jul.
Article in English | MEDLINE | ID: mdl-27747405

ABSTRACT

We have isolated the Drosophila gene skittles (sktl) which shows homology to members of a novel family of phosphatidylinositol-4-phosphate 5-kinases, including the gene product encoded by the human STM-7.I gene which has been assigned to the neurodegenerative disorder Friedreichs ataxia. In situ hybridization reveals sktl expression during oogenesis and spermatogenesis.

15.
Mech Dev ; 57(1): 59-68, 1996 Jun.
Article in English | MEDLINE | ID: mdl-8817453

ABSTRACT

We have found a novel gene (alien) that is expressed exclusively in the muscle attachment sites (apodemes) during embryogenesis in Drosophila. Antibodies raised against the Alien protein enable us to follow the developing attachments from state 11/12 until stage 16/17. The coding region of the Drosophila alien gene is highly conserved to a gene of unknown function, isolated from a plant (Loo et at., 1995), and to the human TRIP15 gene (Lee et al., 1995). Searching for thyroid receptor interacting proteins, TRIP15 was isolated as a negative regulator. Whether there is a functional correlation to Alien remains to be analyzed. Alien expression is independent of muscle formation, as shown in rolling stone mutant embryos. Even in twist and snail mutants, lacking mesodermal development, alien expression is fairly normal, showing a rather autonomous development of the apodemes. The conservation of alien suggests an important role in differentiation.


Subject(s)
Adaptor Proteins, Signal Transducing , Drosophila/embryology , Drosophila/genetics , Genes, Insect/physiology , Insect Proteins/genetics , Proteins , ATPases Associated with Diverse Cellular Activities , Amino Acid Sequence , Animals , Base Sequence , Blotting, Northern , COP9 Signalosome Complex , Cloning, Molecular , Conserved Sequence , Humans , Immunohistochemistry , LIM Domain Proteins , Molecular Sequence Data , Muscles/embryology , Muscles/immunology , Mutation/physiology , Plants/genetics , Proteasome Endopeptidase Complex , Repressor Proteins , Sequence Analysis , Sequence Homology, Amino Acid , Tissue Distribution , Transcription Factors/genetics , Tubulin/immunology
16.
Int J Dev Biol ; 40(1): 189-96, 1996 Feb.
Article in English | MEDLINE | ID: mdl-8735928

ABSTRACT

In Drosophila beta tubulins are encoded by a small gene family whose members are differentially expressed in a highly cell and tissue specific manner. Here we focus on the expression of the beta 3 tubulin isotype during mesoderm differentiation and beta 1 tubulin expression in the apodemes during embryonic development. The beta 3 tubulin isotype is first detectable at the extended germband stage shortly before the separation of somatic and visceral derivatives. Comparing the distribution of the beta 3 mRNA and the beta 3 isotype shows that the transcription of the beta 3 tubulin gene is cell type specifically repressed during differentiation of individual mesodermal derivatives, from which the dorsal vessel remains transcriptionally active until shortly before hatching. In contrast the beta 3 tubulin protein is detectable in all mesodermal derivatives. The beta 3 tubulin is an excellent marker to study mesoderm differentiation on a regulatory and cellular level using both genetics and molecular biology. In the visceral mesoderm, the expression of the beta 3 tubulin gene is regulated by homeotic gene products, while other transactivators regulate expression in the dorsal vessel and the body wall musculature. In the somatic mesoderm, the beta 3 tubulin allows to visualize myotube formation and insertion into the epidermis. This contact to the epidermal attachment sites (apodemes) induces beta 1 tubulin expression, as can be seen in double staining experiments. We determined a 14bp cis-regulatory enhancer element guiding expression of the beta 1 tubulin gene in these attachment sites. Using the beta 1 and beta 3 tubulin isotypes as markers we started to isolate mutants which are disturbed in muscle formation.


Subject(s)
Drosophila/embryology , Drosophila/genetics , Muscles/embryology , Muscles/metabolism , Tubulin/genetics , Animals , Drosophila/metabolism , Gene Expression Regulation, Developmental , Genes, Insect , Mesoderm/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Trans-Activators/genetics
17.
Development ; 121(8): 2611-20, 1995 Aug.
Article in English | MEDLINE | ID: mdl-7671823

ABSTRACT

The development and differentiation of the body wall musculature in Drosophila are accompanied by changes in gene expression and cellular architecture. We isolated a Drosophila gene, termed rolling stone (rost), which, when mutated, specifically blocks the fusion of mononucleated cells to myotubes in the body wall musculature. beta 3 tubulin, which is an early marker for the onset of mesoderm differentiation, is still expressed in these cells. Gastrulation and mesoderm formation, as well as the development of the epidermis and of the central and peripheral nervous systems, appear quite normal in homozygous rolling stone embryos. Embryonic development stops shortly before hatching in a P-element-induced mutant, as well as in 16 EMS-induced alleles. In mutant embryos, other mesodermal derivatives such as the visceral mesoderm and the dorsal vessel, develop fairly normally and defects are restricted to the body wall musculature. Myoblasts remain as single mononucleated cells, which express muscle myosin, showing that the developmental program of gene expression proceeds. These myoblasts occur at positions corresponding to the locations of dorsal, ventral and pleural muscles, showing that the gene rolling stone is involved in cell fusion, a process that is independent of cell migration in these mutants. This genetic analysis has set the stage for a molecular analysis to clarify where the rolling stone action is manifested in the fusion process and thus gives insight into the complex regulating network controlling the differentiation of the body wall musculature.


Subject(s)
Drosophila Proteins , Drosophila/embryology , Genes, Insect , Mesoderm/physiology , Muscle Proteins , Muscles/embryology , Transcription Factors , Animals , Cell Fusion , Drosophila/genetics , Ectoderm/cytology , Epidermal Cells , Epidermis/embryology , Gene Expression , In Situ Hybridization , Morphogenesis/genetics , Mutagenesis , Myosins/analysis , Nervous System/embryology , Proteins/genetics
18.
J Cell Sci ; 108 ( Pt 4): 1443-54, 1995 Apr.
Article in English | MEDLINE | ID: mdl-7615665

ABSTRACT

In a search for mutations affecting embryonic muscle development in Drosophila we identified a mutation caused by the insertion of a P-element, which we called not enough muscles (nem). The phenotype of the P-element mutation of the nem gene suggests that it may be required for the development of the somatic musculature and the chordotonal organs of the PNS, while it is not involved in the development of the visceral mesoderm and the dorsal vessel. Mutant embryos are characterized by partial absence of muscles, monitored by immunostainings with mesoderm-specific anti-beta 3 tubulin and anti-myosin heavy chain antibodies. Besides these muscle distortions, defects in the peripheral nervous system were found, indicating a dual function of the nem gene product. Ethyl methane sulfonate-induced alleles for the P-element mutation were created for a detailed analysis. One of these alleles is characterized by unfused myoblasts which express beta 3 tubulin and myosin heavy chain, indicating the state of cell differentiation.


Subject(s)
DNA Transposable Elements , Drosophila melanogaster/embryology , Mutagenesis, Insertional , Alleles , Animals , Drosophila melanogaster/genetics , Embryo, Nonmammalian/cytology , Embryo, Nonmammalian/physiology , Ethyl Methanesulfonate , Gene Expression , Genes, Insect , Larva , Muscles/embryology , Mutagenesis , Myosins/biosynthesis , Organ Specificity
19.
Mol Gen Genet ; 241(3-4): 263-70, 1993 Nov.
Article in English | MEDLINE | ID: mdl-8246879

ABSTRACT

Stem cell differentiation to mature spermatozoa is a morphogenetic process that is highly dependent on microtubular arrays. In the early, mitotically active stages of spermatogenesis, only the beta 1 tubulin isotype is expressed. Analysis of transgenic flies containing beta 1-lacZ gene fusions revealed that this expression is regulated by sequences located between positions -45 and -191 upstream of the transcription initiation site. Furthermore, beta 1 tubulin is a major component of cyst cells. Expression in these cells is driven by enhancer elements located in the beta 1 tubulin gene intron. These enhancer elements also guide expression in combination with the hsp70 basal promoter. In addition, redundant enhancer elements in the intron drive expression in the testis wall. Our data show that within a single tissue, the male gonad, expression of the beta 1 tubulin gene is under cell-type-specific control mediated by independent cis-acting elements. Therefore in the germ line, control of beta 1 tubulin expression is strictly governed by promoter-proximal elements, while for the somatic parts of the testis, enhancer elements confer less stringent expression control.


Subject(s)
Gene Expression Regulation , Spermatogonia/metabolism , Testis/metabolism , Tubulin/genetics , Animals , Drosophila , Enhancer Elements, Genetic , Heat-Shock Proteins/genetics , Introns , Male , Promoter Regions, Genetic , Spermatogenesis/genetics , Spermatogonia/cytology , Stem Cells/metabolism , Testis/cytology , Transcription, Genetic
20.
Eur J Cell Biol ; 62(1): 66-74, 1993 Oct.
Article in English | MEDLINE | ID: mdl-8269980

ABSTRACT

During Drosophila spermatogenesis transcriptional activity is mainly restricted to premeiotic stages. Translation during sperm morphogenesis, however, proceeds for several days, requiring a high stability for mRNAs translated postmeiotically. We studied expression of the Drosophila beta 2 tubulin gene, which is expressed solely in the male germ line from the primary spermatocyte stage onwards. Cis-acting elements involved in the regulation of mRNA levels were investigated in transgenic fly strains. In adult testes, mRNA amounts from beta 2-lacZ fusion genes in the presence of an 18-bp AT-rich element, termed beta 2DE1, are elevated about threefold. The element is present at about the same position in the 5' untranslated regions of the beta 2 tubulin genes of the distantly related species Drosophila melanogaster and Drosophila hydei. Changing the position of the element on the mRNA reduces the stabilizing effect, while inversion of the beta 2DE1 abolishes its function. The element also acts in a combination with the beta 1 tubulin transcription start site, and the beta 2UE1, which is required to achieve tissue-specific expression. In all experiments done, the comparison of premeiotic with postmeiotic stages strongly implies that this element is involved in regulating mRNA stability. This mRNA stabilizing element acts in a position-independent manner and also on a heterologous mRNA, showing its autonomous functional activity.


Subject(s)
Drosophila/genetics , RNA, Messenger/metabolism , Regulatory Sequences, Nucleic Acid , Spermatogenesis , Tubulin/genetics , Animals , Base Sequence , DNA, Recombinant , Drosophila/physiology , Drosophila melanogaster/genetics , Drosophila melanogaster/physiology , Gene Expression Regulation , Genes, Insect , Male , Meiosis , Molecular Sequence Data , Nucleic Acid Conformation , Protein Biosynthesis , RNA, Messenger/genetics , Spermatocytes/metabolism
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