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1.
EMBO Rep ; 24(9): e56463, 2023 09 06.
Article in English | MEDLINE | ID: mdl-37462213

ABSTRACT

Mitotic chromatin is largely assumed incompatible with transcription due to changes in the transcription machinery and chromosome architecture. However, the mechanisms of mitotic transcriptional inactivation and their interplay with chromosome assembly remain largely unknown. By monitoring ongoing transcription in Drosophila early embryos, we reveal that eviction of nascent mRNAs from mitotic chromatin occurs after substantial chromosome compaction and is not promoted by condensin I. Instead, we show that the timely removal of transcripts from mitotic chromatin is driven by the SNF2 helicase-like protein Lodestar (Lds), identified here as a modulator of sister chromatid cohesion defects. In addition to the eviction of nascent transcripts, we uncover that Lds cooperates with Topoisomerase 2 to ensure efficient sister chromatid resolution and mitotic fidelity. We conclude that the removal of nascent transcripts upon mitotic entry is not a passive consequence of cell cycle progression and/or chromosome compaction but occurs via dedicated mechanisms with functional parallelisms to sister chromatid resolution.


Subject(s)
Chromatids , Drosophila , Mitosis , Animals , Cell Cycle Proteins/metabolism , Chromatids/metabolism , Chromatin , DNA Topoisomerases, Type II/genetics , Drosophila/cytology , Drosophila/genetics
2.
PLoS Biol ; 17(2): e3000016, 2019 02.
Article in English | MEDLINE | ID: mdl-30794535

ABSTRACT

Studying aneuploidy during organism development has strong limitations because chronic mitotic perturbations used to generate aneuploidy usually result in lethality. We developed a genetic tool to induce aneuploidy in an acute and time-controlled manner during Drosophila development. This is achieved by reversible depletion of cohesin, a key molecule controlling mitotic fidelity. Larvae challenged with aneuploidy hatch into adults with severe motor defects shortening their life span. Neural stem cells, despite being aneuploid, display a delayed stress response and continue proliferating, resulting in the rapid appearance of chromosomal instability, a complex array of karyotypes, and cellular abnormalities. Notably, when other brain-cell lineages are forced to self-renew, aneuploidy-associated stress response is significantly delayed. Protecting only the developing brain from induced aneuploidy is sufficient to rescue motor defects and adult life span, suggesting that neural tissue is the most ill-equipped to deal with developmental aneuploidy.


Subject(s)
Aneuploidy , Drosophila melanogaster/physiology , Longevity/physiology , Neural Stem Cells/physiology , Stress, Physiological , Animals , Brain/physiology , Cell Cycle , Cell Cycle Proteins/metabolism , Cell Self Renewal , Chromosomal Instability , Chromosomal Proteins, Non-Histone/metabolism , Chromosomes, Insect/metabolism , Karyotype , Kinetics , Larva/physiology , Mitosis , Neural Stem Cells/cytology , Time Factors , Wings, Animal/physiology , Cohesins
3.
Curr Biol ; 28(17): 2837-2844.e3, 2018 09 10.
Article in English | MEDLINE | ID: mdl-30122528

ABSTRACT

The fidelity of mitosis depends on cohesive forces that keep sister chromatids together. This is mediated by cohesin that embraces sister chromatid fibers from the time of their replication until the subsequent mitosis [1-3]. Cleavage of cohesin marks anaphase onset, where single chromatids are dragged to the poles by the mitotic spindle [4-6]. Cohesin cleavage should only occur when all chromosomes are properly bio-oriented to ensure equal genome distribution and prevent random chromosome segregation. Unscheduled loss of sister chromatid cohesion is prevented by a safeguard mechanism known as the spindle assembly checkpoint (SAC) [7, 8]. To identify specific conditions capable of restoring defects associated with cohesion loss, we screened for genes whose depletion modulates Drosophila wing development when sister chromatid cohesion is impaired. Cohesion deficiency was induced by knockdown of the acetyltransferase separation anxiety (San)/Naa50, a cohesin complex stabilizer [9-12]. Several genes whose function impacts wing development upon cohesion loss were identified. Surprisingly, knockdown of key SAC proteins, Mad2 and Mps1, suppressed developmental defects associated with San depletion. SAC impairment upon cohesin removal, triggered by San depletion or artificial removal of the cohesin complex, prevented extensive genome shuffling, reduced segregation defects, and restored cell survival. This counterintuitive phenotypic suppression was caused by an intrinsic bias for efficient chromosome biorientation at mitotic entry, coupled with slow engagement of error-correction reactions. Thus, in contrast to SAC's role as a safeguard mechanism for mitotic fidelity, removal of this checkpoint alleviates mitotic errors when sister chromatid cohesion is compromised.


Subject(s)
Drosophila melanogaster/cytology , M Phase Cell Cycle Checkpoints/physiology , Mitosis/physiology , Sister Chromatid Exchange/physiology , Animals
4.
EMBO Rep ; 19(8)2018 08.
Article in English | MEDLINE | ID: mdl-30037897

ABSTRACT

The transition from fertilized oocyte to totipotent embryo relies on maternal factors that are synthetized and accumulated during oocyte development. Yet, it is unclear how oocytes regulate the expression of maternal genes within the transcriptional program of oogenesis. Here, we report that the Drosophila Trithorax group protein dMLL3/4 (also known as Trr) is essential for the transition to embryo fate at fertilization. In the absence of dMLL3/4, oocytes develop normally but fail to initiate the embryo mitotic divisions after fertilization. This incapability results from defects in paternal genome reprogramming and maternal meiotic completion. The methyltransferase activity of dMLL3/4 is dispensable for both these processes. We further show that dMLL3/4 promotes the expression of a functionally coherent gene subset that is required for the initiation of post-fertilization development. Accordingly, we identify the evolutionarily conserved IDGF4 glycoprotein (known as oviductin in mammals) as a new oocyte-to-embryo transition gene under direct dMLL3/4 transcriptional control. Based on these observations, we propose that dMLL3/4 plays an instructive role in the oocyte-to-embryo transition that is functionally uncoupled from the requirements of oogenesis.


Subject(s)
Drosophila Proteins/metabolism , Drosophila melanogaster/genetics , Drosophila melanogaster/metabolism , Fertilization/genetics , Genome , Histone-Lysine N-Methyltransferase/metabolism , Zygote/metabolism , Animals , Drosophila melanogaster/cytology , Embryo, Nonmammalian/cytology , Embryo, Nonmammalian/metabolism , Embryonic Development/genetics , Female , Germ Cells/metabolism , Glycoproteins/metabolism , Intracellular Signaling Peptides and Proteins , Male , Meiosis , Oocytes/cytology , Oocytes/metabolism , Oogenesis
5.
Nat Commun ; 7: 12331, 2016 08 10.
Article in English | MEDLINE | ID: mdl-27507044

ABSTRACT

Oocytes are arrested for long periods of time in the prophase of the first meiotic division (prophase I). As chromosome condensation poses significant constraints to gene expression, the mechanisms regulating transcriptional activity in the prophase I-arrested oocyte are still not entirely understood. We hypothesized that gene expression during the prophase I arrest is primarily epigenetically regulated. Here we comprehensively define the Drosophila female germ line epigenome throughout oogenesis and show that the oocyte has a unique, dynamic and remarkably diversified epigenome characterized by the presence of both euchromatic and heterochromatic marks. We observed that the perturbation of the oocyte's epigenome in early oogenesis, through depletion of the dKDM5 histone demethylase, results in the temporal deregulation of meiotic transcription and affects female fertility. Taken together, our results indicate that the early programming of the oocyte epigenome primes meiotic chromatin for subsequent functions in late prophase I.


Subject(s)
Chromatin Assembly and Disassembly/genetics , Drosophila/physiology , Epigenesis, Genetic/physiology , Meiotic Prophase I/genetics , Oocytes/physiology , Animals , Chromatin/metabolism , DNA Demethylation , Drosophila Proteins/genetics , Drosophila Proteins/metabolism , Female , Fertility/genetics , Histone Demethylases/genetics , Histone Demethylases/metabolism , Histones/genetics , Histones/metabolism , Oogenesis/physiology
6.
Bioessays ; 37(5): 514-24, 2015 May.
Article in English | MEDLINE | ID: mdl-25823409

ABSTRACT

The development of living organisms requires a precise coordination of all basic cellular processes, in space and time. Early embryogenesis of most species with externally deposited eggs starts with a series of extremely fast cleavage cycles. These divisions have a strong influence on gene expression as mitosis represses transcription and pre-mRNA processing. In this review, we will describe the distinct adaptations for efficient gene expression and discuss the emerging role of the multifunctional NineTeen Complex (NTC) in gene expression and genomic stability during fast proliferation.


Subject(s)
Gene Expression/physiology , RNA Precursors/genetics , Spliceosomes/genetics , Animals , Cell Proliferation/genetics , Cell Proliferation/physiology , Humans , RNA Splicing/genetics
7.
Development ; 139(3): 503-13, 2012 Feb.
Article in English | MEDLINE | ID: mdl-22223679

ABSTRACT

Epithelial cells mostly orient the spindle along the plane of the epithelium (planar orientation) for mitosis to produce two identical daughter cells. The correct orientation of the spindle relies on the interaction between cortical polarity components and astral microtubules. Recent studies in mammalian tissue culture cells suggest that the apically localised atypical protein kinase C (aPKC) is important for the planar orientation of the mitotic spindle in dividing epithelial cells. Yet, in chicken neuroepithelial cells, aPKC is not required in vivo for spindle orientation, and it has been proposed that the polarization cues vary between different epithelial cell types and/or developmental processes. In order to investigate whether Drosophila aPKC is required for spindle orientation during symmetric division of epithelial cells, we took advantage of a previously isolated temperature-sensitive allele of aPKC. We showed that Drosophila aPKC is required in vivo for spindle planar orientation and apical exclusion of Pins (Raps). This suggests that the cortical cues necessary for spindle orientation are not only conserved between Drosophila and mammalian cells, but are also similar to those required for spindle apicobasal orientation during asymmetric cell division.


Subject(s)
Cell Division , Drosophila Proteins/metabolism , Drosophila melanogaster/enzymology , Protein Kinase C/metabolism , Spindle Apparatus/enzymology , Animals , Cell Cycle Proteins , Cell Polarity , Drosophila Proteins/genetics , Drosophila melanogaster/cytology , Epithelial Cells/metabolism , Female , Guanine Nucleotide Dissociation Inhibitors/metabolism , Male , Mutation , Protein Kinase C/genetics
8.
Article in English | MEDLINE | ID: mdl-20079453

ABSTRACT

Sex steroids are known to be involved in gonadal differentiation in fish, but whether androgens are early mediators of testis differentiation remains unclear. We studied the sex-related developmental variations in the gene expression of two key enzymes involved in steroids and androgen synthesis (cyp11a1 and cyp11b1) in trunks and isolated gonads of pejerrey (Odontesthes bonariensis) larvae during and after the sex determination period. Also, and in order to have a better characterization of this process we studied the expression of Sertoli (dmrt1, amh, sox9) and Leydig (nr5a1 or sf-1) cell markers as well as a gene with higher expression in females (cyp19a1a). No clear differences were observed in the expression of cyp11a1 and cyp11b1 during the temperature-sensitive window in the trunk of pejerrey larvae. Nevertheless, a clear increase of cyp11b1 was observed in isolated gonads taken from fish reared at the male producing temperature. In these gonads we also confirmed the trends of genes with higher expression in males (dmrt1, amh) and females (cyp19a1a) as previously described in larval trunks of pejerrey. Our results showed that the expression of cyp11b1 was positively associated with the morphological differentiation of the testis. Nevertheless the involvement of 11-oxygenated androgens during the temperature-sensitive window could not be clearly established.


Subject(s)
Cholesterol Side-Chain Cleavage Enzyme/genetics , Smegmamorpha/growth & development , Smegmamorpha/genetics , Steroid 11-beta-Hydroxylase/genetics , Amino Acid Sequence , Androgens/biosynthesis , Animals , Base Sequence , DNA Primers/genetics , Female , Gene Expression Profiling , Gene Expression Regulation, Developmental , Gene Expression Regulation, Enzymologic , Larva/growth & development , Larva/metabolism , Male , Molecular Sequence Data , Ovary/growth & development , Ovary/metabolism , Sequence Homology, Amino Acid , Sex Differentiation/genetics , Sex Differentiation/physiology , Smegmamorpha/metabolism , Steroids/biosynthesis , Temperature , Testis/growth & development , Testis/metabolism
9.
J Mol Evol ; 64(6): 614-27, 2007 Jun.
Article in English | MEDLINE | ID: mdl-17557168

ABSTRACT

Most vertebrates express two gonadotropin releasing hormone (GnRH) variants in brain tissue but there is an increasing number of fish species for which a third GnRH form has been detected. We characterized the precursors (cDNAs) of all three forms expressed in the brain of the pejerrey (silverside) fish, Odontesthes bonariensis (Atheriniformes): type I (GnRH-I; 440 bp), type II (GnRH-II; 529 bp), and type III (GnRH-III; 515 bp). The expression of these GnRHs precursors was also observed in peripheral tissues related to reproduction (gonads), visual and chemical senses (eye and olfactory epithelium), and osmoregulation (gill), suggesting that in teleost fish and possibly other vertebrates GnRH mediates directly or indirectly many other functions besides reproduction. We also present a comprehensive phylogenetic analysis including representatives of all chordate GnRH precursors characterized to date that supports the idea of two main paralogous GnRH lineages with different function. A "forebrain lineage" separates evolutionarily from the "midbrain lineage" as a result of an ancient duplication (ca. 600 million years ago). A third, fish-only clade of GnRH genes seems to have originated before the divergence of fish and tetrapods but retained only in fish. Phylogenetic analyses of GnRH precursors (DNA and protein sequences) under different optimality criteria converge on this result. Although alternative scenarios could not be statistically rejected in this study due to the relatively short size of the analyzed molecules, this hypothesis also receives support from chromosomal studies of synteny around the GnRH genes in vertebrates.


Subject(s)
DNA, Complementary/genetics , Evolution, Molecular , Fishes/genetics , Gonadotropin-Releasing Hormone/genetics , Protein Precursors/genetics , Amino Acid Sequence , Animals , Base Sequence , Brain/metabolism , Cloning, Molecular , Gene Expression Profiling , Gene Expression Regulation , Gonadotropin-Releasing Hormone/chemistry , Gonadotropin-Releasing Hormone/metabolism , Likelihood Functions , Models, Genetic , Molecular Sequence Data , Organ Specificity , Phylogeny , Protein Isoforms/genetics , Protein Isoforms/metabolism , Reverse Transcriptase Polymerase Chain Reaction
10.
Article in English | MEDLINE | ID: mdl-16716622

ABSTRACT

Gonadotropin-releasing hormone (GnRH) is the final common signaling molecule used by the brain to regulate reproduction in all vertebrates. Until now, a total of 24 GnRH structural variants have been characterized from vertebrate, protochordate and invertebrate nervous tissue. Almost all vertebrates already investigated have at least two GnRH forms coexisting in the central nervous system. Furthermore, it is now well accepted that three GnRH forms are present both in early and late evolved teleostean fishes. The number and taxonomic distribution of the different GnRH variants also raise questions about the phylogenetic relationships between them. Most of the GnRH phylogenetic analyses are in agreement with the widely accepted idea that the GnRH family can be divided into three main groups. However, the examination of the gnathostome GnRH phylogenetic relationships clearly shows the existence of two main paralogous GnRH lineages: the ''midbrain GnRH" group and the "forebrain GnRH" group. The first one, represented by chicken GnRH-II forms, and the second one composed of two paralogous lineages, the salmon GnRH cluster (only represented in teleostean fish species) and the hypophysotropic GnRH cluster, also present in tetrapods. This analysis suggests that the two forebrain clades share a common precursor and reinforces the idea that the salmon GnRH branch has originated from a duplication of the hypophysotropic lineage. GnRH ligands exert their activity through G protein-coupled receptors of the rhodopsin-like family. As with the ligands, multiple GnRHRs are expressed in individual vertebrate species and phylogenetic analyses have revealed that all vertebrate GnRHRs cluster into three main receptor types. However, new data and a new phylogenetic analysis propose a two GnRHR type model, in which different rounds of gene duplications may have occurred in different groups within each lineage.


Subject(s)
Evolution, Molecular , Gonadotropin-Releasing Hormone/physiology , Phylogeny , Receptors, LHRH/physiology , Vertebrates/physiology , Animals , Gonadotropin-Releasing Hormone/chemistry , Gonadotropin-Releasing Hormone/classification , Ligands , Receptors, LHRH/chemistry , Vertebrates/classification
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