The conserved LEM-3/Ankle1 nuclease is involved in the combinatorial regulation of meiotic recombination repair and chromosome segregation in Caenorhabditis elegans

<div><p>Homologous recombination is essential for crossover (CO) formation and accurate chromosome segregation during meiosis. It is of considerable importance to work out how recombination intermediates are processed, leading to CO and non-crossover (NCO) outcome. Genetic analysis in budding yeast and <i>Caenorhabditis elegans</i> indicates that the processing of meiotic recombination intermediates involves a combination of nucleases and DNA repair enzymes. We previously reported that in <i>C</i>. <i>elegans</i> meiotic joint molecule resolution is mediated by two redundant pathways, conferred by the SLX-1 and MUS-81 nucleases, and by the HIM-6 Bloom helicase in conjunction with the XPF-1 endonuclease, respectively. Both pathways require the scaffold protein SLX-4. However, in the absence of all these enzymes, residual processing of meiotic recombination intermediates still occurs and CO formation is reduced but not abolished. Here we show that the LEM-3 nuclease, mutation of which by itself does not have an overt meiotic phenotype, genetically interacts with <i>slx-1</i> and <i>mus-81</i> mutants, the respective double mutants displaying 100% embryonic lethality. The combined loss of LEM-3 and MUS-81 leads to altered processing of recombination intermediates, a delayed disassembly of foci associated with CO designated sites, and the formation of univalents linked by SPO-11 dependent chromatin bridges (dissociated bivalents). However, LEM-3 foci do not colocalize with ZHP-3, a marker that congresses into CO designated sites. In addition, neither CO frequency nor distribution is altered in <i>lem-3</i> single mutants or in combination with <i>mus-81</i> or <i>slx-4</i> mutations. Finally, we found persistent chromatin bridges during meiotic divisions in <i>lem-3; slx-4</i> double mutants. Supported by the localization of LEM-3 between dividing meiotic nuclei, this data suggest that LEM-3 is able to process erroneous recombination intermediates that persist into the second meiotic division.</p></div

[Regulation of germline stem cells: the niche expands in Drosophila]

International audienceOur fascination for stem cells originates from their ability to divide asymmetrically in order to self-renew and produce daughter cells which can differentiate and replenish tissues. Stem cells could thus represent an unlimited source of differentiated cells that could be used to repair malformed, damaged or ageing tissues. Understanding how their behaviour is regulated is then of paramount medical interest. Specific microenvironments surrounding the stem cells, termed "niches", were proposed to play a major role in the balance between self-renewal and differentiation. However, it is only recently, in the case of the stem cells producing the germline (GSGs) in Drosophila, that the cells and signals creating a niche were identified for the first time. Here, we review how this niche has been defined at the cellular and functional levels in vivo, thanks to the powerful genetic tools available in Drosophila. Such studies have revealed adhesive interactions, cell-cycle modifications and intercellular signals that control the GSC behavior. Extracellular signals from the niche activate the BMP or JAK-STAT pathways in the GSCs and are necessary for their maintenance. Strikingly, both signaling pathways are also sufficient to convert differentiated germ cells into functional GSCs, demonstrating in vivo that a niche has the capacity to regenerate stem cells from differentiated cells. Rapid progresses have further identified direct links between these signaling pathways and the transcriptional regulation of the GSCs, providing a simple paradigm for stem cells regulation. Many of these features and signals are conserved in stem cells niches from Drosophila to mammals. We can thus hope that research on the GSCs in Drosophila will benefit therapeutic approaches to human degenerative diseases

Régulation des cellules souches de la lignée germinale

L’intérêt médical des cellules souches provient de leur capacité à se diviser de manière asymétrique donnant naissance à une nouvelle cellule souche, et à une cellule capable de se différencier. Cette division est contrôlée à la fois par une identité spécifique des cellules souches et un microenvironnement complexe, appelé une niche, qui reste souvent inaccessible chez les mammifères. Au contraire, les outils génétiques disponibles chez la drosophile ont permis récemment d’identifier in vivo non seulement les cellules constituant la niche des cellules souches de la lignée germinale, mais également les signaux émis par celles-ci ainsi que les gènes cibles dans les cellules souches elles-mêmes. La conservation de ces mécanismes chez les mammifères, pour les cellules souches hématopoïétiques ou épithéliales, laisse envisager des applications thérapeutiques potentielles

Crible génétique afin d'identifier de nouveaux gènes requis durant l'ovogenèse précose chez Drosophila melanogaster

L ovogenèse de drosophile offre l opportunité d étudier une grande variété de processus biologiques. En effet la détermination cellulaire, la mitose et la méiose, l établissement et le maintien d une polarité cellulaire, l adhérence, la migration cellulaire, la localisation d ARNm sont des processus requis pour la formation d un gamète femelle fonctionnel. Pour mieux comprendre les différents mécanismes mis en œuvre durant l ovogenèse précoce, nous avons réalisé un crible génétique afin d établir une collection de mutants affectant la formation du follicule ovarien. L étude d un mutant identifié durant le crible qui affecte le gène D12 est en cours. Il s agit d un membre du complexe ATAC qui permet l acétylation des histones. Il est ainsi possible de lier, pour la 1ère fois, l acétylation des histones et différenciation de l ovocyte. Par ailleurs, nous avons pu montrer que le suppresseur de tumeur lethal giant larvae était impliqué lors de la polarisation précoce de l ovocyte.PARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

lethal giant larvae is required with the par genes for the early polarization of the Drosophila oocyte.

International audienceMost cell types in an organism show some degree of polarization, which relies on a surprisingly limited number of proteins. The underlying molecular mechanisms depend, however, on the cellular context. Mutual inhibitions between members of the Par genes are proposed to be sufficient to polarize the C. elegans one-cell zygote and the Drosophila oocyte during mid-oogenesis. By contrast, the Par genes interact with cellular junctions and associated complexes to polarize epithelial cells. The Par genes are also required at an early step of Drosophila oogenesis for the maintenance of the oocyte fate and its early polarization. Here we show that the Par genes are not sufficient to polarize the oocyte early and that the activity of the tumor-suppressor gene lethal giant larvae (lgl) is required for the posterior translocation of oocyte-specific proteins, including germline determinants. We also found that Lgl localizes asymmetrically within the oocyte and is excluded from the posterior pole. We further demonstrate that phosphorylation of Par-1, Par-3 (Bazooka) and Lgl is crucial to regulate their activity and localization in vivo and describe, for the first time, adherens junctions located around the ring canals, which link the oocyte to the other cells of the germline cyst. However, null mutations in the DE-cadherin gene, which encodes the main component of the zonula adherens, do not affect the early polarization of the oocyte. We conclude that, despite sharing many similarities with other model systems at the genetic and cellular levels, the polarization of the early oocyte relies on a specific subset of polarity proteins

Combinatorial regulation of meiotic holliday junction resolution in C. elegans by HIM-6 (BLM) helicase, SLX-4, and the SLX-1, MUS-81 and XPF-1 nucleases

Holliday junctions (HJs) are cruciform DNA structures that are created during recombination events. It is a matter of considerable importance to determine the resolvase(s) that promote resolution of these structures. We previously reported that C. elegans GEN-1 is a symmetrically cleaving HJ resolving enzyme required for recombinational repair, but we could not find an overt role in meiotic recombination. Here we identify C. elegans proteins involved in resolving meiotic HJs. We found no evidence for a redundant meiotic function of GEN-1. In contrast, we discovered two redundant HJ resolution pathways likely coordinated by the SLX-4 scaffold protein and also involving the HIM-6/BLM helicase. SLX-4 associates with the SLX-1, MUS-81 and XPF-1 nucleases and has been implicated in meiotic recombination in C. elegans. We found that C. elegans [mus-81; xpf-1], [slx-1; xpf-1], [mus-81; him-6] and [slx-1; him-6] double mutants showed a similar reduction in survival rates as slx-4. Analysis of meiotic diakinesis chromosomes revealed a distinct phenotype in these double mutants. Instead of wild-type bivalent chromosomes, pairs of "univalents" linked by chromatin bridges occur. These linkages depend on the conserved meiosis-specific transesterase SPO-11 and can be restored by ionizing radiation, suggesting that they represent unresolved meiotic HJs. This suggests the existence of two major resolvase activities, one provided by XPF-1 and HIM-6, the other by SLX-1 and MUS-81. In all double mutants crossover (CO) recombination is reduced but not abolished, indicative of further redundancy in meiotic HJ resolution. Real time imaging revealed extensive chromatin bridges during the first meiotic division that appear to be eventually resolved in meiosis II, suggesting back-up resolution activities acting at or after anaphase I. We also show that in HJ resolution mutants, the restructuring of chromosome arms distal and proximal to the CO still occurs, suggesting that CO initiation but not resolution is likely to be required for this process

A Mosaic Genetic Screen for Genes Involved in the Early Steps of Drosophila Oogenesis

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Depletion of LEM-3 and MUS-81 leads to formation of dissociated bivalents.

<p>(A) Images of DAPI-stained chromosomes in –1 oocytes at diakinesis in wild type, <i>lem-3</i>, <i>mus-81</i> and <i>mus-81 lem-3</i> mutants. Red arrows indicate dissociated bivalents. Chromosome fragment is highlighted with a red arrowhead. Scale bars: 2 μm. (B) Quantification of bivalents, ‘dissociated bivalents’ and fragments observed in indicated genotypes. Overlapping chromosomes that could not be assigned to the above categories were scored as “n/d”. Sample sizes of indicated genotype are as follows: wild type n = 40; <i>lem-3</i> n = 36; <i>mus-81</i> n = 36; <i>mus-81 lem-3</i> n = 42.</p

Absence of chiasmata on ‘dissociated bivalents’ revealed by HTP-3 staining.

<p>(A) Projections of representative nuclei of diakinesis oocytes stained with α-HTP-3 antibody and DAPI. Open arrowheads indicate the linked chromosomes shown in close-ups. The upper right panels show SIM images of a representative ‘dissociated bivalents’ of an <i>[slx-1; him-6]</i> oocyte. Linked univalent are encircled in blue. Scale bars are shown in white (4 µm). (B) Schematic representation of the proximal germ line, spermatheca and early embryogenesis in the <i>C. elegans</i> germ line.</p