Dynamics of Notch signaling in a self-renewing tissue, the C. elegans germline

Notch signaling was in many stem cell systems shown to play not only an important role in self-renewal but also in differentiation. It is not surprising that perturbations within the pathway are associated with a variety of diseases. These range from loss- of-function mutations to activating mutations within the Notch signaling pathway and are mostly located within the Notch receptor itself. Hyperactivating mutations of Notch are often associated with auto-activation of the receptor or a failure of the activated receptor to be degraded. In the C. elegans stem cell system - the germline - the Notch receptor GLP-1 was shown to be absolutely essential for the maintenance of germline stem cells. Similar to the described hyperactivation found in cancer, hyperactivation of GLP-1 in the C. elegans germline is associated with the formation of a tumor. As in other stem cell systems the output of the signaling pathway has to be tightly controlled, which can generally happen on the various levels that assure receptor availability and its activation. To study GLP-1 dynamics in the C. elegans germline we constructed a functional GFP knock-in within the intracellular domain of GLP-1, which is the form of Notch that translocates into the nucleus to activate gene expression. We found considerable differences in the receptor activation between the larval and adult germline and could show that this is likely due to differential turnover of the NICD by the ubiquitin- proteasome-system. In Notch signaling contexts in other organisms but also in Notch cell fate decisions in C. elegans, which are distinct from the germline stem cell fate decision, nuclear turnover of the activated Notch receptor was shown to be mediated by FBXW7/SEL-10. In contrast to the mechanism proposed for other Notch signaling events, we could show that activated GLP-1 is likely not turned over by SEL-10 but by a mechanism involving the U-box-containing E3 ligase PRP-19

Multiplex DNA fluorescence in situ hybridization to analyze maternal vs. paternal C. elegans chromosomes

Recent advances in high-throughput microscopy have paved the way to study chromosome organization at the single-molecule level and have led to a better understanding of genome organization in space and time. During development, distinct maternal and paternal contributions ensure the formation of an embryo proper, yet little is known about the organization of chromosomes inherited from mothers versus fathers. To tackle this question, we have modified single-molecule chromosome tracing to distinguish between the chromosomes of two well-studied strains of C. elegans called Bristol and Hawai'ian. We find that chromosomes from these two strains have similar folding patterns in homozygous hermaphrodites. However, crosses between Bristol and Hawai'ian animals reveal that the paternal chromosome adopts the folding parameters of the maternal chromosome in embryos. This is accomplished by an increase in the polymer step size and decompaction of the chromosome. The data indicate that factors from the mother impact chromosome folding in trans. We also characterize the degree of intermixing between homologues within the chromosome territories. Sister chromosomes overlap frequently in C. elegans embryos, but pairing between homologues is rare, suggesting that transvection is unlikely to occur. This method constitutes a powerful tool to investigate chromosome architecture from mothers and fathers

Increasing Notch signaling antagonizes PRC2-mediated silencing to promote reprograming of germ cells into neurons

Abstract Cell-fate reprograming is at the heart of development, yet very little is known about the molecular mechanisms promoting or inhibiting reprograming in intact organisms. In the C. elegans germline, reprograming germ cells into somatic cells requires chromatin perturbation. Here, we describe that such reprograming is facilitated by GLP-1/Notch signaling pathway. This is surprising, since this pathway is best known for maintaining undifferentiated germline stem cells/ progenitors. Through a combination of genetics, tissue-specific transcriptome analysis, and functional studies of candidate genes, we uncovered a possible explanation for this unexpected role of GLP-1/Notch. We propose that GLP-1/Notch promotes reprograming by activating specific genes, silenced by the Polycomb repressive complex 2 (PRC2), and identify the conserved histone demethylase UTX-1 as a crucial GLP-1/Notch target facilitating reprograming. These findings have wide implications, ranging from development to diseases associated with abnormal Notch signaling

The round goby genome provides insights into mechanisms that may facilitate biological invasions

Background: The invasive benthic round goby (Neogobius melanostomus) is the most successful temperate invasive fish and has spread in aquatic ecosystems on both sides of the Atlantic. Invasive species constitute powerful in situ experimental systems to study fast adaptation and directional selection on short ecological timescales and present promising case studies to understand factors involved the impressive ability of some species to colonize novel environments. We seize the unique opportunity presented by the round goby invasion to study genomic substrates potentially involved in colonization success. Results We report a highly contiguous long-read-based genome and analyze gene families that we hypothesize to relate to the ability of these fish to deal with novel environments. The analyses provide novel insights from the large evolutionary scale to the small species-specific scale. We describe expansions in specific cytochrome P450 enzymes, a remarkably diverse innate immune system, an ancient duplication in red light vision accompanied by red skin fluorescence, evolutionary patterns of epigenetic regulators, and the presence of osmoregulatory genes that may have contributed to the round goby's capacity to invade cold and salty waters. A recurring theme across all analyzed gene families is gene expansions. Conclusions: The expanded innate immune system of round goby may potentially contribute to its ability to colonize novel areas. Since other gene families also feature copy number expansions in the round goby, and since other Gobiidae also feature fascinating environmental adaptations and are excellent colonizers, further long-read genome approaches across the goby family may reveal whether gene copy number expansions are more generally related to the ability to conquer new habitats in Gobiidae or in fish

Long-read sequencing of benthophilinae mitochondrial genomes reveals the origins of round goby mitogenome re-arrangements

Genetic innovation may be linked to evolutionary success, and indeed, the invasive round goby mitochondrial genome sequence carries two novel features not previously described in Benthophilinae. First, the round goby mitochondrial genome carries a rearrangement of the tRNA cluster Ile-Glu-Met. Second, the round goby mitochondrial genome features a 1250 bp non-coding sequence insertion downstream of the D-loop region. In this publication, we test where in the goby phylogeny the novel tRNA arrangement first arose and whether the sequence insertion is associated with invasive populations only or a genuine feature of the species. We sequence native and invasive populations in Europe and North America, and show that all round gobies carry the sequence insertion. By sequencing the tRNA cluster in selected Gobiidae, we show that the tRNA arrangement arose at the root of the Benthophilinae species radiation

Multiplex DNA fluorescence in situ hybridization to analyze maternal vs. paternal C. elegans chromosomes

Abstract Recent advances in microscopy have enabled studying chromosome organization at the single-molecule level, yet little is known about inherited chromosome organization. Here we adapt single-molecule chromosome tracing to distinguish two C. elegans strains (N2 and HI) and find that while their organization is similar, the N2 chromosome influences the folding parameters of the HI chromosome, in particular the step size, across generations. Furthermore, homologous chromosomes overlap frequently, but alignment between homologous regions is rare, suggesting that transvection is unlikely. We present a powerful tool to investigate chromosome architecture and to track the parent of origin

Distinct functions and temporal regulation of methylated histone H3 during early embryogenesis

During the first hours of embryogenesis, formation of higher-order heterochromatin coincides with the loss of developmental potential. Here, we examine the relationship between these two events, and we probe the processes that contribute to the timing of their onset. Mutations that disrupt histone H3 lysine 9 (H3K9) methyltransferases reveal that the methyltransferase MET-2 helps terminate developmental plasticity, through mono- and di-methylation of H3K9 (me1/me2), and promotes heterochromatin formation, through H3K9me3. Although loss of H3K9me3 perturbs formation of higher-order heterochromatin, embryos are still able to terminate plasticity, indicating that the two processes can be uncoupled. Methylated H3K9 appears gradually in developing; C. elegans; embryos and depends on nuclear localization of MET-2. We find that the timing of H3K9me2 and nuclear MET-2 is sensitive to rapid cell cycles, but not to zygotic genome activation or cell counting. These data reveal distinct roles for different H3K9 methylation states in the generation of heterochromatin and loss of developmental plasticity by MET-2, and identify the cell cycle as a crucial parameter of MET-2 regulation

Increasing Notch signaling antagonizes PRC2-mediated silencing to promote reprograming of germ cells into neurons

Cell-fate reprograming is at the heart of development, yet very little is known about the molecular mechanisms promoting or inhibiting reprograming in intact organisms. In the C. elegans germline, reprograming germ cells into somatic cells requires chromatin perturbation. Here, we describe that such reprograming is facilitated by GLP-1/Notch signaling pathway. This is surprising, since this pathway is best known for maintaining undifferentiated germline stem cells/progenitors. Through a combination of genetics, tissue-specific transcriptome analysis, and functional studies of candidate genes, we uncovered a possible explanation for this unexpected role of GLP-1/Notch. We propose that GLP-1/Notch promotes reprograming by activating specific genes, silenced by the Polycomb repressive complex 2 (PRC2), and identify the conserved histone demethylase UTX-1 as a crucial GLP-1/Notch target facilitating reprograming. These findings have wide implications, ranging from development to diseases associated with abnormal Notch signaling

PRP-19, a conserved pre-mRNA processing factor and E3 ubiquitin ligase, inhibits the nuclear accumulation of GLP-1/Notch intracellular domain

The Notch signalling pathway is a conserved and widespread signalling paradigm, and its misregulation has been implicated in numerous disorders, including cancer. The output of Notch signalling depends on the nuclear accumulation of the Notch receptor intracellular domain (ICD). Using the Caenorhabditis elegans germline, where GLP-1/Notch-mediated signalling is essential for maintaining stem cells, we monitored GLP-1 in vivo. We found that the nuclear enrichment of GLP-1 ICD is dynamic: while the ICD is enriched in germ cell nuclei during larval development, it is depleted from the nuclei in adult germlines. We found that this pattern depends on the ubiquitin proteolytic system and the splicing machinery and, identified the splicing factor PRP-19 as a candidate E3 ubiquitin ligase required for the nuclear depletion of GLP-1 ICD

Additional file 1 of Multiplex DNA fluorescence in situ hybridization to analyze maternal vs. paternal C. elegans chromosomes

Additional file 1: Figure S1-S4