Memar, Nadin et al. (2022) MicroPubl Biol

Memar, Nadin, Lambie, Eric J, Sethi, Aditya, Luehr, Sebastian, Conradt, Barbara

[MicroPubl Biol, 2022]

Visualization of genomic loci with open chromatin state has been reported in mammalian tissue culture cells using a CRISPR/Cas9-based system that utilizes an EGFP-tagged endonuclease-deficient Cas9 protein (dCas9::EGFP) (Chen et al. 2013). Here, we adapted this approach for use in Caenorhabditis elegans . We generated a C. elegans strain that expresses the dCas9 protein fused to two nuclear-localized EGFP molecules (dCas9::NLS::2xEGFP::NLS) in an inducible manner. Using this strain, we report the visualization in live C. elegans embryos of two endogenous repetitive loci, rrn-4 and rrn-1 , from which 5S and 18S ribosomal RNAs are constitutively generated.

Memar, Nadin et al. (2009) International Worm Meeting "A view on the embryogenesis of the Caenorhabditis Family."

Martin, Katharina, Schnabel, Ralf, Memar, Nadin

[International Worm Meeting, 2009]

The molecular differences of the four Caenorhabditis species C. elegans, C. briggsae, C. remanei and C. brenneri are currently of great interest, however little is known about development. Zhao et al. (2008) reported an automatic lineage of C. briggsae and came - based mostly on the cleavage pattern and cell positions - to the conclusion that the embryogenesis of the two species is very similar. We now present detailed 4D analyses of the species including the terminal differentiation patterns. All analyses including bioinformatical quantifications of cell behaviour show a huge similarity between those species. Immunochemical analyses of the tissue distributions only reveal a difference in the intestinal differentiation of C. brenneri. Interestingly hybrid embryos always appear to fail in different ways in embryogenesis.

Memar, Nadin et al. (2015) International Worm Meeting "Never change a running system? A surprising similarity in nematode embryogenesis."

Schiemann, Sabrina, Conradt, Barbara, Chiesielski, Hanna, Memar, Nadin, Luthe, Katharina, Hennig, Christian, Schnabel, Ralf

[International Worm Meeting, 2015]

An important justification of genome projects in general is the notion that there should be a strong correlation between genotype and phenotype. C. elegans and C. briggsae show more differences at the genomic level than humans compared to mice [1]. However, the behavior and anatomy of these nematodes are overall very similar. C. elegans and C. briggsae shared the last common ancestor 80-100 million years ago [2]. Here we present a detailed analysis of the embryogenesis of C. elegans, C. briggsae, C. remanei and C. brenneri. Microscopic 4D analyses, including the terminal differentiation patterns and bioinformatical quantifications of cell behavior show that the embryogenesis of these nematodes cannot be distinguished. Thus, significantly differing genomes lead to morphological processes that are almost completely identical. To determine whether this process is also almost identical in more distantly related nematodes, we analyzed Pristionchus pacificus, which separated from C. elegans more than 280-430 million years ago [3]. We found that up to the pre-morphogenetic stage, when embryos contain 400 cells and are about to undergo morphogenesis, the Caenorhabditis species and P. pacificus share (beside some minor differences such as, for example, an additional cell death after the 9th cleavage round) identical cell positions and fates. However, starting with the pre-morphogenetic stage, development in P. pacificus and the Caenorhabditis species analyzed takes two different paths. We propose that in nematodes, the pre-morphogenetic stage may be equivalent to the so called 'phylotypic stage' that Klaus Sander proposed to be a conserved embryonic stage in other animals [4]. 1. Stein, L.D., et al., The genome sequence of Caenorhabditis briggsae: a platform for comparative genomics. PLoS Biol, 2003. 1(2): p. E45.2. Hillier, L.W., et al., Comparison of C. elegans and C. briggsae genome sequences reveals extensive conservation of chromosome organization and synteny. PLoS Biol, 2007. 5(7): p. e167.3. Dieterich, C., et al., The Pristionchus pacificus genome provides a unique perspective on nematode lifestyle and parasitism. Nat Genet, 2008. 40(10): p. 1193-8.4. Sander, K., Specification of the Basic Body Pattern in Insect Embryogenesis. Advances in Insect Physiology, 1976. 12: p. 125-238.

Memar, Nadin et al. (2009) International Worm Meeting "A screen for temperature-sensitive embryonic lethal mutations affecting cell focussing in Caenorhabditis elegans."

Wiekenberg, Anne, Schnabel, Ralf, Martin, Katharina, Memar, Nadin

[International Worm Meeting, 2009]

Schnabel et al. (2006) proposed cell focussing as mechanism for global cell sorting by cell identity in the C. elegans embryo. It should be helpful to find mutants to define the mechanism of this process. Interestingly a very large collection of nonconditional embryonic lethal mutants did not contain a mutant affecting cell focussing. We supposed that genes affecting this process might be highly pleiotropic, since it should also keep larvae and adults in shape and/or maternal rescue may occur in the embryo. Therefore, ts mutants may be the tool of choice to identify genes. We designed a new screen for ts mutants using the worm sorter, since a very large number of mutants may be required but the yield is less than 1%. Identification of specific mutants is very cumbersome since candidates have to be subjected to a careful 4D analysis. Only mutants in which fates are wild-type - but the positioning of subsets of cells is wrong - qualify as potential cell focussing mutants. A first screen yielded two candidates in approx. 1000 ts mutants. In the next round we isolated again 1000 mutants, which now await analysis. We expect that up 10000 ts mutants may be required to define a cell focussing pathway. However, the mutant collection may be also a valuable resource for the worm community.

Memar, Nadin et al. (2021) International Worm Meeting "The loss of psf-2 GINS leads to the inappropriate survival of cells programmed to die during C. elegans development"

Schnabel, Ralf, Conradt, Barbara, Memar, Nadin, Sherrard, Ryan, Lambie, Eric

[International Worm Meeting, 2021]

The genetic pathway required for programmed cell death during C. elegans development is highly conserved. The key activator of this pathway is the pro-apoptotic BH3-only gene egl-1. Earlier studies showed that egl-1 activity is controlled at the level of gene expression. Specifically, egl-1 expression is controlled both at the transcriptional level through lineage-specific transcription factors and at the post-transcriptional level through microRNAs. In this study, we present evidence that egl-1 expression is controlled at an additional level. The gene psf-2 encodes a subunit of the GINS complex, which is essential for DNA replication. We identified a temperature-sensitive loss-of-function mutation of psf-2, t3443ts, and found that it does not only cause a cell cycle defect but a general block in cell death i.e. a Ced phenotype. Furthermore, we provide evidence that the Ced phenotype exhibited by psf-2(t3443ts) mutants is independent of these animals' cell cycle defects. Based on these observations we propose that psf-2 GINS is required for programmed cell death during C. elegans development. To determine whether psf-2(t3443ts) affects the expression of egl-1, we quantified the number of egl-1 transcripts in cell death lineages i.e. lineages in which a cell death occurs. We found that psf-2(t3443ts) abolishes a spike in the number of egl-1 transcripts normally observed in cells programmed to die. This suggests that the loss of psf-2 GINS blocks programmed cell death by abolishing the transcriptional up-regulation of egl-1in cells programmed to die. Based on our findings, we propose that the GINS complex is necessary for changes in chromatin state at the egl-1 locus that permit the transcriptional up-regulation of the egl-1 gene in cells programmed to die.

Nadin Memar et al. (2007) International Worm Meeting "News from the Schnabel Laboratory: 4D-Microscopic Lineage analysis of embryos until hatching and a comparison of the embryonic development of different Caenorhabditis species."

Ralf Schnabel, Nadin Memar, Henning Schmidt

[International Worm Meeting, 2007]

Using 4D-microscopy it is now "easy" to analyse the development of Caenorhabditis elegans embryos at great detail. Because wild-type embryos start with muscle contractions after the embryonic comma stage, it was not possible to follow cells further than approximately to the 10th cleavage of cells. However, now many labs are interested to lineage further. We learned that it is possible to follow cells until the end of embryonic development by stopping embryonic movement using RNA interference (feeding) against pat-4. With this approach it is possible to analyse any interesting mutant or transgene without much effort. Genomics, transcriptomics and proteomics are used to analyse and compare different species of Caenorhabditis. Considering the molecular differences it is an intriguing question how similar the development of the worms proceeds. A lineage and topological analysis of C. briggsae embryos did, to our surprise, not reveal any differences between the two species so far. We are currently proceeding with the analysis of C. remanei ssp. vulgaris.

Martin, Katharina et al. (2010) C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany "A screen for temperature-sensitive embryonic lethal mutants affecting cell migration in Caenorhabditis elegans."

Wiekenberg, Anne, Martin, Katharina, Memar, Nadin, Schnabel, Ralf, Hennig, Christian, Schiemann, Sabrina

[C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany, 2010]

Schnabel et al. (2006) proposed cell focussing as mechanism for global cell sorting by cell identity in the C. elegans embryo. A collection of embryonic lethal mutants could give us a deeper understanding of this barely described process. As genes affecting cell focussing may be highly pleiotropic, temperature-sensitive mutants are the tool of choice. Our group designed a large screen for ts mutants. Unfortunately a very strict and consistent inspection leads to a yield of less than 1 %. Nevertheless, we have almost 2000 mutant strains by now with a great variety of interesting phenotypes. So far 538 strains were analysed via 4D microscopy and 82 of them seem to have defects in morphogenesis and cell adhesion, which are the most valuable phenotypes for our project. After a short analysis of a defined subset of cells with SIMI Biocell, our bioinformatical tools show if cells or whole regions migrate not correctly. Continuative methods give us a deeper insight in cell migration, cell fates, time of development and many more aspects of the embryogenesis. This way, we hope to find mutants with defects in cell migration and sorting, identify corresponding genes and thus get a molecular view about cell focussing. Moreover the mutant collection will be hopefully a valuable source for the worm community.

Tobin, David V et al. (2013) International Worm Meeting "The C. elegans UBE2Q2 homolog, UBC-25, Promotes Cell Cycle Quiescence by Inhibiting Cyclin E Expression."

Saito, R. Mako, Tobin, David V, Roy, Sarah H, Conradt, Barbara, Memar, Nadin

[International Worm Meeting, 2013]

Cell cycle quiescence is important for coordinating differentiation with development. In C. elegans, vulva precursor cells (VPCs) remain quiescent for over two larval stages before re-entering the cell cycle to generate vulva and hypodermal tissues. A forward genetic screen identified several novel regulators of VPC quiescence, including the cdc-14 phosphatase. cdc-14 promotes VPC quiescence through stabilization of cki-1, contrary to its requirement for mitosis in yeast. We conducted an RNAi screen to identify additional genes necessary to maintain VPC quiescence. We focus on the screen positive ubc-25: it is highly conserved with human UBE2Q2 and ubc-25(lf) produces severe extra intestinal nuclear divisions. Additionally, studies of human UBE2Q2 have provided conflicting functions, which may be clarified from our studies in C. elegans. Lineage analysis of predicted null ubc-25(ok1732) embryos found that the extra nuclei originate from shorter cell cycles in the E lineage. Genetic analyses suggest that ubc-25 acts in a linear pathway with SCF component cul-1, in parallel to cki-1 and lin-35 to restrict intestinal nuclear divisions. Consistent with the role of cul-1 in limiting Cyclin-E expression, animals lacking ubc-25 display 1.5 to 8.7 fold increased CYE-1 expression by western blot analysis. Interestingly, the phenotype of ubc-25(lf) is not as severe as cul-1(lf). Other ubiquitin-conjugating enzymes compensate for ubc-25(lf) since inhibition of several ubc genes substantially enhanced intestinal nuclei number in ubc-25(lf) animals. Broad expression of a mCherry::UBC-25 fusion protein suggests that ubc-25 activity may promote quiescence in diverse tissues. This hypothesis is supported by the dramatic enhancement of rare ectopic VPC divisions in ubc-25(lf) animals treated with lin-35 RNAi. Our experiments support a model in which ubc-25 activity broadly supports cell cycle quiescence though inhibition of CYE-1 expression, but through the action of parallel processes only appears rate limiting in the intestine.

Kalinski, Cristine et al. (2021) International Worm Meeting "Connectomic comparison of P. pacificus and C. elegans anterior nervous system structure"

Kalinski, Cristine, Schnabel, Ralf, Cook, Steven J., Sommer, Ralf J., Majeed, Maryam, Hobert, Oliver, Stephen, S. Rebecca, Memar, Nadin, Conradt, Barbara

[International Worm Meeting, 2021]

Generating connectomes can lead to an increased understanding of how nervous system structure and connectivity can dictate behavior. To expand upon this growing body of knowledge, here we use connectomics to describe how the nematode nervous system has evolved by comparing the divergent nematode species P. pacificus and C. elegans. As the complete connectome of C. elegans has already been established, we reconstruct serial-section electron micrographs (EM) to generate volumetric reconstructions of multiple P. pacificus samples. We extend our analysis of P. pacificus nervous system ultrastructure beyond the pharyngeal (Bumbarger et al 2013) and amphidial (Hong et al 2019) circuits to include the complete nerve ring and surrounding anterior nervous system (defined as the tip of the nose to the deirid commissures). We have completed our reconstruction of these samples, and find the overall structure of the nervous system is conserved, and we can unambiguously identify all neurons whose cell bodies or commissures are present within these EM series. Through embryonic lineaging, we observe only one difference in neuron number - the AVH neurons are absent in P. pacificus due to an extra cell death. Despite a gross anatomic similarity, approximately 20% of neurons are structurally different between species. These differences (examples in parenthesis) include axonal extensions (ALM), axonal absences (AVM), somatic protrusions (ASJ), dendritic absences (URB), somatic dislocations (IL2), and local branching (RIS). Beyond structural differences at the neuron level, we explored whether fundamental aspects of neighborhood or strata formation, as described by Brittin et al 2021 and Moyle et al 2021, are conserved across species. The number of neighboring or adjacent processes is similar between species and is also nearly equivalent in its constancy. We find, however, that the set of individual neuronal adjacencies differ between species, leading to a more lateralized partitioning of left-right homologous neurons into distinct groups. Together, our results suggest the nerve rings of P. pacificus and C. elegans are structurally more different than what is seen across development or between the sexes. We continue to analyze our results in the context of synaptic connectivity, behavior, and molecular markers.

Koehler, Fabian et al. (2015) International Worm Meeting "The loss of mma-1 LRPPRC function induces the mitochondrial unfolded protein response."

Koehler, Fabian, Rolland, Stephane, Mueller-Rischart, Kathrin, Conradt, Barbara

[International Worm Meeting, 2015]

In a genetic screen for Caenorhabditis elegans mutants with abnormal mitochondrial morphology, we identified mma-1 (mitochondrial morphology abnormal-1), a C. elegans homolog of the French Canadian Leigh Syndrome gene LRPPRC (leucine-rich pentatricopeptide repeat containing). Inactivation of mma-1 in C. elegans or LRPPRC in mammalian cells results in a decrease in the production of mitochondria-encoded subunits of cytochrome c oxidase (COX) and consequently a reduction in COX activity. We have previously shown that in this context, mitochondria form a hyperfused network that transiently maintains cellular ATP levels (Rolland et al, 2013). We now demonstrate that the inactivation of mma-1 LRPPRC also leads to an imbalance between mitochondria- and nuclear-encoded COX subunits, which triggers a conserved mitochondrial unfolded protein response (UPRmt). We also show that the activation of UPRmt leads to the restoration of mitochondrial proteostasis. Finally, we present evidence that UPRmt and the mitochondrial hyperfusion response are coordinated but mediated by genetically distinct pathways. We propose that in the context of mma-1 LRPPRC knock-down, mitochondrial hyperfusion helps to transiently maintain cellular ATP levels enabling UPRmt to restore mitochondrial proteostasis.Rolland, S.G., Motori, E., Memar, N., Hench, J., Frank, S., Winklhofer, K.F. , and Conradt, B. (2013). Impaired complex IV activity in response to loss of LRPPRC function can be compensated by mitochondrial hyperfusion. PNAS 110, E2967-2976.Barbara Conradt and Stephane Rolland are co-corresponding authors.