Tang, Ngang Heok et al. (2017) International Worm Meeting "A conserved RNA binding protein regulates axon regeneration and integrity."

Chisholm, Andrew, Tang, Ngang Heok

[International Worm Meeting, 2017]

Axonal regeneration after injury involves a suite of signaling pathways and cytoskeletal regulators such as DLK-1 and EFA-6. Intriguingly, DLK-1 has also been implicated in maintenance of axon integrity in the mammalian nervous system (1). Conceptually, maintenance of axonal integrity is crucial to prevent neurodegeneration whereas axon regeneration provides potential therapeutic routes to recovery from neurodegeneration. From protein interaction screens with known regeneration regulators we have identified a conserved RNA binding protein that regulates axon regeneration. Loss of function in this protein results in increased regeneration, if injury occurs prior to adulthood. Longitudinal analysis of axon regeneration suggests this protein controls the axon extension phase of regrowth. Intriguingly, upon entry into adulthood, we observe progressive axonal breakage in mechanosensory and GABAergic motor neurons in this mutant. Importantly, the axonal breakage observed in this mutant can be reversed by loss of function in an axonal transport protein. Our data suggest this protein acts as a double-edged sword in controlling axon regeneration and integrity. We will describe our efforts to dissect the biochemical and functional roles of this pathway in axon regeneration and integrity. 1. A. S. Ghosh et al., DLK induces developmental neuronal degeneration via selective regulation of proapoptotic JNK activity. The Journal of cell biology 194, 751-764 (2011).

Tang, Ngang Heok et al. (2019) International Worm Meeting "Regulation of axon regeneration by the mRNA decay factors."

Tang, Ngang Heok, Kim, Kyung Won, Yeo, Gene, Blazie, Stephen, Chisholm, Andrew, Jin, Yishi, Xu, Suhong, Yee, Brian

[International Worm Meeting, 2019]

mRNA decay factors regulate mRNA turnover by recruiting non-translating mRNAs and targeting them for translational repression, decapping and degradation, yet it remains poorly understood how mRNA decay factors function in vivo to regulate specific cellular processes. We found that mRNA decay factors form cytoplasmic puncta in C. elegans neurons and have opposing roles in axon regrowth. While decapping enzymes such as DCAP-1/2 promote axon regrowth after injury, translational repressors such as CAR-1 and CGH-1 inhibit axon regrowth in adult animals. We find that enhanced axon regeneration in a translational repressor mutant is dependent on mitochondrial functions. We show that axon injury triggers a transient mitochondrial calcium influx, which is more sustained in animals lacking translational repressors. We use genome-wide seCLIP to identify mRNA targets of translational repression in neurons, among which a regulator of mitochondrial calcium influx appears to be a direct target. Loss of translational repression leads to elevated mRNA levels for the mitochondrial calcium regulator. Moreover, the enhanced axon regrowth and sustained axonal calcium elevation observed in translational repressor mutants is dependent on mitochondrial calcium influx regulators. Our results reveal specific roles for mRNA decay regulation in neurons and a novel pathway that controls axon regeneration through mitochondrial calcium uptake.

Lyu, Xiaohui et al. (2021) International Worm Meeting "Identification of factors regulating the localization of a microtubule regulator EFA-6"

Chisholm, Andrew, Jin, Yishi, Meng, Xuefeng, Tang, Ngang Heok, Wan, Xinghaoyun, Lyu, Xiaohui, Xie, Jing

[International Worm Meeting, 2021]

The EFA6 (Exchange Factor for Arf6) family is conserved from yeast to human, originally named after the Sec7 homology domain that has GEF activity specific to ARF6 GTPases. Functional studies have shown that EFA6 family proteins can also regulate neuronal microtubule (MT) dynamics, involving a conserved 18 aa motif embedded in an intrinsic disordered region in the EFA6 N-terminus (O'rourke et al., Nat. Cell Biol., 2010). EFA6 proteins additionally contain a pleckstrin homology (PH) domain and a coiled-coil domain that enable their association with plasma membrane and actin cytoskeleton. In C. elegans, both expression level and localization of EFA-6 are critical for its function in neurons (Chen et al., Neuron, 2011; Chen et al., Elife, 2015). However, endogenous expression of EFA-6 remains unknown. To dissect mechanisms regulating EFA-6, we first generated a GFP knock-in to tag endogenous EFA-6. Wild-type GFP::EFA-6 is found in multiple tissues, including neurons, pharynx, epidermal cells and germline. We then carried out a visual genetic screen and isolated several classes of mutants that affect GFP::EFA-6 localization in various tissues. Among these we focused on the ones showing EFA-6 mis-localization in touch receptor neurons (TRNs) or adjacent epidermal cells. Combining whole-genome sequencing and genetic mapping, we identified several components of the specialized extracellular matrix (ECM) for TRNs, known as the mantle, which has been well studied for its role in touch sensitivity. The mantle consists of MEC-1, MEC-5, MEC-9, and HIM-4 (Emtage et al., Neuron, 2004). MEC-1 is essential for accumulation of the collagen MEC-5 and other ECM components. MEC-5 and MEC-9 do not affect the general structure of mantle and attachment of the neurons to the epithelia by themselves. The hemicentin HIM-4 functions separately from the MEC ECM proteins, and is needed for TRN attachment but not touch sensitivity. Our results suggest that mec-1, mec-5, and him-4 have similar effects on EFA-6 localization and may act in a common pathway. We also find that disruption of the TRN specific tubulins MEC-7 and MEC-12 affect EFA-6 localization. We are currently investigating how ECM interacts with MTs to regulate EFA-6 localization and function in neurons and other tissues.

Sarinay Cenik, Elif et al. (2019) International Worm Meeting "Maternal Ribosomes Are Sufficient for Tissue Diversification during Embryonic Development in C. elegans."

Hall, Richard Nelson, Tang, Ngang Heok, Cenik, Can, Jin, Yishi, Arribere, Joshua A., Fire, Andrew, Meng, Xuefeng, Sarinay Cenik, Elif

[International Worm Meeting, 2019]

Caenorhabditis elegans provides an amenable sys- tem to explore whether newly composed ribosomes are required to progress through development. Despite the complex pattern of tissues that are formed during embryonic development, we found that null homozygotes lacking any of the five different ribosomal proteins (RPs) can produce fully functional first-stage larvae, with similar developmental competence seen upon complete deletion of the multi-copy ribosomal RNA locus. These animals, relying on maternal but not zygotic contribution of ribosomal components, are capable of completing embryogen- esis. In the absence of new ribosomal components, the resulting animals are arrested before progression from the first larval stage and fail in two assays for postembryonic plasticity of neuronal structure. Mosaic analyses of larvae that are a mixture of ribosome-competent and non-competent cells suggest a global regulatory mechanism in which ribosomal insufficiency in a subset of cells triggers organism- wide growth arrest.

Svensk, Emma et al. (2013) International Worm Meeting "PAQR-2 Regulates Fatty Acid Desaturation During Cold Adaptation in C. elegans."

Pilon, Marc, Andersson, Carl-Henrik, Stahlman, Marcus, Johansson, Maja, Svensk, Emma, Boren, Jan

[International Worm Meeting, 2013]

Adiponectin is an adipokine with insulin-sensitizing and antiatherogenic actions, which also influences energy homeostasis via the hypothalamus (Kadowaki et al. 2008). The human adiponectin receptors AdipoR1 and AdipoR2 are PAQR proteins: 7-transmembrane domain proteins with topologies reversed that of GPCRs, i.e. their C-terminus is extracellular (Tang et al. 2005). We have previously shown that a loss of function mutant of the C. elegans adiponectin receptor homolog paqr-2 is unable to adapt to growth at 15 deg C (Svensson et al. 2011). We have now leveraged the paqr-2 cold adaptation defect in a suppressor screen with the aim of identifying downstream receptor targets. The screen produced nine suppressor mutations that have all been identified through whole genome sequencing and experimental verification. The nine mutants fall into two categories: 1) Mutations that decrease synthesis of phosphatidylcholine and 2) Mutations that influence fatty acid metabolism. The two categories are tied together by phosphatidylcholine being a regulator of the fatty acid metabolism transcription factor sbp-1, a homolog of the mammalian SREBP (Walker et al. 2011). Consequently we have been able to connect all nine paqr-2 suppressor mutants into one pathway, the paqr-2 target pathway. Also, fat-6 or -7 RNAi can completely abolish the suppression in mutants from both categories pointing towards an increase in fatty acid desaturation as the common suppressive mechanism. We conclude that paqr-2 regulates cold adaptation and that this process is highly dependent on the D9 desaturases. However, despite having identified the paqr-2 target pathway, the direct action point of this receptor remains unknown. References: Kadowaki T. et al. (2008) FEBS Lett 582:74-80; Svensson E. et al. (2011) PLoS One 6:e21343; Tang Y. et al. (2005) J Mol Evol 61:372-380; Walker A. et al. (2011) Cell 147:840-852.

Kislyak Y et al. (2000) East Coast Worm Meeting "Biochemical Characterization of C. elegans RNA Helicase A and Malate Dehydrogenase"

Kislyak Y, Walstrom KM, Do IP

[East Coast Worm Meeting, 2000]

In my laboratory, we are overexpressing and biochemically characterizing two different proteins from C. elegans. RNA helicase A (RHA-1) is essential for proper development in many organisms, including C. elegans. Human RHA is involved in RNA metabolism. For example, it is capable of mediating an association between CREB binding protein (CBP) and RNA polymerase II and is suggested to be required for the activation of transcription by CBP (Nakajima et al. (1997) Cell 90, 1107-1112). Human RHA is also required for RNA export from the nucleus (Tang et al. (1999) MCB 19, 3540-3550). We are attempting to determine the RNA binding targets of RHA-1 in the cell in order to elucidate its function in C. elegans. In another project, malate dehydrogenase (MDH), the last enzyme in the citric acid cycle, has been overexpressed and purified. This enzyme is active, and the enzyme kinetics of C. elegans MDH are being studied. Recent results from both projects will be presented. We are grateful to Yuji Kohara for providing the cDNA clones of these enzymes.

Katherine M Walstrom et al. (2002) Mid-west Worm Meeting "Yet Another Germline Helicase"

Byram Ozer, Jens-Christian Paul, Katherine M Walstrom

[Mid-west Worm Meeting, 2002]

RNA helicase A (RHA) is conserved in many organisms and has numerous cellular functions. Human RHA is required for RNA export from the nucleus (Tang et al. (1999) MCB 19, 3540) and the Drosophila homolog, MLE, is required for dosage compensation in male flies (Kuroda et al. (1991) Cell 66, 935). We are characterizing tm329, a strain containing a deletion in rha-1, the C. elegans homolog of RHA. These worms develop normally between 17-20C, but above 23C hermaphrodites accumulate oocytes in the uterus or have atrophied gonads (small, few germ cells). In males, sperm development occurs at lower temperatures but appears to be defective at 25C. Double-mutant worms, gld-1 (q266); rha-1 (tm329), were constructed since human RHA and the human homologue of GLD-1 (Sam68) interact (Reddy et al. (2000) Oncogene 19, 3570), but no epistasis or enhancement was observed in hermaphrodites between 17-25 C. We are continuing experiments to identify the relationship between RHA-1 and other known germline and spermatogenesis proteins. We are grateful to the C. elegans Gene Knockout Consortium for the tm329 strain and to T. Schedl for the rha-1 (q266) strain and for helpful advice.

Henriksson, Johan et al. (2011) International Worm Meeting "A new model of C. elegans embryogenesis with cell contacts and spatio-temporal gene expressions."

Henriksson, Johan, Luppert, Martin, Abou-Zied, Akram, Hench, Jurgen, Burglin, Thomas R., Baillie, David

[International Worm Meeting, 2011]

The events that specify cell fates during embryogenesis are highly dynamic. To understand them, one would ideally like to obtain precise expression levels for each developmental control gene in each type of cell. We have developed a multi-channel spatio-temporal (4D) microscopy framework (poster: J.Henriksson "Endrov - an open source framework for image processing and analysis", www.endrov.net) to monitor live GFP expression throughout embryogenesis. We have applied this to homeobox gene transcription factors and have so far recorded 60 homeobox gene expression patterns in 200 recordings using DIC and DIC/mCherry::His timelapse microscopy. One of these, ceh-5, we selected for in depth analysis (abstract: Lois Tang et al.). Analysis of expression patterns shows that they are very reproducible. We have mapped the expression patterns onto a model of normal C. elegans development previously made in our lab (Hench et al. PMID:19527702), and have compared patterns in terms of similarity, and shown when and where genes are turned on. We have developed this technique both on a simplified view which is the most reproducible, and on an approximate single-cell level. Our model allows quantitative comparison of many expression patterns simultaneously, and may elucidate how transcription factors regulate development and cell differentiation.

Henriksson, Johan et al. (2010) C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany "Spatio-temporal gene expression studies using the Endrov framework with VW-Base plug-ins"

Henriksson, Johan, Luppert, Martin, Baillie, David, Burglin, Thomas, Hench, Jurgen, Abou-Zied, Akram

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

About 150 different cell types are generated in C. elegans during ontogeny. To understand development, one would ideally like to obtain precise expression levels for each developmental control gene in each type of cell. We have developed a multi-channel spatio-temporal (4D) microscopy framework, called Endrov (www.endrov.net). Within Endrov, we have built a series of plug-in modules to implement our ideas for VirtualWorm-Base (VW-Base). This framework has now been expanded to also allow acquisition of images from standardized microscopes. As previously reported, we have been monitoring live GFP expression throughout embryogenesis. We have applied this to homeobox gene transcription factors and other developmental control genes, having now over 250 recordings using DIC and reporter-GFPs or DIC/mCherry::His and reporter GFPs. For a specific application with the homeobox gene ceh-5 , see abstract by Lois Tang. We have previously generated a new model for normal, uncompressed C. elegans embryos. This new 4D model is suitable to map the spatio-temporal gene expression patterns onto. We have lineaged some expression patterns but found that much analysis can be done without the complete lineage. We have found that agarose-mounted embryos are not suitable representatives for 4D models due to compression artifacts and random rotational movements. However, recordings can easily be normalized if the embryo is not compressed and hence not rotating. Hence, it is possible to segment the embryo into voxel regions and derive a numerical map of expression intensities from this for each recording. In turn, pairwise comparisons of the recordings can be clustered using clustering algorthims and a tree can be obtained that represents how similar expression patterns are. We have assessed our method to be reproducible using the same method, ensuring that recordings of the same strain end up in the same cluster.

Henriksson, Johan et al. (2011) International Worm Meeting "Endrov - an open source framework for image processing and analysis."

Burglin, Thomas R, Hench, Jurgen, Henriksson, Johan

[International Worm Meeting, 2011]

During our work on Virtual-Worm Base (a database of spatio-temporal gene expression patterns during C. elegans development), we found that the current tool chain for microscopy is insufficient. Commercial software did not provide the necessary flexibility and could not adapted for our purposes. We also evaluated free solutions. But we found that, for example, ImageJ, the de facto-standard image software, is not designed for demanding 4D microscopy needs. Adding the necessary code would be difficult, and still not result in the desired end product.To solve these problems we have developed a new platform - Endrov (www.endrov.net) - that covers the entire microscopy chain. It is a plug-in framework consisting of 160 000 lines of Java, running on all operating systems. The following are Endrov's features <ul> It has advanced control of the microscope hardware (via Micro-manager) It can view, annotate, process and analyze recordings. New functional modules can be implemented either with a graphical programming language (flows) if they are simple, with interpreted Java, or with new plugins. Endrov can also be used as a library, allowing therefore batch processing of large data sets. Presently, there are about 130 image processing filters and 80 plugins available in Endrov. An important aspect is image data handling; a new scheme for data storage was developed which handles arbitrary metadata, 6D recordings, mixed resolutions and compression (both lossy and lossless). This allows handling of large data sets, and - given todays hardware limitations such as hard-drive data transfer speeds - the speed of most operations is unaffected by the size of datasets. Endrov supports most file formats (via Bio-formats). </ul> Our framework is open and free of charge. It is highly flexible and can be adopted to any needs in current research, in fact it is not limited to C. elegans. As an application example, we have used our framework to quantify the expression levels of a large number of genes (see abstract: J. Henriksson et al,) and to study one of them in particular detail (see abstract: Lois Tang).