Rahman, Maisha et al. (2021) International Worm Meeting "Specific N-glycans fine-tune somatosensory dendrite patterning"

Diaz-Balzac, Carlos, Rahman, Maisha, Bulow, Hannes

[International Worm Meeting, 2021]

Dendrites are essential for the transmission and processing of stimuli through the nervous system, and their development requires the precise orchestration of many proteins. For example, the C. elegans PVD somatosensory neurons require a conserved cell-adhesion 'menorin' complex - comprised of skin-derived MNR-1/Menorin and SAX-7/L1CAM, muscle secreted LECT-2/Chondromodulin II, and the transmembrane receptor, DMA-1/LRR-TM, in PVD - for their stereotyped arborization. We found that a key enzyme in the N-glycosylation pathway, AMAN-2/Golgi alpha-mannosidase II, plays a role in fine-tuning PVD dendrite patterning. aman-2/GM-II encodes an evolutionarily conserved enzyme required for the formation of complex and paucimannose N-glycans. Mutations in aman-2/GM-II result in dendritic trees with reduced complexity, and enhance the severity of dendrite defects in hypomorphic alleles of members of the menorin complex. AMAN-2/GM-II requires enzymatic activity in PVD to form higher order branches, suggesting that N-glycosylation of a menorin complex component in PVD itself may be significant. Consistent with this hypothesis, we find that DMA-1/LRR-TM is glycosylated in vivo with primarily high-mannose/hybrid N-glycans, and that DMA-1/LRR-TM carries larger, abnormal N-glycans in aman-2/GM-II mutants. Importantly, we determined that the presence of abnormal N-glycans, rather than the absence of wildtype N-glycans, is the root cause of the observed defects, and likely leads to altered protein-protein interactions. Lastly, we found that specific N-glycosylation sites in DMA-1/LRR-TM are important for PVD dendrite morphogenesis. Collectively, our findings suggest that specific N-glycan structures fine-tune dendrite patterning, possibly by regulating complex formation of the DMA-1/LRR receptor with other components of the menorin complex.

Carrick, Brian et al. (2017) International Worm Meeting "Genomic analyses of interactions that govern an RNA regulatory network in stem cells."

Guthrie, Timothy, Wickens, Marvin, Carrick, Brian, Kimble, Judith, Kroll-Conner, Peggy, Prasad, Aman, Porter, Douglas

[International Worm Meeting, 2017]

mRNA regulation governs when, where, and how much protein an mRNA produces and thereby determines cell identity, function and behavior. A single mRNA regulatory protein can bind to and control hundreds to thousands of mRNAs, forming a "network" that enables control of a biological outcome. This work focuses on C. elegans FBF-1 and FBF-2 (collectively termed FBF), which maintain germline stem cells (GSCs). FBF is a member of the PUF (Pumilio and FBF) family of RNA-binding proteins (RBPs), which bind mRNAs via sequence-specific elements in the 3' untranslated region (3'UTR) and regulate expression. We have now identified the RNAs bound by FBF in vivo in both oogenic and spermatogenic germlines using the technique of iCLIP (individual-nucleotide resolution CrossLinking and ImmunoPreciptation). The goal of this work is to learn FBF targets that function independently of gender, as well as gamete-specific targets and to thereby gain insight into FBF sub-networks. Like other PUF proteins, FBF acts with protein partners which can modulate binding specificity in vitro. The challenge now is to understand how these partners affect FBF regulatory networks in vivo. Currently, we are delineating protein - protein interfaces of FBF with its partners and generating CRISPR-induced mutants that abrogate those partnerships. Ultimately we will analyze how the FBF subnetworks fluctuate in response to changing FBF partnerships.

Stegeman, Gregory et al. (2011) International Worm Meeting "Variation in temperature-dependent behaviours among natural isolates of Caenorhabditis briggsae."

Shin, Jiwon, Ryu, William, Cutter, Asher, Bueno De Mesquita, Matthew, Stegeman, Gregory, Lin, Nan

[International Worm Meeting, 2011]

Natural genetic variation allows the discovery of new gene functions and novel alleles for genes already known to act in biologically important processes. We are applying this approach to temperature-dependent behaviours in nematode worms in order to better understand the genetics behind behaviour. We focus on Caenorhabditis briggsae because most wild caught individuals fall into two genetically distinct clades that correspond approximately with northern temperate or with tropical latitiudes. Interestingly, strains from the tropical clade have higher fecundity when reared at higher temperature than do the temperate strains, suggesting local adaptation to climate variables like temperature (Prasad et al. 2011). Movement through its thermal landscape is the main way for nematodes like C. briggsae to regulate body temperature, so we also expect to see heritable differences in temperature-dependent behaviours. Here we quantify for the first time classic thermal-response behaviours among several C. briggsae wild strains from different haplotype groups using assays like accumulation on a linear thermal gradient, isothermal tracking, and a new droplet based thermal gradient assay. We demonstrate that C. briggsae shows thermotaxis and isothermal tracking similar to C. elegans but with some differences. We also identify heritable differences among strains from wild genetic backgrounds within C. briggsae. We will continue to develop higher throughput assays for temperature-dependent behaviour in order to carry out a quantitative trait loci mapping project using recombinant inbred lines derived from tropical and temperate parental strains. Prasad, A., M. Croydon-Sugarman, R.L. Murray & A.D. Cutter. 2011. Temperature-dependent fecundity associates with latitude in Caenorhabditis briggsae. Evolution. 65: 52-63.

Prajapati, Gaurav et al. (2019) International Worm Meeting "Four new members of the sperm activation pathway."

Austin, Sarojani, LaMunyon, Craig, Aldaco, Fermina, Keung, Steven, Prajapati, Gaurav

[International Worm Meeting, 2019]

Becoming active is a critical event in the life of a sperm cell. Failure to activate at exactly the right time dooms a sperm to failure. Here, we describe four genes that add to the growing list of a set involved in determining sperm activation. These genes were identified from mutations recovered in a suppression screen of spe-27(it132ts) IV. Worms homozygous for the spe-27 mutation are normally sterile at 25C, but a mutation in the identified genes results in a small number of sperm that activate prematurely. Normally, sperm remain as inactive spermatids until an extracellular signal interacts with surface receptors on the spermatid. Two parallel activation pathways exist: (i) a TRY-5 signal in male seminal fluid interacting with a SWM-1 receptor, and (ii) an unknown signal in both seminal fluid and in the hermaphrodite reproductive tract interacting with the SPE-8 group receptor complex (SPE-8, -12, -19, -27, -29, and -43). Inside the cell, the signal to activate unleashes a plethora of responses, including zinc, calcium, and pH spikes, and modification of phosphorylation states. The four new genes were identified through one-step whole genome sequencing and SNP-mapping. One of the genes described here is F55F8.7, which was identified from mutation zq9 and is a predicted tyrosine phosphatase. F55F8.7 has 9 paralogs expressed primarily or exclusively in sperm, which is likely the reason that a knockout of the gene has no phenotype. This gene expands the list of kinases/phosphatases involved in sperm activation, including kinases spe-6 and spe-8, and phosphatases gsp-3 and gsp-4. Another gene, preliminarily identified from mutation zq13, is aman-3, a sperm upregulated gene encoding a predicted alpha-mannosidase. Involvement of aman-3 suggests that modification of protein glycosylation is also involved in sperm control of sperm activation. The mutation zq14 was identified as affecting Y73F8A.14, which has no predicted function and has only nematode orthologs. Finally, the hc202 mutation was determined to affect F29B9.7, another gene with no predicted function or orthologs outside nematodes. While the exact role of these gene products remains unclear, they add to the set of genes that have been functionally determined to be involved in sperm activation.

Chien, Shih-Chieh et al. (2013) International Worm Meeting "Autonomous and nonautonomous regulation of Wnt-mediated neuronal polarity by the C. elegans Ror kinase CAM-1."

Garriga, Gian, Chien, Shih-Chieh, Gurling, Mark

[International Worm Meeting, 2013]

Wnts are a conserved family of secreted glycoproteins that regulate various developmental processes in metazoans. Three of the five C. elegans Wnts (CWN-1, CWN-2 and EGL-20) and the sole Wnt receptor of the Ror kinase family (CAM-1) are known to regulate the anterior polarization of the mechanosensory neuron ALM.1,2,3 Here we show that CAM-1 and the Frizzled receptor MOM-5 act in parallel pathways to control ALM polarity. We also show that CAM-1 has two functions in this process: an autonomous signaling function that promotes anterior polarization and a nonautonomous Wnt-antagonistic function that inhibits anterior polarization. These antagonistic activities can account for the weak ALM phenotypes displayed by cam-1 mutants. Our observations suggest that CAM-1 could function as a Wnt receptor in many developmental processes, but the analysis of cam-1 mutants fails to reveal CAM-1's role as a receptor in these processes because of its Wnt-antagonistic activity. In this model, loss of CAM-1 results in increased levels of Wnts that act through other Wnt receptors, masking CAM-1's autonomous role as a Wnt receptor. 1: Hilliard & Bargmann (2006) Dev Cell 10(3):379-90 2: Prasad & Clark (2006) Development 133(9):1757-66 3: Babu et al. (2011) Neuron 71(1):103-16.

Kim, Grace S et al. (2009) International Worm Meeting "unc-3 is necessary for axon pioneering and guidance in C. elegans."

Hall, David H., White, John G., Xu, Meng, Kim, Grace S

[International Worm Meeting, 2009]

Genetic and phenotypic studies of unc-3 mutants have shown that unc-3 is required for proper organization of ventral cord processes (1,2). The defasciculation and wiring defects seen in unc-3 mutants have been attributed to disruption of pathfinding in the pioneer neurons during embryonic development. We have used Elegance, a 3D reconstruction software program developed at AECOM (3), to analyze the deformed morphology of the unc-3 (e151) ventral cord to unprecedented detail at the EM level. Both the e151/e151 and e151/Df animals display severe axon guidance errors that affect some pioneers (AVKs) and some motor neurons more strongly than the command interneurons. These results support the essential role of the unc-3 gene in axon pioneering and guidance. We present the pattern of severe axon guidance errors in terms of the mispositioning of specific axons, their deviance from their normal neighborhoods, and their synaptic wiring defects. This study, along with molecular genetic studies identifying the unc-3 gene product as a CeO/E transcription factor, should guide future studies of specific axon guidance cues at the molecular level and provide further insight into the role of the mammalian O/E family members. This study used annotated unc-3 TEM datasets received from John White (MRC/LMB and U. Wisconsin) that are now part of the MRC archive in the Hall lab. This work was supported by NIH RR12596 (to DHH) and an AECOM summer research fellowship (to GSK). 1.Wightman, B., Baran R. and Garriga, G. (1997) Development 124: 2571-2580. 2.Prasad, B.C. et al. (1998) Development 125: 1561-1568. 3.Emmons, S.W. et al. (2009) at this meeting.

Crittenden, Sarah et al. (2021) International Worm Meeting "FBF binding elements in the gld-1 3'UTR and their role in germline regulation"

Woodworth, Jennifer, Kimble, Judith, Kroll-Conner, Peggy, Wickens, Marv, Crittenden, Sarah

[International Worm Meeting, 2021]

PUF RNA binding proteins (for Pumilio and FBF) are key regulators of self-renewal in the C. elegans germline. Two nearly identical PUF proteins, FBF-1 and FBF-2 (collectively FBF), have long been known as self-renewal regulators of late larval and adult germline stem cells (GSCs) (1). Genomic studies using iCLIP identified a battery of FBF target RNAs as well as FBF binding elements (FBEs) within them (2). That knowledge of in vivo FBEs provides a powerful inroad for studying the direct effects of FBF binding on regulation of individual target RNAs and germ cell fate. A major target of FBF regulation is gld-1, which promotes meiotic progression and oogenesis (3). Strong evidence has accumulated for FBF repression of gld-1 in GSCs (1), but FBF had also been proposed to activate gld-1 to promote meiotic entry. (1,4). We have now begun to focus on FBEs in the gld-1 3'UTR to assess their molecular and biological roles. To this end, we used CRISPR/Cas9 gene editing to mutate the two canonical gld-1 FBEs, FBEa and FBEb, in the 3'UTR of endogenous gld-1. Phenotypically, FBEa and FBEb single mutants both maintain GSCs and are fertile; an FBEa FBEb double mutant was just recovered and is being characterized. Using GLD-1 immunostaining, we find that the FBEa single mutant makes higher than normal levels of GLD-1 protein in GSCs, while the FBEb single mutant makes wild-type levels in GSCs. We are currently examining gld-1 mRNA levels in these single mutants using smFISH, and soon will start analyzing the double mutant. 1 Crittenden et al.(2002).Nature, 417,660. 2 Porter, Prasad et al.(2019).G3,9,153. 3 Francis et al (1995) Genetics 139, 579 4 Suh et al.(2009).Genetics,181,1249.

Wang, X. et al. (2019) International Worm Meeting "The balance of activities of PUF family proteins FBF-1 and FBF-2 coordinately regulates germline stem cell proliferation and differentiation in C. elegans."

Voronina, E., Hickey, B., Wang, X., Ellenbecker, M., Day, N., Baumgarten, E.

[International Worm Meeting, 2019]

Studies of the molecular mechanisms regulating the balance of stem cell proliferation and differentiation can help us understand the causative factors for ageing, cancer and various degenerative disorders. Pumilio and FBF (PUF) family RNA-binding proteins are highly conserved regulators of stem cell development. Here, we report that FBF-1 and FBF-2, two PUF proteins in C. elegans, coordinately change the rate of both proliferation and differentiation of germline stem cells. FBF-1 and FBF-2 function redundantly in maintaining germline stem cells through translational repression of target mRNAs (Crittenden et al., 2002). FBF-1 and FBF-2 are very similar in primary sequence and share most of their target mRNAs (Prasad et al., 2016, Kershner et al., 2010 and Porter et al., 2018), but recently we found that they have distinct effects on proliferation and differentiation of stem cells. The analysis cell cycle parameters indicated that FBF-1 might slow down cell proliferation while FBF-2 accelerates cell proliferation of germline stem cells. Additionally, estimating the rate of meiotic entry suggested that FBF-1 slows down differentiation in concert with slowing down proliferation. Quantification of FBF target mRNA abundance by qPCR suggested that the presence of FBF-1 correlates with degradation of target mRNAs regulating proliferation and differentiation, while FBF-2 protects these targets from degradation. We propose that the balance of FBF-1 and FBF-2 activities might determine the steady-state abundance of target mRNAs and thus change the rate of both cell cycle and meiotic entry. Furthermore, we found that FBF-1 activity in stem cells requires CCR4-NOT deadenylase machinery that shortens poly(A) tail of mRNA. By contrast, FBF-2 activity in stem cells shows less dependence on CCR4-NOT, which correlated with FBF-2's interaction with its specific cofactor, DLC-1. In conclusion, our study suggested a new mechanism for control of stem cell proliferation in concert with differentiation by PUF proteins through differential cooperation with CCR4-NOT deadenylase machinery.

Gurling, Mark et al. (2009) International Worm Meeting "Frizzled receptor antagonism in neuronal polarity."

Garriga, Gian, Gurling, Mark, Pan, Chun-Liang

[International Worm Meeting, 2009]

Wnt glycoproteins signal through Frizzled receptors to regulate many aspects of neural development such as neuronal migration and polarity1,2,3,4. The two Frizzled receptors LIN-17 and MIG-1 appear to play antagonistic roles in certain developmental contexts3,5. In worms mutant for the Frizzled receptor LIN-17, the polarity of the PLM mechanosensory neuron is reversed. Mutations in lin-44, which encodes one of the Wnt proteins, caused similar PLM phenotypes. Synapses made by the anterior process in wild-type animals are now made by the posterior process of mutant PLMs1,2. A similar phenotype is seen when another Frizzled, MIG-1, is over expressed in the PLM suggesting an antagonistic relationship between lin-17 and mig-1. Consistent with this hypothesis, loss of MIG-1 suppresses the polarity reversal of lin-17 and lin-44 mutants. We propose two explanations for how excess MIG-1 may cause a reversal of PLM polarity. In model 1, increased MIG-1 signaling antagonizes LIN-17 signaling. In model 2, excess MIG-1 competes with LIN-17 for the Wnt ligand LIN-44, thereby reducing signaling through LIN-17, and possibly another Wnt receptor. Model 1 predicts that loss of the MIG-1 Wnt ligand should suppress the PLM polarity defect caused by excess MIG-1. Loss of cwn-1, cwn-2 or mom-2 does not ameliorate the polarity phenotype. This lack of a genetic interaction suggests that MIG-1 might bind LIN-44 to prevent it from interacting with LIN-17, inconsistent with a MIG-1 signaling role proposed in model 1. We are attempting to distinguish between these two models by determining which domains of MIG-1 are necessary and sufficient to generate the PLM polarity defect. 1. Prasad BC, Clark SG. (2006) Development 133: 1757-1766 2. Hilliard MA, Bargmann CI. (2006) Developmental Cell 10: 379-390 3. Pan C-L, et al. (2006) Developmental Cell 10: 367-377 4. Maloof JN, Whangbo J, et al. (1999) Development 126: 37-49 5. Forrester, W.C., Kim, C., Garriga, G. (2004) Genetics 168: 1951-1962.

Prasad Kasturi et al. (2006) European Worm Meeting "MOG-3 Links Sex Determination to GLP-1/Notch Signaling"

Alessandro Puoti, Prasad Kasturi

[European Worm Meeting, 2006]

Prasad Kasturi and Alessandro Puoti. The hermaphroditic nematode Caenorhabditis elegans sequentially produces both sperm and oocytes from a single pool of germ cell precursors. After the brief period of spermatogenesis during the fourth larval stage, the adult produces oocytes for the rest of its life. The events that regulate the fate of germ cells are at least in part controlled post-transcriptionally: While the entry into meiosis and its progression require the silencing of the glp-1 mRNA, the switch from spermatogenesis to oogenesis is dependent on post-transcriptional repression of the fem-3 mRNA.. To understand the molecular mechanisms that govern fem-3 repression, several trans-acting factors have been identified in genetic and biochemical screens. The mog genes have been shown to repress fem-3 mRNA through its 3 untranslated region. Four cloned mog genes code for nuclear proteins that are homologous to RNA processing factors. In addition these MOG proteins directly bind to the nuclear zinc finger protein MEP-1 that is also required for fem-3 repression. We have cloned the mog-3 by SNP mapping and candidate gene approach. mog-3 encodes a conserved nuclear protein that shares weak homology to a spliceosomal complex protein in fission yeast. A mog-3::gfp transgene is expressed in the nuclei of many somatic cells. MOG-3 binds to MEP-1 in a similar fashion as the other MOG proteins. Similar to other mog genes mog-3 also play a role in the mitosis to meiosis switch.. To investigate the molecular function of MOG-3, a yeast two-hybrid system was used to screen the interacting factors. One of the interacting protein (CIR) is a co-repressor of LAG-1. CIR also interacts with MEP-1. Since MEP-1 is a part of the NuRD complex, we speculate that CIR and MOG-3 with MEP-1 may form a complex to regulate target genes in the GLP-1/Notch pathway.. Keywords & Abbreviations: Germline, Sex determination, Post-transcriptional regulation, mog (masculanization of germ line), fem (feminization), mep (mog interacting and ectopic P granules), gld (germ line defective), SNP (Single Nucleotide Polymorphism) and NuRD (nucleosome remodeling and deacetylation), glp (germline proliferative) and Notch signaling.