Takao Inoue et al. (2002) West Coast Worm Meeting "lin-17, lin-18 and patterning of the P7.p lineage"

Rashmi Deshpande, Paul W Sternberg, Russell Hill, Takao Inoue

[West Coast Worm Meeting, 2002]

We are interested in patterning of the 2 (secondary) vulval lineages P5.p and P7.p. In the wild-type, P5.p and P7.p produce stereotyped ABCD and DCBA patterns respectively. In lin-17 and lin-18 mutants, the polarity of the P7.p lineage becomes altered. We examined the P7.p lineage in lin-17 (encoding Frizzled Wnt receptor), lin-18 (encoding RYK receptor tyrosine kinase-related protein; W. Katz and P.W.S.), and double mutant using POP-1 (TCF/LEF) antibody staining and cell fate markers ceh-2::yfp and cdh-3::cfp.

Rand JB et al. (2000) East Coast Worm Meeting "UNC-13 in the C. elegans Nervous System"

Kohn RE, Rand JB, McManus JR, Moulder GL, Duke A, Barstead RJ, Duerr JS

[East Coast Worm Meeting, 2000]

C. elegans unc-13 and its homologues in vertebrates and Drosophila are involved in neurotransmitter release. UNC-13 has several regions homologous to PKC regulatory domains; these domains confer it with calcium, phorbol ester and phospholipid binding properties (Maruyama and Brenner, PNAS 88, 1991). A 5.9kb transcript coding for a 200kDa protein was initially identified (now designated L-R for left and right regions). We have identified two additional types of transcripts. One transcript includes a 1kb novel exon (L-M-R, M for middle region) and another transcript lacks the 5' region included in the other two transcripts (M-R). All three transcripts are identical at the 3' end (R). C. elegans with mutations in the 5' end of the gene (L) alter two types of transcripts (L-R and L-M-R) resulting in an uncoordinated coily phenotype and resistance to the anti-cholinesterease, aldicarb. A 2.7kb deletion near the 3' end (R) (identified by Bob Barstead using PCR analysis) affects all unc-13 transcripts and results in a lethal phenotype. Antibodies recognizing the N-terminal region of UNC-13 (L) label synapses, but not synaptic vesicles, of most or all neurons; many mutations in L and R remove staining with this antibody. Supported by grants from the NIH and OCAST.

Abergel, Z. et al. (2017) International Worm Meeting "Adaptation of the nematode C. elegans to hypoxia and reoxygenation stress reveals an unexpected function of the neuroglobin GLB-5 in innate immunity."

Zuckerman, B., Zelmanovich, V., Abergel, Z., Abergel, R., Gross, E., Smith, Y., Romero, L., Livshits, L.

[International Worm Meeting, 2017]

Deprivation of oxygen (hypoxia) followed by reoxygenation (H/R stress) is a major component in several pathological conditions such as vascular inflammation, myocardial ischemia, and stroke. However how animals adapt and recover from H/R stress remains an open question. Previous studies showed that the neuroglobin GLB-5(Haw) is essential for the fast recovery of the nematode Caenorhabditis elegans (C. elegans) from H/R stress. Here, we characterize the changes in neuronal gene expression during the adaptation of worms to hypoxia and recovery from H/R stress. Our analysis shows that innate immunity genes are differentially expressed during both adaptation to hypoxia and recovery from reoxygenation stress. Moreover, we reveal that the prolyl hydroxylase EGL-9, a known regulator of both adaptation to hypoxia and the innate immune response, inhibits the fast recovery from H/R stress through its activity in the O2-sensing neurons AQR, PQR, and URX. Finally, we show that GLB-5(Haw) acts in AQR, PQR, and URX to increase the tolerance of worms to bacterial pathogenesis. Together, our studies suggest that innate immunity and recovery from H/R stress are regulated by overlapping signaling pathways.

A V Bicknell et al. (2002) European Worm Meeting "The C. elegans F47F2.1 gene encodes a cyclic AMP-dependent protein kinase (PK-A) catalytic subunit"

A V Bicknell, M J Fisher, M Tabish, R A Clegg, H H Rees

[European Worm Meeting, 2002]

PK-A mediates all known effects of cyclic AMP on cellular activity in eukaryotes. The holoenzyme is an inactive tetramer of two regulatory (R) subunits and two catalytic (C) subunits. Following binding of cyclic AMP to the R subunits, dissociation of active C-subunits occurs. In mammals, , and isoforms of C-subunit, encoded by different genes, have been identified.

Angelo RG et al. (1997) International C. elegans Meeting "DISCOVERY OF A POTENTIAL ANCHOR/SCAFFOLD PROTEIN FOR C. ELEGANS PROTEIN KINASE A (PKA)"

Angelo RG, Rubin CS

[International C. elegans Meeting, 1997]

C. elegans PKA is composed exclusively of catalytic and regulatory (R) subunits encoded by the kin-1 and kin-2 genes. Since C. elegans lacks other PKA isoforms, this enzyme must disseminate signals carried by cAMP to all cell compartments. Approximately 60% of total R (PKA) is in the particulate fraction of disrupted C. elegans, indicating that protein/protein interactions may diversify PKA signaling by anchoring the kinase at specific intracellular locations. Co-localization of PKA with upstream activators and/or downstream effectors can create target sites for cAMP action. To understand the mechanism of PKA localization in C. elegans, a cDNA expression library was screened with recombinant radiolabeled-R (0.5 nM) to obtain high-affinity binding proteins. A cDNA encoding a novel, 143 kDa A kinase anchor protein (AKAP1) was retrieved and sequenced. The corresponding gene (rap-1) is located in LGII and contains 17 exons. AKAP1 is an acidic protein (pI=4.4) that is unrelated to previously characterized proteins. C. elegans R is avidly bound by both soluble and immobilized fragments of AKAP1. Competition binding studies indicate that C. elegans R is a preferred ligand, whereas mammalian RII and RI isoforms are only weakly sequestered. Scatchard analysis yielded a Kd value of ~10 nM for the R/AKAP1 complex. Deletion mutagenesis, coupled with protein expression in E. coli and in vitro assays, demonstrated that residues 235 to 255 govern high-affinity binding of R by AKAP1. A hydrophobic surface generated by amino acids with branched aliphatic side chains may be a key determinant of R binding activity. Site directed mutagenesis will pinpoint essential residues involved in PKA binding. Studies aimed at identifying the AKAP1 binding site on R are also in progress. High-affinity anti-AKAP1 IgGs are being used to determine (a) whether AKAP1 expression is developmentally-regulated and cell- specific and (b) the relationship between R (PKA) and AKAP1 in vivo.

Katz, David et al. (2021) International Worm Meeting "The Pipeline CURE: lowering institutional barriers to research by reiteratively incorporating original C. elegans experiments throughout a biology curriculum"

Lee, Teresa, Schmeichel, Karen, Katz, David, Carpenter, Brandon

[International Worm Meeting, 2021]

Participation in research provides personal and professional benefits for undergraduates. However, some students face institutional barriers that prevent their entry into research, particularly those from underrepresented groups who may stand to gain the most from research experiences. Course-based undergraduate research experiences (CUREs) effectively scale research availability, but many only last for a single semester, which is rarely enough time for a novice to develop proficiency. To address these challenges, we established a Pipeline CURE. The Pipeline CURE integrates C. elegans epigenetics research being conducted by the Katz Lab at Emory University throughout the biology curriculum at Oglethorpe University, a nearby liberal arts college. Students are introduced to C. elegans with their first course in the major. After revisiting the research system in several subsequent courses, students can choose to participate in an upper-level research experience, led by an NIH Institutional Research and Academic Career Development Award (IRACDA) postdoctoral fellow from the Katz Lab. Preliminary data taken during implementation of The Pipeline CURE suggests that the reiterative CURE strategy can recapitulate benefits of a traditional apprenticeship lab experience entirely within the curriculum in a classroom setting. These benefits associated with The Pipeline Cure include including replacing beginner confidence with mastery and resilience via repeated exposure to the same research system. As a result, the Pipeline CURE levels the playing field by giving underrepresented students the chance to experience original research, and potentially conduct honors thesis research, without having to seek out a traditional apprenticeship lab experience. The research experience gained by students can serve as an entry to STEM careers, or provide a platform for competing for admission to graduate programs. In addition, the data generated can open new areas of research and help facilitate publication of manuscripts. As an added benefit, The Pipeline CURE also exposes postdoctoral fellows to challenges that underrepresented groups face in the classroom. Thus, by uniting evidence-based teaching methods with ongoing scientific research, the Pipeline CURE provides a new model for overcoming barriers to participation in undergraduate research, that is flexible enough to be implemented at a range of institutions using a variety of research questions.

Downs WD et al. (1997) International C. elegans Meeting "THE GENE MOM-1 IMPLICATES A WNT/WINGLESS PATHWAY IN ESTABLISHING CELL POLARITY AND ASYMMETRIC LINEAGES IN C. ELEGANS"

Cha Y-H, Mello CC, Priess JR, Downs WD

[International C. elegans Meeting, 1997]

During the development of C. elegans, the majority of cell divisions are asymmetric, resulting in daughter cells that will assume different fates. Our work indicates that the gene mom-1 (MOre Mesoderm) is necessary to induce polarity in a cell that will lead to an asymmetry in its lineage. Two of these mom-1 dependent asymmetries rely upon cell signaling in C. elegans: 1) induction of the intestine in the 4-cell stage embryo (Goldstein, 1992) and 2) patterning of the vulva during postembryonic development (Katz & Sternberg, 1990). Cell signaling events involving glp-1/lin-12 (Notch-related), and lin-3 (EGF) mediated pathways remain operational in mom-1 mutants. However, the vulva defects observed in mom-1 mutants are also observed in loss of function alleles of lin-17, a gene previously implicated in wnt signaling in C. elegans and homologous to the putative Wingless receptor of Drosophila (Sawa & Horvitz, 1996). We have cloned the mom-1 gene and found that it is homologous to porcupine, a gene of the Drosophila wingless pathway. Based on these findings we now believe that mom-1 is acting through a wnt/wingless cell-signaling pathway.

Hicks, Tara et al. (2021) International Worm Meeting "R-loop-induced irreparable DNA damage in C. elegans meiosis"

Hicks, Tara, Koury, Emily, Williams, Cameron, Smolikove, Sarit

[International Worm Meeting, 2021]

Most DNA-RNA hybrids are formed naturally during transcription and are composed of a nascent RNA strand hybridized to DNA as part of R-loops. The accumulation of these structures in S-phase can result in replication-transcription conflict, an outcome which can lead to the formation of double strand breaks (DSBs). While R-loops' role in mitotically dividing cells has been characterized, there are only a handful of studies describing the effect of R-loops in meiosis and these studies present a complex picture of the outcome of R-loop formation on germ cells. Here we show that DSBs formed by R-loops trigger an altered cellular response to DNA damage. RNase H is an enzyme responsible for degradation of the RNA strand in DNA-RNA hybrids and plays an essential role in preventing this outcome and its deleterious consequences. Using null mutants for the two Caenorhabditis elegans genes encoding for RNase H1 and RNase H2 (hereby rnh mutants), our studies explore the effects of replication stress-induced DNA-RNA hybrid accumulation on meiosis. As expected, rnh mutants exhibit an increase in R-loop formation. Consequently, an elevation of DSBs in germline nuclei is evidenced by the accumulation of RAD-51 foci. Despite no repair mechanism abrogation, rnh mutants fail to repair all DSBs generated, leading to a fragmentation of chromosomes in diakinesis oocytes. By combining our double mutant with a spo-11 null mutation, we show that although replicative defects are the main contributor to the phenotype, R-loops formed in meiosis are likely contributors as well. We present evidence that while rnh mutants accumulate DNA-RNA hybrids and subsequent DSBs may signal a degree of checkpoint activation in mitosis, some damaged nuclei prevail past the checkpoint, enter into meiosis, and remain unrepaired throughout. Moreover, we find no evidence of an increase in apoptosis, which indicates that DNA damage generated by R-loops remain undetected by an apoptotic checkpoint. This data altogether points to DSBs initiated by R-loops representing an irreparable type of DNA damage that evades cellular machineries designed for damage recognition.

Grant KA et al. (1998) West Coast Worm Meeting "ROLE OF SEM-4 IN VULVAL FATE SPECIFICATION"

Grant KA, Han M

[West Coast Worm Meeting, 1998]

We conducted a screen to discover genes downstream of ras signaling involved in formation of the vulva. We have identified several alleles with abnormal vulval lineages, including 3 alleles that map to a small region in the cluster of LG-I that was not previously shown to contain genes affecting the vulval lineage. In wild type worms P5.p, P6.p, and P7.p divide three times in a specific manner (2?, 1?, 2? fates, respectively) creating the 22 cells that will form the vulva. In contrast, approximately 80% of worms with the mutation exhibit an abnormal lineage in P7.p. The mutant P7.p phenotypes include: not induced (3? fate), hybrid fate, and normal 2? pattern of division where the cells do not integrate with the P5.p and P6.p descendants. P5.p has a similar but much less penetrant defect, manifested in only 10% of the mutant worms. Additionally, the planes of division of P6.p are altered in the mutant. We determined that our mutants were allelic to sem-4. sem-4 has been recognized to have defects in sex myoblasts, coelomyctes and several neurons, as well as precocious divisions in the Pn.ps (Basson & Horvitz, 1996); however, an under-induced vulval phenotype had not been described. Our alleles; ku200, ku206, and ku210; are most similar to n1378, in that they display defects in the vulva and sex myoblasts, but the defecation machinery remains functional (Basson & Horvitz, 1996). The lesions in ku200 and ku206 are both premature stop codons, presumeably deleting the last 4 zinc fingers. In an effort to determine SEM-4s role in the vulval formation pathway, we have analyzed its interactions with genes in the ras pathway. In the case of the double with let-60ras(n1046gf) neither gene could fully suppress the other. Additionally, sem-4(ku200) enhanced the weak loss of function allele of lin-45raf(ku112). In ku200; ku112 we saw a marked decrease in induction of P5.p and a slight decrease for P7.p. Together, these results indicate that SEM-4 does not function directly in the ras pathway and that other factors may be affecting SEM-4s activity in P5.p. We have also made a double mutant with lin-39(n709ts). LIN-39 has recently been shown to function downstream of RAS to promote induction of the vulva precursor cells (Clandinin, Katz, Sternberg, 1997; Maloof & Kenyon, 1998), as well as playing an earlier role in L1. The doubles show a synergistic interaction, whereby induction of both P5.p and P7.p is more severely reduced than in the presence of either allele alone. We are continuing to investigate the possible interaction between these genes. Basson, M. & Horvitz, R. Genes and Development. 10:1953-1965. 1996. Clandinin, T., Katz, W., & Sternberg, P. Developmental Biology. 182:150-161. 1997. Maloof, J. & Kenyon, C. Development. 125:181-190. 1998.

Birsoy B et al. (2010) Biology of the C. elegans Male, Madison, WI "Novel Left-Right Asymmetries of Male C.elegans"

Rothman J H, Choi S, Birsoy B, Downes J C, Bloss T A

[Biology of the C. elegans Male, Madison, WI, 2010]

While the mechanism that breaks left-right (L-R) anatomical asymmetry has been described in C. elegans, we have found that at least one additional mechanism must exist to generate L-R differences in the deployment of alternative cell death pathways in the male tail, male mating behavior and patterning of the male gonad. Upon recognizing a hermaphrodite, males initiate backward locomotion and continuously scan along the hermaphrodite's body. When their tails reach either the head or tail of the hermaphrodite, males turn to maintain contact and then continue backward locomotion on the opposite side of the hermaphrodite. We found that this turning behavior shows a distinct right-handed bias: when individual males were allowed to mate with lin-2(e1309) vulvaless hermaphrodites, >70% of the turns when made over the hermaphrodites' body (away from the agar surface) and >55% of turns when made under the hermaphrodites' body (toward the agar surface) are right-handed. To determine whether this bias in turning direction correlated with L-R asymmetry in the male mating structure, which results from stochastic EGL-1-dependent loss of sensory rays, we examined the turning bias in egl-1(n1084n3082) mutants. Wild-type males lack rays more frequently on the right and egl-1 mutant males have no ray loss but still display the right-hand turning bias. To assess whether this L-R behavioral bias is dependent on anatomical asymmetry, we analyzed gpa-16(it143) mutants in which the L-R asymmetry of the internal organs is reversed as a result of the reversal in the early embryonic symmetry break. Males with reversed anatomical asymmetry showed a virtually identical right hand bias in turning, demonstrating that an asymmetry-determining system that is independent of the previously described L-R symmetry breaking event must control this behavior. During the characterization of the gonad reversals of males, we also observed a previously unreported temperature sensitive reversal of L-R asymmetry of the male gonads in N2 (Bristol) and a Hawaiian wild isolate. We will describe our recent findings on these novel L-R asymmetries of the C.elegans male anatomy and behavior.