Attner*, M. et al. (2019) International Worm Meeting "lin-12/Notch-mediated lateral specification: new insights from the AC/VU decision in gonadogenesis."

Benavidez, J., Greenwald, I., Attner*, M., Keil*, W.

[International Worm Meeting, 2019]

Notch-mediated lateral specification is utilized during the development of all animals to generate different cell fates from initially equivalent cells. The anchor cell (AC)/ventral uterine precursor (VU) cell fate decision is a simple lateral specification paradigm in which two initially equivalent cells, Z1.ppp and Z4.aaa, interact via LIN-12/Notch such that one becomes the AC and one becomes the VU (reviewed in Greenwald 2012). Studies using this paradigm have illuminated many fundamental features about how differences in Notch signaling between equivalent cells arise and are amplified during a cell fate decision. During the AC/VU decision, Z1.ppp and Z4.aaa initially express both LIN-12/Notch and its ligand LAG-2; a stochastic small difference in ligand or receptor activity is amplified by feedback mechanisms that restrict transcription of lin-12 to the presumptive VU cell and lag-2 to the presumptive AC. The AC/VU decision is random, as 50% of animals have an AC derived from Z1.ppp and 50% have an AC derived from Z4.aaa (Kimble and Hirsh 1979). However, the first-born of these cells is biased towards the VU fate (Karp and Greenwald, 2003). By using a microfluidic device to image somatic gonad lineages in large numbers, we have achieved a better understanding of the relationship between birth-order and cell fate, revealing a temporal distinction: at short birth-orders, < 24 minutes, there is no cell fate bias, but at longer birth orders, the first-born cell always becomes the VU. To understand the basis for this difference, we have performed lineage studies in mutants and visualized the expression of fluorescently tagged endogenous proteins, generated using CRISPR/Cas9, that mediate the AC/VU decision. Our analysis suggests that the bias observed for long birth-orders contributes to the robustness of the AC/VU decision and reveals a potential mechanistic link between birth-order, LIN-12/Notch activity, and cell-fate specification.

Attner*, M. A. et al. (2017) International Worm Meeting "Investigating the relationship between birth order and cell fate in the AC/VU decision, a simple lateral specification paradigm for stochastic cell fate specification."

Attner*, M. A., Greenwald, I., Shaham, S., Keil*, W., Siggia, E. D.

[International Worm Meeting, 2017]

Notch-mediated lateral specification is utilized during the development of all animals to generate different cell fates from initially equivalent cells. The anchor cell (AC)/ventral uterine precursor (VU) cell fate decision is a simple lateral specification paradigm in which two initially equivalent cells interact via LIN-12/Notch such that one becomes the AC and one becomes the VU (reviewed in Greenwald 2012). Studies using this paradigm have illuminated many fundamental features about how differences in Notch signaling between equivalent cells arise and are amplified during a cell fate decision. During the AC/VU decision, Z1.ppp and Z4.aaa initially express both LIN-12/Notch and its ligand LAG-2; a stochastic small difference in ligand or receptor activity is amplified by feedback mechanisms that restrict transcription of lin-12 to the presumptive VU cell and lag-2 to the presumptive AC cell. The AC/VU decision is random, as 50% of animals have an AC derived from Z1.ppp and 50% have an AC derived from Z4.aaa. However, the first-born of these cells is biased towards the VU fate (Karp and Greenwald, 2003), suggesting that stochasticity in birth order or a process correlated with birth order is at play. A recent advance in microfluidic technology for imaging C. elegans larvae (Keil et al., 2017) has allowed us, for the first time, to image dozens of somatic gonad lineages and to probe the relationship between lineage patterns, birth-order, and cell fate specification in the AC/VU decision. We fluorescently labeled Z1, Z4, and their descendants, followed Z1 and Z4 lineages at 8-min intervals through their three rounds of division and somatic primordium formation, and observed how the timing of cell divisions in the Z1 and Z4 lineages correlates with the AC/VU decision. We find that Z1.ppp and Z4.aaa birth-order differences generally reflect accumulating birth order differences higher up in each respective lineage. Importantly, we find that at 20 deg C, when the birth-order difference between Z1.ppp and Z4.aaa is longer than 16 minutes, the first-born cell always becomes the VU (n=19). When the birth-order difference between Z1.ppp and Z4.aaa is less than or equal to 16 minutes, birth-order no longer biases cell fate (n=24). Thus, above a threshold in birth-order time difference, the AC/VU decision becomes predictable. We are currently investigating how birth-order controls the AC/VU decision, testing specifically whether birth-order bias is affected by environmental conditions. We hypothesize that the stochastic pattern of cell divisions in the Z1 and Z4 lineages contributes to robustness in the AC/VU decision.

Wypijewska A et al. (2012) Biochemistry "7-methylguanosine diphosphate (m(7)GDP) is not hydrolyzed but strongly bound by decapping scavenger (DcpS) enzymes and potently inhibits their activity."

Wypijewska A, Darzynkiewicz E, Davis RE, Jemielity J, Bojarska E, Lukaszewicz M, Stepinski J

[Biochemistry, 2012]

Decapping scavenger (DcpS) enzymes catalyze the cleavage of a residual cap structure following 3' 5' mRNA decay. Some previous studies suggested that both m(7)GpppG and m(7)GDP were substrates for DcpS hydrolysis. Herein, we show that mononucleoside diphosphates, m(7)GDP (7-methylguanosine diphosphate) and m(3)(2,2,7)GDP (2,2,7-trimethylguanosine diphosphate), resulting from mRNA decapping by the Dcp1/2 complex in the 5' 3' mRNA decay, are not degraded by recombinant DcpS proteins (human, nematode, and yeast). Furthermore, whereas mononucleoside diphosphates (m(7)GDP and m(3)(2,2,7)GDP) are not hydrolyzed by DcpS, mononucleoside triphosphates (m(7)GTP and m(3)(2,2,7)GTP) are, demonstrating the importance of a triphosphate chain for DcpS hydrolytic activity. m(7)GTP and m(3)(2,2,7)GTP are cleaved at a slower rate than their corresponding dinucleotides (m(7)GpppG and m(3)(2,2,7)GpppG, respectively), indicating an involvement of the second nucleoside for efficient DcpS-mediated digestion. Although DcpS enzymes cannot hydrolyze m(7)GDP, they have a high binding affinity for m(7)GDP and m(7)GDP potently inhibits DcpS hydrolysis of m(7)GpppG, suggesting that m(7)GDP may function as an efficient DcpS inhibitor. Our data have important implications for the regulatory role of m(7)GDP in mRNA metabolic pathways due to its possible interactions with different cap-binding proteins, such as DcpS or eIF4E.

Ali Khan L et al. (1994) Worm Breeder's Gazette "cej-1 Encodes a Novel Protein With Poly-Threonine Motif"

Siddiqui SS, Fukushige T, Ali Khan L, Tsukita Sh, Tabish M, Itoh M

[Worm Breeder's Gazette, 1994]

cej-1 Encodes a Novel Protein with Poly-Threonine Motif M. L. A. Khanl, M. Tabish, T. Fukushigel1 S. Tsukita2, M. Itoh , Sh. Tsukita , and S. S. Siddiqui. (1): Lab. of Molecular Biology, Dept of Ecological Engg. Toyohashi Univ. Technology, Toyohashi 441, and (2). National Institute for Physiological Sciences, Okazaki 444, Japan.

Wood WB (1976) Worm Breeder's Gazette "maternal effects among ts zygote-defective (zyg) mutants."

Wood WB

[Worm Breeder's Gazette, 1976]

We have studied maternal effects in 23 zyg ts mutants to estimate the times of expression of genes whose products are required in embryogenesis. We have used the following three tests, called arbitrarily A, B, and C. A test: Heterozygous (m/+) L4's are shifted to 25 C and allowed to self-fertilize. If 100% of their eggs yield larvae (25% of which express the mutant phenotype as adults), then the mutant is scored as maternal (M). If 25% of the F1 eggs fail to hatch, then the mutant is scored as non-maternal (N). An M result indicates that expression of the + allele in the parent allows m/m zygotes to hatch and grow to adulthood. A result of N indicates the opposite: that the + allele must be expressed in the zygote for hatching to occur. Out of 23 zyg mutants tested, 3 were scored N and 20 were scored M in the A test. Therefore, for most of the genes defined by these mutants, expression in the parent is sufficient for zygote survival, even if the gene is not expressed in the zygote. B test: Homozygous (m/m) hermaphrodites reared at 25 C are mated with N2 (+/+) males. If eggs fail to hatch at 25 C, but mated hermaphrodites shifted to 16 C produce cross progeny to give proof of mating, then the mutant is scored M. If cross progeny appear in the 25 C mating, then the mutant is scored N. An M result indicates that expression of the + allele in the zygote is not sufficient to allow m/+ progeny of an m/m hermaphrodite to survive. Conversely an N result indicates either that zygotic expression of the + allele is sufficient for survival, or that a sperm function or factor needed for early embryogenesis can be supplied paternally (see C test below). Out of the 23 zyg mutants tested, 11 were scored M and 12 were scored N. The combined results of A and B tests and their simplest interpretation are as follows. Ten mutants are M,M; the genes defined by these mutants must be expressed in the hermaphrodite parent for the zygote to survive. Ten mutants are M,N; these genes can be expressed either in the parent or in the zygote. Two mutants are N,N; these genes must be expressed in the zygote. One mutant is N,M; this gene must be expressed both in the maternal parent and in the zygote. C test: Homozygous (m/m) hermaphrodites reared at 25 C are mated with heterozygous (m/+) males. If rescue by a +/+ male in the B test depends on the + allele, then only half the cross progeny zygotes of a C test mating (m/+ male x m/m hermaphrodite) should survive. However, if rescue depends on a function or cytoplasmic component from the male sperm, then all the cross progeny zygotes in a C test should survive. Of the 10 M,N mutants, 6 have been C tested; one exhibited paternal rescue independent of the + allele. The A and B tests also were carried out on 16 mutants that arrest before the L3 molt (acc mutants). In the A test on 2 of these mutants, all m/m progeny of m/+ parents grew to adulthood at 25 C. Therefore, parental contributions are sufficient to overcome a progeny mutational block as late as the L2 stage. All 16 acc mutants scored N in the B test.

Lemire BD et al. (2009) Mech Ageing Dev "C. elegans longevity pathways converge to decrease mitochondrial membrane potential."

Behrendt M, Decorby A, Lemire BD, Gaskova D

[Mech Ageing Dev, 2009]

Energy production via oxidative phosphorylation generates a mitochondrial membrane potential (DeltaPsi(m)) across the inner membrane. In this work, we show that a lower DeltaPsi(m) is associated with increased lifespan in Caenorhabditis elegans. The long-lived mutants daf-2(e1370), age-1(hx546), clk-1(qm30), isp-1(qm150) and eat-2(ad465) all have a lower DeltaPsi(m) than wild type animals. The lower DeltaPsi(m) of daf-2(e1370) is daf-16 dependent, indicating that the insulin-like signaling pathway not only regulates lifespan but also mitochondrial energetics. RNA interference (RNAi) against 17 genes shown to extend lifespan also decrease DeltaPsi(m). Furthermore, lifespan can be significantly extended with the uncoupler carbonylcyanide-3-chlorophenylhydrazone (CCCP), which dissipates DeltaPsi(m). We conclude that longevity pathways converge on the mitochondria and lead to a decreased DeltaPsi(m). Our results are consistent with the 'uncoupling to survive' hypothesis, which states that dissipation of the DeltaPsi(m) will extend lifespan.

DiBartolomeis SM et al. (2002) Mol Genet Genomics "Molecular analysis of the Drosophila miniature-dusky ( m-dy) gene complex: m-dy mRNAs encode transmembrane proteins with similarity to C. elegans cuticulin."

Jackson FR, Akten B, Roberts MA, DiBartolomeis SM, Genova G

[Mol Genet Genomics, 2002]

Mutations in the Drosophila miniature-dusky ( m-dy) gene complex were first reported by Morgan and Bridges about 90 years ago. m-dy mutants have abnormally small wings, a phenotype attributed to a cell-autonomous reduction in the size of the epidermal cells comprising the differentiated wing. Using a molecular genetic approach, we have characterized the m-dy chromosomal interval and identified a pair of adjacent transcription units corresponding to m and dy. A dy mutant known as dy (And) has a single base substitution within the protein-coding region that is predicted to result in an amber stop codon and premature translational termination. We show that dy mRNA is expressed at two discrete periods during the life cycle - one during embryonic development and early larval instars, the second during adult development, coincident with wing differentiation. In agreement with the phenotypic similarity of m and dy mutants, sequence comparisons reveal a similarity between the predicted MINIATURE and DUSKY proteins, and indicate that the m and dy genes are members of a larger Drosophila gene family. Both m and dy, as well as other members of this superfamily, are predicted to encode transmembrane proteins with similarity to C. elegans cuticle proteins known as cuticulins. We postulate that m, dy and other members of this protein superfamily function as structural components of the Drosophila cuticulin layer. Such a role for m and dy products in wing differentiation is sufficient to explain the morphological phenotypes associated with m-dy mutants.

Nirav Amin et al. (2008) Development & Evolution Meeting "Systematic analysis of transcription factors important in C. elegans postembryonic mesodermal development"

Zach Via, Jun Liu, Nirav Amin

[Development & Evolution Meeting, 2008]

The C. elegans postembryonic mesodermal lineage, the M lineage, is a powerful model system to study mesodermal patterning and cell fate specification at single cell resolution. The M lineage arises from a single pluripotent cell, the M mesoblast, during embryogenesis. In hermaphrodites, the M cell undergoes a series of postembryonic cell divisions to produce 18 cells: 14 body wall muscles (BWMs), 2 coelomocytes (CCs), and 2 sex myoblasts (SMs). We and others have previously identified a handful of transcription factors important for the proper development of this lineage. In order to identify additional transcription factors that play a role in the M lineage, we have generated a feeding RNAi library that targets a majority of the predicted transcription factors encoded in the C. elegans genome and conducted an RNAi screen using cell type-specific GFP reporters in the M lineage. From this screen, we identified a novel set of 32 transcription factors that, upon RNAi knockdown, give reproducible phenotypes in the M lineage. Among these 32 transcription factors, four are important for patterning and fate specification of the early M lineage, while the rest appear to play a role in fate decisions in the SM lineage. We have primarily focused on let-381, which encodes a forkhead transcription factor that is essential for C. elegans development. let-381(RNAi) causes a dorsal to ventral fate transformation in the M lineage. We have found that a let-381::gfp translational fusion is expressed in the dorsal M lineage. Previous studies from our lab have shown that SMA-9, the Sma/Mab TGF-beta and LIN-12/Notch signaling pathways are involved in dorsal/ventral patterning of the M lineage. We are currently investigating the relationship between let-381 and these pathways.

Almuhanna SA et al. (2024) Cytoskeleton (Hoboken) "Sequences in the myosin A rod interact with UNC-89/obscurin and the zinc-finger protein UNC-98 during thick filament assembly and M-line formation in C. elegans striated muscle."

Lee KM, Oishi HZ, Almuhanna SA, Hoppe PE

[Cytoskeleton (Hoboken), 2024]

The M-line of striated muscle is a complex structure that anchors myosin-containing thick filaments and also participates in signaling and proteostasis. While the physical associations among many M-line components have been defined, the mechanism of thick filament attachment is not completely understood. In Caenorhabditis elegans, myosin A is essential for viability and forms the site of M-line attachment at the center of the filament, whereas myosin B forms the filament arms. Using a mutant myosin A that forms ectopic filaments, we examined interactions between myosin A and M-line proteins in intact muscle cells. Ectopic myosin A recruits the giant kinase UNC-89/obscurin, a presumed scaffolding protein, in an interaction that requires the zinc-finger protein UNC-98, but not UNC-82/NUAK, UNC-97/PINCH, or UNC-96. In myosin A mutants, UNC-89/obscurin patterning is highly defective in embryos and adults. A chimeric myosin containing 169 residues of the myosin A C-terminal rod, coincident with the UNC-98/ZnF binding site, is sufficient for colocalization of UNC-89/obscurin and UNC-98/ZnF in M-line structures whereas a myosin chimera lacking these residues colocalizes with UNC-89/obscurin in M-lines that lack UNC-98. Thus, at least two myosin A rod regions contribute independently to M-line organization. We hypothesize that these M-line-organizing functions correspond to the essential "filament initiation function" performed by this isoform.

Seo J et al. (2005) Arch Environ Contam Toxicol "Biodegradation of the insecticide N,N-diethyl-m-toluamide by fungi: identification and toxicity of metabolites."

Kim SD, Cha CJ, Hur HG, Lee YG, Seo J, Ahn JH

[Arch Environ Contam Toxicol, 2005]

Fungi (Cunninghamella elegans ATCC 9245, Mucor ramannianus R-56, Aspergillus niger VKMF-1119, and Phanerochaete chrysosporium BKMF-1767) were tested to elucidate the biologic fate of the topical insect repellent N,N-diethyl-m-toluamide (DEET). The elution profile obtained from analysis by high-pressure liquid chromatography equipped with a reverse-phase C-18 column, showed that three peaks occurred after incubation of C. elegans, with which 1 mM DEET was combined as a final concentration. The peaks were not detected in the control experiments with either DEET alone or tested fungus alone. The metabolites produced by C. elegans exhibited a molecular mass of 207 with a fragment ion (m/z) at 135, a molecular mass of 179 with an m/z at 135, and a molecular mass of 163 with an m/z at 119, all of which correspond to N,N-diethyl-m-toluamide-N-oxide, N-ethyl-m-toluamide-N-oxide, and N-ethyl-m-toluamide, respectively. M. ramannianus R-56 also produced N, N-diethyl-m-toluamide-N-oxide and N-ethyl-m-toluamide but did not produce N-ethyl-m-toluamide-N-oxide. For the biologic toxicity test with DEET and its metabolites, the freshwater zooplankton Daphnia magna was used. The biologic sensitivity in decreasing order was DEET > N-ethyl-m-toluamide > N,N-diethyl-m-toluamide-N-oxide. Although DEET and its fungal metabolites showed relatively low mortality compared with other insecticides, the toxicity was increased at longer exposure periods. These are the first reports of the metabolism of DEET by fungi and of the biologic toxicity of DEET and its fungal metabolites to the freshwater zooplankton D. magna.