Keil W et al. (2016) Dev Cell "Long-Term High-Resolution Imaging of Developing C.elegans Larvae with Microfluidics."

Shaham S, Keil W, Kutscher LM, Siggia ED

[Dev Cell, 2016]

Long-term studies of Caenorhabditis elegans larval development traditionally require tedious manual observations because larvae must move to develop,and existing immobilization techniques either perturb development or are unsuited for young larvae. Here, we present a simple microfluidic deviceto simultaneously follow development of ten C.elegans larvae at high spatiotemporal resolution from hatching to adulthood (3days). Animals grownin microchambers are periodically immobilized bycompression to allow high-quality imaging of even weak fluorescence signals. Using the device,we obtain cell-cycle statistics for C.elegans vulval development, a paradigm for organogenesis. Wecombine Nomarski and multichannel fluorescence microscopy to study processes such as cell-fate specification, cell death, and transdifferentiation throughout post-embryonic development. Finally, we generate time-lapse movies of complex neural arborization through automated image registration. Our technique opens the door to quantitativeanalysis of time-dependent phenomena governing cellular behavior during C.elegans larval development.

Keil, W. et al. (2017) International Worm Meeting "Long-Term High-Resolution Imaging of Developing C. elegans Larvae with Microfluidics."

Keil, W., Shaham, Shai, Siggia, E. D., Kutscher, L. M.

[International Worm Meeting, 2017]

Decades of research on C. elegans development have shed light on a large variety of developmental phenomena; from cell-fate decisions and transdifferentiation to cell migration and cell death. However, long-term high-resolution in-vivo imaging of these processes has been accessible only in embryos, because larvae must move and feed to develop, and existing immobilization techniques perturb development and compromise animal viability. To overcome this problem, we have developed a microfluidic setup to simultaneously follow development of ten C. elegans larvae at high spatiotemporal resolution from hatching to adulthood (~3 days). Animals grown in microchambers are periodically immobilized by compression to allow high-quality imaging of even weak fluorescence signals. We show that this technology can be used to follow cell-fate decisions, cell death programs, and transdifferentiation events with Nomarski and multichannel fluorescence microscopy. We also obtain cell-cycle timing statistics during the formation of the C. elegans vulva in a large number of animals, exposing the limits of fidelity and robustness of C. elegans development. Finally, we develop algorithms for automated image registration to generate time-lapse movies. For the first time, these movies enable us to visualize and quantify highly-diverse processes such as neural arborization or cell divisions during gonadogenesis in a feeding, moving, and growing animal. Our technique opens the door to quantitative analysis of time-dependent phenomena governing cellular behavior during C. elegans larval development.

Keil, W. et al. (2019) International Worm Meeting "BMLP-1 and ELT-3 amplify transcriptional output to ensure developmental robustness in C. elegans."

Keil, W., Hammell, C. M., Dorfel, K., Ercan, S., Stec, N.

[International Worm Meeting, 2019]

Precise and robust dynamic changes in gene expression are a hallmark of developmental systems. In the C. elegans developmental timing pathway, microRNAs (miRNAs) mediate such changes through the successive post-transcriptional down-regulation of RNA-binding proteins and transcription factors that establish earlier gene expression programs. Results from our labs and others indicate that the transcription of miRNAs is pulsatile throughout larval development and coupled to the molting cycles. Mutations in the C. elegans Period ortholog, lin-42, lead to the overexpression of several miRNAs and precocious developmental timing phenotypes - suggesting that LIN-42 functions as a negative regulator of transcriptional output. We leveraged the phenotypes of lin-42 mutants to identify transcription factors that antagonize LIN-42 activity in transcriptional regulation. These efforts identified BLMP-1 and ELT-3, a conserved zinc-finger and a GATA binding protein transcription factor, respectively. Both genes are expressed in the hypodermis throughout larval development and are bound to the putative regulatory elements of hundreds of protein coding and miRNA genes. Importantly, the regulatory binding sites of BLMP-1 and ELT-3 correlate with open chromatin, suggesting that these proteins may function to remodel the accessibility and therefore the activity of their target genes. Analysis of blmp-1(0); elt-3(0) synthetic developmental phenotypes indicate that these genes function redundantly during development to control aspects of animal morphology and to limit the normal molting cycles to the L1-L4 stages. Through long-term time-lapse microscopy with microfluidics, we demonstrate that transcription of BLMP-1 and ELT-3 targets is maintained in blmp-1(0); elt-3(0) mutants but transcriptional output is reduced. Importantly, BLMP-1 and ELT-3 modulate amplitude and duration of transcriptional pulses for cyclically expressed mRNAs and miRNAs, known to mediate temporal cell-fate decisions in dosage-dependent manners during normal development. Other dynamical aspects of transcription such as periodicity and phasing are not altered, suggesting that BLMP-1 and ELT-3 directly modulate transcriptional output. Finally, we demonstrate that the ability to regulate transcription through BLMP-1 and ELT-3 is essential in rapidly changing environmental conditions or when nutritional status during development is altered. Our studies provide a novel molecular mechanism for developmental adaptation and robustness.

K Omata et al. (1999) International C. elegans Meeting "Characterization of the neural circuit of C. elegans: model analysis of the touch response"

F Yonezawa, K Omata, K Kawamura

[International C. elegans Meeting, 1999]

In order to characterize the neural circuit of C. elagans, we construct a simple model by making use of the data table completed recently by Oshio et al . [1]. We assume that the signal of a neuron is calculated by the product of the signals from the neighboring neurons, and we investigate the touch sensitivity to continuous stimuli described by sinusoidal functions as defined in the rage from 0.0 to 1.0. We calculate the responses of the motor neurons by changing the frequencies of the stimuli. In our calculations, we change only the frequency w PLM for the input signal to the sensory neuron PLM, while the frequency for the other sensory neurons ALM, AVM and PVM is fixed to be a same value w 0 . We show that the output signals from the motor neurons A and B oscillate in time. We measure the minima of the oscillation for each w PLM value. The plot of the minima versus w PLM shows different hehaviors for the case of the neuron A and B. As for the signals from the neuron A, the values of the minima are widely distributed between 0.0 and 1.0 for all w PLM . As for the signals from the neuron B, on the other hand, the features are different for different w PLM values. (a) In the high frequency region of w PLM / w 0 0.4, the oscillation is simple harmonic and there exists only one minimum value (I min = 0.0). (b) As w PLM / w 0 is decreased, another minimum appears at a certain frequency, and the bifurcation takes place discontinuously. This behavior is different from usual continuous bifurcation observed in nonlinear systems. After a few discontinuous branching occur, signals with five periods can be seen in the intermediate frequency region of 0.3 w PLM / w 0 w PLM / w 0 [1] K. Oshio et al. ; C. elegans synaptic connectivity data'', Technical Report, CCEP, Keio Future No.1 (1998).

Wang C et al. (2018) Nat Commun "Author Correction: Small-molecule TFEB pathway agonists that ameliorate metabolic syndrome in mice and extend C. elegans lifespan."

Zhao T, Oswald NW, Li Y, Lin R, Wang C, Jaramillo J, Zhou A, McMillan EA, Douglas PM, MacMillan JB, Huang G, Luo M, Gao J, Mendiratta S, Lin Z, Wang Z, Niederstrasser H, Posner BA, Brekken RA, White MA

[Nat Commun, 2018]

The originally published version of this Article contained an error in the spelling of the author Nathaniel W. Oswald, which was incorrectly given as Nathaniel W. Olswald. This has now been corrected inboth the PDF and HTML versions of the Article.

Nawa Y et al. (1985) Parasite Immunol "Defective protective capacity of W/Wv mice against Strongyloides ratti infection and its reconstitution with bone marrow cells."

Kotani M, Nawa Y, Kiyota M, Korenaga M

[Parasite Immunol, 1985]

The susceptibility of congenitally anemic, and mast cell deficient W/Wv mice to infection with Strongyloides ratti was examined. After a primary infection, W/Wv mice showed greater and more persistent peak larval counts than did normal littermates. Worm expulsion was also slower in W/Wv mice than in +/+ mice. Furthermore, difference in susceptibility was expressed as early as 24 h after infection, suggesting not only that protective mechanisms of the gut but also of the connective tissue were defective in W/Wv mice. Reconstitution with bone marrow or spleen cells from +/+ mice was effective in restoring the protective response in W/Wv mice, whereas thymocytes or mesenteric lymph nodes had no effect. Both connective tissue and mucosal mast cells were repaired in W/Wv mice after marrow reconstitution and infection. Since relatively long incubation period was required for the expression of such reconstituting activities, bone marrow cells seem to contain precursor cells of the effector and/or regulator cells.

Casiraghi M et al. (2004) Int J Parasitol "Mapping the presence of Wolbachia pipientis on the phylogeny of filarial nematodes: evidence for symbiont loss during evolution."

Guerrero R, Bandi C, Franceschi A, Pocacqua V, Gardner SL, Casiraghi M, Martin C, Bain O

[Int J Parasitol, 2004]

Wolbachia pipientis is a bacterial endosymbiont associated with arthropods and filarial nematodes. In filarial nematodes, W. pipientis has been shown to play an important role in the biology of the host and in the immuno-pathology of filariasis. Several species of filariae, including the most important parasites of humans and animals (e.g. Onchocerca volvulus, Wuchereria bancrofti and Dirofilaria immitis) have been shown to harbour these bacteria. Other filarial species, including an important rodent species (Acanthocheilonema viteae), which has been used as a model for the study of filariasis, do not appear to harbour these symbionts. There are still several open questions about the distribution of W. pipientis in filarial nematodes. Firstly the number of species examined is still limited. Secondly, it is not clear whether the absence of W. pipientis in negative species could represent an ancestral characteristic or the result of a secondary loss. Thirdly, several aspects of the phylogeny of filarial nematodes are still unclear and it is thus difficult to overlay the presence/absence of W. pipientis on a tree representing filarial evolution. Here we present the results of a PCR screening for W. pipientis in 16 species of filariae and related nematodes, representing different families/subfamilies. Evidence for the presence of W. pipientis is reported for five species examined for the first time (representing the genera Litomosoides, Litomosa and Dipetalonema); original results on the absence of this bacterium are reported for nine species; for the remaining two species, we have confirmed the absence of W. pipientis recently reported by other authors. In the positive species, the infecting W. pipientis bacteria have been identified through 16S rDNA gene sequence analysis. In addition to the screening for W. pipientis in 16 species, we have generated phylogenetic reconstructions based on mitochondrial gene sequences (12S rDNA; COI), including a total of 28 filarial species and related spirurid nematodes. The mapping of the presence/absence of W. pipientis on the trees generated indicates that these bacteria have possibly been lost during evolution along some lineages of filarial nematodes.

Kutscher LM et al. (2018) Dev Cell "RAB-35 and ARF-6 GTPases Mediate Engulfment and Clearance Following Linker Cell-Type Death."

Kutscher LM, Keil W, Shaham S

[Dev Cell, 2018]

Clearance of dying cells is essential for developmentand homeostasis. Conserved genes mediate apoptotic cell removal, but whether these genes control non-apoptotic cell removal is a major open question. Linker cell-type death (LCD) is a prevalent non-apoptotic developmental cell death process with features conserved from C.elegans to vertebrates. Using microfluidics-based long-term invivo imaging, we show that unlike apoptotic cells, the C.elegans linker cell, which dies by LCD, is competitively phagocytosed by two neighboring cells, resulting incell splitting. Subsequent cell elimination does not require apoptotic engulfment genes. Rather, we find that RAB-35 GTPase is a key coordinator of competitive phagocytosis onset and cell degradation. RAB-35 binds CNT-1, an ARF-6 GTPase activating protein, and removes ARF-6, a degradation inhibitor, from phagosome membranes. This facilitates phosphatidylinositol-4,5-bisphosphate removal from phagosome membranes, promoting phagolysosome maturation. Our studies suggest that RAB-35 and ARF-6 drive a conserved program eliminating cells dying by LCD.

Lewis JA (1981) Worm Breeder's Gazette "Modified Ficoll Method for Cleaning Worms"

Lewis JA

[Worm Breeder's Gazette, 1981]

I have made two modifications in the Ficoll method I originally described in WBG, vol. 3, #2. First, all Ficoll solutions are made 0. 1 M in NaCl to avoid shocking osmoticalIy sensitive worms like unc-29( e1072). Second, after sedimenting worms through 15% w/w Ficoll 400 ( 15', 300 x g), the worms are diluted with an equal volume of 0.1 M NaCl and floated on 35% w/w Ficoll (15', 300 x g) before washing 2x with 0.1 M NaCl. The Ficoll sedimentation removes dead or degenerated worms, cuticles, bacteria. The flotation removes crystalline debris sometimes occurring in worm cultures. We also find that special care to pipette out the last residue of bacterial medium (e.g. 3XD) before resuspending bacteria for worm growth is most conducive to obtaining uncontaminated worms.

Li Z et al. (2018) Pak J Pharm Sci "Cellulase-assisted extraction and anti-ultraviolet activity of polysaccharides from the root of Flammulina velutipes on Caenorhabditis elegans."

Chen M, Lin L, Zhang Y, Wang T, Cui H, Wang C, Li Z

[Pak J Pharm Sci, 2018]

We investigated the cellulase-assisted extraction and anti-ultraviolet activity of water-soluble polysaccharides from the root of Flammulina velutipes on Caenorhabditis elegans. A Box-Behnken design experiment with three factors and three levels, including enzymolysis temperature, microwave time, and microwave power, was designed on the basis of the results of single-factor experiments. For improving the polysaccharide yield of F. velutipes root, the following optimal extraction conditions were used: 52.67C enzymolysis temperature, 80s microwave time, and 144 W microwave power. Under optimal conditions, the actual measured value of the yield was 2.01% (w/w) and the predicted value was 2.06% (w/w). One fraction (FRP-2) was isolated and purified, and its characteristics were analyzed. The average mean molecular weight of FRP-2 was measured to be 2.60x10⁵ Da, and its monosaccharide composition is mainly glucose. The sugar units are present both in the -configuration and -configuration. Moreover, FRP-2 exhibited certain anti-ultraviolet activity to C. elegans when the polysaccharide concentration ranged between 0.05mg/mL and 0.20mg/mL.