[
1983]
Nematodes are a very large group of animals. The estimated 500,000 species represent an independent phylum, and a very successful one, since they are found, with the exception of the pelagic and aerial habitats, in every type of environment. The great majority of nematodes are free-living and inhabit in large numbers the top few centimeters of the ocean's bed, fresh water muds, and a variety of soils. In the soil, where it has been measured, their biomass is comparable to that of insects. A few hundred species are extremely important in human health and agriculture because of their parasitic relationship to plants and animals. In humans, parasitic nematodes can cause very severe diseases, such as filariasis and river blindness (Oncocercus)...
[
Worm Breeder's Gazette,
1980]
Following the suggestion of Tony Otsuka at the worm meeting in May, we have been using chitinase to digest the shells of eggs that have been treated with NaClO. We found that the commercially available enzyme works in Egg Buffer (Hepes pH 7.2 5mM, NaCl 110mM, KCl 4mM, Mg Acetate 5mM, CaCl2 5mM); in this solution blastomeres survive and stay reasonably healthy. As suggested by Tony Otsuka, the hypochlorite treatment is required in order for chitinase to have effect; the concentration of NaClO and the length of treatment vary somewhat depending on the purpose of the experiment and on whether one starts from gravid worms or from eggs. For large scale preparations gravid worms are treated for 2 to 3 minutes with 2% NaClO with frequent mixing. The NaClO concentration is then reduced to 0.2% and disruption of the carcasses and freeing of the eggs is accomplished with the use of a homogenizer loosely fitted with a Teflon pestel. After 7 minutes in 0.2% NaClO, the eggs are collected and washed in Egg Buffer. Chitinase is added to the egg suspension. Because of the low specific activity, 5 to 10mg of protein per ml have to be used. Digestion is followed by taking samples for microscopic observation and is interrupted by washing several times with cold Egg Buffer. We have noticed that prolonged digestion damages the embryos. At this point most of the embryos have assumed a spherical shape and are still surrounded by the 'inner membrane' which is also the main permeability barrier of the embryo. To break the inner membrane and disaggregate the cells, embryos are suspended in a medium composed of one part of Ascaris coelemic fluid and four parts of Egg Buffer lacking Ca++ and Mg++. Embryos are gently homogenized (2-3 strokes) with a loose fitting Teflon pestel and Mg+ and Ca++ are restored immediately. Filtration and differential centrifugation yield fairly clean preparations of blastomeres. Cells prepared by this method appear reasonably healthy; but the fraction of them that divide a few more times has varied substantially in different experiments. Large quantities of embryos can be easily processed and the shells used as starting material for a variety of biochemical studies. In these suspensions of disaggregated blastomeres gut cells containing polarizing granules can be easily distinguished from other cells with the use of crossed polarizers. We want to instruct a cell sorter to use this property to separate E cells from non-E cells. We have used chitinase digestion on small amounts of eggs cut out of gravid hermaphrodites. In this case the 2% NaClO treatment cannot exceed 1 minute. The rest of the treatment is the same. After chitinase digestion embryos can be fixed for EM with excellent results. Eggs as early as the 2-cell stage can be fixed. We are identifying a number of ultrastructural markers of differentiation by looking at different stages of N2 embryos and plan to examine their appearance in some ts embryonic lethals. We are also using the chitinase method as another means to obtain isolated early blastomeres (e.g., Pl, P2, AB, EMST, C, etc.) to study their intrinsic cleavage pattern and their potential for differentiation and would like to confirm with this method the results obtained by the burst egg technique.
[
European Worm Meeting,
2000]
The molecular mechanisms through which cells and neuronal growth cones migrate, adhere to the substrate and find their targets are varied, appear to often work in redundant pathways, and appear to be conserved in evolution. Although many molecules have already been identified and studied, especially using the invertebrate model systems, it is clear that many other molecules involved in these processes still need to be discovered. Kallmann syndrome (KS) is a human inherited disorder defined by the association of anosmia and hypogonadism. The human gene responsible for the X-linked form of the disease has been identified. The chicken, the quail and recently two zebra fish homologs, but not the mouse one have also been identified. These genes code for novel secreted proteins, KALs, which contain a new combination of domains: the four-disulfide-core domain together with fibronectin type III domains. The vertebrate genes are expressed in the olfactory bulb and are required for proper neuronal connectivity and cell or axonal migration in this region both during development and in adult life. Studies in the chicken suggest that the KAL proteins may be new components of the mechanisms of cell/axon migration or cell adhesion. A C. elegansgene, identified by the Sequencing Consortium, has significant homology to the human gene for X-linked Kallmann syndrome. The gene, which we have named
kal-1, maps on LGI near
unc-54. A cDNA clone from the Kohara collection has been obtained and sequenced. Comparison with the genomic sequence shows that the mRNA is composed of 6 exons. The open reading frame codes for 700 aa. The domain topology of the protein, is conserved between nematode and vertebrates. The main difference is the presence at the C-terminus of the C. elegansprotein of a stretch of hydrophobic residues. We have studied the expression of
kal-1 with the use of a series of lac-Z and GFP reporter constructs. The reporter experiments indicate that 6-8 cells begin expressing the gene in 200-cell-stage embryos. In larval stages three groups of cells express the gene. A group of neurons (15-20) in the nerve ring region surrounding the pharinx. A group of 4-5 neurons in the tail ganglia. The two canal associated neurons (CANs) at mid body. In adults the pattern does not change but it is more restricted. Sequences upstream of the ATG and in the first intron appear to have a role in modulating expression. We have isolated a deletion mutant,
kal-1(
gb503), using TMP + UV as mutagen and screening by PCR. The deletion is 2.2 Kb long and eliminates the 440 terminal residues. RT-PCR analysis of the mature mRNA present in the mutant strain suggests that the mutant is a functional null. The mutant shows no major visible phenotype. More subtle defects present at low penetrance in the mutant will be discussed. To help identify the function of the gene we have injected worms with constructs overexpressing either the full length or truncated forms of the KAL-1 protein fused to GFP. Worms transformed with these constructs show various abormalities both during embryogenesis and in larval development. An interpretation of these phenotypes will be presented. We have also identified and sequenced cDNAs with significant homology to Kal genes in Drosophilaand in the silk worm. The N-terminal regions of the predicted proteins are strongly conserved but the insect proteins are shorter than the nematode and vertebrate ones. By in situ hybridization the Drosophila gene appears to be expressed in embryos in the stomatogastric ganglia. Thus also in this system the homolog of the Kallmann syndrome gene appears to be expressed by neurons.
[
International C. elegans Meeting,
1999]
Kallmann syndrome (KS) is an inherited disorder defined by the association of anosmia and hypogonadism. The human, chicken and quail genes have been identified. They code for novel secreted proteins, KALs, which contain a new combination of domains: the four-disulfide-core domain together with fibronectin type III domains. The vertebrate genes are expressed in the olfactory bulb and are required for proper neuronal connectivity and cell or axonal migration in this region both during development and in adult life. The C. elegans gene K03D10.1, identified by the Sequencing Consortium, codes for a protein with significant homology and colinear domain topology to vertebrate KALs. We named the gene
kal-1 . A corresponding cDNA clone from the Kohara collection has been obtained and sequenced. The C. elegans protein presents at its C-terminus a hydrophobic trans-membrane domain which is absent in the vertebrate counterparts. RT-PCR and experiments with reporters indicate that the gene is transcribed beginning with late embryos and during larval stages. Specific antibodies to fragments of CeKAL-1 have been generated. The nematode protein is detected in worm extracts as a cleaved C-terminal fragment with an apparent mass of 42 kD. Similarly the vertebrate KALs, secreted by COS cells, are present in the medium as cleaved C-terminal fragments of 42-45 kD. We have isolated a deletion mutant of
kal-1 using TMP + UV as mutagen and screening by PCR. The deletion is 2.2 Kb long and eliminates 440 terminal aminoacids. Transcriptional analysis and the use of specific antibodies indicates that the deletion mutant (
gb503) is a functional null. We are characterizing the phenotype of
kal-1(
gb503) worms which show no major visible abnormality. Mutants appear to chemotax normally to several odorants to avoid soluble repellents and respond to mechanic stimuli. We will present further data on the behavioral characterization of
kal-1(
gb503) and on experiments with GFP constructs in which the path followed by several neurons in this strain has been studied.
[
Worm Breeder's Gazette,
1980]
We have continued experiments on the cleavage patterns of partial embryos and isolated blastomeres obtained by crushing early N2 eggs in embryonic culture medium supplemented with Ascaris coelomic fluid (W.B. G. June 1978). Neither isolated somatic precursor cells (blast cells) nor isolated P cells give rise to the spatial patterns formed by their progeny in intact embryos. However, all our results so far are consistent with the following simple hypothesis. The blast cells, and their progeny, are internally determined to cleave in a simple helical pattern. The P cells are internally determined to cleave linearly, maintaining the spindle orientation of preceding P-cell divisions parallel to the anterior-posterior axis. In the intact embryo, interactions of cells with each other (and perhaps with the eggshell) combine with these two intrinsic cleavage patterns to dictate normal cellular organization, which therefore depends upon both extracellular and intracellular cues. Cleavage of an isolated P2 blastomere results in an approximately linear chain of cells in the order C, C, D, P4. This result, together with the approximately anterior-posterior orientation of the preceding cleavages that give rise to the AB, EMSt, E, and MSt cells suggests the possibility that in the zygote, determinants for blast-cell primary differentiation are arranged along the posterior-anterior axis in the order C (ectoderm), D (mesoderm), P4 (germ line), E (endoderm), MSt (mesoderm), AB (ectoderm). This arrangement corresponds to that of a fate map proposed for the Ascaris des zygote by Zur Strassen (1896). It differs from that of the fate map proposed for the C. elegans zygote by Schierenberg (1978), who postulated the order P4, D, C, E, MSt, AB based on observations of division orientation in intact embryos,cytoplasmic streaming,and cell movements during early cleavage.
[
European Worm Meeting,
2006]
Chemical sensitivity allows animals to identify and respond appropriately to the chemical composition of the environment. Noxious water-soluble compounds that are avoided by C. elegans are generally sensed as bitter by humans and are discarded in double choice test by mice. We have used C. elegans to focus on the molecular mechanisms involved in primary sensing of quinine, a molecule detected as bitter by humans. ASH is the main sensory neuron involved in sensing quinine. Two G? subunits, GPA-3 and ODR-3 are necessary for the response of ASH to repellent stimuli (Hilliard et al., 2004 and 2005). In addition the TRPV channel proteins, OSM-9 and OCR-2, are also necessary for the ASH avoidance responses (Colbert et al 1997, Tobin et al 2002). Finally we identified a novel protein, QUI-1, as an essential components of the response to quinine (Hilliard et al., 2004). With regard to the molecular function of QUI-1, we demonstrate that QUI-1 function is required in ASH for the response to quinine and, using specific antibodies, that the protein is localized to the sensory cilia. These results, together with the discovery that QUI-1 contains an RGS (Regulator of G protein Signaling) domain, strongly suggest that this novel protein might be involved in quinine signaling.. Are there other components of the quinine signal transduction pathway?. We are using a best candidate approach and a variety of behavioral assays to identify new molecules involved in sensing repellent chemicals and in particular quinine. We analyzed behaviorally loss of function and overexpression mutants in several molecules known to act in the G protein signaling pathways (G? subunits, G? subunits, RGS proteins, etc.). The results obtained will be discussed.. Colbert, H. A., Smith, T. L. and Bargmann, C. I. (1997). OSM-9, a novel protein with structural similarity to channels, is required for olfaction, mechanosensation, and olfactory adaptation in Caenorhabditis elegans. J Neurosci 17, 8259-69.. Hilliard, M. A., Apicella, A. J., Kerr, R., Suzuki, H., Bazzicalupo, P. and Schafer, W. R. (2005). In vivo imaging of C. elegans ASH neurons: cellular response and adaptation to chemical repellents. Embo J 24, 63-72.. Hilliard, M. A., Bergamasco, C., Arbucci, S., Plasterk, R. H. and Bazzicalupo, P. (2004). Worms taste bitter: ASH neurons, QUI-1, GPA-3 and ODR-3 mediate quinine avoidance in Caenorhabditis elegans. Embo J 23, 1101-11.. Tobin, D., Madsen, D., Kahn-Kirby, A., Peckol, E., Moulder, G., Barstead, R., Maricq, A. and Bargmann, C. (2002). Combinatorial expression of TRPV channel proteins defines their sensory functions and subcellular localization in C. elegans neurons. Neuron 35, 307-18.
[
International C. elegans Meeting,
1997]
We are studying chemoreception in C.elegans focusing our attention on the avoidance of water soluble substances. We have used a new test, "drop test", which consists in delivering a small drop of the chosen solution behind a worm freely moving on the plate surface. By capillarity the solution reaches the anterior end of the animal where the amphids are located. If the solution contains a repellent, the worm stops immediately and moves backward for a few seconds; if no repellent is present the worm continues its forward movement. Using this test we have identified, including those already known, 16 chemicals acting as repellent at concentrations between 0.1 and 10 mM. We have chosen quinine, at a concentration of 10 mM, to screen the F2 progeny of EMS mutagenized worms. Out of 70.000 worms screened we have found 15 mutants which fail to avoid quinine. After preliminary behavioral characterization we have focused our attention on two,
gb404 and
gb408. These are allelic and identify a new gene,
qui-1, which has been positioned on the right arm of the LG IV using STS mapping. The amphid neurons in both alleles stain normally with FITC. They respond normally to light touch and to some other repellent e.g. copper ions, zinc ions, veratridin, levamisole, garlic extract, high osmotic strenght. We hope we have identified either the receptor for quinine, if it exists, or some other component acting relatively early in the sensory transduction pathway used by C.elegans to avoid quinine.