EP1137754A2 - Methode de construction de bibliotheques de profils phenotypiques - Google Patents

Methode de construction de bibliotheques de profils phenotypiques

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Publication number
EP1137754A2
EP1137754A2 EP99963460A EP99963460A EP1137754A2 EP 1137754 A2 EP1137754 A2 EP 1137754A2 EP 99963460 A EP99963460 A EP 99963460A EP 99963460 A EP99963460 A EP 99963460A EP 1137754 A2 EP1137754 A2 EP 1137754A2
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EP
European Patent Office
Prior art keywords
worm
compound
library
profiles
phenotypic
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EP99963460A
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German (de)
English (en)
Inventor
Titus Kaletta
Richard Feichtinger
Jonas Van Poucke
Anton Van Geel
Saskia Appelmans
Wim Van Criekinge
Thierry Bogaert
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Devgen NV
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Devgen NV
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43536—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from worms
    • C07K14/4354—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from worms from nematodes
    • C07K14/43545—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from worms from nematodes from Caenorhabditis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1079—Screening libraries by altering the phenotype or phenotypic trait of the host

Definitions

  • the present invention is concerned with the field of ⁇ genetic pharmacology' . Specifically, it relates to methods which can determine, among other things, whether a compound has potential pharmacological activity, whether a compound interacts with a particular gene or biochemical pathway in man or animals, what side effects are likely to be associated with a particular pharmaceutical compound and/or the mode or modes of action of any compound with biological activity. Additional uses for the methods of the invention include the assignment of function to particular genes or assignment of genes and their encoded proteins to particular biochemical pathways.
  • the invention relates to the use of a microscopic nematode worm, for example Caenorhabdi tis elegans, and libraries of such worms in the aforementioned methods.
  • the test against the target might be carried out in vivo, for example by use of animal models of a human disease.
  • the compounds could be tested for interaction with the molecule in vi tro .
  • the limitations of such methods are that in the event of a negative result no other information about the pharmaceutical potential of the compound tested is gained.
  • an in vi tro test might show a compound to have no inhibitory action against a particular target enzyme but that compound might have an inhibitory action against another enzyme in the same biochemical pathway as the target enzyme and therefore, in fact, have potential in treatment of the target disease.
  • C. elegans is a microscopic nematode worm which occurs naturally in the soil but can be easily grown in the laboratory on nutrient agar inoculated with bacteria, preferably E . coli , on which it feeds. Each worm grows from an embryo to an adult worm of about 1 mm long in three days or so.
  • C. elegans genome is now almost entirely sequenced as a result of the C. elegans genome project, carried out at the Sanger Center and Washington University School of Medicine. The sequence is available in a public database at http://www.sanger.ac.uk/projects/C_ elegans/. As a result of this it has emerged that C. elegans comprises genes which have equivalents that are widely distributed in most or all animals including humans.
  • C. elegans might be useful for establishing links between compounds and specific C. elegans genes by virtue of comparison of phenotypes generated by exposure to particular compounds and by selected mutations is considered by Rand and Johnson in Methods of Cell Biology, Chapter 8, vol 84, Caenorhabditis elegans: Modern Biological Analysis of an Organism Ed. Epstein and Shakes, Academic Press, 1995 and J. Ahringer in Curr. Op. in Gen. & Dev. 1_; 1997; 410-415.
  • phenotypic characteristics have all been described differently by different workers in the C. elegans field.
  • Phenotype descriptions in the literature largely omit aspects not directly related to or not recognised to be related to the principle interest of the individual researcher. There is no standard nomenclature to identify a specific change. Without this it is impossible to equate newly observed phenotypes with particular known phenotypes for comparison purposes.
  • each worm a 'phenotype profile' or 'fingerprint' is established based on looking for plurality of changed characteristics in a particular mutant or worm which has been exposed to an environmental change or a compound. Furthermore, each profile is scored by following a strict standard protocol of measurement and a standard description is applied to each characteristic. The determination of a phenotypic profile in this way for a plurality of mutants or worms exposed to compounds illuminates differences between different mutants or otherwise treated worms which would not be apparent based on prior art methods.
  • libraries of reference profiles can be established for mutant worms -and for worms exposed to particular environmental changes or different sorts of compounds.
  • Such libraries allow complex patterns of linkage to be established between particular compounds and particular genes or biochemical pathways and between individual compounds of known or unknown biochemical or pharmacological activity.
  • a method of constructing a library of phenotypic profiles of nematode worms which comprises the steps of:
  • Caenorhabdi tis elegans is the preferred nematode worm although the method could be carried out with other nematodes and in particular with other microscopic nematodes, preferably microscopic nematodes belonging to the genus Caenorhabditis .
  • microscopic nematode encompasses nematodes of approximately the same size as C. elegans, being of the order 1mm long in the adult stage.
  • Microscopic nematodes of this approximate size are extremely suited for use in mid- to high-throughput screening as they can easily be grown in the wells of a multi-well plate of the type generally used in the art to perform such screening. It is preferred to establish the phenotypic profile on the basis of the measurement and scoring of at least three different characteristics, preferably at least six characteristics and more preferably at least ten characteristics. It will be appreciated that the more differences which can be scored between a worm with a genetic defect and a worm without the defect the better the resolution between different mutants. Although not limited to such, at least one of the plurality of changed characteristics which can be measured and scored may be selected from the list shown in Table 1, and possibly each of all the changed characteristics scored is one of those shown in Table 1.
  • the method used to establish the phenotypic profile comprises measurement and scoring of two or more characteristics selected from the group consisting of: viability, life cycle, body shape, movement behaviour, mechanotransduction, pharynx pumping, defecation and fertility.
  • This list provides a core set of measurable characteristics which can be used to establish an informative phenotypic profile for any type of worm.
  • each of these characteristics is measurable using technical measuring apparatus, such as video image analysis, multiwell plate reader, and/or a technical assay procedure.
  • the method used to establish the phenotypic profile comprises measurement and scoring of all eight of the listed core characteristics.
  • Measuring and scoring this set of core characteristics allows meaningful comparisons to be made between phenotypic profiles for worms subjected to diverse interventions.
  • comparisons can be drawn between profiles for two different mutant worms and between profiles for mutant worms and profiles for worms exposed to compound.
  • the terms "measuring” or “measurement” as used in connection with any of the methods described and claimed herein are to be interpreted as including not just absolute quantitative measurement wherein a numerical value is assigned to the characteristic but also comparative measurement, wherein characteristics of a worm which has been subject to an intervention (i.e.
  • the scored characteristics are represented in the same order for each profile.
  • measurement and scoring of the characteristics could be carried out in a predetermined order according to a standard protocol. However, this is not essential to the operation of the method.
  • the characteristics are recorded in a binary manner as 'present' or 'not present' based on deviations from wild-type worms.
  • the known or newly generated genetic defects may manifest themselves, for example, as the absence of expression of a gene, the reduction in expression of a gene, the over-expression of a gene, the expression of a functionally defective protein, the mis-expression of a protein, the ectopic mis-expression of a protein, the expression of a protein of altered stability or the alteration of gene expression as a function of time.
  • C. elegans can be carried out on wild- type worms or worms with existing single or multiple mutations. It may be desirable to genetically manipulate C. elegans carrying a reporter gene construct.
  • the reporter molecule might be LacZ or green fluorescent protein but many other reporter molecules are known to those skilled in the art. Reporter gene constructs for C. elegans are described in Chalfie et al, 1994, Science 263 pp 802-805. It can also be desirable to genetically manipulate and then profile a transgenic worm, preferably a worm carrying a human gene, particularly where the gene is associated with, or is a candidate for association with a human disease and therefore a putative drug target.
  • a form of the worm which may show a change in phenotype and may therefore be subject to profiling as described above is one in which the genetic defect and/or transgene and/or reporter gene is only present in a sub-set of the cells of the worm. It is possible for just the cells of a particular tissue to be the subject of a genetic manipulation.
  • the worm which is to be subject to determination of its phenotypic profile can be cultured by methods well-known in the art.
  • C. elegans can grow on nutrient agar which has first been inoculated with bacteria on which the worms feed. Suitable culture methods are described in Rand and Johnson (see above) and in the examples given herein.
  • Measurement of any changed characteristics which will determine the profile may be carried out using light microscopy, differential interference contrast optics or fluorescence microscopy.
  • immuno-chemical detection, colorimetric detection or detection of fluorescence, luminescence or radioactive labels may be used.
  • the changed characteristics may be biochemical only and might be detected, for example by a pH change in the growth media or a change in electrical potential.
  • Different characteristics may need to be determined at different points in the growth cycle of the worm. For example, some phenotypic characteristics may be manifested only in the larvae while others are only detectable in the adult worm. In some cases it may be necessary to make several measurements of the same characteristic at predetermined time intervals.
  • Phenotypic profiles generated by the methods described above can be collated into a library of profiles which are stored electronically on a database.
  • the invention also provides a method of constructing a physical library or bank or worms each identifiable by their individual phenotypic profile.
  • a worm library can be created using any or all of the methods described above and used for comparative purposes.
  • the worms may be maintained by the culture methods described herein and/or frozen for long term storage by methods known to those skilled in the art.
  • Libraries of phenotypic profiles or fingerprints of mutant worms or mutant worm libraries can be used to determine linkages between different genes and hence identify biochemical pathways. A particularly important use is the profiling of several mutations of the same gene and several genes of the same pathway.
  • the present invention provides a method of constructing a library of phenotypic profiles of nematode worms which comprises the steps of:
  • the worms which are exposed to the compound may be wild-type worms, mutant worms, transgenic worms and/or worms carrying reporter gene constructs as already described herein. Further the measurement and scoring of a plurality of changed characteristics is carried out by exactly the same procedures as already described herein for the phenotypic profiling of mutant worms. This must be a standard format in order that direct comparisons can be made between profiles obtained on exposure to compounds and profiles exhibited by mutants. With compound screening it is possible to build up a series of different libraries depending on the compounds being tested. For example one library can comprise profiles generated in respect of each of the known compounds in a Pharmacopoeia, in other words compounds with known pharmacological activity.
  • Another library can comprise profiles generated by compounds known to interact with a particular biochemical pathway, which may or may overlap with those compounds from the Pharmacopoeia.
  • Other libraries could include profiles for known compounds but with no known biological activity or compounds which are completely new molecules such as might be generated by combinatorial chemistry.
  • the present invention is not limited to the production of phenotypic profile libraries but includes libraries or banks of worms whose phenotypic profile has been altered by exposure to compounds.
  • assays may be carried out with several concentrations of the same compound, and/or with mixtures of compounds . For example compounds from compound libraries may each be tested individually or with one or more other influencing compounds.
  • Such compound testing protocols may be executed against identical worms or multiple mutant and/or transgenic backgrounds.
  • a panel of worm strains covering a wide range of biochemical pathways and cellular activities by means of mutations in particular pathways, as well as reporter genes, is used for testing compounds.
  • a profile is recorded for the measurable phenotypes of each of the worm strains, either in parallel or sequentially.
  • the invention provides a method of constructing a library of phenotypic profiles of nematode worms which comprises the steps of:
  • the environmental change may be, for example, a change in pH, osmolarity, temperature, exposure to radiation or exposure to bacteria or viruses.
  • Each of these external influences may result in the manifestation of a different phenotypic profile of characteristics so that libraries of said profiles and affected worms can be constructed. Again, measurements and scoring of the profile should follow a standard protocol in order that valid comparisons can be made between these profiles and those in mutant and compound libraries.
  • the construction of worm and phenotypic profile libraries by the methods described above using the novel phenotypic profiling method described herein provides a very powerful tool for the discovery of new drugs. Profiles in each of the different libraries can be compared and links established between C. elegans genes and pathways, compounds and environmental effects.
  • the process of measuring and scoring the changed characteristics which go to make up the phenotypic profile is automated, making use of technical measuring apparatus.
  • the profiles so generated may advantageously be stored electronically. Libraries of profiles can then be searched by computer which can identify identical or similar profiles, either within or between the different libraries. Quantitative data calculations, optionally in combination with boolean operations can be used. A comparison of the profile generated by a particular compound with the profiles of particular mutants may indicate the likely gene or biochemical pathway with which the compound interacts in the worm. Other databases can then be searched for a match of the worm gene with an equivalent human gene. The human gene might already be associated with a human disease as could be determined for example, from the OMIM database mentioned above. Thus, by use of the worm screen a potential candidate drug can be identified.
  • the discovery of the mode of action of a compound with known pharmacological or biochemical activity is facilitated by comparing its phenotypic profile in the worm with the mutant library or environmental change library of profiles to identify possible targets for the compound, other possibilities include finding a new potential medical indication of a known compound, a medical indication for a novel compound, an alternative method of treatment of a known disease or an indication of the reason for the side effect exhibited by some known pharmaceuticals.
  • Testing worms with compounds, scoring the phenotypic profile in the novel manner described herein and then searching previously established libraries of profiles can potentially achieve all those goals.
  • Once a compound has been identified as having the potential to be a therapeutic agent it can be processed through the more traditional drug discovery routes.
  • the compound can be tested in more specific in vitro tests based on the new knowledge of the target for the compound and in animal models of the target disease. Structural variants then can be generated by medicinal chemistry with a view to improving activity.
  • Figure 1 is a schematic diagram of the left lateral view of the body of C. elegans .
  • the body of C. elegans is divided into a head, a body and a tail region.
  • the head region stops at the end of the pharynx, the body stops at the rectum and the tail includes the tail whipe.
  • C. elegans usually crawl on the right side.
  • the ventral located vulva defines the ventral side of C. elegans .
  • Figure 2 is a schematic diagram of C. elegans showing the characteristics "hypertrophy of the head and "extensions on head”.
  • elegans (L4 stage) per plate is put in the bacterial lawn. Worms are checked after some hours, plates are incubated at 21°C and worms screened for phenotypes (control have LI progeny growing) . Plates are checked again after 4 days for phenotypes of FI progeny (control shows all stages up to gravid hermaphrodites) . Plates which have to be looked at again on subsequent days because of slow growth or for further checks are put aside. A plate protocol sheet such as that shown in Table 2 is completed deciding on one of the following routes: no effect/unspecific effect/needs to be applied at lower concentrations/needs to be profiled.
  • the stock solution is preferred as lOOmM in DMSO and the experiment is started ab ini tio with a concentration series.
  • concentration series is used as described below. In one series of concentrations 15 or so worms (for a reasonable number of short term effects) are placed in the agar. In three series 1 worm each is placed on the agar to score a reasonable number of progeny. Lost worms of the latter three series of concentrations can be replaced from the large pool where worms have been exposed to the compound in the same way.
  • concentrations can be used:
  • a set of compounds from the pharmacopoeia have been profiled using the general protocol (all compounds were of known activity and are described in Martindale: The Complete Drug Reference, 32nd edition, Pharmaceutical Press 1999) .
  • the plate drop assay was compared against standard of pouring compounds into the agar as described in literature which method is designated agar assay.
  • the compounds were added to the worm in a variety of concentrations, and the survival of the worm was scored as well as the phenotypic profile induced by the compound. The lowest concentration of a compound, still resulting in the death of the nematode was designated minimal lethal dose.
  • the maximal concentration of a compound that did not result in the death of the nematode was designated maximal nonlethal dose.
  • the minimal concentration of a compound that still resulted in a measurable phenotype was designated minimal effective dose.
  • the concentrations of the compounds in the agar assay were compared to the concentrations in the drop assay. From this observation one may conclude that the newly described drop assay protocol turns out to be far more efficient for most compounds.
  • Minimal lethal dose rate between the lowest concentration in which the compound is lethal to the worm in both assays Maximal non-lethal dose: rate between the highest concentration in which the compound is not lethal in both assays Minimal effective dose: rate between the lowest concentration in which the compounds results m a phenotype in both assays
  • Worms are examined for viability at all stages of the life cycle, being embryogenesis, larval stages 1 to 4 and adulthood.
  • Dead embryos are defined by not hatching within 24h and dead worms are defined by not moving, by lack of pharynx pumping, by sick or pale appearance and by lack of response to mechanical stimulation.
  • Embryonic lethality is measured by counting the amount of unhatched worms after 24 hours (Elispot, Zeiss) . Counting of unhatched worms could also be automated using the FANS device, described below. Viability of larvae and adults is measured by dye uptake.
  • Life cycle Progeny are examined for the length of the generation cycle in comparison to control progeny (of a wild-type worm) .
  • the stage of a synchronized progeny will be compared to the stage of a synchronized control progeny (N2, Bristol strain) after three days at 20°C.
  • the developmental stages can be distinguished by vulva development, expression of stage-specific markers, such as collagen IV, body length and transparency.
  • stage-specific markers can be examined using antibodies of the appropriate specificity, by way of example an antibody that recognizes an antigen on the surface of C. elegans LI larvae has been described by He mer et al . , (1991) J Cell Biol , 115(5) : 1237-47.
  • Body shape Worm size is determined by measuring worm length and worm diameter.
  • the body length of a synchronized progeny of adult worms is compared to the body length of a synchronized control progeny (N2, Bristol strain) .
  • Measurement of body length can be achieved using a 'worm dispenser apparatus' which is commercially available from Union Biometrica, Inc, Somerville, MA, USA.
  • This apparatus has properties analogous to flow cytometers, such as fluorescence activated cell scanning and sorting devices (FACS) . Accordingly, it may be commonly referred to as a "FANS" apparatus, for fluorescence activated nematode scanning and sorting device (FANS) .
  • the FANS device enables the measurement of properties of microscopic nematodes, such as size, optical density, fluorescence, and luminescence.
  • Body size may also be measured via image analysis, in which case the measurements recorded may include worm diameter and deviation from the typical tube shape of a wild-type worm.
  • Movement behaviour can include measurement of the speed of movement , or of the pattern of movement (e.g. direction) or both.
  • a wild-type worm moves in a sinusoidal way forward and pauses or moves backward occasionally. Any deviation from this wild-type pattern of movement can be scored as a 'changed' characteristic.
  • An assay based on the following principles may be used to determine the speed of movement of a worm culture:
  • Nematode worms that are placed in liquid culture will move in such a way that they maintain a more or less even (or homogeneous) distribution throughout the culture. Nematode worms that are defective in movement will precipitate to the bottom in liquid culture. Due to this characteristic of nematode worms as result of their movement phenotype, it is possible to monitor and detect the difference between nematode worms that move and nematodes that do not move.
  • Advanced multi-well plate readers are able to detect sub-regions of the wells of multi-well plates. By using these plate readers it is possible to take measurements in selected areas of the surface of the wells of the multi-well plates. If the area of measurement is centralized, so that only the middle of the well is measured, a difference in nematode autofluorescence (fluorescence which occurs in the absence of any external marker molecule) can be observed in the wells containing nematodes that move normally as compared to wells containing nematodes that are defective for movement.
  • autofluorescence measurements can be taken in two areas of the surface of the well, one measurement in the centre of the well, and on measurement on the edge of the well. Comparing the two measurements gives analogous results as in the case if only the centre of the well is measured but the additional measurement of the edge of the well results in an extra control and somewhat more distinct results.
  • specialist software such as SIMI Scout (designed for movement study of an athlete) may be used to determine speed of movement, deviation from sinusoidal movement and even the overall pattern of movement of the worm.
  • the phenotypes "Pumping frequency reduced, Pharynx pumping irregular” etc. describe the activity of the cyclic contraction of the pharynx muscles that occurs in a feeding adult about 3 times in a second.
  • the contraction cycle can be descried as the nearly simultaneously contraction of the corpus, anterior isthmus, and terminal bulb, followed by relaxation.
  • Pharynx pumping characteristics may be analyzed by image analysis: The frequency of pumping by counting the pharynx contraction. Pharynx contraction can be measured visibly by the opening and closing of the anterior corpus. The time of opened anterior corpus and the diameter of the opened corpus is used to measure hypercontraction, relaxation and strength of a contraction.
  • the pumping rate of the pharynx is measured indirectly by adding a marker molecule precursor such as calcein- AM to the medium and measuring the formation of marker dye in the C. elegans gut.
  • Calcein-AM is cleaved by esterases present in the C. elegans gut to release calcein, which is a fluorescent molecule.
  • the pumping rate of the pharynx will determine how much medium will enter the gut of the worm, and hence how much calcein-AM will enter the gut of the worm. Therefore by measuring the accumulation of calcein in the nematode gut, detectable by fluorescence, it is possible to determine the pumping rate of the pharynx.
  • a concentration of between 1 and lOO ⁇ M calcein-AM is added into the medium.
  • C. elegans The defecation of C. elegans is a recurrent event comprising of the following steps: pBoc, aBoc and expulsion.
  • Defecation in nematodes such as C. elegans is achieved by periodically activating a defined sequence of muscle contractions. These contractions are started in the anterior body wall muscles. At the zenith of the anterior body contractions the four anal muscles also contract. The four anal or enteric muscles are the two intestinal muscles, the anal depressor and the anal sphincter.
  • specific neurons are also involved in the regulation of defecation, including the motor neurons, AVL and DVB.
  • phenotypic profile In order to construct a phenotypic profile, well-fed adults are typically examined after one day for constipation. The time between two pBocs is also scored.
  • the rate of defecation of C. elegans can also be quantitatively measured using an assay based on the following principles:
  • the rate of defecation of nematodes such as C. elegans can be easily measured using a marker molecule which is sensitive to pH, for example the fluorescent marker BCECF.
  • This marker molecule can be loaded into the C. elegans gut in the form of the precursor BCECF-AM which itself is not fluorescent. If BCECF-AM is added to nematode culture medium in the wells of a multi- well plate the worms will take up the compound which is then cleaved by the esterases present in the C. elegans gut to release BCECF.
  • BCECF fluorescence is sensitive to pH and under the relatively low pH conditions in the gut of C. elegans (pH ⁇ 6) the compound exhibits no or very low fluorescence.
  • the BCECF is expelled into the medium which has a higher pH than the C. elegans gut and the BCECF is therefore fluorescent.
  • the level of BCECF fluorescence in the medium is therefore an indicator of the rate of defecation of the nematodes.
  • a wild-type adult hermaphrodite C. elegans lays about 8 eggs per hour.
  • the amount of eggs laid by 20 hermaphrodite C. elegans during at least 60 min is counted.
  • the amount of eggs may be counted by simple visual inspection or using a FANS device, described above.
  • Mutant worms have been profiled according to the general profile protocol.
  • Table 4 shows a summary of the profile, also called fingerprints, of one mutation of the indicated genes. Entries are binary with empty fields indicating a phenotype (deviation from negative control, here wild-type) not found assuming that it could have been measured. Any other entry including comments or quantitative data is read as measured phenotype in this binary scheme and indicated by *.
  • the table lists only phenotypes that do have a positive entry, not necessarily complete, leaving pages of empty fields alongside and arranged according to a particular enquiry.
  • the upper half consists of the hierarchical categories "dauer formation phenotypes" and "body shape phenotypes” as well as their relevant sub-phenotypes.
  • the lower part consists of a set of hierarchically unrelated phenotypes subsumed under the enquiry categories, "increased activity” and "decreased activity”. The complete list of characteristics is to be found in Table 1.
  • the point of including the lower part is to show the principle of recording all observed phenotypes, that they can be used to distinguish similar phenotypic profiles in detail and that they can be arranged in order to make comparisons. In this case it is seen that the dichotomy of long versus short body length does not correlate to the dichotomy of increased versus decreased activity.
  • the upper part shows 5 genes (i.e. a mutation in that gene) affecting dauer formation as well as 5 genes affecting body shape in a particular combination.
  • a mutation in one gene, daf-4, is unique in sharing the characteristics of both phenotypic groups.
  • the following picture illustrates the phenotypic overlap as found by comparing entries in the phenotypic profiles.
  • the DAF-4 protein probably acts as a type II receptor in both pathways.
  • the similarity of phenotypic profiles allows one to hypothesize mechanistic relationships in a manner analogous to sequence similarity of genes. For example a compound which induces the phenotypes: longer or shorter body length in combination with 2 or 3 of pale, thin and variable egg size, in worms exposed to it, is very likely to act on a protein of the TGF ⁇ pathway.
  • Wild type C. elegans adults have been exposed to acetylcholine esterase inhibitors at various concentrations.
  • the worms have been profiled over two generations, meaning four profiles have been generated.
  • All phenotypes from the phenotype list are displayed that have been measured in this experiment.
  • Two phenotypes “loopy head movement” and “body dragged by head” are shared by most of the esterase inhibitors. This is called phenotype activity relationship (PAR, by analogy to structure activity relationship SAR) .
  • the shared phenotypes are used to identify the action of a new compound.
  • the unshared phenotypes are used to distinguish drugs or unravel side effects when these phenotypes are part of another PAR.
  • unc-4 looks like a snail.
  • the fingerprint of unc-4 adds for "coiler” the details "ventral side out” and “spiralling inwards posteriorly”. This occurs when a set of neurons that control the forward movement of the ventral part of the worm (VA2 - VA10) gets the same input than another set of neurons that controls the backward movement of the ventral part (VB2 - VB10 ) .
  • Wild-type C. elegans adults have been exposed to GABA agonists (Muscimol) and GABA antagonists (Ivermectin and Fipronil) at various concentrations. Worms have been profiled and the scored phenotypes are displayed as fingerprints.
  • the phenotype "shrinker” is present in all fingerprints (see Table dark grey) . This phenotype is used as marker or diagnostic phenotype to identify activity of a compound or gene in the GABAnergic pathway. There are further phenotypes only shared by some compounds and mutants (see Table light grey) . These phenotypes are used to build a phenotype activity relationship (PAR) .
  • PAR phenotype activity relationship
  • the shared phenotypes are used to identify the action of a new compound when "shrinker" cannot be used or to reveal more details on a compound action.
  • all compounds and unc-25 fingerprints contain constipation phenotypes but not the fingerprint of unc-49, although GABA is used for the defecation process. This is coincident with earlier findings that the UNC-49 gene product is not required for defecation.
  • Aberrations of the body shape of C. elegans can be the result of mutations in a vast amount of genes. These genes may be required directly for the formation of the hypodermis, the hydroskeleton and the correct patterning of the worm body plan, e.g., collagen or even-skipped. They could be involved in the control of growth or metabolism like genes of the TGF ⁇ pathway or genes required for feeding. Eventually, mutations in certain genes that cause primary defects, e.g., absence of head muscle, cause secondary defects in the body shape like dystrophy in the head region. Body shape phenotypes are all visible or measurable deviations of the body shape, colour and content.
  • Phenotypes are comparatively measured against wild- type (N2, Bristol strain) and scored as deviation of wild type in the corresponding developmental stage, sex and preparation. The scored phenotype comes with the percentage of worms positive for that phenotype within a population.
  • Table 8 Scientific definition of body shape phenotypes. The phenotypes listed in the left column are described and defined in the right column. Some phenotypes are derived from the classical worm jargon like “dumpy”, which is still shorter than “short and thick worm”.
  • Table 10 list of scientific body shape phenotypes, together with their corresponding technical definitions , in terms of characteristics which can be comparatively measured relative to wild-type characteristics using automated measuring apparatus.
  • C. elegans as described in Table 1 can be easily monitored, either automatically by image analysis, microtiter plate readers, or visual means, e.g. by normal microscopy or by Nomarski microscopy. Some features of C. elegans are more difficult to visualize. For these characteristics transgenic animals expressing a marker gene are very useful. Moreover, even for characteristics that are rather easily to score, the use of a nematode expressing a marker gene, such as GFP, LacZ, or luciferase, enhances the fingerprinting of C. elegans .
  • the C. elegans can be a wild type, a mutant, or a strain subjected to a compound or environmental stress, or a combination of those.
  • C. elegans mutant unc-23 has a fingerprint, which comprises “jerky movement”, “tend to coil”, “bent head” and “egl”. Expressing GFP in the muscle cells of the animal could result in identification and scoring of additional characteristics such as “improperly folded muscles”, and/or “detached muscles in head region”, and/or “no muscles in head region”, and/or “defective muscle attachment", and/or “vulva muscle defects” (data not shown) .
  • C. elegans mutant unc- 71 has a fingerprint which comprise "reduced movement”, “weak amplitude”, “strong kinker”, and “slightly egl”.
  • GFP GFP
  • a closer look at the neurons of this mutant worm revealed at least following extra phenotypes: "fasculation defects", “VD/DC connection defects” (data not shown) .
  • GFP-phenotypes are hence very important in allowing phenotypes which are not otherwise visible to be measurable with Nomarski or dissection microscopy. GFP-phenotypes are further important in the pinpointing of defects to certain tissues and cells, and moreover GFP-phenotypes are important in distinguishing between similar defects with different causes .

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Abstract

L'invention porte sur des méthodes qu'on met en oeuvre pour construire des bibliothèques de profils phénotypiques chez un ver nématode, tel que C. elegans. Les méthodes consistent à mesurer des caractéristiques identifiables du ver et à établir un pointage systématique de ces caractéristiques. L'invention porte en outre sur des méthodes d'identification de composés ayant une activité pharmacologique potentielle pour déterminer le mode d'action d'un composé donné et pour affecter des gènes à des voies biochimiques particulières.
EP99963460A 1998-12-07 1999-12-07 Methode de construction de bibliotheques de profils phenotypiques Withdrawn EP1137754A2 (fr)

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