Hope IA (1999) "Background on Caenorhabditis elegans."

Hope IA

[1999]

Hope IA (1994) "Caenorhabditis elegans, the Nematode Worm."

Hope IA

[1994]

The current interest in the nematode Caenorhabditis elegans began approximately 25 years ago when Sidney Brenner selected this species as the most suitable for studies of metazoan development and nervous system. The basis of this selection rested on the anatomical simplicity of nematodes, which nevertheless possess the major differentiated cell types of higher animals, and the tractability of C. elegans to the genetic approach. Over the past two decades or so, progress has been impressive: the cell lineage from egg to adult and the anatomy of the nervous system have been completely described, genetic investigations of numerous developmental problems are co-ordinated within a universally-agreed, systematic nomenclature, a physical map of the C. elegans genome is nearing completion and a project to sequence the entire genome is underway. Furthermore, the number of laboratories seeking to understand the mechanisms controlling animal development through genetic and molecular investigations of C. elegans is rising rapidly as the advantages of this organism become

Hope IA (1999) "C. elegans. A Practical Approach."

Hope IA

[1999]

Hope IA (1991) Development "'Promoter trapping' in Caenorhabditis elegans."

Hope IA

[Development, 1991]

A screen of gene expression patterns has been developed for the nematode Caenorhabditis elegans. Promoter-reporter gene fusions were constructed in vitro by ligating C. elegans genomic DNA fragments upstream of a lacZ gene. Patterns of beta-galactosidase expression were examined by histochemical staining of C. elegans lines transformed with the constructs. beta-galactosidase expression depended on translational fusion, so constructs were assayed in large pools to expedite detection of the low proportion that were active. Expression in a variety of cell types and temporal patterns was observed with different construct pools. The most striking expression patterns were obtained when the beta-galactosidase activity was localized to subcellular structures by the C. elegans portion of the fusion protein. The active constructs of three selected pools were identified subsequently by an efficient combinatorial procedure. The genomic locations of the DNA fragments from the active constructs were determined and appear to define previously uncharacterized genetic loci.

Hope IA (1996) European Worm Meeting "EXPRESSION OF PES-1/LACZ IN ISOLATED BLASTOMERES."

Hope IA

[European Worm Meeting, 1996]

The pes-1/lacZ fusion gene expresses b-galactosidase in the early C.elegans embryo; within part of the AB cell lineage (28-200 cell embryo), in the D cell lineage (100-550 cell embryo) and in Z1/Z4 (late embryogenesis from the start of elongation). To probe the mechanisms generating this expression pattern, and thereby the mechanisms for cell fate specification in the early embryo, pes-1/lacZ expression was examined in isolated blastomeres. Embryos were prepared from the C.elegans strain UL1 which has the pes-1/lacZ fusion gene chromosomally integrated. AB and P1 were separated and incubated at 20oC, usually for 4 hours, before staining for b-galactosidase. EMS and P2 were isolated from a divided P1. C and P3 were isolated from a divided P2. Isolated cells were only considered viable if they demonstrated appropriately timed cell division. No pes-1/lacZ expression was seen either for isolated AB or isolated EMS. However, placing MS up against an isolated AB cell cluster induced expression in AB derived cells. This is consistent with previous observations that the AB component of the pes-1/lacZ expression is dependent on glp-1 activity. pes-1/lacZ expression was detected in cells derived from isolated P1 and isolated P2 but before expression in the D lineage would have been expected. An isolated C cell that had divided to give 4 cells also showed expression, whereas an isolated P3 cell that had divided to give 3 cells (P4, Da and Dp) failed to show expression. The position of cells expressing pes-1/lacZ in isolated P1 and P2 was also more consistent with expression within the C lineage than within the D lineage. These observations suggest that the C lineage would express pes-1/lacZ autonomously but a signal, possibly from cells of the AB lineage, prevents this expression in the intact embryo. The reliability of these experiments was much higher than had been expected based on results of similar experiments of others using differentiation markers expressed later during C.elegans embryogenesis. Perhaps this is because the early pes-1/lacZ expression is dependent on relatively few regulatory steps. Further experiments are underway to examine; i. the timing of the MS induction of pes-1/lacZ expression in the AB cell lineage, ii. the autonomy of pes-1/lacZ expression in the D cell lineage, and iii. the inhibition of pes-1/lacZ expression in the C cell lineage in the intact embryo.

Hope IA et al. (1988) Worm Breeder's Gazette "transformation to the rescue."

Thierry-Mieg D, Hoskins R, Hope IA, Sulston JE, La Volpe A, Spence AM

[Worm Breeder's Gazette, 1988]

In order to make good use of the genome map (see update, this issue), we need an efficient means for searching out loci that have been shown genetically to lie within a limited region. Transformation rescue, developed by A. Fire (EMBO J. 5(10) pp.2673-2680 1986), offers a potentially more powerful, and also more stringent, identification of loci than has previously been available. It is, of course, also central to the identification of functional elements by reverse genetic analysis (eg Fire, WBG 10(1)). Since hearing from Andy Fire that he had been successful in obtaining transformants with DNA minipreps, we have been using a very simple protocol to prepare DNA for injection. Cosmid DNA is extracted from 2ml bacterial culture by a standard alkaline lysis procedure, ethanol precipitated and redissolved in 200 l TE. An equal volume of 4.4M LiCl is added, and the mixture is left on ice for an hour. RNA and other contaminants are pelleted by centrifugation and the DNA is precipitated from the supernatant with two volumes of ethanol. The DNA is reprecipitated from 100mM KOAc (Fire,1986) and dissolved in 10 l of injection solution. The size of the cosmid DNA should always be checked on an agarose gel to guard against large deletions. We use the injection set-up and procedure of Fire (1986). We are following his advice in injecting under paraffin oil rather than Voltalef. We find that it is unnecessary to have Lucifer Yellow in the injection solution as successful injection is easily visible with Nomarski optics. We periodically verify that the needle is flowing by touching it to the agarose pad. It is possible to obtain rescued animals by injecting nuclei or by actively avoiding them, but we do not know what influence the site of injection has on the transmission of the rescuing DNA. We have observed rescue arising in the offspring of unrescued progeny of injected animals. Whenever possible, when searching for a gene, we inject a set of cosmids which overlap to the extent that each region is represented in two cosmids. This should control against the presence of small rearrangements in any given cosmid and increase the chance of having the entire gene on at least one cosmid. Our experience is admittedly limited, and some mutants may well prove difficult to rescue, but we have not yet encountered a gene refractory to transformation or a DNA clone which is known to span a gene and yet fails to rescue mutants in that gene. It is worth pointing out that unc-32 and unc-30 are, as far as we know, the first cases of nematode genes being cloned on the basis of genetics, physical linkage, and rescue, without the involvement of polymorphisms in mutants. As the physical map becomes more crowded, this approach to cloning loci can be expected to become increasingly general and convenient. [See Figure 1]

Hope IA (1991) International C. elegans Meeting "'PROMOTER TRAPPING' IN C. ELEGANS."

Hope IA

[International C. elegans Meeting, 1991]

An approach has been developed for screening gene expression patterns in C.elegans. C.elegans is particulariy suited to this type of approach because patterns of gene expression can be fully analyzed at cellular resolution. Initial attempts with strategies related to Drosophila enhancer trapping were unsuccessful, so promoter/reporter fusions were constructed in vitro by ligating random C.elegans genomic DNA fragments upstream of lacZ. Expression patterns were assayed after transformation of C.elegans with the plasmid constructs. Seventeen expression patterns have been obtained so far in the initial screen. , B-galactosidase expression has been observed in muscle cells, nerve cells, cells of the gonad, hypodermal cells and undifferentiated cells. Examples of expression have also been observed from early in embryogenesis, throughout development to the adult. -galactosidase expression depends on translational fusion and for some constructs the C.elegans encoded portion of the fusion protein appears to localize the -galactosidase activity to specific subcellular structures. The genomic origins of three tagged genes were determined using the C. elegans physical genome map and appear to define previously uncharacterized genetic loci. One of the early embryonic expression patterns has been defined with cellular resolution. Expression begins in a subset of AB descendants as the AB lineage divides in the 28/44 cell embryo and -galactosidase activity persists for a couple of cell divisions. This observation reveals that AB descendants have distinct identities at an early stage of development. -galactosidase activity is also detected in all descendants of the founder cell D as the D lineage increases from 2 to 16 cells. From the beginning of elongation until shortly after hatching expression is restricted to Zl and Z4, the somatic gonad precursors. No subsequent expression was detected. The mechanism by which this intriguingly complex expression pattern is generated will be pursued.

Hope IA (1990) Worm Breeder's Gazette "A screen of patterns of gene expression in C. elegans."

Hope IA

[Worm Breeder's Gazette, 1990]

One way to screen patterns of expression for a large number of genes might be to place a reporter, whose expression can be easily monitored, under the control of the promoter elements of many individual genes. Attempts (over the preceding 2 years) to generate such fusions in C. elegans through transposition of Tc1 and derivatives had failed. So instead, fusions were created in vitro using conventional molecular procedures and patterns of expression assayed after germ-line transformation of C. elegans.A library of potential promoter fusions was constructed by ligating 5-10 kb partial Sau3A C. elegans genomic DNA fragments into the BamHI site of pPD22.11 (an Andy Fire vector). The lacZ gene of this vector lacks promoter elements so expression of - galactosidase should depend on promoter elements within the insert and translational fusion with a coding region reading out from the insert. Therefore only 1 in about 120 inserts (about 5% coding, 3 frames, 2 orientations) was expected to drive -galactosidase expression. So, DNA pools each prepared from 96 independent clones were introduced into C. elegans by syncytial injection and rol-6 selection, which gives a high rate of co-transformation (as described by Craig Mello). (Pooled DNA at 180 g/ml, pRF4 at 20 g/ml). Transmitting lines were established before staining for -galactosidase using a modification of Andy Fire's procedure (see below). Patterns of expression observed reproducibly so far (31 pools examined): Pool 2. staining in the nerve ring observed post- embryonically. Pool 4. Nuclear localized staining in adult body wall, probably a subset of muscle cells. Pool 9. Cytoplasmic staining of cells forming the uterus, appearing as the gonad develops, L4 onwards. Pool 23. Staining of the tips of head and tail in adult. Pool 24. Nuclear localized staining, subset of cells (maximally <20) in embryos at approximately the 50-200 cell stage. Initially thought expression turned off completely in later embryos but -galactosidase may persist until L1 in 2 cells (possibly Z1 and Z4). Pool 26. Nuclear localized, 2 cells in rectal region (possibly blast cells F and U) observed post- embryonically. Pool 29. Nuclear localized staining in ventral cord observed post-embryonically. Pool 38. Staining of the spermathecal valve (L4 onwards) and the intestinal-rectal valve (late embryo onwards). Staining is subcellularly localized in an annulus, possibly the filamentous structure seen by EM. Pool 42. A ring of staining in the nerve ring observed post-embryonically. (Note; pPD22.11 lacZ has a nuclear localization signal, though this signal may be overridden for some translational fusions). 14 pools gave expression within the pharynx, many in distinct patterns. This high rate suggests that in these cases -galactosidase is not being expressed to accurately reflect the pattern of expression of a gene to which lacZ has been fused. Many explanations are possible, but at least for the moment these pools have been put aside. To identify the individual plasmids responsible for the expression patterns of pools 2, 4, 9 and 24, further DNA pools were prepared. The original clones had been picked into 8x12 multi well plates. Now, DNA pools were prepared for all clones in each row and each column of all 4 multiwell plates, generating 12 new pools of 32 clones and 8 new pools of 48 clones. Each new pool was assayed as described above. Each pattern observed for the original pools should be observed for one row and one column, unambiguously identifying the individual plasmid responsible. This was successful for pools 4, 9 and 24, but not for pool 2. Transformed lines have been established using the identified plasmids or pools 4 and 24, (not yet for pool 9), and stain with the appropriate pattern. The locations on the genomic physical map of the DNA inserts for the 3 identified plasmids have been determined by a combination of probing a 'YAC polytene grid (provided by Alan Coulson) and fingerprinting (carried out by Ratna Shownkeen). None coincide with a likely genetic locus. Comments: No constitutive expression has been observed yet. Perhaps a weak, cryptic enhancer on the vector plasmid causes constitutive or incomplete promoters to give -galactosidase expression only within the pharynx, hence the preponderance of pharyngeal patterns. (Other explanations possible). The non-pharyngeal patterns observed probably accurately reflect the expression of the genes to which lacZ has been fused because translational fusion should be necessary for expression. However any enhancers downstream from the point of fusion will have been lost. Confirmation of expression patterns will require independent work with the identified genes. This screen is labor intensive, particularly compared to the approaches that had been initially intended utilizing Tc1. However, the first 22 pools were screened in one month and interesting patterns of expression have already been obtained. A larger screen has just begun. Staining procedure: 3 l aliquots of worms were placed in each well of an 8 well multi-well microscope slide, a coverslip was applied and the worms frozen on dry ice. The coverslip was flicked off and the slide placed in acetone, 5 min. -20 C. Worms were then air dried and mounted in 25 l/slide of staining solution under a coverslip sealed on with nail varnish. This procedure is convenient for examining large numbers of lines but preservation of structure is variable.

Hope IA (2001) Trends Genet "Broadcast interference - functional genomics."

Hope IA

[Trends Genet, 2001]

Four recent papers mark a major shift in functional genomic analysis for multicellular organisms. RNA-mediated interference was applied to inactivate individual genes systematically on a genomic scale. These studies subjected a third of the genes in the genome of Caenorhabditis elegans to reverse genetic analysis.

Hope IA et al. (1995) Worm Breeder's Gazette "Request For Gene Expression Pattern Information for ACEDB."

Durbin RM, Martinelli SD, Hope IA

[Worm Breeder's Gazette, 1995]

Request for gene expression pattern information for ACEDB.