Newman AP et al. (1992) Worm Breeder's Gazette "The Anchor Cell Induces Specialization in the Ventral Uterus"

Newman AP, Sternberg PW

[Worm Breeder's Gazette, 1992]

The cells of the ventral portion of the C. elegans hermaphrodite uterus result from divisions of three precursors, W1 ,W2 ,and W3 (Kimble and Hirsh, Dev. Biol. 70: 396-417; 1979). Each W cell generates slightly different sets of intermediate precursors of differentiated progeny. One intermediate precursor produced by the W cells (d/v) divides dorsal-ventrally and undergoes one less round of division than the other precursor types. The three W lineages differ in the number of d/v's they produce (VU1, 2 and 3 produce one, two, and three d/v's respectively). Four bipotential cells generate the 3 W cells and the anchor cell; Z1 .pppand Z4 .aaacan each become either an anchor cell or VU3 ,and Z1 .ppaand Z4 .aapcan each become W1 or W2 .Kimble has demonstrated several types of regulation among these cells. Ablation of the presumptive anchor cell results in its replacement by the alternative precursor (which would otherwise become W3 ).Ablation of W3 results in W1 adopting a W2 lineage, while ablation of W1 leads W3 to adopt a novel lineage or change its polarity (Dev. Biol. 87: 286-300; 1981). Results from our ablation experiments suggest that the anchor cell may be necessary for the d/v fate. We ablated the anchor cell during L2 lethargus/ early L3 ,after the time during which anchor cell regulation can occur. We found that all three W cells now follow a "VUO" lineage, in which no d/v's are produced, i.e. all cells undergo the fourth round of division characteristic of non-d/v's (13 VU lineages followed in 6 animals). In presumptive W1 and W2 lineages, all cells appear indistinguishable in terms of size and timing of division. However, in the presumptive W3 lineage, the cells that would normally be d/v's divide later than other cells. When the ablations were done after the first W division (during L3 ),approximately half of the presumptive d/v's underwent an extra round of division (3/3 animals). Therefore, cell fate is not irrevocably determined at the level of the W precursors. We have determined the ventral uterine lineages in highly penetrant vulvaless mutants of let-23 (sy97 )and lin-3 (n3781 n1059 heterozygotes). In neither case was there a defect in d/v fate specification (4/4 animals for each strain). Thus the anchor cell does not appear to use the identical signal transduction pathway in inducing VPCs and d/v's. Furthermore, since d/v's are present in Vul mutants, the W0 lineages observed in anchor cell-ablated animals are presumably not an indirect consequence of the absence of vulval tissue. We are examining animals with VPCs ablated to confirm this result. We infer that a signal from the anchor cell induces specialization of the ventral uterus. We are currently determining the ventral uterine lineages in lin-12 (d),lin-12 (0),and additional mutants to identify potential signaling molecules. We also plan to screen for mutants defective in specification of the d/v fate. [See Figure]

White JG et al. (1975) Worm Breeder's Gazette "Re-wiring of Motor Neurones"

White JG, Thomson JN, Southgate E

[Worm Breeder's Gazette, 1975]

We have been studying the structure of the N2 hermaphrodite ventral and dorsal nerve cords for some time and have recently extended these studies to L1 larvae prior to the post-embryonic development that takes place. The adult has 5 classes of motor neurone in the ventral cord designated A, B, C, AS and D. Classes A, B and D can be divided into two sub-classes; those that innervate the ventral muscles and those that have axones that run in the dorsal cord and innervate the dorsal muscles. Class AS only innervate dorsal muscles and class C only innervate the ventral muscles (predominantly the sex specific muscles). Classes A, B, and AS are driven from interneurones that come from the nerve ring. Class D neurones that innervate the ventral side have a dendritic process on the dorsal which is driven by all the other motor neurones active in its locality. Conversely, dorsal type D receive their innervation from ventral motor neurones and innervate the dorsal body muscles. If all the motor neurones which are known to develop post-embryonically are subtracted one is left with the complement of motor neurones which is present in the L1 larva and these turn out to be dorsal A, B, and D. This seemed paradoxical, but when the nerve cords of an L1 were looked at it was seen that the cells which were dorsal type D's in the adult innervate the ventral side of the L1, receiving their innervation from a dendrite on the dorsal side which is driven by the dorsal type A and B motor neurones. Thus it seems as if these cells 're-wire' in the course of development. We are now trying to find out whether this re-wiring is induced by the arrival of the new motor neurones or is autonomous.

Greenwald IS (1990) Worm Breeder's Gazette "SEQUENCE ANALYSIS OF LIN-12(D) MUTATIONS."

Greenwald IS

[Worm Breeder's Gazette, 1990]

The accompanying figure shows sequence alterations associated with eight lin-12(d) alleles. All eight lin-12(d) mutations examined are missense alterations of the protein coding region and are therefore likely to be modifying protein activity or increasing protein stability. Thus, the fact that lin-12(d) mutations behave like genetic hypermorphs (Greenwald et al., Cell 34: 435 -444,1983) would be consistent with two possibilities: either the lin-12(+) allele contributes a basal level of lin-12 activity that can augment lin-12(d) activity, or the lin-12(d) protein transactivates the lin-12(+) protein. We favor the second possibility, in light of genetic studies suggesting that lin-12 interacts with itself (Geraldine Seydoux and I. G., unpublished observations) and biochemical evidence that Notch can form dimers (Kidd et al., Genes & Dev. 3:1113-1129,1989). Our current favorite working hypothesis is that intercellular signals promote self-association of lin-12, thereby activating a signal transduction mechanism. This model is plausible in view of what is known about biochemically-defined receptors: in many cases, the association state of receptors in the membrane controls their activity, and association state is in turn controlled by ligand-binding ( reviewed in Andersen, Nature 337:12,1989). The lin-12(d) mutations could lead to activation in the absence of signals necessary to activate lin12(+) protein. Consistent with this explanation is the observation that if all gonadal cells except for Z1.ppp or Z4.aaa are ablated in a lin-12(n137) hermaphrodite, it becomes a VU; in contrast, in wild type, it would become an AC, presumably because it lacks a necessary signal (Seydoux and Greenwald, Cell 57:1237-1245,1989). [See Figure 1] Predicted amino acid changes (residue number: change) deduced from sequence analysis of different lin-12(d) alleles are shown on a schematic depiction of the lin-12 product (Yochem et al., Nature 335: 547-550,1988). EGF = EGF-like motifs; T = 'T + Y' encoded motif; LNR = lin12/Notch repeated motif; cdc10 = cdc10/SWI6 motif; C = position of a pair of cysteines that is conserved in lin-12, h. Kidd et al. (1989) propose that these cysteines mediate the dimerization of Notch, and it is curious that they are conserved in a region of the lin-12 protein that shows no overt sequence similarity to Notch.

Ward S et al. (1980) Worm Breeder's Gazette

Ward S, Hogan E, Roberts TM

[Worm Breeder's Gazette, 1980]

A polypeptide which comigrates with worm muscle actin on 1-D and 2-D gels can be isolated from purified sperm. Careful quantitation of the Coomassie blue staining of this peptide using rabbit actin as a standard reveals that this putative actin is only 0.02% of the sperm's protein. It can be detected easily on autoradiograms of 2-D gels of 35s labelled sperm, it is barely detectable on Coomassie blue stained gels, but is strongly stained with the new silver stain. It comigrates exactly with muscle actin on the gel systems we have tried so far. It is probably in the sperm because it is not labelled by external surface labelling. Greg Nelson and Yair Argon found that by indirect immunofluorescence staining of spermatozoa with anti-actin antibodies, only the pseudopods stained. We do not know how it contributes to sperm motility.

Royal DC et al. (1997) Worm Breeder's Gazette "Death Defying Acts I"

Royal DC, Royal MA, Driscoll MA

[Worm Breeder's Gazette, 1997]

mec-4 encodes a candidate subunit of a mechanosensitive ion channel that plays a key role in touch transduction. Unusual dominant mec-4(d) alleles cause the swelling and necrotic-like death of the six touch receptor neurons. Neurodegeneration is thought to be initiated by elevated ion influx allowed by the mutant MEC-4 channel. A time course study of mec-4(d)-inducedneurodegeneration revealed that remarkable infoldings of the plasma membrane occurs, membranous whorls are internalized, vacuoles appear and the chromatin clumps (Hall et al., 1997). To learn more about mechanosensory ion channel function and about mechanisms of degenerative cell death, we are isolating suppressor mutations that block mec-4(d) mediated neurodegeneration. The mutagenesis scheme involves the expression of the green fluorescent protein in the touch receptor neurons under the control of the mec-4 promoter. ZB134, a strain in which a pmec-4GFP construct is integrated into a mec-4(+) background, exhibits 6 brightly fluorescent touch receptor neurons. When a mec-4(d) allele, mec-4(u231), is crossed into this background (strain ZB137), fluorescent touch cells are no longer evident as they have undergone cell death. We mutagenize strain ZB137 and screen for rare animals that have glowing touch cells and thus bear candidate suppressor mutations. To date we have mutagenized and screened 12,000 haploid genomes for death suppressing mutations. Our preliminary screen has resulted in seventeen non-mec-4 alleles that suppress touch cell death. Surprisingly, many of these exert dominant effects. Mapping and determination of linkage groups of the suppressors is underway. We are also testing the three recessive suppressors to determine whether they fall into distinct complementation groups. The recessive suppressors are being tested for complementation with mec-6 (mec-6 is the only known suppressor of mec-4(d)-induced degeneration) and a combinatorial matrix is being constructed to determine which alleles, if any, possess discernible epistatic or synergistic effects. We will also determine the touch sensitivity of the suppressor alleles in the absence of mec-4(d). We are further testing the suppressors for the ability to block the formation of vacuoles induced by expression of human Ab4 peptide (we thank C. Link for providing this strain) in order to help distinguish between general necrotic-like death disrupters and mec-4 channel disrupters. We are optimistic that our suppressors will identify new genes that affect channel function, cellular osmotic balance and necrotic-like cell death.

Wissmann AK et al. (1994) Worm Breeder's Gazette "Characterization of the let-502 Gene"

Mains PE, Wissmann AK, McGhee JD

[Worm Breeder's Gazette, 1994]

Characterization of the let-502 gene Andreas Wissmann, James D. McGhee and Paul E. Mains, Dept. of Medical Biochemistry, University of Calgary, Calgary, Alberta, Canada T2N 4N1

Xu K et al. (1997) Worm Breeder's Gazette "Death Defying Acts II"

Tavernarakis NN, Driscoll MA, Xu K

[Worm Breeder's Gazette, 1997]

mec-4 encodes a subunit of a touch receptor ion channel that is normally needed for mechanosensation. Dominant mec-4 alleles induce swelling and degeneration of the touch cells via a mechanism proposed to involve increased ion import through this channel. Touch receptor degeneration is accompanied by an unusual sequence of subcellular events that includes striking apparent internalization of plasma membrane-like structures and unusual intracellular trafficking/degradative processes. We are interested in identifying genes involved in the degenerative process and in genes needed for function of members of the degenerin family. We have previously reported that ectopic expression of mec-4(d) allele can induce degeneration in several cell types. For example, we have found that expression of mec-4(d) from the unc-8 promoter the toxic causes swelling and degeneration of some interneurons and many ventral cord neurons, resulting in severe paralysis. We are using this behavioral phenotype to identify suppressor mutations that block the deleterious effects of mec-4(d)--suppressors restore normal or near normal locomotion by preventing cell death. We have constructed a strain that harbors an integrated array of punc-8mec-4(d) on chromosome II. These animals are severely paralyzed and exhibit swollen neurons in the nerve ring and in the ventral cord. We have crossed mec-6(u450) into the background of this array to verify that the deleterious effects of ectopically expressed mec-4(d) can be suppressed by a known suppressor mutation. We have mutagenized 20,000 haploid genomes with EMS and identified 16 independent candidate suppressor strains. All proved to retain the integrated punc-8mec-4(d) array. Of these, 7 appear to be new mec-6 alleles. Nine alleles complement mec-6 both for touch insensitivity and death-suppressing defects, thus they may identify previously unknown means of genetically blocking degenerative cell death. Seven of these suppressor alleles have phenotypes independent of the toxic array (either weak Unc or Mec), two alleles do not have an obvious phenotype independent of death suppression. We are currently testing the new alleles to determine if they can suppress death of the touch receptor neurons as assayed in the Is[pmec-4GFP] mec-4(d) background (described by Royal et al., this issue). Thus far, one of the suppressor alleles has been shown to also block mec-4(d)-induced neurodegeneration. We are currently focusing on assigning the novel death-suppressor alleles to linkage groups. Encouraged by the success of this pilot mutagenesis we plan to mutagenize more extensively in the hope of identifying all non-lethal loss-of-function mutations that exhibit death suppressing mutations.

Hough DM et al. (1993) Worm Breeder's Gazette "Search for RXR Homologs in Nematodes"

Hough DM, Maina CV, Richer J

[Worm Breeder's Gazette, 1993]

We are studying the genetic regulation of molting in the nematodes C. elegans and Dirofilaria Immitis through the use of ecdysone and its receptor, EcR (see abstract by Richer et al., this issue). EcR is a member of the nuclear hormone receptor (NHR) superfamily. It requires the presence of ecdysone and another Drosophila protein ultraspiracle, usp, in order to activate transcription. usp, also a member of the NHR superfamily (Yao et al. 1992, Cell 71:63-72), is a homolog of the vertebrate retinoid X receptor (RXR, Oro et al. 1990, Nature 347:298-301). To determine if a nematode EcR behaves as does the Drosophila EcR, we have initiated a search for nematode usp homologs. We have used the Drosophila usp to search for D. immitis and C. elegans homologs using several approaches, the most fruitful being PCR with degenerate primers to the DNA binding domain. Two degenerate primers (C1 and C3 )encoding the amino acids shown in the figure were used in PCR experiments with genomic DNA from both D. immitis and C. elegans. All PCR products were cloned into pUC19 and sequenced. The C1 /C3primer combination amplified 2 DNA fragments from C. elegans (crf-6 and crf-7 ,in keeping with the nomenclature for nematode NHR s established by Sluder et al. WBG 11, #3) and 1 from D. immitis (dirf-4), ranging in size from 137 to 190 bps. The predicted amino acid sequence for each of the 3 shows significant similarity to the NHR superfamily. 1 of the 3 contains a putative intron of 50 bps at the site indicated by the arrow. The sequence of dirf-4 shows 93% similarity at the amino acid level to the Drosophila usp. While the crf-4 NHR shows less similarity to usp (68%), we are pursuing it further because it shows cross hybridization to the D. immitis dirf-4 done and most of the matches in a TFASTA search of GenBank</EMBL are to RXR sequences. Neither of the C. elegans NHR s have been described previously and they have been mapped to the genome (see figure). crf-6 maps to chromosome III and cosmid DF12 .crf-7 shows hybridization to 2 noncontiguous regions of the genome, strong hybridization to chromosome II and weak hybridization to chromosome IV. We have obtained a cDNA for 1, crf-6 ,from a screen of a mixed stage cDNA library (Stratagene). The D. immitis RXR homolog is the first to be described for a nematode and we are presently screening an adult female D. immitis cDNA library (Grandea et al 1989, Mol Biochem Parasitol 35:31-42) for a cDNA copy of this gene.

Crossgrove K et al. (1996) Worm Breeder's Gazette "Search for homologs of Drosophila ecdysone response genes in the parasitic nematode Dirofilaria immitis"

Maina CV, Crossgrove K

[Worm Breeder's Gazette, 1996]

Filariasis afflicts over 300 million people worldwide causing severe and debilitating disease with symptoms such as lymphoedema, elephantiasis and blindness. Filariasis is caused by several species of parasitic nematodes including Brugia malayi, Brugia timori, Wuchereria bancrofti, and Onchocerca volvulus, transmitted to man by mosquitoes or black flies. Knowledge of the molecular mechanisms involved in parasite development may suggest strategies for parasite control. A useful model system for the study of these parasites is the closely related parasite, Dirofilaria immitis, the causative agent of dog heartworm disease. D. immitis undergoes four molts from the microfilarial to the adult stage. Many insects undergo a superficially similar series of molts during their development. The steroid hormone 20-hydroxyecdysone (hereafter referred to as ecdysone) has a well-characterized developmental role in insects, where it is necessary for molting and metamorphosis. The molecular basis of this response during metamorphosis has been extensively studied and many of the relevant genes have been cloned (reviewed in Andres and Thummel. 1992, TIG 8:132-138). Briefly, ecdysone exerts its effect by binding to the functional ecdysone receptor, which is composed of a heterodimer between the Ecdysone Receptor and Ultraspiracle gene products. This complex activates a small set of early genes, many of which also encode transcription factors. These transcription factors negatively autoregulate their own expression as well as activating a large set of late genes which are thought to encode the structural proteins responsible for metamorphosis. Ecdysone has also been shown to have an effect on the development of D. immitis. Specifically, molting from the L3 larva, the infective form, to the L4 larva can be prematurely induced in culture by ecdysone (Warbrick et al. 1993, Parasitology 107: 459-463). Homologs of both the ecdysone receptor and ultraspiracle genes have been identified in D. immitis (Richer et al. 1993, WBG 13: 86; Hough et al. 1993, WBG 13:87). In order to determine how much of the gene hierarchy is conserved between Drosophila and D. immitis, we initiated a search for homologs of known Drosophila early genes. A number of these genes are members of the nuclear hormone receptor (NHR) superfamily. NHRs are thought to play a role in the development of a wide variety of multicellular organisms. Over 40 putative NHRs have been identified in C. elegans so far. We used degenerate primers in the highly conserved DNA binding domain to perform PCR on D. immitis genomic DNA. Primers derived from the E75 and E78 ecdysone primary response genes were successful in identifying putative homologs in D. immitis. The E75 homolog, dinhr6, exhibits 83% amino acid identity to Drosophila E75 in the DNA binding domain (Figure 1A). The E78 homolog, dinhr7, exhibits 83% amino acid identity to Drosophila E78 in the DNA binding domain (Figure 1B). In addition, a C. elegans E78 homolog, CNR14, has been identified (Kostrouch et al. 1995, PNAS 92: 156-159) which exhibits 79% identity to E78 and 71% identity to dinhr7 in the DNA binding domain. It is interesting to note that CNR14 and dinhr7 are more closely related to Drosophila E78 than to each other. The presence of these genes in D. immitis and in C. elegans supports the ever expanding role of both nuclear hormone receptors and steroid-regulated gene cascades in development. We are currently cloning full-length cDNAs for dinhr6 and dinhr7, determining their expression patterns and asking whether they are responsive to ecdysone in D. immitis. Figure 1: Alignment of dinhr6 and dinhr7 DNA binding domains to Drosophila E75 and E78. A. E75 CGDKASGFHYGVHSCEGCKGFFRRSIQQKIQYRPCTKNQQCSILRINRNRCQYCRLKKCIAVGM ||| |||||||| ||||||||||||||||||||||| ||| | |||||| ||| ||| ||| dinhr6 CGDRASGFHYGVFACEGCKGFFRRSIQQKIQYRPCTKSQQCIVARNNRNRCQHCRLQKCIRVGM B. E78 CKVCGDKASGYHYGVTSCEGCKGFFRRSIQKQIEYRCLRDGKCLVIRLNRNRCQYCRFKKCLSAGM ||||||| || ||||| ||||||||||||||| |||||||||| |||||||| ||| ||| || dinhr7 CKVCGDKSSGFHYGVTACEGCKGFFRRSIQKQMEYRCLRDGKCHIHRLNRNRCQFCRFRKCLAVGM

Kinnell M et al. (1993) Worm Breeder's Gazette "Killer on the Loose: Ectopic Expression of mec-4(d)"

Fire A, Driscoll MA, Singh H, Xu SQ, Kinnell M

[Worm Breeder's Gazette, 1993]

mec-4 mutations that encode substitutions of large side chain amino acids for a conserved Ala (position 442 in the truncated mec-4 cDNA clone) induce swelling and degeneration of the touch receptor neurons, the cells in which mec-4 is normally expressed. We have begun testing the effects of ectopic expression of mec-4 (d)by fusing mec-4 cassettes to various C. elegans promoters. The Genomic mec-4 cassette. Our deletion analysis of the mec-4 gene established that removal of the first 1066 nucleotides from the 5' end of our published genomic sequence abolishes function of the gene in complementation assays. This deletion removes all S' regulatory sequences and the beginning of the putative coding region such that the first 19 amino acids of Mec 4 would be missing if the protein was translated. Expression of mec-4 alleles from the mec-7 promoter. We first used the mec-7 promoter to demonstrate that genomic mec-4 sequences after nucleotide 1066 can function as a mec-4 cassette. The mec-7 gene is expressed at high levels in the touch cells and at lower levels in the PVD cells, the ELP cells, the BDU cells, a pair of cells in the ventral ganglion and a few cells in the tail (Hamelin et al., EMBO J 11:2885, 1992; M. Chalfie and colleagues, pers. comm.). Placing the mec-7 promoter in front of mec-4 ^1066restores mec-4 expression of the mec-7 mec-4 (+)fusion gene complements mec-4 (r) mutations. Note that this implies that the first 19 amino acids of Mec-4 are dispensable for function of this protein in the touch cells. A mec-4 (d)cassette fused to the mec-7 promoter induces death of the touch receptor neurons and the other cells in which mec-7 is expressed. The observed ectopic killing of some of these cells was in part expressed from results of studies of double mutants of mec-4 (d)and egl-44 ,egl-46 and sem-4 (M Chalfie and colleagues, pers. comm.) Expression of mec-4 (d) from a heat shock promoter. We have fused the mec-4 (d)cassette to the hsp16 /2promoter and examined animals transformed with this gene for degenerations induced after heat shock Animals observed after four hours of recovery from four hours of heat shock at 34 exhibited many dying cells throughout their bodies. Eggs also exhibit many swollen cells. Because transformants are mosaic for the presence of the fusion gene it is not clear what cells, if any, are refractory to the deleterious effects of mec-4 (d).We have constructed several integrated lines that we can now use to study the types of cells that suffer degenerative death. These lines are fairly sickly even when reared at low temperature. Expression of mec-4 (d) from the unc-54 promoter. In order to examine the consequences of expression of mec-4 (d)in muscle cells, we have fused the gene to the unc-54 promoter. In control experiments with a unc-54 p-mec-4 /lacZfusion, we observe -galactosidase in muscle but not in touch cells. In lines expressing mec-4 (d)from the unc-54 promoter we observe a range of phenotypes: animals that roll as larvae often become nonrollers (rol-6 (su1006)is coinjected in these experiments), some animals appear hypercontracted and some animals bag. A clearlike phenotype is often observed--the gut remains prominent, but the gonad and muscles appear transparent (if they are there at all). At higher magnifications we can see many small vacuoles at positions consistent with body wall muscle. Thus, it appears that muscles expressing mec4 (d)contain multiple small vacuoles rather than the single large swelling exhibited by degenerating neurons. We are using antibody staining to clarify what happens to the muscle. We conclude that ectopic mec-4 (d)expression can have drastic consequences for multiple cell types. mec4 (d)may prove generally useful for eliminating the function of cells for which regulated cell-specific promoters have been characterized.