Newman AP et al. (1996) Worm Breeder's Gazette "Morphogenesis of the hermaphrodite uterus."

Newman AP, Sternberg PW, White JG

[Worm Breeder's Gazette, 1996]

We have followed the uterine nuclei from the completion of the lineages in early L4 (Kimble and Hirsh, 1979) through the beginning of L4 lethargus. Differentiated uterine cell types have been defined by EM reconstruction (White, Southgate, and Kershaw, WBG 11(3) p.75); they are the toroidal ut cells that make structural epithelium and specialized utse and uv cells that make the connection between the uterus and the vulva. Comparison of Nomarski and EM data enabled us to correlate differentiated fate with lineal position. The 60 uterine cells are produced by the dorsal uterine (DU) and ventral uterine (W) precursor cells. The anchor cell (AC) induces adjacent W granddaughters (which are intermediate precursor cells) to adopt the fate rather than the ground state p (Newman, White, and Sternberg, Development 1995). For convenience, we have assigned names to the DU and W great grandprogeny: Dl-8 (from anterior to posterior) and Vl-12 (see diagram). The cells that contribute to each of the differentiated cell types are as follows:

Wright DJ et al. (1976) Worm Breeder's Gazette "acetylcholinesterase in C elegans."

Awan FA, Wright DJ

[Worm Breeder's Gazette, 1976]

Staining of small nematodes for cholinesterase activity gives very inconsistent results due to poor penetration of the reagents, notably the substrate. Although formaldehyde-fixation and cutting of the nematodes can help, staining is still too unreliable for substrate specificity-inhibitor studies. We have used mild ultrasonic treatment of the nematodes to improve staining consistency in wild-type C. elegans and four other species . C. elegans were sonicated for 20 seconds in distilled water (15 cubic cm) at 12 m (probe dia. 9mm) and then fixed in cold formalin for 2 hours. Worms severed just below the basal bulb were stained by the 'direct-coloring' thiocholine method . Under these conditions 80 of the nematodes showed intense staining in the region of the nerve ring which was identified as due to AChE (E.C.3.1.1.7) activity. Similar results were obtained with Panagrellus redivivus, Prionchulus punctatus, Aphelenchus avenae and Ditylenchus dipsaci, although the tylenchids required more rigorous ultrasonic treatments. The ultrasonic treatments used do not appear to change the structure of the pharyngeal region at the light microscope level (or at the EM level in P. redivivus). Further work is proceeding on the EM localization of AChE in the pharyngeal region of C. elegans.

Yasuda H et al. (1990) Worm Breeder's Gazette "Trans-splicing in Tubulin?"

Siddiqui SS, Yasuda H

[Worm Breeder's Gazette, 1990]

At the last worm meeting (CSH, 1989), we reported the nucleotide sequence of the alpha-1 tubulin gene AT3 that was cloned by direct screening of a C. elegans cDNA expression library in a lambda gt11 vector (Bob Barstead), using a tubulin specific monoclonal anti-body 3A5 (provided by M. Fuller). This gene was previously identified by Linda Gremke using a chicken tubulin probe (Don Cleveland). Our clone SQ#TbA77 was cytologically mapped on chromosome I of C. elegans (Alan Coulson and John Sulston, MRC, LMB, UK). As Dr. Tom Blumenthal walked by our poster at the meeting, he pointed out the presence of an eight nucleotide non-coding sequence TTCAGGTT, which corresponds to the acceptor sequence of C. elegans in the trans-splicing reaction of mRNA. This is a rather novel feature for the tubulin gene, as the other alpha or beta tubulin genes, from C. elegans or other metazoans have not been reported to be trans-spliced at the mRNA level. We are currently trying to sequence the mRNA to test if this mRNA is indeed spliced. Figure below shows the physical map of the alpha-1 tubulin gene, and the nucleotide sequence showing the acceptor sequence ( marked by arrowed box), in the non-coding sequence of the tubulin gene. [See Figure 1]

Klass MR (1980) Worm Breeder's Gazette "More on Sperm Protein 15K"

Klass MR

[Worm Breeder's Gazette, 1980]

The major sperm protein found in isolated sperm from C. elegans males is a 15,000 mwt monomer with a 30,000 dimer form and PI of 8.6. Using an affinity purified antibody preparation it was shown to be specific to sperm. Antibody localization shows 15K in clusters around the inside perimeter of the cell. It is not found in the pseudopodia. PQ1Y A mRNA coding for 15K is first detected immediately prior to the time of synthesis of 15K and we are now investigating the possibility of transcriptional control using actinomycin and alpha amanitin. We have attempted EM localization using the antibody but have encountered some problems. One of them is that 15K is so soluble that it appears if sperm are fixed in a concentrated form with 2% glutoraldehyde that 15K is fixed to the outside of the sperm and all the antibody reaction takes place there. We have also had some permeability problems and are using techniques to alleviate.

Miller A et al. (1998) Worm Breeder's Gazette "A microwave fixation protocol for immunoEM"

Miller A, Hall DH

[Worm Breeder's Gazette, 1998]

EM methods for C. elegans generally require the specimens to be cut open with a razor blade during the primary fixation in order to allow the fixative solution to get past the cuticle. Embryos have been particularly difficult to process, as they are too small to dissect mechanically. Microwave energy somehow enhances penetration of fixatives in many EM protocols (1), and has been shown to be helpful in fixing intact adult C. elegans (2). We have tested a new microwave fixation protocol to improve preservation of uncut animals at all stages for immunoEM, where there is need to achieve uniform processing of hundreds of individual specimens at once. Using a specialized microwave oven (Ted Pella) with adjustable energy levels and programmable pulse regimes, aldehyde fixation is achieved within minutes. To preserve antigenic sites, specimens are chilled during irradiation. A 1 cc sample in a glass multiwell dish is positioned at a microwave "hotspot", bathing the dish in 100 cc of ice. A beaker holds a large water load (600 cc) away from the sample. Microwave energy is set at the 20% or 50% level, with a regime of 2 min ON, 2 min OFF, etc. repeated over total period of ten minutes. The ice load rechills the sample between each microwave cycle, so that the worms never become heated above 10oC. After aldehyde fixation, samples are washed in cold buffer, and embedded into agarose as a dense "pellet" just before the agarose sets (Seaplaque or Sigma type VII agarose; gelling temp is 30oC). After the agarose cures at 4oC overnight, small blocks are cut out, dehydrated and embedded into LR Gold resin. Specimens are placed into gelatin capsules and cured under UV in a Pella cryochamber at -20oC as described previously (3). Thin sections of the pellet are collected onto nickel mesh grids and treated with primary and gold-linked secondary antibodies. Adults, larval stages and embryos are adequately preserved and also become better embedded than without the microwave treatment. We use different buffered aldehyde mixtures, depending upon antigen sensitivity (3).

Oliver Hobert et al. (2002) Worm Breeder's Gazette "Biased asymmetry of the RID motor axon"

Hannes Buelow, Oliver Hobert, Adam S Wenick

[Worm Breeder's Gazette, 2002]

The nervous system of C.elegans displays significant patterns of bilateral symmetry (Hobert et al., 2002). Deviations from this symmetry are usually highly stereotyped. For example, the unilateral RIS neuron is always located on the left side of the animal, but never on the right side (White et al., 1986; this observation, based on a limited number of EM-reconstructed animals, has now been confirmed with gfp reporters). One major exception to this stereotyped laterality is represented by the expression of the putative odorant receptor gene str-2, which occurs stochastically in either the left or right AWC neuron (the distribution is about 50:50; Troemel, et al., 1999). Are there other examples of stochastic laterality in the nervous system? In their EM reconstruction work, White et al. (1986) noted that the unilateral RID motor neuron (see Fig.), whose cell body is located on the dorsal midline, sends its axon along the right side of the nerve ring in one reconstructed animal, but along the left side in another reconstructed animal. Could this observation reflect a stochastic choice of the growth cone when encountering the dorsal side of the nerve ring? The sample size of two obviously did not allow one to derive such a statement, but the advent of gfp reporter technology allows us to address this question. Several previously described gfp reporter strains show RID expression, yet the presence of GFP in the axons of many other neurons precluded the tracing of the RID axon. A promoter fragment from a putative serotonin-like receptor gene that we study in the lab drives expression of gfp in RID and only one additional pair of neurons, allowing us to trace the axon of RID. Taking advantage of this, we found that in 56 out of 58 animals, the axon traveled along the left side of the nerve ring, while in 2/58 animals, the axon migrated on the right side. In another genetic background in which a gfp marker is ectopically expressed in RID (Is[unc-119::ttx-3; ttx-3::gfp]), we found a similar distribution; namely 46 out of 48 animals have their RID axon on the left side.

(1982) Worm Breeder's Gazette "Goettingen worm group activities 1981 and prospects 1982"

[Worm Breeder's Gazette, 1982]

After Gunter von Ehrenstein's death of a heart attack December 26, 1980, the 6 directors of the Max-Planck-Institute for Experimental Medicine decided to close the Department of Molecular Biology as of the end of 1982. We are continuing work to finish up and publish work in progress with gradual attrition as people find new positions elsewhere and with correspondingly diminishing budget. A list of our recent papers and manuscripts is given below. Eddi Isnenghi completed his paper on the phenotypes of the Gottingen emb mutants (including TSP's and maternal tests) and is trying to microinject cloned DNA to rescue worm mutants. He is also trying to transform yeast mutants with worm DNA. Randy Cassada is still trying to map embs on LG III with deficiencies and duplications and is looking for new strains of C. elegans in the wild. Eddi has received a 6-months' extension of his DFG (= NSF) fellowship until July. Ken Denich is writing up his work on emb mutant lineages in Gottingen for a paper and as PhD Thesis for the Department of Zoology, University of Manitoba and will be returning there this summer to finish up PhD requirements. Einhard Schierenberg has continued with laser-induced cell fusion experiments. If the membrane between 2 cells is disrupted at the right time, the division of both nuclei results in 4 more or less normal cells. Development continues to a twitching lima bean monster. Fusing cells in later stages results in uncoordinated animals. All treated embryos show timing defects in the E-cell line similar to emb mutants, perhaps due to remixing of cytoplasm. A copy of Einhard's film on embryogenesis is available (with commentary) from Scott Emmons for those who want to show it to students. Einhard is going to Bill Wood's lab this fall. Khosro Radnia has left for a job in industry. Ulli Certa and Franz Scharfenberg are characterizing histone H2A variants in emb mutants. They have also identified several histone H4 DNA clones in a C. elegans genomic library using a sea urchin probe obtained from Mike Grunstein. Ulli has finished his PhD and plans to go to Grunstein's lab at UCLA this spring for a post- doc on yeast histones. Ulli and the other gene cloners have been working successfully with similar minded members of the Institute's Department of Chemistry. Franz hopes to do his PhD in that department. Shahid Siddiqui has just joined Joe Culotti at Northwestern. Tom Cole is very happy with his new Zeiss EM 109 electron microscope. Our computer-aided reconstruction program with color display now works for LM and EM input. We are trying to transfer the programs and data to the MRC, so they will still be available after our department has closed. Chris Carlson has gone to the EMBL in Heidelberg as of Jan 1 to work on computer graphics of molecules. Ursula Reuter also has a position at EMBL as of Jan 1 as an EM technician. Ed Hedgecock visited for 3 weeks this fall to show us microinjection, antibody staining (with anti-actin from Mary Osborne and Klaus Weber), post- embryonic lineage techniques and the like. It was a very useful and pleasant visit. We hope to have some of you as visitors in 1982!

Yasuda H et al. (1988) Worm Breeder's Gazette "a tubulin tale from Toyohashi: two genes one polypedtide."

Siddiqui SS, Yasuda H

[Worm Breeder's Gazette, 1988]

Previously we have described our efforts to clone tubulin genes by direct screening of C. elegans expression libraries using nerve specific anti-tubulin antibodies(WBG 10, 1:59, 1987). For alpha- tubulin genes, 14 lambda gt11 clones were identified with a monoclonal antibody 3A5 (kindly given by M. Fuller), which is specific for alpha- tubulin and stains C. elegans neurons. Two of the 14 clones were subcloned in pUC 118, and sequenced by dideoxy method of Sanger and Coulson. One of these clones SQ#TbA77 (insert size 1.0 Kb) contained a DNA which is 79% homologous to the chicken alpha-tubulin DNA sequence; and its amino acid sequence deduced from DNA sequence is 88% homologous to the C-terminal region of the chicken alpha-tubulin. The second putative clone on sequencing revealed DNA that has no homology with tubulin DNA. Similarly, we have identified 15 lambda gt11 clones using E6B6, ( kind gift of T. Arai) a monoclonal antibody which is specific for alpha-tubulin and also stains C. elegans nervous system. Three of the 15 clones were subcloned and sequenced as stated above. One of these SQ#TbB03 has a sequence 76% homologous to the chicken tubulin DNA. We compared the nucleotide sequence of SQ#TbB03 with Nw#B8, the only other -tubulin cloned and sequenced in C. elegans (L. Gremke, Ph.D. Thesis,1987). The two nucleotide sequences and the deduced amino acid sequence is given below. It is quite remarkable that the two -tubulin genes have 100% homology at the level of amino acid sequence. Currently, we are trying to get the complete DNA sequence data of the tubulins identified by SQ#TbA77 and SQ#TbB03. [See Figure 1]

Fukushige T et al. (1990) Worm Breeder's Gazette "Cloning and sequencing of the tubulin alpha-2 gene"

Siddiqui SS, Fukushige T

[Worm Breeder's Gazette, 1990]

Tubulin isotypes in C. elegans are coded by a small family of alpha and beta tubulin genes identified by using heterologous probes from chicken tubulin genes (Don Cleveland). These have been named as alpha- 1 and alpha-2, and -1, -2, -3 and -4.(Linda Gremke, Ph.D. thesis, 1987). We have obtained the alpha-2 gene genomic DNA clone from J. Culotti and L. Gremke, and subcloned it into a pUC119 vector. The 4. 5 kb insert has been cut by SalI, appropriate deletions made, and its nucleotide sequence has been partially determined. The restriction map of the alpha-2 tubulin gene is given below. Amino acid sequence deduced from the nucleotide sequence of this gene shows a 94% amino acid sequence homology with the alpha-1 tubulin gene from C. elegans, cloned by direct screening of a cDNA expression library using anti- tubulin monoclonal antibody 3A5 (Kindly provided by M. Fuller). Other sequence homologies with alpha tubulin amino acid sequences are: Human, 89%; pig brain, 88%; Drosophila A3, 86%; Drosophila A1, 87%; S. pombe A2, 77%, and S. pombe A1, 76%. [See Figure 1] One remarkable observation in the amino acid sequence of the alpha-2 tubulin gene from C. elegans is that a stretch of nine amino acids ( #280-288) i.e. 27 nucleotides are missing from the coding region of the sequence, shown here by the dashes in the sequence. A simple explanation is that an internal deletion may have spontaneously arisen in the sequencing vector, eliminating these 27 bases. We are now reisolating this region of the sequence from another cDNA library and double checking our observation. If the nine amino acids are indeed missing, this will be a novel alpha tubulin in metazoans with a truncated tubulin sequence at this position. [See Figure 2] We thank the support provided by Dr. Johji Miwa of NEC, Tsukuba Lab, in nucleotide sequencing of this gene, and the discussions with Mr. Hachiro Yasuda, Drs. Y. Tabuse, K. Nishiwaki, and T. Sanno.

Cassada RC et al. (1976) Worm Breeder's Gazette "studies on development by the Goettingen C e-group (GCE)."

Von Ehrenstein G, Cole TS, Cassada RC, Deppe U, Schmitt D, Schierenberg E, Krieg C, Yoder B

[Worm Breeder's Gazette, 1976]

We are continuing our study on the development of C. elegans, concentration on embryogenesis. Cell lineage on living eggs has been followed past the 170-cell stage, using Nomarski optics and recordings on video tape. Selected cell types have been followed further, including the gut cells taking advantage of their large size and characteristic autofluorescence. We were able to identify cells uniquely by recording cell division, position of nuclei and cell size. Mitoses proceed in waves from anterior to posterior. From the 16-cell stage onwards groups of cells are clearly distinguishable. The members of each group form a clone. Again, these groups of cells divide largly sequentially from anterior to posterior. Within each group, cell division is essentially synchronous. Every complete wave starts with division of the AB descendants (classically designated Ectoderm), strictly alternating with the mitoses of the P1 descendants (classically called EMST, C and D). After four rounds of cell division a new cycle is initiated before the previous one has been completed. The length of the cell cycle in early cleavage is 15 to 20 minutes (at 22 C). The cycle lengthens gradually to 30 to 40 minutes at the 170-cell stage. We did not observe any cell death in the stages investigated. A coeloblastula with 2-3 very small cavities has been formed at the 24- cell stage. Cleavage and the formation of the three germ layers are not clearly separated in time. Gastrulation starts after the 24-cell stage when the two precursor cells of the endoderm begin to slide into the center. This movement is complete at the 51-cell stage. This type of gastrulation, known as morula delamination, is different from the types of gastrulation so far known in nematodes, as listed in Chitwood. Morula delamination also occurs in some Cnidaria (Clava Laomedea etc.) and also in some insects (e.g. some Collembola). Between the 24- and the 170-cell stage, bilateral symmetry as has been described for Ascaris is not apparent. In the fertilized egg the male and the female pronuclei meet in that part of the egg which at the 2- cell stage becomes the smaller P1-cell (situated at the posterior end). This allows early assignment of the anterior-posterior axis of the egg. The dorso-ventral assignment is possible at this stage by the asymmetry of the membrane of the first cell division. This membrane begins to form dorsally and the final hole in this 'asymmetric iris' closes well in the ventral part of the egg. So far 14 eggs in various stages have been serially sectioned for EM and are being reconstructed by hand or by computer for correlation with and extension of the Nomarski results. The fixation-embedding procedure works on all stages of embryogenesis, including prehatching. Computer capabilities have been increased this year by major hardware additions and by the installation of a new operating system ( RSX). Special attention is being given to computer-aided reconstruction of the embryo from EM series, from video film of light microscopic data, and to the correlation of the EM and LM data. Major enhancement in computer capabilities are in process along two lines. On the one hand the mechanical details associated with the recording process will be made as simple as possible for the user. On the other hand the analysis of the data involving abstraction and simplification will become much more efficient by organizing and storing the data in a 'tree-structured' data base. This will facilitate study not only of the cellular anatomy but also of subcellular structures. For example, we have begun to analyze the distribution of mitochondria in eggs and early cleavage stages. One ultimate goal is to display the important events in embryogenesis in a highly schematized movie on the screen of the graphics display. Some 10,000 EMS mutagenized clones have been tested for temperature sensitive mutants by methods similar to those of Hirsh and Vanderslice (1976), except that about half of the clones came from mutagenized hatchees. Over 100 ts mutants have been selected (designated ts G1, ts G2 etc. according to David Hirsh's nomenclature proposal, where G denotes Gottingen), including some 20% embryological arrest. Our first interest in these mutants is microscopic characterization. Dr. Johji Miwa joined the group at the end of the summer.