Schnabel R et al. (1987) International C. elegans Meeting "embryonic lethal mutations of C elegans."

Schnabel H, Schnabel R

[International C. elegans Meeting, 1987]

Schnabel R et al. (2000) European Worm Meeting "Proteome-analysis of C. elegans germ cells"

Glocker MO, Ashman K, Schnabel R, Thiesen H-J, Ringel B

[European Worm Meeting, 2000]

We are using germ cell formation during maturation of C. elegans as a model to explore the potentials of a proteome approach with a completely sequenced organism. C. elegansis also regarded as a model sys-tem to facilitate the purification of low abundant proteins involved in regulatory protein networks. In this project we are studying protein expression patterns of the temperature-sensitive C. elegansmutant glp-1(e2144) ( ). This mutant presents a normal phenotype when grown at *C. However, when young larvae (L1) are shifted to 25*C, germ cells are lacking in adult worms. Differential proteome analyses ( - ) of the adult C. elegansmutant grown at the different temperatures were performed and char-acteristic alterations in protein expression were observed by 2D-gel electrophoresis. The protein extracts (1.5 ml) contained a total of 4.1 mg protein, each. 2D-gel electophoresis (30 x 23 cm2) was performed with 15 l aliquots, applying high resolution conditions [ ]. Isoelectric focussing was carried out using a pH gradient ranging from 4 to 10. After staining with silver approx. 3000 high abundant protein spots were detected by computer assisted image analysis. Proteins found to be differentially expressed are analyzed by mass spectrometry after in-gel digestion. Subsequent data base searches identify specific germline proteins.

Schnabel R et al. (1989) International C. elegans Meeting "the cib genes -- determination of P cells in the early embryo."

Schnabel R, Schnabel H

[International C. elegans Meeting, 1989]

Schnabel R et al. (1996) West Coast Worm Meeting "LINEAGE ANALYSIS OF THE C. ELEGANS EMBRYO WITH A 4D-MICROSCOPE"

Schnabel H, Moerman DG, Hutter H, Schnabel R

[West Coast Worm Meeting, 1996]

We have developed an improved version of John White's 4-dimensional microscope which permits a 3-dimensional time lapse analysis of embryogenesis. A new software program, Biocell, permits a rapid and thorough analysis of the embryonic lineages as well as the reconstruction of various embryonic stages. The analysis is stored in the form of an annotated lineage. Embryonic stages and cell movements can be viewed as 3-D models in which cells can be coloured to better visualize the relative position of cells or, groups of cells. The models can be rotated to provide different views of the embryo as well as to allow easy comparison of different embryos. This feature should also faciliate identification of cells in immunostainings or in situ hybridizations. We used the new program to create a set of data (cell positions at various time points during development) for the reconstruction of embryonic stages of wild type embryos and the analysis of cell migrations in the embryo. Analysis of this data set reveals the dynamics of cell migrations in the embryo, e.g. in the establishment of left-right asymmetry, or during the migration of muscle precursors to form the four muscle qudrants. In addition, analysis of the development of several wild type embryos shows that the timing of cleavages and cell movements (migrations) are quite variable although the assignment of cell fates appears absolutely invariant.

Schnabel R et al. (1997) International C. elegans Meeting "LINEAGE ANALYSIS OF THE C. ELEGANS EMBRYO WITH A 4D-MICROSCOPE"

Schnabel R, Schnabel H, Moerman DG, Hutter H

[International C. elegans Meeting, 1997]

We have developed an improved version of John White's 4dimensional microscope which permits a 3-dimensional time lapse analysis of embryogenesis. A new software, "Biocell", permits a rapid and thorough analysis of the embryonic lineages as well as the reconstruction of various embryonic stages. The analysis is stored in the form of an annotated lineage. Embryonic stages and cell movements can be viewed as 3-D models in which cells can be coloured to better visualize the relative position of cells or groups of cells. The models can be rotated to provide different views of the embryo as well as to allow an easy comparison of different embryos. These features should also facilitate the identification of cells in immunostainings or in situ hybridizations. We used the new program to create a set of data (cell cleavages, cell positions at various time points during development) for the reconstruction of embryonic stages of normal embryos. We observe an unexpected variability in the timing of cleavages, cell positions, cell-cell contacts and of cell movements in normal embryos. The analysis reveals a regional organisation of the lineage.

Schnabel R et al. (1991) International C. elegans Meeting "AN ATTEMPT TO 'SATURATE' CHROMOSOME II OF C. ELEGANS FOR EMBRYONIC LETHAL MUTATIONS"

Schnabel H, Schnabel R

[International C. elegans Meeting, 1991]

To extend our studies on embryonic lethal genes we isolated 213 maternal effect and 885 zygotic embryonic lethal mutations using mnCI as balancer chromosome. Both types of mutations were isolated by screening 27400 chromosomes after 'standard' mutagenesis with EMS. A subset of 100 zygotic mutations mapping under the deficiencies mnDf88 & 89 defines 56 genes. Extrapolation to the whole screen suggests that we missed at most a few embryonic lethal genes located in the balanced region of chromosome II. The maternal effect mutations define 92 genes. Thereof 28 genes are represented by more than one mutation in average with 5 per gene. For half of those genes only strict maternal effect alleles were isolated. To test the hypothesis that the 64 mutations which stayed single are rare alleles of genes with a zygotic function we complemented a subset, those under mnDf88 & 89, of the zygotic against the maternal effect lethal genes. Only 10% of the rare mutations seem to be alleles of zygotic embryonic lethal genes. Thus the rare mutations either correspond to genes only required after embryogenesis or represent pure maternal genes mutating only with an extremly low frequency. We also included 23 mutations isolated in a similar screen (Kemphues et al. (1988) Genetics 120, 977-986) in our analysis. The two sets overlap in 5 genes. The 'forward mutation rate' for the zygotic genes in our screen was only 1.4x10-4 and thus deviated significantly from the commonly accepted rate of 5xl0-4 . We estimate that the genome contains less than 200 pure maternal effect, at least 400 genes which mutate rarely to maternal effect, and about 1000 zygotic embryonic lethal genes. Phenotypic analysis of the mutations in the pure maternal effect genes suggests that 6 genes are required for developmental decisions, e. g. the specification of early blastomeres. Mutations in 6 other genes affect cell cycles at specific stages of embryogenesis. One of these, sud-l, is required to repress cell cycles at the end of the general proliferation of cells. Three genes are required for the formation of a proper eggshell. Five genes, 3 of which ( zyg-1, -9, -II) were identified earlier by others, are required for the organisation of the first cleavage.

Schnabel R et al. (2000) European Worm Meeting "OOC-3, a novel putative transmembrane protein, required for establishment of cortical domains and spindle orientation in the P1 blastomere of C. elegans embryos"

Pozniakowski A, Schnabel H, Pichler SC, Ashford A, Gonczy P, Schnabel R, Hyman AA

[European Worm Meeting, 2000]

In the P1 blastomere of the two-cell stage C. elegans embryo, the orientation of the mitotic spindle is the most obvious manifestation of the properly established antero-posterior axis. To identify genes required for the establishment of polarity after cell division we screened a collection of maternal-effect lethal mutations on chromosome II by scoring for mutants defective in spindle orientation. We identified one mutation, ooc-3, in which spindle orientation is defective in 100% of the embryos. Indirect immunofluorescence of fixed embryos revealed that ooc-3 is required for the establishment of the antero-poster axis in P1 by localizing PAR-2 and PAR-3 to the posterior and anterior cortical domains, respectively. Furthermore, the organization of the actin, but not the microtubule cytoskeleton, is affected in ooc-3 mutant embryos. We cloned ooc-3 by germline transformation and RNA interference and found that it encodes a novel transmembrane protein with two PEST sequences and a negatively charged cluster at its C-terminus. Sequencing of two mutant alleles revealed that the C-terminus is essential for the function of ooc-3. OOC-3 localizes to a recticular structure that resembles the ER and is enriched at cell-cell boundaries in two- and four- cell stage embryos. We propose a model in which OOC-3 is delivered via the ER to cell-cell boundaries where it is required to re-establish the cortical domains after cell division and thereby the antero-posterior axis by localizing PAR-2 and PAR-3.

Granato M et al. (1991) International C. elegans Meeting "CLONING OF pha-1, A GENE REQUIRED FOR MORPHOGENESIS AND TERMINAL DIFFERENTIATION OF THE PHARYNX"

Granato M, Schnabel H, Schnabel R

[International C. elegans Meeting, 1991]

Mutadons in the zygotic gene pha-l affect morphogenesis and terminal differentiation of the pharynx in an organ-specific manner. In embryos homozygous for mutations in the pha-l gene, pharynx development is blocked at the onset of morphogenesis, before the cells have initiated terminal differentiation. Therefore all tested pharyngeal cell types ( marginal, muscle and glands) of mutant embryos do not express specific markers. However, development outside the pharynx is not affected by the mutation (for details see H. Schnabel & R. Schnabel, Science, Vol. 250, p.686-688,1990). We started to clone pha-l by the molecular identification of the left eDf20 breakpoint. The small deficiency eDf20 fails to complement pha-l and tra-l, which maps to the right of pha-l. The gene is thus located between the left deficiency breakpoint and tra-l. We localized the deficiency breakpoint in one of the clones we obtained from Jonathan Hodgkin. We mapped the clones around the deficiency breakpoint and microinjected them into the gonad of pha-l hermaphrodites. With one of these clones we could rescue the mutant pha-l phenotype. By subcloning this clone we were able to identify a 6kb fragment which rescues the pha-l phenotype and is therefore likely to contain the entire gene. The fragment detects one transcript of about 2.4kb on a developmental Northern blot of embryonic and larval stages. The expression of the message is strongest during mid- embryogenesis, which is the tsp for pha-l . Screening of a cDNA library (kindly provided by J. Ahringer and J. Kimble ) resulted in one unfused cDNA clone of 1.2kb. Sequencing and comparison of this part of the coding region to databases revealed no significant similarity to any known protein.

Zeyu Zheng et al. (2006) East Asia C. elegans Meeting "Towards a quantitative 4D analysis of early embryogenesis using subcellular GFP markers"

Takafumi Konishi, Fumio Motegi, Asako Sugimoto, Nobuko Uodome, Zeyu Zheng

[East Asia C. elegans Meeting, 2006]

One of the challenges for analyses of developmental process is that manipulation of gene function can affect not only fate of cells but also their location, size, movement and developmental timings. Therefore, to understand the genes function comprehensively, 4-dimensional (= 3-dimension + time) description of cellular information is crucial. The C. elegans embryo is one of the best model systems for the 4D analysis of developmental processes, because of its transparency and invariant cell lineage, as well as amenability to genetic and cellular manipulations. Recent advancement of 4D-microscopy and computer technology has led to the development of several approaches to 4D data analysis (1-5), mainly focusing on nuclear divisions and lineage analysis. Here, to study cellular dynamics of early embryogenesis in C. elegans at a higher spatiotemporal resolution, we aim to establish a new 4D image analysis algorithm by incorporating GFP markers that highlight specific subcellular components. We have been testing GFP markers available in the community (e.g., histone, β-tubulin, and γ-tubulin), and also constructing potentially useful new markers. We will present our progress on the optimization of fluorescent 4D image capturing with a high-speed laser scanning confocal microscope, and the algorithms for quantitative analysis of the 4D datasets. 1. Bao, Z., et al. (2006) PNAS 103, 2707-2712. 2. Hamahashi, S., Onami, S. and Kitano, H. (2005) BMC Bioinformatics 6, 125-140. 3. Heid P., Voss E. and Soll D. (2002) Developmental Biology 245, 329-347. 4. Schnabel R., et al. (2006) Developmental Biology 294, 438-431. 5. Bischoff M. and Schnabel R. (2006) Developmental Biology 294, 432-444.

Borgonie G et al. (1998) European Worm Meeting "The cell lineage of the nematode Halicephalobus sp. (Panagrolaimidae)"

Schnabel R, Borgonie G

[European Worm Meeting, 1998]

In order to analyze the evolution of the nematode lineage we have started lineaging embryos of nematode species other than C. elegans. To this end the lineage of three embryos of Halicephalobus sp. has been determined from zygote to first movment and compared. During this interval 516 cells are formed of which 31 undergo programmed cell death. In the early development the polarity of the P3 division is reversed in comparison with C. elegans necessitating considerable early blastomere movement. However, at the onset of gastrulation the blastomeres have a spatial arrangment much like C. elegans. Further in development some deviations do occur in comparsion with C. elegans. Two prominent cell deaths occur in Ea, D makes only 16 body wall muscles but MS makes 33 body wall muscles, at the expense of nerve cells, coelomocyte and intestinal muscle as compared to C. elegans) therfore compensating not only the muscles not made by D but also the single body wall muscle made in AB which is not present in Halicephalobus. As a result Halicephalobus has the same number of body wall muscles as in C. elegans. Although there are few early cell deaths in MS and E, most of the early cell deaths in C and AB are missing in Halicephalobus Generally the cell deaths are occurring at different positions than in C. elegans. The lineage in Halicephalobus appears much more organised in blocks of tissues than the rather chaotic cell lineage of C. elegans.