Gunter von Ehrenstein

Cell lineages and developmental defects of temperature-sensitive embryonic arrest mutants in Caenorhabditis elegans.

The cellular phenotypes of 11 temperature-sensitive mutants in nine genes (emb-1-emb-9) arresting embryogenesis in Caenorhabditis elegans are described, including early embryonic cell lineages and developmental defects and the terminal phenotypes at the stage of arrest at the nonpermissive temperature (25°C), as well as residual phenotypes at the permissive temperature (16°C). By Nomarski microscopy of living embryos, the behavior of individual cells in mutant embryos is compared to that of the same cells in wild-type embryos. All mutants but one (emb-9) have visible defects before the 50-cell stage, even if they arrest much later. Cell proliferation continues in the absence of normal morphogenesis. The terminal phenotype of the late-arresting mutants looks grossly abnormal. The overall rate of cell division is faster than in the wild-type in two mutants, emb-3 and emb-5(hc67), and slower in five mutants, emb-2, emb-4, emb-5(hc61), emb-6, and emb-7. In five of these mutants, emb-3, emb-4, emb-5 (both alleles), and emb-7, the rate and the sequence of divisions of specific cell lines is altered. For all mutants with timing defects, maternal gene expression is at least sufficient. Gastrulation is abnormal in the two emb-5 mutants. A premature division of the intestine precursor cells occurs in close temporal coincidence with the defective execution stage (determined by temperature-shift experiments). The order of migration and division of the intestine precursor cells is reversed in gastrulation, and the cell pattern of the intestine primordium is abnormal. It seems possible that the timing error is responsible for the pattern defect. The cleavage behavior of emb-3 eggs indicates that germ line determinants are prelocalized in the egg of C. elegans.

Genetic analysis of temperature-sensitive embryogenesis mutants in Caenorhabditis elegans

Abstract A large set of temperature-sensitive (ts) embryonic arrest mutants in the nematode Caenorhabditis elegans has been isolated. Following ethylmethane sulfonate mutagenesis, ts mutants were identified from 10,000 segregated clones by replica plating from 16 to 25°C. At least 54 independent ts developmental mutants showed embryonic arrest when the appropriate developmental stage was shifted up. These represent 28% of the independent developmental mutants from 294 ts clones from 155 mutagenized animals. Initial characterization of this subset of emb mutants is reported. Most of them arrest in middle or late embryogenesis with abnormal terminal phenotypes. Additional nonembryonic arrest stages (depending on shift-up regimen) suggest that many emb genes are also required for pre- or postembryonic development. For most of the emb mutants parental gene expression is sufficient for embryonic development at 25°C. Based on complementation analysis of 37 new emb mutants together with 32 isolated by other investigators, 25 new emb genes are reported. This brings the total of embryonic arrest genes defined in C. elegans to 54. The new genes have been mapped approximately. In the present set, 10 emb genes map in the middle of linkage group III. The frequency of occurrence of second alleles gives one estimate (by Poisson analysis) for the number of genes required for embryogenesis of about 200. A second estimate is obtained from the fraction of 28% emb mutants among all ts lethal mutants in the screen; for an estimated 2000 essential genes in C. elegans, this corresponds to 560 genes required for embryogenesis. A small subset of highly mutable genes is also described. Results of the present screen for emb mutants are compared to those of others. Several ts mutants gave complex linkage results (apparently not due to double mutations), suggesting chromosome rearrangements.

Genetics and mode of expression of temperature-sensitive mutations arresting embryonic development in Caenorhabditis elegans

Eleven temperature-sensitive mutations causing arrest of embryogenesis in Caenorhabditis elegans have been mapped. The mutations define nine genes (emb-1 to emb-9) on four chromosomes. The functions of six genes seem to be required exclusively for embryogenesis. Mutants in these genes have no other detectable phenotype at the permissive (16°C) or nonpermissive (25°C) temperature. The function of the other three genes is also required for postembryonic development. As shown by progeny tests for parental effects, for seven genes, maternal gene expression is necessary and sufficient for normal embryogenesis; for one gene, emb-2, either maternal or zygotic expression is sufficient; for one gene, emb-9, zygotic expression is necessary and sufficient. The high proportion of emb genes with maternal expression is consistent with the model of intracellular preprogramming of the egg of C. elegans (U. Deppe, E. Schierenberg, T. Cole, C. Krieg, D. Schmitt, B. Yoder, and G. von Ehrenstein, 1978; Proc. Nat. Acad. Sci. USA 75, 376–380). Two developmental stages have been defined by temperature-shift experiments: (1) the normal execution stage indicating the time of execution of the normal event at the permissive temperature; (2) the defective execution stage indicating the time of the execution of an irreversible defect at the nonpermissive temperature. The classes of mutants defined by the progeny tests have corresponding execution stages, but the maternal necessary and sufficient class is subdivided into mutants executing during oogenesis or embryogenesis.

The cellular anatomy of embryos of the nematode Caenorhabditis elegans: Analysis and reconstruction of serial section electron micrographs

Abstract As described from light microscopy, embryogenesis of the free-living soil nematode Caenorhabditis elegans follows a strictly determinate cleavage pattern, producing a newly hatched juvenile with about 550 cells arranged quite predictably. In this communication we present results on the electron microscopy of C. elegans embryos and introduce methods for fixing, embedding, and serially sectioning embryos encased in the egg shell. Fixation at elevated temperature either with osmium tetroxide alone or with glutaraldehyde followed by osmium tetroxide gives reproducible results with embryos in all developmental stages so far tested, from the fertilized egg to hatching. Eighteen wild-type eggs at various stages have been sectioned to date. We have achieved using newly developed procedures for analyzing electron micrographs of serial sections detailed reconstructions of the cellular anatomy of complete embryos of a metazoan organism. Three-dimensional serial section reconstructions were made with a computer system. We characterize and map the 24 cells of an early-stage embryo in this report. Additionally, we can specify the lineage history of all cells of this embyro by matching the reconstructed three-dimensional arrangement of this series to a living embryo at this stage, where cell lineage has been observed with Nomarski optics and analyzed using videotape ( U. Deppe, E. Schierenberg, T. Cole, C. Krieg, D. Schmitt, B. Yoder, and G. von Ehrenstein, 1978 , Proc. Nat. Acad. Sci. USA 75, 376–380). In addition, cytoplasmic and nuclear morphological features such as incomplete membranes between sister cells, rounding-off of the cytoplasm, and chromatin condensation patterns have been correlated with cell division. Mapping of such structures presents a new method by which supplementary lineage information can be obtained directly from an electron micrographic series.

Reversed-phase high-performance liquid chromatography of histones.

Abstract Calf thymus histones are separated into the five classical components H1, H2A, H2B, H3, and H4 using reversed-phase high-performance liquid chromatography. This method is fast and sensitive; a single run takes 80 min and protein quantities ranging from 3 μg up to 1 mg can be separated. The primary structure of the proteins is not affected, as demonstrated by peptide mapping and gel electrophoresis. Yeast, nematode, and calf thymus histones are compared to each other and have similar retention times for individual peaks, thus demonstrating the evolutionary stability of these proteins by using this different approach.

Embryonic Cell Lineages and Segregation of Developmental Potential in Caenorhabditis elegans

Embryogenesis of the nematode Caenorhabditis elegans is determinate and virtually invariant from individual to individual. The fertilized egg develops into an anatomically relatively simple juvenile animal having a constant number of only 550 cells (or nuclei) at hatching. The complete embryonic cell lineage up to the 220-cell stage has been described previously, and some lineages have been followed considerably further (Deppe et al. 1978; Krieg et al. 1978; Schierenberg 1978; von Ehrenstein and Schierenberg 1980). During postembryonic development, the cell number increases to about 950 in mature hermaphrodites (including the 143 of the somatic gonad structures) and to about 1025 in males. These cells arise from about 50 blast cells which resume division after hatching. The lineages of all of these cells have been described (Sulston 1976; Sulston and Horvitz 1977; Kimble and Hirsh 1979; Sulston et al. 1980).