Metazoan Model Research Articles

Commitment to the CD4 Lineage Mediated by Extracellular Signal-Related Kinase Mitogen-Activated Protein Kinase and Lck Signaling

The development of T cells results in a concordance between the specificity of the TCR for MHC class I and class II molecules and the expression of CD8 and CD4 coreceptors. Based on analogy to simple metazoan models of organ development and lineage commitment, we sought to determine whether extracellular signal-related kinase (Erk) mitogen-activated protein (MAP) kinase pathway signaling acts as an inductive signal for the CD4 lineage. Here, we show that, by altering the intracellular signaling involving the Erk/MAP kinase pathway, T cells with specificity for MHC class I can be diverted to express CD4, and, conversely, T cells with specificity for MHC class II can be diverted to express CD8. Furthermore, we find that activation of the src-family tyrosine kinase, p56lck is an upstream mediator of lineage commitment. These results suggest a simple mechanism for lineage commitment in T cell development.

Dissection of cell division processes in the one cell stage Caenorhabditis elegans embryo by mutational analysis.

To identify novel components required for cell division processes in complex eukaryotes, we have undertaken an extensive mutational analysis in the one cell stage Caenorhabditis elegans embryo. The large size and optical properties of this cell permit observation of cell division processes with great detail in live specimens by simple differential interference contrast (DIC) microscopy. We have screened an extensive collection of maternal-effect embryonic lethal mutations on chromosome III with time-lapse DIC video microscopy. Using this assay, we have identified 48 mutations in 34 loci which are required for specific cell division processes in the one cell stage embryo. We show that mutations fall into distinct phenotypic classes which correspond, among others, to the processes of pronuclear migration, rotation of centrosomes and associated pronuclei, spindle assembly, chromosome segregation, anaphase spindle positioning, and cytokinesis. We have further analyzed pronuclear migration mutants by indirect immunofluorescence microscopy using antibodies against tubulin and ZYG-9, a centrosomal marker. This analysis revealed that two pronuclear migration loci are required for generating normal microtubule arrays and four for centrosome separation. All 34 loci have been mapped by deficiencies to distinct regions of chromosome III, thus paving the way for their rapid molecular characterization. Our work contributes to establishing the one cell stage C. elegans embryo as a powerful metazoan model system for dissecting cell division processes.

Second-site noncomplementation identifies genomic regions required for Drosophila nonmuscle myosin function during morphogenesis.

Drosophila is an ideal metazoan model system for analyzing the role of nonmuscle myosin-II (henceforth, myosin) during development. In Drosophila, myosin function is required for cytokinesis and morphogenesis driven by cell migration and/or cell shape changes during oogenesis, embryogenesis, larval development and pupal metamorphosis. The mechanisms that regulate myosin function and the supramolecular structures into which myosin incorporates have not been systematically characterized. The genetic screens described here identify genomic regions that uncover loci that facilitate myosin function. The nonmuscle myosin heavy chain is encoded by a single locus, zipper. Contiguous chromosomal deficiencies that represent approximately 70% of the euchromatic genome were screened for genetic interactions with two recessive lethal alleles of zipper in a second-site noncomplementation assay for the malformed phenotype. Malformation in the adult leg reflects aberrations in cell shape changes driven by myosin-based contraction during leg morphogenesis. Of the 158 deficiencies tested, 47 behaved as second-site noncomplementors of zipper. Two of the deficiencies are strong interactors, 17 are intermediate and 28 are weak. Finer genetic mapping reveals that mutations in cytoplasmic tropomyosin and viking (collagen IV) behave as second-site noncomplementors of zipper during leg morphogenesis and that zipper function requires a previously uncharacterized locus, E3.10/J3.8, for leg morphogenesis and viability.

Teratogenic development in chicken embryos associated with a major deletion in the rRNA gene cluster.

A new strain of chickens (mPNU) that segregates a severely deleted rDNA cluster was studied. Individuals heterozygous (+/p2 ) and homozygous (p2 /p2 ) for the deletion were found to have 56 and 27%, respectively, of the normal complement of rRNA genes (290 copies/cell). Morphogenesis, cellular rRNA levels, and nucleolar sizes, were investigated and compared in normal +/+, +/p2 , and p2 /p2 embryos. Cellular rRNA contents were similar among the three genotypes at stage X, but subsequently during gastrulation, p2 /p2 levels were reduced to 56% and eventually to 43% of +/+. Viability and morphogenesis were normal in p2 /p2 embryos until the initial primitive streak stage of gastrulation. However, further development was abnormal and characterized by disrupted axis formation. In +/+ and +/p2 embryos, rRNA levels and nucleolar sizes increased during early development; however, the profile of these increases differed temporally and quantitatively between the genotypes. The +/p2 embryos, at the full streak stage of gastrulation, exhibited reduced rRNA levels and nucleolar sizes (80% of +/+), yet the +/p2 embryos developed normally. These studies establish a minimum copy number requirement lower than previously demonstrated, that is, a rDNA genotype with only 56% of the normal gene complement (∼160 genes) is compatible with early embryonic viability. Also, a rRNA threshold was detected: rRNA levels that were 56% of +/+ failed to support normal gastrulation; however, even under the circumstance of reduced rRNA levels (43% of control), some aspects of gastrulation apparently continue (cell migration and invagination). The teratogenic development of p2 /p2 embryos is a biological consequence unique from that found in other metazoan models of rDNA-deficiency, and will be useful as a model to investigate mechanisms of vertebrate gastrulation and axis formation.

Evolution of Cyst and Noncyst-Forming Heteroderinae

Evolution and diversity of Heteroderinae are of special interest because worldwide the subfamily includes some of the most destructive plant-parasitic nematodes of agriculture. Some Heteroderinae form resistant cysts which may protect eggs in the soil for years. Other Heteroderinae lack a cyst, but typically these forms are not a threat to agriculture. Seventeen genera and more than one hundred species are recognized (Table I). Phylogenetic analysis of this diverse group is important to a sound taxon­ omy of Heteroderinae. Because a natural classification that reflects genealogi­ cal history has predictive value it will readily adapt to accommodate newly discovered taxa and characters. Classifications that attempt to reflect evolu­ tionary history have a basis for stability not inherent to artificial classifica­ tions. Heteroderinae may provide a useful metazoan model for interpreting evolu­ tion in other small invertebrates. Global knowledge of extant genera and species of Heteroderinae is undoubtedly more complete, and living specimens of most species are more readily available than specimens of species of invertebrate groups often considered to be of lesser economic importance

Genotoxicity and teratogenicity of diphenyl and diphenyl ether: A study of sea urchins, yeast, andSalmonella typhimurium

This study was designed to investigate the possible genotoxic and teratogenic actions of diphenyl (DP), diphenyl ether (DPE), and their eutectic mixture, in a comparative approach including different test systems. Two microbial systems and a metazoan model were used: (1) diploid D7 strain of Saccharomyces cerevisiae; (2) Salmonella typhimurium strains TA100, TA98, TA1535, TA1537, TA1538, TA1532, TA2636; and (3) sea urchins (Paracentrotus lividus and Sphearechinus granularis). Both compounds resulted in severe toxicity in all of test organisms at levels greater than or equal to 10(-5) M (approximately 2 ppm). DP caused genetic effects in yeast with and without activating system, while the two chemicals appeared to be ineffective in Salmonella up to toxic levels. The action of DP and DPE on sea urchins resulted in developmental defects and mitotic abnormalities, following exposure of embryos or by pretreatment of sperm or eggs. In this system DPE appeared to be more effective than DP by about one order of magnitude (minimal active concentrations: 10(-5) M vs 10(-4) M). The eutectic mixture, industrially used as a heat transfer medium, was tested in its virgin and used form, for genotoxicity and embryotoxicity. The latter appeared to be more effective than the virgin eutectic. This increase in the embryo- and genotoxicity of the used eutectic may be related to the appearance of newly formed compounds in the heat transfer process. These compounds have been separated by high-pressure liquid chromatography and detected by fluorimetry.

Sensory Mechanisms in Nematodes

There have recently been unprecedented advances in our knowledge of senses and responses in nematodes. As this review is being published data on sensory mecha­ nisms in nematodes continue to flood in from laboratories all over the world. Developments have not come directly from electrophysiology, but rather indirectly, through detailed neuroanatomy and through interpretations based on the behavior of the entire organism. Studies of the free-living bacteriophagous species Caenorhab­ dill's elegans have led to many of these advances and are the basis for a major part of this review. Caenorhabditis elegans, the subject of a classical study by Nigon (51), was used as a metazoan model for various biological studies by Dougherty & Calhoun (26). Although C elegans and the closely related C briggsae had become familiar, they were little studied until the late 1960s and 1970s. C elegans and C briggsae are now used as models for physiology (7), gerontology (67), genetics (3, 24, 30, 64), and behavior ( 1417, 27-30, 53, 58). During the late 1960s and early 1970s, Dr. Sydney Brenner selected C elegans to explore the genetic basis of behavior. In 1974 Brenner (3) wrote, Behaviour is the result of a complex and ill-under­ stood set of computations performed by nervous systems and it seems essential to decompose the problem into two: one concerned with the question of the genetic speciation of the nervous systems and the other with the way nervous systems work to produce behaviour. Both require that we must have some way of analysing the structure of the nervous ·system. While C elegans is now studied all over the world, the inspiration for these studies and the direction of the current work comes from Dougherty and more recently from Brenner. Details of laboratory handling of C elegans, mutagens, storage, and culturing are available (3, 30). The fundamental objectives outlined by Brenner have not yet been met but the results achieved so far are of critical importance in the study of nematode behavior.