Caenorhabditis elegans (nematode)
A true metazoan – free living soil nematode (roundworm), eats bacteria, amoebae.
Selected by Seymour Benzer as a model organism for study of neural development.
Adult has 945 cells with 959 somatic nuclei; 302 neuronal cells derived from 407 neural precursor cells.
Adult is 1 mm long, can be grown in Petri dishes with bacteria; 104-105 animals per dish.
Generation time of only 3 days from zygote to fertile adult.
300 progeny per adult.
Sexually dimorphic – hermaphrodite (XX), male (XO)
Hermaphrodites may self-fertilize or may mate with males.
Fertilization takes place in spermatheca, zygotes enter uterus, embryogenesis occurs in utero.
Hatched L1 larvae leave through vulval opening.
- L1 – 558 cells; 55 continue cell division
- L3 – under adverse conditions will form Dauer (no mouth, no anus)
Genome: 97,000 kb per haploid genome (15X E. coli; 2X D. discoideum; .02X human) – encodes approx. 18,000 proteins
5 autosomes and X chromosome
C. elegans genome was first multicellular genome to be sequenced; prototype for human genome project
Cell Lineage: Transparent body permits tracing of nuclei through embryogenesis via DIC microscopy.
- Lineages invariant from embryo to embryo.
- Lineage known for each adult nucleus.
- First cleavage divisions are asymmetric and asynchronous.
- 1st division produces larger AB blastomere and smaller P1 blastomere. AB divides first and faster.
- Cell fates determined from earliest division: only the P1 cell can produce germ cells or intestinal cells.
- Laser ablations of individual cells show that neighboring cell fates unaffected.
- Extreme example of mosaic embryo development – virtually each cell of embryo has predetermined fate that cannot be altered.
- Cytoplasmic determinants may segregate through cell divisions; e.g., P granules are germ-line specific, initially distributed uniformly in oocyte, but associate at posterior pole just before 1st cleavage, so all segregate with P1, P2, P3, P4.
C. elegans WWW Server http://elegans.som.vcu.edu
Gilbert, SF, “Early Development of the Nematode, Caenorhabditis elegans,”Developmental Biology, 6th ed., 2000, NCBI Bookshelf http://www.ncbi.nlm.nih.gov/books/NBK10011/
Worm Atlas http://wormatlas.psc.edu/ver1/index.htm
Worm Book http://www.wormbook.org/
Worm Browser http://browser.openworm.org/
Maternal Effect Mutants Defective in Asymmetric Division & Cytoplasmic Localization:
Mechanisms for localization of cytoplasmic determinants expected to be made in oocytes.
Thus maternal genes responsible for proper oocyte construction.
Effects visible only in progeny.
Look for maternal effect mutations: phenotype determined by genotype of mother only, not of the father, or the zygote.
- Start with strains with an egg-laying (egl) defect: vulvaless mutant.
- Larvae hatch within adult, eat through the body wall – mother dies.
- If mother lays defective eggs so that no viable progeny hatch, mother lives.
- Mutagenize adult hermaphrodites with eggs. Some eggs will become mutant.
- Resulting F1 progeny will have some heterozygotes (m/+).
- Self the F1 progeny – F2 genotypes will be 25% +/+; 50% m/+; 25% m/m.
- Self the F2 – all the F2 will die except the mutant homozygotes, which have no viable progeny.
- If mutation was zygotic lethal, the F2 homozygote would be dead.
Large numbers (over 100) maternal effect lethal mutants found; most had nuclear abnormalities or defects in cytokinesis or very slow cleavages, resulting in cleavage stage arrest.
Rare mutants (par 1-4) arrest at late stages of cell proliferation & differentiation.
- equal first cleavage
- altered orientation of second cleavage
- synchronous early cleavages
- defective segregation of P granules – granules destroyed or uniformly distributed
- cell differentiation without organization: arrest as amorphous masses of fully differentiated cells, with more cells (809) than wild type (533) – includes pharyngeal muscle, body wall muscle, and neuronal cells, but lacks intestinal cells.
- occasional survivors that hatch are sterile, with no gametes.
- mutants do not map to any of known actin gene loci.
Par genes identify a conserved signaling pathway for cell polarity
Par-1 encodes a protein kinase that is asymmetrically distributed in the posterior cortex of the fertilized egg. Par-1 has homology to human MARK1 and MARK2, shown to phosphorylate microtubule associated proteins and trigger microtubule disruption (Drewes et al.., 1997). Strong par-1 alleles have defects in kinase domain, indicating that kinase activity is essential for wild type Par-1 function. Defect in par-1 causes equal first cleavage, uniform distribution of P granules, and uniform distribution of transcription factor SKN-1.
Par-2 is the only Par protein with no homologues outside the nematodes. Par-2 is also localized to the posterior cortex, and recruits Par-1 to the posterior cortex.
Par-3 and Par-6 form a complex with aPKC (atypical protein kinase C) and CDC42 (small GTPase), localized to the anterior cortex.
The unfertilized egg starts with posterior Par proteins uniformly localized to the membrane, with anterior Par proteins in the cytoplasm. Upon fertilization, the egg completes meiosis, and then the anterior Par proteins become membrane localized, displacing the posterior Par proteins to the cytoplasm, still with no evident polarity. Symmetry is broken when the sperm centriole forms a microtubule organizing center (MTOC) to the posterior, and initiates cortical contractions and flow of the actomyosin cytoskeleton towards the anterior. The sperm MTOC recruits Par-2 to the posterior cortex, and the actomyosin flows sweep the Par-6 complex towards the anterior. Par-2/Par-1 and Par-3/Par-6/aPKC/CDC42 complexes exclude each other to establish and maintain distinct posterior and anterior cortical domains (Bastock and St. Johnston 2011).
Similar systems establish cell polarity in diverse animal cells, such as fly embryos, mammalian neurons (Goldstein and Macara 2007).
Search for maternal effect mutants defective in pharynx development:
- cell autonomous development of pharyngeal cells segregates with P1, to EMS, to MS.
- mex-1 mutants: both AB and P1 produce MS-like descendents
- pie-1 mutants: both daughters of P1 (EMS and P2) produce MS-like descendents; required for germ-line specification.
- skn-1 mutants: MS cells produce hypodermal cells instead of pharyngeal and intestinal cells. SKN-1 is similar to bZIP transcription factors, localized to P1. Both daughters of P1 have SKN-1, so SKN-1 cannot be the only determinant for pharyngeal cell development. (See description of PAL-1 in Gilbert, 7th ed., p. 255.)
Formation of some organs (pharynx, vulva) require cell-cell interactions.
Laser ablation of key cells prevents pharynx development or vulva formation.
Mutants defective in pharynx development or vulva formation turn out to be defective in genes involved in cell-cell signalling.
Mutations in lin-44 reverse polarity of certain cells named T and B cells near the tail. Tail morphology is aberrant as a result. Lin-44 gene cloned by mapping mutation, and rescuing mutation with YAC clones, fragments of YAC clones, and finally phage genomic clones. A 1.2 kb cDNA clone isolated; lin-44 mutant alleles have mutations in coding sequence of this cDNA clone. Expression of cDNA clone rescues lin-44 phenotype.
Analysis of cDNA sequence and data base search showed homology (30% identity) to DWnt-2 and wg proteins from Drosophila and Wnt7a and 7b from mouse.
Lin-44 is expressed in tail hypodermis cells hyp 8-11, posterior to cells whose polarity is reversed in the mutants.
Mosaic analysis: introduced an extrachromosomal DNA array that contains wild type lin-44, ncl-1 and unc-36 genes in a strain with lin-44, ncl-1 and unc-36 defects. The extrachromosomal DNA is mitotically unstable, and becomes lost randomly and spontaneously. A cell that loses the DNA array during embryogenesis will generate progeny cells with mutant phenotype, resulting in a mosaic animal. T and B cells are descendents of the AB blastomere, but hyp11 descends from the P1 blastomere. Animals that have lost the DNA array in AB but retained it in P1 have normal phenotype! Animals that have lost the DNA array in both AB and precursors of hyp-11 among P1 descendents have abnormal polarity of T and B cells.
Han 1997, Rocheleau et al. 1997, Thorpe et al. 1997.
Recent articles by Brangwynne et al. (2009), with an accompanying Perspective by LeGoff and LeCuit (2009) provide insight into the mechanism of P-granule localization. However, Gallo et al. (2010) find that P-granules per se are not required for germline specification!
The tao kinase Kin-18 is shown to play a roll in C. elegans contractility and polarity establishment in this study by Spiga et al. (2013) http://www.devbio.biology.gatech.edu/?page_id=8212
Bastock, R. and D. St. Johnston, 2011. Going with the flow: an elegan model for symmetry breaking, Dev Cell 21: 981-982. http://dx.doi.org/10.1016/j.devcel.2011.11.015
Bowerman et al., 1993. The maternal gene skn-1 encodes a protein that is distributed unequally in early C. elegans embryos, Cell 74:443-452.
Brangwynne, CP, CR Eckmann, DS Courson, A Rybarska, C Hoege, J Gharakhani, F Julicher, AA Hyman, 2009. Germline P granules are liquid droplets that localize by controlled dissolution/condensation, Science 324:1729-1732. http://dx.doi.org/10.1126/science.1172046
Drewes, G., A. Ebneth, U. Preuss, E.-M. Mandelkow and E. Mandelkow, 1997. MARK, a novel family of protein kinases that phosphorylate microtubule-associated proteins and trigger microtubule disruption, Cell 89:297-308.
Gallo, CM, JT Wang, F Motegi, G Seydoux, 2010. Cytoplasmic partitioning of P granule components is not required to specify the germline in C. elegans, Science 330:1685-1689. http://dx.doi.org/10.1126/science.1193697
Goldstein, B. and I.G. Macara, 2007. The Par proteins: fundamental players in animal cell polarization, Dev Cell 13: 609–622. doi:10.1016/j.devcel.2007.10.007.
Guo, S. and K.J. Kemphues, 1995. par-1, a gene required for establishing polarity in C. elegans embryos, encodes a putative ser/thr kinase that is asymmetrically distributed, Cell 81:611-620.
Han, M., 1997. Gut reaction to Wnt signaling in worms, Cell 90:581-584.
Herman, M.A. and H.R. Horvitz, 1994. The Caenorhabditis elegans gene lin-44 controls the polarity of asymmetric cell divisions, Development 120:1035-1047.
Herman, M.A., L.L. Vassilieva, H.R. Horvitz, J.E. Shaw and R.K. Herman, 1995. The C. elegans gene lin-44, which controls the polarity of certain asymmetric cell divisions, encodes a Wnt protein and acts cell nonautonomously, Cell 83:101-110.
Hill and Sternberg, 1992. The gene lin-3 encodes an inductive signal for vulval development in C. elegans, Nature 358:470-476.
Kemphues et al., 1988. Identification of genes required for cytoplasmic localization in early C. elegans embryos, Cell 52:311-320.
LeGoff, L and T Lecuit, 2009. Phase transition in a cell, Science 324:1654-1655. http://dx.doi.org/10.1126/science.1176523
Rocheleau, C.E., W.D. Downs, R. Lin, C. Wittman, Y. Bei, Y.-H. Cha, M. Ali, J.R. Priess and C.R. Mello, 1997. Wnt signaling and an APC-related gene specify endoderm in early C. elegans embryos, Cell 90:707-716.
Sawa, H., L. Lobel and H.R. Horvitz, 1996. The Caenorhabditis elegans gene lin-17, which is required for certain asymmetric cell divisions, encodes a putative seven-transmembrane protein similar to the Drosophila frizzled protein, Genes Dev. 10:2189-2197.
Thorpe, C.J., A. Schlesinger, J.C. Carter and B. Bowerman, 1997. Wnt signaling polarizes an early C. elegans blastomere to distinguish endoderm from mesoderm, Cell 90:695-705.