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Chapter 19: Using Genetics to Study Development

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  1. Chapter 19: Using Genetics to Study Development

  2. DEVELOPMENT • An organism arises from a fertilized egg as the result of three related processes • Cell division • Cell differentiation • Morphogenesis

  3. DIFFERENTIATION • Cells may initially remain undifferentiated • Embryonic stem cells • Cells ultimately differentiate • Become specialized in structure and function

  4. DIFFERENTIATION • Virtually all cells within a multicellular organism are genetically identical • Differences between cells are due to differences in gene expression • Different subsets of genes are “on” and “off” • Different cell types make different proteins

  5. GENE EXPRESSION • Much of the regulation of gene expression occurs at the level of transcription • Transcriptional regulation of gene expression is directed by • Maternal molecules in the cell’s cytoplasm • Signals from other cells

  6. PATTERN FORMATION • The development of a spatial organization in which an organism’s tissues and organs are all in their characteristic places • In animals, it begins in early embryo • Basic body plan is established • Major axes are established early

  7. Most commonly used model organisms Yeast (S. cerevisiae) Worms (C. elegans) Fruitfly (D. melanogaster) Zebrafish (D. rerio) Mustard Weed (A. thaliana) Mouse (M. musculus)

  8. Two important feature of Model Organism • The availability of powerful tools of and study the organism genetically • Ideas, methods, tools, and strains could be shared among scientists investigating the same organism, facilitating rapid progress.

  9. The choice of a model organism depends on what question is being asked.

  10. 一、Saccharomyces cerevisiae

  11. BAKER’S YEAST, the best studied unicellular eukaryote is the budding yeast S. cerevisiae. • Unicellular eukaryotes offer many advantages as experimental model systems.

  12. S. cerevisiae exists in three forms. • Two haploid cell types, a and α • The diploid product of mating between these two.

  13. The lifecycle of the budding yeast S. cerevisiae

  14. These cell types can be manipulate to perform a variety of genetic assays. • Genetic complementation can be performed the two mutations whose complementation is being tested. • If the mutations complement each other, the diploid will be a wild type for mntations can be made in haploid cells in which there is only a single copy of that gene.

  15. Generating precise mutations in yeast is easy • The genetic analysis of S. cerevisiae is further enhanced by the availability of techniques used to precisely and rapidly modify individual genes.

  16. Recombinational transformation in yeast

  17. The ability to make such precise changes in the genome allows very detailed questions concerning the function of particular genes or their regulatory sequences to be pursued with relative ease.

  18. S. cerevisiae has a small, well-characterized genome • Because of its rich history of genetic studies and its relatively small genome, S. cerevisiae was chosen as the first eukaryotic ( nonviral ) organism to have its genome entirely sequenced. This landmark was accomplished in 1996.

  19. The availability of the complete genome sequence of S. cerevisiae has allowed “genome-wide” approaches to studies of this organisn.

  20. S. cerevisiae cells change shape as they grow • As S. cerevisiae cells progress through the cell cycle. They undergo characteristic changes in shape.

  21. The mitotic cell cycle in yeast

  22. Simple microscopic observation of S. cerevisiae cell shape can provide a lit of information about the events occurring inside the cell. • A cell that lacks a bud has yet to start replicating its genome. A cell with a very large bud is almost always in the process of executing chromosome segregation.

  23. 二、NEMATODE WORM, caenorhabditis elegans

  24. In 1965 Sydney Brenner settled on the small nematode worm caenorhabditis elegans to study the important questions of development and the molecular basis of behavior, because it contained a variety of suitable characteristics. • And due to its simplicity and experimental accessibility, it is now one of the most completely understood metazoan.

  25. it can be handled like a microbe – very amenable togenetic analysis - WHY? • on agar plates • in liquid medium • as frozen stocks • self-fertilization • crosses with males • short life cycle • genome sequence known Dec 11 1998 Vol 282: 5396 C. elegans: Sequence to Biology

  26. easy to observe under microscope- • easy to make mutants- • small size (1 mm) • transparent body • invariant cell number • mutagenesis • DNA microinjection • RNA interference

  27. Scorecard C.elegans H.sapiens Chromosomes 5 + 1 22 + 2 Genome Size 97 million 3000 million Encoded Proteins 19,099 ~30,000 Life Span 0.06 years ~80 years Sexes male, herm. male, female Somatic cells 1031, 959 ??? Neurons 381, 302 100,000,000,000 Synaptic connections 5,000 100,000,000,000,000 Body size 1 mm ~170 cm Body weight 5 g ~75 kg Food E.coli Omnivore

  28. C. elegans has a very rapid life cycle • At 25℃ fertilized embryos of C. elegans complete development in 12 hours and hatch into free-living animals capable of complex behaviors. • The first stage juvenile(L1) passes through four juvenile stages(L1-L4) over the course of 40 hours to become a sexually mature adult.

  29. The life cycle of the worm, C.elegans

  30. Under stressful conditions, the L1 stage animal can enter an alternative developmental stage in which it forms what is called a dauer. • Dauers are resistant to environmental stresses and can live many months while waiting for environmental conditions to imptove.

  31. C. elegans has a simple body plan. Its lineages is relatively few and well studied.

  32. The body plan of the worm

  33. THE WORM In case of self-fertilization there are ~ 0.1 - 0.3% male worms in the population.

  34. Males Males (5AA;X0) arise from fusion of nullo-X gametes and normal X-bearing gametes. Nullo-X gametes are generated by spontaneous non-disjunction of the X chromosome during meiosis in the germ line.

  35. Nervous system

  36. Worm and neurobiological studies • Depression • Neurodegeneration • Schizophrenia • Insomnia • Addiction • Memory • Learning • etc.

  37. The cell death pathway was discovered in C. elegans • The most notable achievement to date in C. elegans research has been the elucidation of the molecular pathway that regulates apoptosis or cell death. • Analysis of the ced mutants showed that, in all but one case, developmentally programmed cell death is cell autonomous, that is, the cell commits suicide.

  38. Nobel Prize in Physiology or Medicine2002 Sydney Brenner John Sulston Robert Horvitz "for their discoveries concerning genetic regulation of organ development and programmed cell death"

  39. Apoptotic pathways of C. elegans and vertebrates are conserved

  40. RNAi was discovered in C. elegans • In 1998 a remarkable discovery was announced. The introduction of dsRNA into C. elegans silenced the gene homologous to the dsRNA. It significant in two respects.

  41. Nobel Prize in Physiology or Medicine2006 "for their discovery of RNA interference - gene silencing by double-stranded RNA" Andrew Z. Fire Craig C. Mello

  42. One is that RNAi appears to be universal since introduction of dsRNA into nearly all animal, fungal, or plant cells leads to homology-directed mRNA degradation. • The second was the rapidity with which experimental investigation of this mysterious process revealed the molecular mechanisms.

  43. Long term storage of the worm C. elegans can be stored indefinitely at very low temperature (-70 ~ -100 °C freezer) Freezingsolution: S Buffer [129 ml 0.05 M K2HPO4, 871 ml 0.05 M KH2PO4, 5.85 g NaCl] + 30% glycerin 1:1 with M9 containing worms (preferably starved L1) In the dauer larval stage, it can also be kept at 16 °C for months

  44. 三、FRUIT FLY, Drosophila melanogaster

  45. Drosophila has a raid life cycle • The salient features of the Drosophila life cycle are a very rapid period of embryogenesis, followed by three period of larval growth prior to metamorphosis.

  46. The Drosophila life cycle

  47. One of the key processes that occurs during larval development is the growth of the imaginal disks, which arise from invaginations of the epidermis in mid-stage embryos. • Imaginal disks differentiate into their appropriate adult structures during metamorphosis (or putation).

  48. Imaginal disks in Drosophila

  49. The first genome maps were produced in Drosophila • genes are located on chromosomes; • each gene is composed of two alleles that assort independently during meiosis; • genes located on separate chromosomes segregate independently, whereas those linked on the same chromosome do not.