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IGCLECE

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05/11 seminar ppt

05/11 seminar ppt

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Transcript

  • 1. Identification of Genes required for Cytoplasmic Localization in Early C. elegans Embryos Kenneth J. Kemphues James R. Priess Diane G. Morton Niansheng Cheng 皓宇。宇瑄。銘崧
  • 2. C. elegans
    • Hermaphrodite & Male
    • Research was begun in 1974 by Sydney Brenner.
    • It has since been used extensively as a model organism.
    • http://herkules.oulu.fi/isbn9514267567/html/i183412.html
  • 3.
    • http://www.sfu.ca/biology/faculty/hutter/hutterlab/research/Celegans.html
  • 4. Development
    • Fertilization
    • First Cleavage
    • Second Cleavage
    • Axe Determination
    • Anterior & Posterior
    • Dorsal & Ventral
    • Left & Right
  • 5. Fertilization
    • Fertilization determines AP axis.
    • After fertilization, the two pronucleis join together.
    • http://www.mbg.cornell.edu/cals/mbg/research/kemphues-lab/movies.cfm/kjk1_wt
  • 6. First Cleavage
    • Then the mitotic spindle forms, and it migrates posteriorly.
    • Owing to the migration, the two daughter blastomeres are produced in different sizes after the first cleavage.
    • http://www.mbg.cornell.edu/cals/mbg/research/kemphues-lab/movies.cfm/kjk1_wt
  • 7. Second Cleavage
    • At the second cleavage, AB divides transversely and P 1 divides longitudinally. AB always divides before P 1 .
    • http://www.mbg.cornell.edu/cals/mbg/research/kemphues-lab/movies.cfm/kjk1_wt
  • 8. Second Cleavage
    • Cell-Cell Interaction determines the DV axis.
    • Principles of Development (Lewis Wholper) Chapter 5
  • 9. Third Cleavage
    • The LR axis.
    • Principles of Development (Lewis Wholper) Chapter 5
  • 10. Principles of Development (Lewis Wholper) Chapter 5
  • 11. P Granule
    • Large ribonucleoprotein complexes destined to the germ line.
    • One of the cell fate determinants.
  • 12.
    • Mitotic spindle migrates posteriorly.
    • Daughter cells are produced in different sizes after the first cleavage.
    • AB > P1
    • P granules are localized to P1.
    • AB divides transversely and P1 divides longitudinally at second cleavage.
    • AB always divides before P1.
    • P granules are localized to P2.
  • 13.  
  • 14. Background
    • There must be some genes controlling the developmental progresses described before.
  • 15. Aim
    • To identify the genes required
    • for cytoplasmic localization in
    • early C. elegans embryos
  • 16. Aim-1 Identifying the genes
  • 17. Aim-1
    • Genes required for cytoplasmic localization in the early cleavages are expected to be expressed during oogenesis.
    • -> Maternal Genes
    • Mutations in such genes are likely to be maternal effect lethal mutations.
    • Screening method
  • 18.
    • +: egl-23 or lin-2
    • m: recessive maternal effect
    • lethal mutation
    • egl-23 ( lin-2 ):
    • fertilize but don’t lay egg
  • 19. Screening
    • egl: egl-23 egl/egl
    • EMS
    • egl/ egl
    • Self-fertilization
    • egl/egl egl/ egl egl / egl
    • examine defects
  • 20. Defects
    • Equal First Cleavage
    • Altered Second Cleavages
    • Abnormal Localization of P Granule
    • Abnormal Differentiation
    • Grandchildless Phenotype
  • 21. Genes
  • 22. Aim 2 Observing the defects
  • 23. Equal First Cleavage
  • 24. Equal First Cleavage
    • Blastomere Size Measurement
      • Zeiss Photomicroscope III (PM III)
      • Planimeter
    • Spindle Movement Measurement
    http://www.microscopy-uk.org.uk/mag/artnov07/dw-pm3.html
  • 25. DIC=NIC
    • Differential interference contrast microscopy ( DIC )
    • Nomarski Interference Contrast ( NIC )
    • Nomarski microscopy
    • Unstained, transparent samples
    • Appearing black to white on a grey background
    • Similar to phase contrast microscopy (without the bright diffraction halo)
    • Emphasizing lines and edges though not providing a topographically accurate image
    http://en.wikipedia.org/wiki/Differential_interference_contrast_microscopy
  • 26. Table-2 Relative Sizes of AB Blastomeres 37 57±3 par-4(it33) 21 52±1 par-3(e2074) 35 51±2 par-2(it5) 39 53±2 par-1(b274) 57 57±2 N2(wild type) No. of embryos AB/Total Genotype
  • 27. Percent egg length
  • 28. Altered Second Cleavages
  • 29. Altered Second Cleavages
  • 30. Altered Second Cleavages
  • 31. Conclusion
    • Abnormal positioning of the early mitotic spindles  size
    • Altered timing of early cleavage
    • The par embryos contribute significantly to later pattern abnormalities.
  • 32. Abnormal Localization of P Granule
  • 33. P granule localization
    • Normal: the posterior pole
    • Par mutants:
    • immunofluorescence of 4-cell embryos
    • stained with anti-P granule antibody
    http://www.mun.ca/biology/scarr/4241_Devo_Germ_Celegans.html
  • 34. Wild type par-1 mutant par-2 mutant par-3 mutant par-3 mutant par-4 mutant
  • 35. Result
    • par -1:
    • P granules are distributed everywhere
    • par -2:
    • no or incomplete localization
    • par -3:
    • in either 2 middle or 2 polar blastomere
    • par -4:
    • resemble par-1, but more P granules are in posterior-most cells
    WT
  • 36. Conclusion
    • par mutants are fail to localize P granules properly.
  • 37. Abnormal Differentiated Cells Production
  • 38.
    • Intestinal differentiation is most severely affected.
    • The failure to produce intestinal cells correlates with the strength of the mutation.
  • 39.
    • Method:
    • Normarski micrograph
    • Result:
    • par mutation may affect the location of the original cells
  • 40.
    • Method:
    • Immunostaining with different antibodies.
    • Figure C,D
    • Sensory neuron
    • Figure E,F
    • Pharyngeal muscles
    • Figure G,H
    • Birefringent granules
  • 41. Conclusion
    • Detailed cell lineage analysis of par embryos has not yet been undertaken.
    • par mutants may affect differentiated cell types.
  • 42. Grandchildless Phenotype
  • 43.
    • Observation: Normarski microscopy
    • Result:
    • Many par mutant larvae develop into morphologically normal adults but lack mature gametes.
  • 44. Wild type hermaphrodite par -3 mutant hermaphrodite O : oocyte S : spermatheca E : embryos V : vulva I : intestine GS : somatically derived gonad sheath http://www.wormatlas.org/handbook/fig.s/ReprodFIG1.jpg
  • 45. Conclusion
    • Mutations at the four par loci lead to abnormalities, such as cleavage pattern , timing of cleavages , and partitioning of P granules .
    • At terminal stage, par embryos exhibit different phenotypes of the differentiated cells, such as neuron and muscle.
  • 46.
    • The germ line seems also be specially sensitive to mutations in the par genes. All incompletely expressed mutations result in a grandchildless phenotype.
    • The par genes function in a common process requires for proper timing and pattering of cleavages, intestinal differentiation, and P granule localization.
  • 47. Continued Research
    • Actin microfilament have been shown to be required for the pattern of P granule location and the proper positioning of the mitotic spindle…….
    • So par genes may connect to actin…….