IGCLECE

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05/11 seminar ppt

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IGCLECE

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

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