The document summarizes research identifying genes required for cytoplasmic localization in early C. elegans embryos. Key findings include: 1) Mutations in maternal effect genes disrupted cytoplasmic localization, leading to defects like equal first cleavage, altered second cleavages, abnormal P granule localization, abnormal cell differentiation, and sterility. 2) Genes identified included par-1, par-2, par-3, and par-4. Mutations caused mispositioning of spindles, altered cleavage timing, and improper P granule localization. 3) The par genes appear to function in a common process governing cleavage patterning, intestinal differentiation, and P granule localization, and their mutation

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.

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…….