March 29, 2012

1. The cell cycle : key determinant of metazoan development • March 29, 2012

2. An accurate cell cycle is essential for complex metazoan organisms Accurate duplication limits genome size and hence organismal complexity Accurate cell production limits organismal size and lifespan

3. Growth is beneficial Non-dividing cell ages and dies Dividing cells can live much longer

4. Fundamental constraints on cell structure and growth R.I.P. A diploid nucleus can support only so much cytoplasm R.I.P. A minimum amount of cytoplasm is needed to maintain a diploid nucleus, depending on its activity state

5. World’s largest cell* Acetabularia mediterranea *from one diploid nucleus

6. Making big cells: oogenesis Requires: • Time • A stressed and often altered genome

7. Big diploid cells have to overwork their genomes Lampbrush chromosomes: Newt (N. viridescens) oocyte Acetabularia nucleus with lampbrush chromosomes

8. Big cells frequently amplify rDNA genes Turnover proportional to cytoplasm size Multiple rDNA genes Number of rDNA genes limits the amount of cytoplasm that can be supported ribosomes Many large ooctyes (Xenopus, crickets, etc) amplify rDNA, and so does Acetabularia

9. Cell cycle 101 G2 Normal cell cycle G1 S G1 cytoplasmic growth S nuclear growth M division Typical cycle length: 15-30 hr

10. Periodic expression and degradation of cdk kinases and inhibitors drive the cell cycle

11. Accurate cell duplication is achieved using a hodgepog of systems Duplication accuracy: replicate each region exactly once Licensing and replication fork checkpoints Sequence accuracy: minimize copying mistakes and correct errors Proofreading and repair Segregational accuracy: prevent chromosome non-disjunction or loss Control intrinsic instability: control transposons and illegitimate recombination

12. TF Controlling replication by licensing origins Cdk activity Pre-replication complex: geminin ORCs cdc6 Cdt1 MCMs MCM helicase loaded onto DNA Replication origin 1. Licensing requires low genimin and low cdk activity 2. Licensing terminates as cdk activity rises 3. Origin firing requires increase in CycE/cdk2

13. Replicating the genome once per cell cycle Pre-replication complex formation 1. License many origins low cdk activity due to high turnover, low geminin 2. Terminate licensing Cyclin E 3. Fire some origins high cdk activity, high geminin 4. Don’t divide until replication complete Inhibits licensing by promoting Cdt1 turnover, geminin binding, ORC phosphorylation, etc. Link low cdk state to completion of M (also geminin degradation) 5. Don’t license again until after M

14. Initiate mitosis by activating cdk1 kinase cdc25string • Replication fork checkpoint ensures S phase completion Dividing the cell once per cell cycle Timing onset of M • Degrade securin to initiate anaphase • degrade G2 cyclins to initiate G1 cycA degraded in metaphase cycB degraded in anaphase cycB3 degraded in late anaphase Guarantees no new licensing until mitosis completed

15. Variations in the cell cycle are common Alterations in basic cell cycle mechanisms are often involved.

16. Cleavage sea urchin Many large eggs initially undergo rapid cleavage divisions.

17. Egg size help dictate cleavage properties Xenopus cleavage Constitutive high cyclin E levels

18. Multiple origins needed for fast replication EM of DNA from cleavage stage Drosophila Replication “bubbles” Origins every ~2 kb, instead of every 80-100 kb during “normal” S 5 kb

19. Cleavage and high cell cycle protein levels 1. High CycE drives immediate S phase 2. High levels of initiator proteins load MCMs at many DNA sites 3. Replication still uniform: so some form of licensing still functions M phase quickly ensues after S; cdc25 high, may reduce function of replication completion checkpoint

20. Rapid cleavage precludes gene transcription Transcription time: gene size / 2 kb/min > 10 min for a 20 kb gene Drosophila segmentation genes don’t come on until after cell cycle slows down at blastoderm formation

21. Slow cleavage of mammalian embryos Asynchronous divisions compaction Rotational cleavage Slow cycle time allows zygotic gene activation at 4-cell stage

22. min Drosophila cleavage Cycles 1-9 S: 8 min; M: 2 min Cycles 10-13 S increases gradually; Allows onset of zygotic transcription

23. M S A checkpoint slows cleavage cycles 10-13 Lower DNA/cytoplasmic ratios reduce origin number. Completing S takes longer. P The DNA completion checkpoint now becomes important to ensure full genome replication cdc25 Grapes = Chk1 Mei-41 = ATM DNA replication completion grp and mei41 mutant embryos have shorter cycles 10-13, and chromosomes arrest at M phase of cycle 13

24. An DNA repair checkpoint coordinates recombination with oocyte development Spo11/mei-W68 Meiotic DNA breaks spnA/rad51, spnB/xrcc3, spnC/hel308, spnD/rad51C, okra/rad54, Brca2 Grk Atm/atr (mei41) Chk2 Breaks not repaired Vasa-PO4 grk Completion of oogenesis

25. Acquisition of G2 after cleavage (transcription and phosphorylation) Degradation of maternal string terminates cleavage and contributes to the mid-blastula transition (MBT); also tribbles and frustart genes

26. Acquisition of G1 phase dap

27. Withdrawing from the cycle Exit genes: salavador, lats/warts Cycle exit appears to result from a coordinated shut off of the cell cycle machinery: cyclins, cdks, etc.

28. Asymmetric divisions function at many stages of development

29. Alterning cytokinesis Incomplete cytokinesis in germ cells Cytokinesis mechanisms And many epithelial tissues

30. Endocycles Block cytokinesis Grow 2X Grow 2X Relieves stringent requirement for accurate chromosome segregation

31. Endocycle examples Liver, myocardium Rcho-1 cell line Mega- karyocyte Trophoblast cell Salivary gland cell Nurse cell Follicle cell

32. The endocycle: a truncated normal cell cycle M late S turn off G2 cyclins, cdk1 G2 S G G1 early S S A simple oscillator driven by out-of-phase CycE and APC/C Fzr needed to activate APC/C at entry

33. Incomplete replication in endocycles Cell cycle can reset without finishing S or executing M Replication checkpoint lost M late S turn off G2 cyclins, cdk1 G2 S G G1 early S Replication checkpoint Heterochromatic 30% of genome fails to replicate; Forms chromocenter S Likely inactivates centromeres, precluding subsequent mitotic division

34. Polyploid vs polytene endocycles Sister chromatids Anaphase glue If no M phase at all: (Diptera, legumes, ciliates) Then no separase Polytene chromosome If partial M phase: (endocycles in most organisms) Then dispersed chromatids Megakaryocyte endomitosis

35. Altering once per cell cycle replication Chorion gene amplification Endocycling cells seem less tied to once per cell cycle replication Polyploid Drosophila cells lack the p53-dependent apoptosis response to DNA damage Cancer cells frequently become polyploid and acquire specific gene amplification

36. The pupal Drosophila rectum contains polyploid mitotic cells Vertebrate endodermal tissues and cancers are rich in polyploid cells. PH3 Polyploid mitosis claimed to be less stable; source of chromosome instability in cancer.

37. 8c polyploid mitosis in rectal precursor cell 8C 2C DAPI

38. Polyploid mitoses exhibit an elevated frequency of lagging chromosomes DAPI PH3 Culex Metaphases with lagging chromosomes N=74 N=326 Culex N=56 N=326 Normal Lagging

39. Amitosis in ciliate macronuclei Random segregation? Assisted by nuclear envelope or MTs?

40. Regulating growth: the G1-S transition • Activate CycD/cdk4 • Phosphorylate RB • Upregulate E2F, CycE Timing G1 High cdk turnover cdk activity These processes determine the length of G1

41. Coordinating patterning and cell proliferation Developmental signals Nutritional signals string CycE

42. Regulation of mitotic domains by cdc25string During cycle 14-16 stg expressed in dynamic patterns that presage M by 10-20 min Other factors: cdks, cyclins, etc. remain in excess Improper cell cycle timing can disrupt gastrulation, and cause neural defects

43. Where are string regulatory sequences? String/cdc25 3’ 5’ Regulatory DNA? 100% conservation

44. Problems with introduced DNA Multi copy insertions (tandem arrays) DNA injection Expression level/gene: low variable Transposon-mediated integration Single copy insertions Expression level/gene: normal* Observed normal 1.0 expression* total DNA/gene size 1.0 2.0 3.0 4.0

45. neo Homologous recombination allows changes within a more normal chromosomal context Target gene 5’ 3’ Linearized DNA Problems: tedious, slow internal position effects: need double recombination Few cis-regulators have been mapped by homologous recomb.

46. Constructs to analyze string Vector end Test fragment Minimal promoter Reporter gene marker Vector end

47. Mapping string regulatory elements string RNA lacZ RNA

48. Today’s paper: Cytogenetic analysis of human blastocysts with the use of FISH, CGH and aCGH: scientific data and technical evaluation Elpida Fragouli1,2,*, Samer Alfarawati1,2, Danny D. Daphnis3, N-neka Goodall4, Anastasia Mania5, Tracey Griffiths6, Anthony Gordon7, and Dagan Wells1,2 Human reproduction doi:10.1093/humrep/deq344 NATURE MEDICINE VOLUME 15 [ NUMBER 5 [ MAY 2009

49. Background: chromosome instability in human reproduction 1. Spontaneous embryo inviability 2. Aneuploidy syndromes Down’s syndrome Most common human genetic defect; most common form of mental retardation

50. Down’s syndrome 1. Maternally influenced; 90% of extra chromosomes are from female 2. Maternal age effect; >30X increase between age 30 and 50 High recurrence in young women with a DS child;