Cleavage and Breaking Symmetry

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Cleavage and Breaking Symmetry
E. G. Conklin, 1905

• Goals of Cleavage
• Make lots of cells
• Begin making cells different from one another

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How to give daughter cells DIFFERENT Identities
Extrinsic Mechanisms
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Cytoplasmic Rearrangement in Ascidian Zygote
A) Before fertilization Uniform cortical yellow
cytoplasm Gray, yolky cytoplasm inside B)
After sperm entry Yellow cytoplasm streams
towards sperm nucleus C and D) Yellow
cytoplasm moves with migrating sperm
nucleus Yellow crescent is formed
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Fate Mapping the Ascidian Embryo
Yellow Crescent
E. G. Conklin, 1905
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Blastomere Isolation Experiments
Muscle
No Muscle
Whittaker, 1977
-Blastomere pair containing yellow crescent can
form muscle when cultured in isolation -Muscle
markers come on at appropriate time relative to
control embryos -Muscle markers come on even when
cell division is blocked -Remainder of embryo
does not form muscle
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The Ultimate in Blastomere Isolation

• Nishida (Rouxs Archives, 1992)
• -Separate blastomeres after each division until
  110 cell stage
• -Allow blastomeres to develop separately
• -Get many individual cell fates muscle, gut,
  epidermis
• BUT NOT notochord or nervous system
• Thus muscle is AUTONOMOUSLY SPECIFIED
• notochord is CONDITIONALLY SPECIFIED

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Autonomous specification Cell fate determined
by intrinsic mechanism (e.g. cytoplasmic
determinant) Mosaic Development Cells of embryo
retain discrete fates when separated (e.g.
Ascidian embryo) The European Plan You
develop according to where you were born and
lineage is important.
As opposed to- Conditional specification Cell
fate determined by extrinsic mechanism (e.g.
signalling) Regulative Development Cells of
embryo can modify cell fate to form new
structures when isolated (e.g. human identical
twins) The American Plan Cells start off
underdetermined, albeit with some biases. They
are determined by their environment and tend to
behave similarly to their neighbors.
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Asymmetric cell division segregates cytoplasmic
determinants Need to 1) Break Symmetry 2)
Establish cell polarity 3) Localize
cytoplasmic determinants 4) Control mitotic
spindle
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Asymmetric segregation of P granules to P cell
lineage
Susan Strome
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Asymmetric Cell Division in C. elegans
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• C. elegans first cleavage asymmetries
• 1) Cytoplasmic flow toward posterior
• P granules/PIE-1 at posterior
• Asymmetric cleavage (post. cell smaller)
• Different cell fates

P Granules (Stome Lab)
PIE-1-GFP (Seydoux Lab)
Goldstein Lab
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Asymmetric Cell Division

• Break Symmetry
• Establish Cell Polarity
• Localize Cytoplasmic Determinants
• 4) Control Mitotic Spindle

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The C. elegans Hermaphrodite Gonad
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Sperm Entry Point Determines Posterior in C.
elegans (Goldstein and Hird, 1996)
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Sperm Entry Point Determines the D/V Axis in
Xenopus
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Sadler and Shakes, 2000
wt
No sperm nucleus
The paternal pronucleus is not required for A/P
patterning in C. elegans
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spd-2 RNAi
Control
Spd-2 disrupts centrosome function but not MT
nucleation
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Laser ablation of centrosomes disrupts polarity
Time of ablation (relative to establishment of
polarity)
after
before
during
GFP-PAR2
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Asymmetric Cell Division

• Break Symmetry
• Establish Cell Polarity
• Localize Cytoplasmic Determinants
• 4) Control Mitotic Spindle

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PAR6-GFP
PAR2-GFP
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Anterior Complex PAR3 PDZ Domain
(prot-prot) PAR6 PDZ Domain PKC3 Atypical Prot.
Kinase C Posterior Complex PAR2 Ring
Finger PAR1 Ser/Thr Kinase Not
Localized PAR4 Ser/Thr Kinase PAR5 14-3-3

• Who needs who for localization
• Anterior complex required for initial
• localization of PAR2
• PAR2 NOT required for initial localization
• of anterior complex
• PAR2 required for maintenance of anterior complex
• ALL required for PAR1 and PAR1
• required for none of others

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A Common PARt of Cell Polarity
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Breaking Symmetry A Model

• 1) Sperm contributes centrosomes
• 2) Centrosomes create asymmetry in cortical
  actin/myosin contraction
• 3) Cortical contraction creates posteriorly
  directed cytoplasmic flow
• 4) Cytoplasmic flow helps reinforce cortical
  asymmetry and localize P granules to posterior
• (essentially by magic)

Nance, 2005
Goldstein Lab
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Asymmetric Cell Division

• Break Symmetry
• Establish Cell Polarity
• Localize Cytoplasmic Determinants
• 4) Control Mitotic Spindle

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P1 Cell P granules
AB Cell glp-1
Mello, 1996
PIE-1 is Essential for P1 vs AB Cell Fate
Seydoux, 1996 Mello, 1996
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Centrosomes
Actin/myosin
ZIF-1 Dependent Degradation
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Asymmetric Cell Division

• Break Symmetry
• Establish Cell Polarity
• Localize Cytoplasmic Determinants
• 4) Control Mitotic Spindle

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Control of the Mitotic Spindle
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Heterotrimeric G Proteins Regulate the Mitotic
Spindle
-Regulate spindle orientation (e.g. in later C.
elegans divisions) -Regulate spindle asymmetry
Bellaiche and Gotta, 2005
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The posterior pulls harder on the spindle to
generate different size daughter cells
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Asymmetric Cell Division

• Break Symmetry
• Establish Cell Polarity
• Localize Cytoplasmic Determinants
• 4) Control Mitotic Spindle

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Asymmetric Cell Division in Neuroblasts
Baz Par3
Wodarz, 2005
Bellaiche and Gotta, 2005
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Of course, this couldnt be relevant for
Drosphila, since A/P polarity is pre-established
during oogenesis
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PARs and Oocyte Patterning in Drosophila
St. Johnston Lab
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Of course, this couldnt be relevant for the
mouse, since everyone KNOWS that mouse oocytes
dont have maternal determinants
The prevailing view Mouse eggs are
boring Evidence that mouse eggs/zygotes dont
have localized determinants -twins -early
bastomeres are totipotent when used in
chimeras -no evidence for germ plasm -transcriptio
n starts early--no need to invoke maternal factors
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Mouse Blastocyst 4.5 dpc (days post coitum)
OCT-4 DNA
Rossant Lab
ICM inner cell mass (makes embryo proper) TE
trophectoderm (m, mural, p, polar) PE primitive
endoderm
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Label each cell of 2-cell bro with different
fluorescent dye
One cell of 2-cell bro inherits sperm entry
point That cell is more likely to divide
first Decendents of that cell more likely to give
rise to embryonic territory
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(Our)

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May Issue
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CDX2 expressed in TE Genes normally expressed in
ICM but not TE (Oct4, Nanog) now expressed in
most or all cells in CDX2 mutants
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CDX2 Protein is asymmetrically localized to late
dividing cell of 2- cell bro that gives rise to
TE
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Cdx2 RNA is localized to one pole of the mouse
oocyte!
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Exon 1
Exon 2
Exon 3
Untranslated Region
Open Reading Frame