CLEAVAGE:

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CLEAVAGE
BECOMING MULTICELLULAR
BY KAREN CHOI
BIOL 430
January 30, 2002
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What is CLEAVAGE?

• Occurs after fertilization
• First step in embryonic development
• Division of fertilized egg into a large number
• of smaller cells (blastomeres)
• - Leads to hollow blastocyst

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Cleavage Divisions

• Mitotic
• Nuclear divisions are typical
• Cell cycles are short!
• No periods of growth between cell division
• and mitosis

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Cleavage Divisions

• MASS OF EMBRYO DOES NOT
• INCREASE DURING CLEAVAGE

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Compaction

• Occurs at the 8 cell stage
• Is the change in shape of the embryo
• Blastomeres flatten against each other and
• form tight junctions
• Original cell mass shape changes to
• mulberry-shaped solid cell mass ( morula)

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Polarization of Cells at Compaction Stage

• Microvilli are confined to the apical surface
• Expression of membrane transport pumps on
• apical surface (e.g. Na pumps)

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Compaction
Compaction of Mouse embryo. (Fig. 8.9 Wolpert
1998)
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Formation of the Blastocyst
(Blastocyst - mammalian embryos) (Blastula -
other animal embryos)

• Cell adhesion molecules joining blastomeres
• give rise to an inside and an outside
• Inside will eventually be fluid-filled
• A blastocyst is a hollow ball of cells, made of
  an
• epithelial layer that surrounds a fluid-filled
  cavity

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Further Cleavages

• Radial Cleavage
• gives 2 polarized cells
• become trophectoderm and
• extra-embryonic structures

• Tangential Cleavage
• gives 1 polarized cell
• and 1 non-polarized cell
• - become inner cell mass

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Blastocoel
- fluid filled cavity inside the blastocyst

• Fluid accumulates inside the embryo
• via active transport of Na and other ions

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Accumulation of the Blastocoel
Tight junction
Blastomere
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Blastocyst Stage

• Blastocoelic fluid accumulates
• 2 distinctive tissues trophoblast cells
  (hollow sphere)
• containing the inner cell mass

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Differentiation of the epithelial apical
junctional complex during mouse preimplantation
development a role for rab13 in the early
maturation of the tight junction
Sheth et al. 2000. Mech Dev. 97(1-2) 93-104
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Importance of Cell Adhesion

• Cell adhesion is critical in trophectoderm
  differentiation
• and the changes in form (morphogenesis) of the
  blastocyst

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Importance of Cell Adhesion
By understanding the mechanisms involved in
preimplantation development, better methods may
be developed to promote or inhibit fertility.
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Objective of Sheth et al. (2000)
To look at how the epithelial apicolateral
junctional complex (AJC) is made during
trophectoderm differentiation in the mouse
blastocyst.
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The Apicolateral Tight Junction

• Consists of
• apical tight junction (TJ)
• (zonula occludens)
• subjacent adherens junction (underneath)
• (zonula adherens)

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Tight Junction

• Permeability seal
• Prevents paracellular transport across
• epithelium
• Proteins occludin, claudins, ZO-1, ZO-2

Fig. 7.30 Karp (1999)
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Adherens Junctions
Fig. 7.26 Karp (1999)

• Binds a cell to its neighbours
• E-cadherin/catenin complex (cell adhesion
  proteins)
• Connect to actin filaments

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Methods

• Mated 4-5 week old mice
• Collected embryos at various stages of cleavage
• Used
• Immunocytochemistry
• Confocal microscopy
• Transmission electron microscopy
• RT-PCR
• Western Blots

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Molecular Maturation of the AJC

• Visualized ZO-1 (tight junction) and
• ? / ? catenin (E-cadherin adherens junction)

Fig. 1. Sheth et al. (2000)
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Result
8-cell stage ZO-1 and catenins co-localized
(apicolaterally)
16-cell stage most contact sites are still
co-localized some separately localized
(ZO-1 more apical)
and
EB (early blastocyst) nearly all contact sites -
ZO-1 and catenins are separately localized (ZO-1
more apical)
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Molecular Maturation of the AJC (cont)

• The molecular organization of the AJC changes
• during trophectoderm differentiation from a
  single
• complex (TJ and adherens junction together)
  to a
• double complex (TJ apical to adherens
  junction)

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Structural and Functional Maturation of the AJC
Examined with TEM the AJC at different stages of
trophectoderm differentiation
Fig. 3 Sheth et al. (2000)
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Result
8-cell stage no membrane specialization seen
16-cell stage e- dense area with clear
intercellular space ? Adherens junctions
EB apical area e- dense region with no
intercellular space ? Tight junctions
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Structural and Functional Maturation of the AJC
During 8,16 and EB stages, incubated in
fluorescent sugar solution ? paracellular
permeability decreases with embryo growth

• The AJC segregates into 2 distinct structures at
  the same
• time a permeability seal is formed at this
  site.

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Rab Proteins

• Establishment and maintenance of AJC - need
  junctional
• proteins at correct place at the membrane
• Rab proteins - GTPases
• Works as part of the intracellular protein
  transport system
• gt 30 Rab proteins known

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Rab13 and the Maturation of the AJC

• what are the potential mechanisms responsible
• for spatial separation of AJC?
• looked at Rab13 mRNA expression at various
• stages of cleavage

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Result

• RT-PCR - shows Rab13 mRNA
• present at all stages of cleavage in
• trophectoderm
• B) RT-PCR - shows Rab13 also present in
• the ICM
• Western Blot analysis -
• - confirmation of (A) protein is made
• - Rab13 expression throughout
• cleavage
• - Post-translational modification

Fig. 5. Sheth et al. (2000)
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Rab13 and the Maturation of the AJC

• Confocal microscopy showed Rab13 assembled at
  the
• membrane at the same time the AJC is created
• Rab13 and ZO-1 co-localizes at junctional sites
• Rab13 associates with the AJC from an early
  stage
• throughout the period of assembly of the TJ

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Summary and Future Directions

• AJC organization changes as cleavage progresses
• As AJC structure changes, it gains function
• Rab13 is involved in AJC maturation
• But
• what does it do - which protein does it
  traffic? ZO-1?
• what other Rab proteins are involved?
• what controls the expression of Rab13?

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References
Fleming T., Sheth B., and Fesenko I. 2001. Cell
adhesion in the preimplantation mammalian embryo
and its role in trophectoderm differentiation and
blastocyst morphogenesis. Front Biosci. 6
D1000-7. Karp G. 1999. Cell and Molecular
Biology. John Wiley and Sons, Inc. New
York. Larue L., Ohsugi J., Hirchenhain J. and
Kemler R. 1994. E-cadherin null mutant embryos
fail to form a trophectoderm epithelium. Proc
Natl Acad Sci USA. 91 8263-8267. Sheth B.,
Fontaine J., Ponza E., McCallum A., Page A., Citi
S., Louvard D., Zahraoui A., and Fleming T.
2000. Differentiation of the epithelial apical
junctional complex during mouse preimplantation
development a role for rab13 in the early
maturation of the tight junction. Mech Dev.
97(1-2) 93-104. Wolpert L. 1998. Principles of
Development. Current Biology Publications
London.