Blastomeres and Gastrulation

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Blastomeres and Gastrulation
Gilbert-- pp. 221-7 pp. 251-258
pp. 363-374 cleavage series of
rapid mitotic divisions where the large egg is
broken down to many smaller cells occurs
extremely rapidly-- G1 and G2 of the cell cycle
are ignored in Drosophila, nuclei divide every
10 minutes for 2 hours in frogs, 37,000 cells
are formed in 43 hours divisons are
synchronous-- all cells divide at essentially the
same time cleavage is controlled by mitosis
promoting factor (MPF)-- initiates cell
division after fertilization protein is high
in M phase, undetectable in S phase MPF is a
protein dimer of cyclin B and cdk2
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Blastomeres and Gastrulation
cyclins family of proteins which regulate the
cell cycle by binding to cyclin dependent
kinases (cdk) and vary depending on the cell
cycle cdks are relatively constant during the
cell cycle- require cyclins to work normally
cyclin B is only active during M phase, then is
degraded during cleavage, cyclin B is made
immediately after S phase translated from mRNA
stored in oocyte cytoplasm requires protein
synthesis to continue in cell cycle
translation inhibitors block cleavage,
transcription doesn't matter most organisms do
not transcribe during cleavage- relies on
stored mRNAs
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Blastomeres and Gastrulation
mid-blastula transition time in blastula stage
where nuclear transcription starts and
maternal mRNAs are exhausted-- G1 and G2 start
appearing since nuclei are now transcribing
genes (at different rates), division are not
synchronous after the transition cell divisions
occur normally with transcriptional blockers to
the transition but cannot undergo
gastrulation cell division during cleavage is a
combination of two processes karyokinesis
separation of chromosomes using the mitotic
spindle cytokinesis division of the cell into
2 cells (skipped in fly syncitia)
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Blastomeres and Gastrulation
cytoskeleton plays an essential role in cell
divisions microtubules make up the mitotic
spindle to pull chromosomes apart
microfilaments (actin) are used to divide the
cell contractile ring microfilaments that
squeeze between the nuclei and will
eventually separate the cell into two forms
around the mitotic spindle and perpendicular to
it cleavage furrow contracted dimple that will
eventually bisect the cell only seen during
cleavage stage embryos-- later divisions are
slower
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Blastomeres and Gastrulation
two major factors determine patterns of
cleavage 1) amount of yolk in the cytoplasm
2) egg cytoplasm compounds that regulate angle
timing of spindle yolk-rich pole is referred to
as the vegital pole other is the animal
pole isolecthal sparse, equally spaced yolk--
requires lots of external food found in sea
urchins, mammals, and snails characteristic
found in holoblastic cleavage embryos meroblastic
cleavage has yolk to feed embryo through its
development telolecthal yolk is separate from
the blastula cells at one end ie. birds
discoidal cleavage cell divisions occur in a
disk, on embryo surface centrolecthal yolk is
in the middle, cells divide on edge around it
superficial cleavage cells divide on the
periphery of the zygote
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Blastomeres and Gastrulation
cell fates in gastrulation can be determined by
1) cell cell interactions-- ie. juxtacrine
factors 2) uneven distribution of proteins
(often transcription factors) a. can be
bound to the cell cytoskeleton-- passively
acquired by cell b. actively transported
into one cell via cytoskeleton c. becomes
associated with one centrosome-- follows in that
cell two major types of cell fate decisions are
autonomous and conditional autonomous--
characterized by C elegans, internal
factors conditional-- ie. mammals depends upon
cell-cell interactions
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C elegans cleavage
undergoes rotational holoblastic cleavage
cleavages aren't planar initial divisions
produce 1 founder cell and one stem cell
founder cell ie progenitor cell-- produces
differentiated descendants stem cell goes on
to form the germ line first division is
asymmetric-- posterior (germ) cell is smaller w/
granules second division, the two cells divide
along different planes-- anterior (AB) cell
divides equitorially, posterior (P1) divides
transversely asymmetrically to give EMS and P2
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C elegans cleavage
posterior cell always divides transversely--
asymmetric cleavage with the most posterior
cell staying the germ cell all the cell
divisions are stereotyped-- always makes 959
somatic cells aanterior, pposterior cells
l left, rright
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C elegans cleavage
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C elegans cleavage
anterior/posterior within C elegans is determined
by the sperm entry site sperm nucleus and
centriole migrate to the posterior after
fertilization centriole small cylindrical
structures found in centrosomes that
help organize microtubules arranged at
right angles to other by organizing
microtubules, other proteins are moved around--
PAR2 is localized into the cortical (outer)
cytoplasm near the sperm nucleus PAR-3 is
localized at at the anterior cortical
cytoplasm PAR-1 is only found in posterior pole
cell keep maternal factors localized
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C elegans cleavage
P granules ribonucleoprotein complexes that
specify germ cells made up of translation
regulators-- helicases, initiation factors,
etc. initially randomly distributed, P-granules
migrate to the posterior cell requires actin
microfilaments, not depedent upon
microtubules stays in the posterior of cells
P1-P4-- marks the germ cell lineage
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C elegans cleavage
every organism has 3 axes anterior-posterior,
dorsal-ventral, left-right when AB divides,
gives an anterior (ABa) and a posterior (ABp)
cell ABp is located above EMS and will form
the dorsal part of the embryo EMS will become
the ventral side left-right determined by the
interaction of MS progeny with ABa C elegans
uses both conditional and autonomous
specification in blastomere development P1
develops autonomously-- always forms the same
group of cell types AB, however, requires
interaction with P1 descendants to form all of
the correct cells
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Autonomous Specification in C elegans
P1 differentiation is autonomous-- determined by
cytoplasmic factors P-granules work as
translational regulators controlling germ cell
fates 3 transcription factors (SKN-1, PAL-1, and
PIE-1) are used to determine cell fates in P1
descendants which do not inherit the
P-granules SKN-1 regulates EMS differentiation
required for posterior pharynx cells becomes
localized to MS cells by differential
localization of that cell related to bZip
family of leucine zipper transcription
factors SKN-1 turns on two other transcription
factors, med-1 and med-2 ectopic expression of
med-1 and -2 convert other cells into EMS
fates cascade of transcription factors
activing particular cell fates
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Autonomous Specification in C elegans
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Autonomous Specification in C elegans
PAL-1 is required in the next two divisions of P
cells- P2 and P3 PAL-1 mutants do not form
cells from the C and D somatic lineages PAL-1
translation is inhibited at the RNA level by
mex-3 mex-3 overexpression blocks PAL-1 in all
cells SKN-1 also inhibits PAL-1 expression,
blocking EMS lineage fates PIE-1 is required for
germline fates-- cells containing P granules
segregates into P cells via PAR-1 protein
inhibits both SKN-1 and PAL-1 PIE-1 knockout
animals give P2 cells that behave like EMS cells
appears to maintain cells in germline state and
represses somatic cells
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Conditional Specification in C elegans
EMS cells require a signal from P2 to form their
correct descendants should produce an E cell
and an MS cell absence of P2 (from PIE-1
mutant), gives two MS cells and no E cells AB
descendants will not substitute for P2 to give E
cells P2 produces a Wnt protein, mom-2 EMS
expresses a frizzled receptor homologue,
mom-5 pop-1 transcription factor is inhibited by
the mom-2 mom-5 pathway required to make MS
cells (pop-1 deletions give two E cells) P2 also
distinguishes ABp from ABa (otherwise the two are
equivalent) AB cell expresses GLP-1, a Notch
homolog P2 expresses APX-1, a Notch ligand
only ABp contacts P2, thus GLP-1 is activated
only in that cell
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Conditional Specification in C elegans
pharynx is derived from two lineages-- EMS and
ABa EMS cell depends upon SKN-1 (maternal
transcription factor) ABa depends upon GLP-1
signaling (notch) both genes activate pha-4
transcription factor microarray experiments with
pha-4 transcription factor deletions show pha-4
turns on most if not all pharynx specific genes
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Gastrulation in C elegans
there is not much of a blastocoel or blastopore
in C elegans E cells (Ea and Ep) migrate in and
form the endothelial cells (gut) P4 follows
Ea and Ep inside, followed by MS descendents
and C/D cells C descendants form the left side
muscles, D descendants form the right E
cadherin on hypodermis (developing from AB cell)
seals embryo
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Early Mammalian Development
mammalian cleavage is fairly different from other
early organisms fertilization occurs in the
oviduct and cleavage occurs every 12-24 hrs
mammalian cleavage is slow compared to other
organisms secondly, forms rotational
cleavage rotational cleavage, first division is
very typical dividing cell in half second
division, 1 cell divides in same plane, other
divides 90 degrees
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Early Mammalian Development
mammalian blastomeres do not divide synchronously
as amphibians do not go through 2, 4, 8 cell
stages, but often have irregular numbers fourth,
mammalian zygotes express their own genes very
early (2 cell) most organisms (ie. amphibians)
go 6-10 divisions before zygotic genes are
expressed lastly, mammalian embryos undergo
compaction compaction process at 8 cell stage
where blastomeres become tightly associated
(expressing the E cadherin adhesion molecule) and
form gap junctions morula 16 cell stage
embryo with few internal cells and mostly
external external cells form trophoblasts
(becomes chorion and fetal placenta) internal
cells form inner cell mass (ICM) which becomes
embryo
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Early Mammalian Development
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Early Mammalian Development
trophoblasts and ICM differentiation is the first
mammalian differentiation event in
cleavage FGF-4 secreted from the ICM supports
growth of trophoblasts Oct4 and Foxd3 are
expressed in the ICM- pluripotential
cells eomesodermin is expressed in
trophoblasts trophoblasts are required to form a
blastocoel (essential for gastrulation) blastocoe
l with ICM on one side form blastocyst
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Early Mammalian Development
zona pellucida extracellular matrix surrounding
the egg important for sperm binding and
blocking adhesion to oviduct walls ectopic
(tubal) pregnancy implantation occurs in the
oviduct where the zona pellucida fails to
block implantation strypsin protease which
degrades the zona pellucida for binding
uterus endometrium uterine epithelial cell
lining contains extracellular matrix that bind
trophoblast integrins other proteases digest
extracellular matrix of the endometrium so that
the blastocyst can move deeper into uterine wall
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Early Mammalian Development
the inner cell mass sits above the blastocoel as
if the egg were sitting on a mass of yolk (ie.
like a bird or reptile) even if there isn't any
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Early Mammalian Development
the biggest difference between mammals and
everything else is that they have a placenta
instead of yolk for food chorion fetal organ
derived from the trophoblasts ( a few
mesodermal cells from the inner cell mass)
used for absorbing nutrients decidua mother's
portion of the placenta that is induced by the
chorion inner cell mass continues to divide into
a blastocyst with equivalent cells first cell
type specialization segregates cells into 2
layers epiblast upper layer of cells
hypoblast lower layer of cells along the
blastocoel (primitive endoderm) 2 layered
structure is called the bilaminar germ disc
occurs during the process of gastrulation-- cells
move into several layers hypoblast is NOT
endoderm-- comes away from the epiblast and
forms the yolk sac (embryonic endoderm) and is
not part of the newborn
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Early Mammalian Development
embryonic epiblast forms when the epiblast sheds
cells separated from the other epiblast cells
by small clefts embyronic epiblast forms all
of the tissues in the adult amniotic cavity sac
formed by cells that separate from the epiblast
fills with amniotic fluid to protect the
developing embryo
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Early Mammalian Development
primitive streak thickening of the embryonic
epiblast starting at the posterior edge of
the epiblast extends forward down the middle
of the embryo primitive groove depression in
the middle of the primitive streak that allows
cells to migrate inside it. endoderm goes first,
moves to ventral migration requires loss of
E-cadherin expression from epiblast
cells Hensen's node (aka 'node' in mammals)
forms at the anterior of embryo thickening
that induces the formation of the notochord and
somites FGF8 knockout embryos never have cells
move into the primitive groove responsible for
turning down E-cadherin expression FGF8
regulates snail, Brachyury, and Tbx6 (controling
mesoderm pattern)
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Early Mammalian Development
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Early Mammalian Development
epiblast cells are still very primitive-- can
form any of the 3 germ layers only start
specializing after migration in through the
primitive groove while the epiblast cells are
moving and forming the 3 germ layers, the
extraembryonic cells start forming tissues to
interact with the uterus cytotrophoblast
proliferating trophoblast cells surrounding
epiblast digests uterine tissue, inducing
formation of maternal blood vessels syncytiotroph
oblast trophoblasts that have undergone nuclear
division but stopped undergoing cytokinesis--
becomes multinucleated cells works with the
extraembryonic mesoderm to bring maternal blood
vessels in touch with embryonic tissue
umbilical cord fully developed connection
between maternal blood vessels and fetal
circulatory system-- blood never actually mixes
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Early Mammalian Development