Download lH---b

MCB 141
(mouse)development
Mammalian
March20,2014
Lecture9
The mouseis a eutherianmammal,an amniotewith full placentaldevelopment.
development;
and
(Non-eutherian
(platypus,
whichhaveyolkyeggsandnoplacental
echidna),
are:1) monotremes
mammals
whichhaveslightlyyolkyeggsandbrieflateplacentation.)
2) marsupials,
Mammalsare unitedby theirmilkproduction,hair,and a few othertraits,but not placentation.
Simitaitiesof mammati and birdsin their developmeni:
.Like birds,mammalshave a blastodisc(flat in humans,althoughin mouse it is bent intoa cup-shape),
.a hypoblast(amphibians'haveno hypoblast),a primitivestreak,and Hensen'snode,
.Like birdsthey have 4 extraembryonictissuesincludinga yolk sac even thoughthere is no volk.
Differencesof mammalsand birds:
.The mammalianegg is very small(100 pm) and containsno volk,few stockpiledmaternalmaterials,
little
granules.
germ
plasm
probably
and
no
or no cell polarityrelatedto axes of the embryo,
.Gene expressionstartsvery early,at the 2 cell stage.Axis specificationoccursby mechanismsinvolving
cell interactionsand early cell function,ratherthan by cytoplasmiclocalizations.
.A mammalianinnov ir is the trophobla
, an extraembryonictissuewhich is specialized
uterine
wall, uptakeof nutrients,and waste removal.
in
the
implantation
placental
i.e.,
for
for
development,
and differentiatesmuchearlier.lt replaces
ectoderm
embrvonic
from
seoarate
comoletely
This ectodermis
and goes beyondthe chorionicectodermpf birds.
&w
Germ cells and oogonesls: mammalian germ cells don't have germ plasm granules. Their origin is not
understood. They arise in prirnitive gut tissue and migrate through the gut lining and into the gonad.
Zou pcllu<ldr
(viblUre
eovelope)
I
I
.
C umul us
cells
I
.t
\mtero'
I
villi
Coosld
r<o
elB
At ovulationthe ooryte is releasedformthefotlicl'ebut is slillmvered
cumuluscells.Theyfall off soonafterfertilization
I
i
Wetine
0uid240 mosm
Att
lH---b
GRAA.FIANFOLUCLE
I
cL'
++Ala+
k*c
HCo;
2<.ll !ta8.
blastocYst
€vitY
(320 boso)
First
Morub
3. hatching
i
Eiffi"tB
ao earlY I cell em-brYo
into 8 ellg. Combine them 2' 3, or
4 at a time io any orieutation.
Soon tbereafter, celle Polarize
their contentr go ag to move
microvilli and certaio trans'
membra.ue proteina away from tbe
site ofcontdct with other cellg.
O ther trangmembrane Proteina
(cadherine) collect at the contact
Jte. Thev egtabliah aa"aPical'
basal polatitY, as have manY
kinttg ol epitbelial cells.
!
I
'Dla3to<Iet
(128 cells)
apiel
ffface
of ell
14.imolantation
FtcuRE2r
|
.
E
OaY
\
\
I
Developmot
of a human embDfo
frpm fertiliatior
ro implantation.
baslateEl
surfacc.
of ell
Gavitation,or ,.formationof the blastocystcavitlt" oecursat the 32, 64, 128cell stages'
cellsaretightlyjoinedin an epithelialsheet,andtheyare polarized(withapicaland
Trophoblast
spages.Cl-followspassively.
Na*intothe intercellular
regions),-pumping
basalrryer,nbrane
fonnation,inflatesthe cavity.
osmoticmovementbfwater,as in blastocoel
Stepsin establishingthe dorsal-ventralpolari'ty
of the mouseembryo
3?-Cell st ge
Compocted
(@ss stion)
&<ell stagr
hlii"or. J
rloM
(pd4, nanog,
on)
sox2genes^
Step 2
Ebst€tsl
.evity+
fom3tion
/
Trophoblast
cell
TiEht
iuftlions
2
Events in mouse development, pre-implantation.
Day 1.5:2 cell stage;geneexpressionstarts.Cleavagecontinues.
r
D3y 2.5: earlyI cellstage,cellstooselyanangedin a morula("bunchot,gr9R9q")-.
'
Althoughthere ls a regularrelationshipof the spermentrypoint to the dorsoventralaxis,thb B cells are developmentally
equivalentrrvtrenisolated;the gctetcan be splitinto 2 quartetsor fodr doubletsin any directionsand all piecesdevelop
"chimeras"having
normally.Atso,you can presstogether2 or 3 embryos(16 or 24 cells)and these give rtormaFsized
parents.
4 or 6
the surfaceand
ln the late'8cellstage,the compaction procesgbegins.Cellspolarize(segregate)
suchthat microvillian-dvarioussurfaceproteinscollectat whateverpart
internalcvtoplasmic-materials
between
of the cel[srirfaceis not in contactwith othercellq. Tightjunctionsand desmosomes'form
ce|ls,makinganepit@betweentheextema|mediumandintercel|ular|iquid.
r Mice fnom single culhred
cells
Plate the cells in a dish of syrtthetic nutrient culhrre medium.
I.ct celle Erow as separate elonesthrough 15 mitoiic generatigns
to EeCthoirsands ofbells, Pick one clone and replate every I0 datc
for-a year or so (nrany hundreds ofcell generations).
black mouse parents
(fvhite recessiveto black)
cellsfrom
d?
ffi:rd
These p.roliferative
innercellrnasscells
are'called
EMBRYONICSTEM
CELLSdTESCEIIS.
reDilovethe 16 inner cell mass riells from a
-thecells'
ili"ei*yii iel' 128cells),disaggrpgace
i{iiec{edEScellscan developto anyembryonic
tkieu€or to hypoblast,but hotto trophoblast.
lfidant bhstocvslIntoa whitemouse'fosler.
m&hef. whlctrihen givesbirthto a black3ndwhite
'pup' (mousebaby).
lv--\
ve
-/
MITJ
baby
^+
Collec0cells from.the dish .
and draw into a pipet. Inject
cells into the blastocyst obtaine<l
from rrhite mouseparents.
r/:,t<1
foster
mother
t
r *r-----\.
/
whiCemouseparents
Grow up the pup ind crossit with a whi0e mate.'
Get some wbolly black mice.and eomewholly white
mice. The black babies come frori germ cells
descendedfrom the cultured cells.
Conclude: 1) These ES cells probabbfdid not maintain specific
cytoplasmlc localizations for the many mitotic generations in culture.
Morb tikely al lhe organizalion ls generated by the'interadion of cells
wilhin the einbryo. 2) This b a g.{ri}dfnetffi ?omake MOSAIC
EMBRYOSones contailtkgi$et er mere ldnds of genetically different
(embryonic
stem
', innercell inasscells
in a
cells,catledEScells)proliferating
, petridishin nutdentmedium.
to strdv cell
Transgenesfisin tfte mouse (Gilbertbook,p. 94)
;I*rH@T @
Inncr ccll
mass
Microinjcct
$ansg€ric
ES cels into
host cmbryo
Clonedgcnc
in wctor
Culturcof
cnrbryonic
stem(ES)cills
Mix cmbryonic
stm ells with
. clgrcdgcoe
(e.9.,me gene
enosding
ore€nfluorescenl
(g/F)and a
i@n
'erotnoter
for its
Crss s.tdmerk
mouseand
I
tl
,@"@,
-'\ffi*'**
|
tr Dsgoicmice
HcteozYgous
fl|\
'eroresston.
Thisgerg
cohesfromieltYd$tl
i
,/
chimericProgenymlce
wild'tYPc
Cni-.ti.
I
Y
& ^. @=
--f--^ep
'uurL-a=:-'--
tiom€E{€lotls
trarcgenic
(!5%l
3
but someare parallelto the
Day 3: divisionto 16 cell stage;mostcleavageplanesare perpendicular,
surface,so mostcet6 1eg.12-13of them)are stillincludedin the surfaceepitheliumbutsome(egem-bryo's
34 of them)are entirelvinternal
Divisions
continueevery12hr.At 32cellstage,if youremovetheoutercelllayer,theinnercellscanforma new
horizontal
to thesurface.
outerlayer;andoutercellscanformstillforma newinnerlayerbydivisions
removed.
the
other
if
replace
no
longer
can
outer
cells
inner
and
By the 64 cellstage,
the outercellsdevelopas the trophoblast
Thusan outerlayerof cellsand an innermassare established:
and the inner cell mass (lCM)cellsdevelopintothe embryoas
(to placentaltissirewhichis extraembryonic)
as symmetry
tissues.Compactionand cleavagehave_acted
weil as to other non-placentalextraembryonic
brcaking mechanisms(onesthat do not requirepre-localizedspatialinputsbut give uniquegeometric
outputsi.Trophoblastcellsexpressthe cdx gene,whereasICM cells expressthe oct4, nanog,and sox2
junctionsand cell polarity,leadingto
of intercellular
genes.The Hippo siqnalinq oathwav respondsto differences
cdx geneactivationin the outercells.
Day 3-S:the blastocystcavity formation processbeginsand continuesin normalenibryo.The "Ner/K
ce1ls,pumpssodiumions
niF""u purp', whicfris locatedon the innersurfaceoi the polarizedtrophoblast
ions
followpassivelythrough
and
bicarbonate
fromthe'cellinteriorsintothe spacebetweencells. Chloride
andso the ion
escaping,
junctions
prevent
from
ions
the
Tight
charge.
Lfectrii
inannetsto balancetfre
concentrationin the intercellularspac-esooneiceeds that of the externalmedium. Waterentersby osmosis
.w"rii the spacebetweencelis,creatingthe btastocystcavity. The surfacelayerof tightlyadjoined
"no
cellsexoands,and the innercellsdelaminateas a clump,attachedto one siteof the trophoblast
trophoblast
tt;r.- ihi" is noill utastocyst. The site of attachmentof the innercell mass to the trophoblastlayeris
prbuautv
random.Cavityformationacts as a symmettybreakingm.echanism.
'
overtheinnercellmass(lCM)andthe
thepolartrophoblast
making
layeinowdifferentiates,
Thetrophoblast
cellsbecomepolyploid'
trophoblast
The
mural
surface.
blastoryst
of
the
muralirophoblastovertheiemainder
verylarge,andflat.
multinucleate,
Day4 and 5: The ICM cellscontinuedividing.ICMcellscontinueto expressthe oct4, n9!og,andsox2
with the others.The gata6-expressing
gJ;"., inO tome additionqtlyexpressthe gita6 genewhile interm.ixed
polar
and then someof them
trophoblast,
the
from
anO
aviray
Olistocoet
th'e
iells then sort out, toward
layer.Thesecellsformthe hypoblast layer,alsocalledthe
ontothe muraltrophoblast
mior,aie
ffimuryonic endodermor.parietalendoderm'.Hypoblastcellsthat stayin contactwiththeinnercell
massare fhe 'visceralendoderm'.Interiorcellsof the innercell massbecomethe epiblastor embryonic
uJtoCerr, makingthe entireembryo(stillexpressingthe oct4,nanog,and sox2 genes).The few cellsof the
muiaflrodnobhsiwhichare mostbisiantfromthe lCM,and thereforenot undedainby innercells(either
'
,
hypoblastor epiblast),produce"hatchingenzyme".
cells
enzymeproducer
andhatching
hypoblast,
epiblast,
Theembryonowhis a clearpolarig:polartrophoblast,
polarityof theeventual
embryolEachICMcell
Thisrelatesdirectlyto thedorsal-ventral
of themuraltrophoblast.
parts(seep.4). .
canstilldevelopto all embryonic
Day 5: The embryo,now at 128cells,hatchesby digesting.aholein the zonapetlucida(thevitelline
end intothe wall'
envelope)and imolantsin the uterinewall,the polartrophoblast
butthiswillbecomemuchstrongerduringimplantalion.
symmetry
mayhavea weakbilateral
fire btastocyst
in the oocyte;theydevelopin
Gonclusions:the poliritiesof the mammaliinembryowere not pre-localized
the courseof cleavageand osmosis,frornsymmetrybreakingoperationswhich invariablygene6te singular
of the ICM).
-spatialarrays(insideToutside
cells;and randompartialdelamination
Cell lineage relations'hip5in the Eutherian mammal early embryo
128 celSblastocyst,
trophoblast
--r(rdpnectooerml'ion\.^
{
'
I
^
L,^
ii^Arg,
^...^_ cefls
'osmotic
o.utet
(6.t!p1ct:on:.
cetl wbization)
1
swellino of
Lurto&st
acvitl
implantation
lrophoblasi giant cells
(>128ploid)
mural topheclod?rm --->
(trophoblaslcells'not
contacting ICM- I
over blastocystclvity)
polar trophectoddrm
(trophoblast cellsl
-/
1------>
contacting ICM) '
8-cell
cleaved
,/
-/
ecloplacental cone
./
./ltotlophobtastcells
invade uterine wall,
implanl, interact with
matemal tiSSUeS
extraembryonic F
----+
ectoderm
chorion ectoderm
amnion ectoderm
embryonicectoderm
"nn \
ICM celts
innercells (no
compaction,no
polarization)
\
inner cell mass
(rcM)
toward
polartroph-/
,r'
-
epiblast \=p6"
primitivt
ectoclem
embryonicmesoderm
parietal
\extraen&ryonicy'
ertraembryonic mesoderm
(cfnrion, amnion)
mesoderm (.
-\
tffiremayTrevisceral
arpther sn,urceas wel/) ' extraembryonic mesoderm
<r
lcnz\
cells
\
exposed to
blaslocyst
cavity
(blastocoel)
EMSRYO
embryonicendoderm
f\ir""rsh
hypoblasl
(primilive
|
---\endoderm) -
I
|
|
gotk sac, ailantbis)
-\.
*ElSiiX'"T"''"=1----:Sffi
\
allantoisendoderm
lmplantation of the 128-cell blastocyst and then after implantation, the role of the anterior visceral
endoderm (AVE) in primitive streak formation. Note similaritiesto the Cerberus-secretinghypoblastof the chick.
Professor Harland will continue with mouse gastrulation on 4/,2113.
Day 5.5: The pro-amnioticcavity opens by programmedcell death (apoptosis)of the deep cells of the epiblastand the
extraembryonic
ectodermof the polar trophoblast.
Day 5.5-7:The anterior-posterior
axis is establishedas the anteriorvisceralendoderm(AVE;part of the hypoblastlayer)moves
from a centerpositionunderlhe epiblastto a positionon one side, probablydrivenby cell migrationand cell proliferationin the
hypoblast.Oppositethat side, the epiblastforms the posteriorprimitivestreak.
Day 6.5: Primitivestreakformationcontinuesin the epiblaston its side oppositethe AVE of the hypoblast(the futureposterior
4
endof theembryo).
begins.
Day7.5Theprimitive
streakreachesfull lengthandgastrulation
implantation.
tn
havea deepinvdsive
andprimates
lmplantationof the blastocyst in the uterinewall. Bothrodents
is moresuperfcial.pp.353-354.
implantation
othermammals,
(B)sDfr.----
7 daYs
Blastocyst,
( D ) l 0- l I D ay s
Uterinelining
a
MaternalcaPillarY
Uterine epitheliun
(endometrium)
Trophoblastic
lacunrc.
(mattrnd
blood supply)
Amnionic
cells
Anrnionic
caYity
Epiblast
Hypoblast
.
Embryonic
cpiplast
Blastocoel
Trophoblast
Human
Formationof
extraembryonic
mesodernr
5'sdavs
The proamniotic
cavttyopensby
-'
apoplosls(programmed
celldeath).
"*
.
Thosedose to .€Eh
Centralcells
Centrat
cellsdie. Those
iisceralendodermtive. trffi
-
6.0 days
q.5 dayl
eO
L,l
I
I
Eg
EISIEG
usa___{egft{
..'dodn
c
o
F
:
d.9
l '.m-ffi"Hstrf,
I
Extraembrfonic
reticulum
6E
W
oo
OE
.bo
s) z,
Onsetof gastrulation
The role of the anterior visceral endoderm(AVE)in establishingthe anteriorposterior axis of the mouse.
( A)
-_
@
Cerberus(anti-Nodal,
anti-Wnt,inti-Bmp),
Dkk (anti'Wnt),
Lefty (antl-Nodal)
Day 5"5
Cylindrical symmetry
Day 5.25
Proximal
I rf-!:T,'
1a
O
rQ
*^..o.e
-Day6.25 Bilateralsymmetry
Extraembryonic
ectoderm
-_.}-
<
Visceral
endodirm
o o Nodal.
o ao cripto (co-receptor
for Nodal)
pistal
1'.'
-*
otx2
cndoderm
What does the AVE do?
Futuresite of
anteriorneural
ectoderm
induetion, '
Primitive streak
It is essentialfor 1) positioning
of the primitivestreakoppositeit,2) preventing
primitive
secondary
streaks,and 3) preservingthe site of developmentof the anteriorneuralplate(future.,forbbraiir
and midbrain).lt
resemblesthe hypoblaslof chicks-bothproduceCerberusand therebypreventNodalsignalingandendomesoderminduction.Bothare displacedto one side,andthenthe primitivesteakformswhereihey areabsent,
whereasanteriorneuraltissueformsnearwheretheyare present.
TheAVE doesnot itselfinduceanteriorneuraldevelopment.
Thatis doneby the nodeand headendomesoderm,the'headorganizer".Whenthe headorganizeris combinedwiththd,AVEandepiblast,muchmore
anteriorneutraltissueis inducedthanwhenthe headorganizeris combinedwith epiblastalone.TheAVE
keepsthe epiblastin a responsivestatefor neuralinductionand anteriorneuraldevelopment
by preventing
endo-inesoderm
inductionand preventingepidermisforinationandposteriorization.