Mammalian Morphogenesis Flashcards
first steps
cleavage division - mitosis w no growt
cant make feeding structures w/out many specialised cells
so need to use resources at hand
large oocyte divides w/out growth
cell cycle just goes:
S
M
S
M
…
get to about 1000 cells where they start to synthsise proteins
when does zygotic gene transcritption begin
4 cell stage
one of first ones is E-cadherin
causes cells to stick together and compact
compaction and trophectoderm formation
E-cadherin expression
causes cells to compact
some cells will be entirely in the middle
some will be at free edge
cells at edge differentiate into different type of epithelium - trophectoderm
rest remain as middle mass
blastocyst formation
trophectoderm forms
inner cell mass inside
trophectoderm begins letting fluid in - forming a blastocoel at the centre
blastocyst hatching
blastocyst forms and then hatches from the Zona Pellucida from the oocyte
the trophoblast of this hatched blastocyst invades the uterine epithelium
-forms interdigitated columns that will develop into placenta
hypoblast/epiblast formation
blastocyst has blastocoel cavity inside
some cells of ICM face this cavity
-causes them to differentiate forming layer called the hypoblast
this will then line the trophectoderm on the inside and surround what will be the yolk sac
the remaining layer of ICM cells touching the hypoblast polarises and lets go of the overlying trophectoderm cells
-forms the epiblast
now have a kind of disk inside with hypoblasr and epiblast formed from the ICM
monozygotic twins cause
most errors at stage of forming hypo/epiblast are lethal
but some subtle and rare errors are embryo tolerable resulting in identical/monozygotic twins
2 ways of this happening
-cells separate inside the zona pellucida (1/3)
-two ICMs form - almost the rest of MZ twins (2/3)
Cells separating inside the ZP
have two trophectoderms form
ICM forms hypoblast and epiblast in each
2 distinct systems with all the structures each
two ICMs form inside same trophectoderm
Two ICMs form
so get two sets of epi/hypoblast wihtin same trophectoderm
will share a placenta
at risk of foetal transfusion syndrome where one twin steals supplpy from the other and one twin turns out smaller
axes of embryo after epi/hypoblast formation
are currently radial like a jellyfish
need to generate other axes for morphogenesis
have top and bottom but need others
need to mark specific part to distinguish this out of radial symmetry
no way to transform 2 coordinate system of a disc to a 3 coordinate system of 3d object
but if the embryo can somehow mark one part of the edge of the disc different to mark “12 o clock” then it can have 3 coordinates
and these can be transofrmed into the body axes
solution for “marking 12 o clock” and generates the beginnnings of the axes
cells at centre of HYPOBLAST express Hex
Hex expressing cells move out to rim - congregating at one point
these cells at one end mark the head end and make inhibitory signals inhibiting progress in the epiblast cells above them at this end
the furthest away cells in the epiblast (so most towards the posterior)
and so are not inhibited
can begin making tail end of the primitive streak
these sites on the discs mark the future trunk and head
3rd and rarest way of making MZ twins
the formation of one body axis depends on the Hex-expressing cells being in ONE point on the rim of the hypoblast
if there ends up being two distinct sites -then two heads will form and maybe two primitive streaks
can end up with conjoined twins
can also get a partial axis duplication if the two head organising areas still agree on one site for the tail end
this can form 2 headed andimals
usually prenatal in humans but has seen survivals
germ layer formation
GASTRULATION
as primitive streak grows forwards on the epiblast
the advancing end is called the NODE
gastrulation fillows the node - organisation of germ layers of the body
gastrulation:
epiblast cells converge on the midline
-some of these cells push through and move the hypoblast aside and form new cell layer - endoderm (epiblast cells that push in form this)
-other cells that push through stay in the middle and spread out in the space between the epi-/hypoblast discs to form the mesoderm
-other epiblast cells remain in this layer and form ectoderm
the middle part of the new endoderm rises to make notochord plate
this notochord plate then detaches to become the notochord
germ layer cell fates determinant
depends on where and when they dived down
-never dive down - ectoderm (CNS, Epidermis)
-dive down firs right through the node - become endoderm and then form the notochord
-cells that dive early - but not directly through node - become endoderm (gut and most abdominal organs)
-cells that dive down later become mesoderm (muscles, connective tissue, urogenital system)
mouse cup peculiarity
mice are annoying
they make a cup shape rather than staying flat
this cup is arranges inside-out
hypo and epiblast - epiblast on inside of cup
-ectoderm on inside
mesoderm between
endoderm inside
neurulation basic
whole of CNS formed by forming a tube
ectoderm over the back
two edge stripes and one centre stripe forms
cells at these stripes deform their shape
centre stripe folds to form infolded valley
edge stripes form ridges and meet at top of tube
neurulation: when the cells at top meet
stick to each other and let go of their neurectodermal neighbours and adhere to each other
pinches off the tube and separates under the sealed ectoderm forming the neural tube
top of the tube “zips” up
failure of neural tube closure
failure to zip up properly can leave it open - vertebrae cannot form where this has happened
causes Spina Bifida
Mesoderm development basic
each side of neural tube the mesoderm is also developing
embryo is lengthening too
-ends of endoderm pulled out into tubes,
-firther lengthening makes these tube sections linger
-connection to the yolk sac eventually appears as just a minor branch from the tubular gut
components of the mesoderm
paired on each side of neural tube under the ectoderm covering
-notochord below neural tube
-somite directly to side of neural tube
-lateral mesoderm next to that
>wolfian duct in it next to somite
>celom inside it
>somatic mesoderm on part above celom
>plachnic mesoderm below celom
endoderm below this
aorta is below somite
formation of structures from somites:
somites scatter cells and reform to make vertebrae, ribs, muscles and dermis
somite location kind of prefigures vertebrae location
first half of one somite and back half of another come together to become vertebrae (similar to drosophila parasegments)
other mesoderm component diagram
-neural tube
-notochord below it (chorda mesoderm)
-paraxial mesoderm next (head and somites formation)
-intermediate mesoderm next to that
-lateral plate mesoderm next
paraxial mesoderm give rise to
head
somites:
sclerotome, syndotome
myotome
dermatome, endothelial cells
intermediate mesoderm
kidney
gonads
lateral plate mesoderm
splanchnic
somatic
extra-embryonic
the neural crest basic
small region of cells formed after neural tube pinches off
some remains neural - others become different
neural crest migration
forms:
peripheral nervous system
endocrine and paraendochrine derivatives
pigment cells (melanocytes)
facial cartilage and bone
connective tissue
point of this is that they go all over
E9 twisting of mouse embryo
at about E9 the embryo turns
ends up bent around the other way - the “normal way”
from endoderm on outside - “belly out”
to ectoderm on outside - back facing out
phylotypic stage
head somawhat distinct
gut tube present
neural tube present
somites present
v early embryogenesis can look different throughout vertebrate phylum (due to environmental adaptation)
BUT it always converges on this v similar (relatively speaking) form
THE PHYLOTYPIC STAGE wit all the same basic architecture
then they go on to diverge again after this
circulatory system basic
forms from 2 layers of endothelium
in the middle - joins to form a simple 2 chambered heart
-left and right endocardial tubes cometogether
-end up with aortic sac
>right and left aortic arches
>right and left Sinus Venosus
then get folding to form 2 (and then 4 sometimes) chambered heart
in mammals this becomes a 4 chambered heart
E12 onwards
mostly organigenesis
endoderm makes a tube
forms:
-gut
-lungs
-liver
-pancreas
branched from the gut bud off and form these organs
bone formation
from mesoderm
commited cartilage cells
compact
form compact nodules
chondrocytes proliferate from this node
as they proliferate hypertrophic chondrocytes eventually form bone
so bone forms in oler parts nearer middle as bone extends
intermediate mesoderm forms:
reproductive system and kidney
pronephros
nephric duct growing
nephrogenic cord next to duct
mesonephros form there
mesonephros attach to nephric duct
pronephros go away??
metanephrogenic mesenchyme elow these - connected to lower part of nephric duct
cloaca at end of duct
gonad forms next to the mesonephros
foetus in fetu
where you get twinning
a delayed embryo can become enveloped by the other twin
end up with a delayed foetus growing within another animal
generation of in vitro cell types needs understanding:
of fate choices
so you can replicate them in culture
understanding developmental decisions is important dor generating in vitro cell types from pluripotent cells
understanding genetic defects:
the more like a mammal you are
the more relevant to mammalian development genetic studies will be
Pax6 mutant similar in mammals
Pax6 mouse mutant homozygote has no eyes
heterozygote small eyes
equivalent defect in humans - Aniridia - lack of iris - from Pax6+/-
small eyes was what we could recognise in mice - couldnt ask them how they say - turns out they also lacked an iris
public health perspective of development study
pregnant people given folic acid supplements to reduce neural tube defects that lead to exposed nerves
curly tail neural tube defect in mice is not rescuable by folic acid - instead inositol treatment protects
-can protect against other defects in humans that folic acid can’t
looking at mouse defects can have impact on human public health issues - like spina bifida and other neural tube defects
mouse as good model animal
short 9wk generations (shirter than zfish)
large litter size for mammal
easy husbandry
experimental embryology is possible at specific stages
excellent for developmental genetics
-mutagenesis screens
-targeted mutations
preimplantation development
in the oviduct:
free floating embryo pre-implantation
-fertilisation
-few rounds of cell division
-passes through oviduct
-emerges in uterus as a blastocyst
-then implants in the uterus
it is easy to culture while in this free floating period
can culture from fertilised egg to blastocyst
can replicate oviduct conditions nicely
can study in detail the things that happen to the embryo ready to implant in nucleus
4 cell stage polar body
extra bump on 4 cell stage
this is polar body
extruded chromosomes not needed (from X inactivation ig??)
8 cell to 9 cell stages
compaction is occurring
flattening
then after this cavity forms - becomes blastocyst
cavity expands
acellular protective membrane surrounds the blastocyst
then hatches out of this in order to implant
mouse preimplantation embryos stages:
cell division w/out growth here - cleavage division
gives the 8 cell stage
the sides of the cell flatten into each other - compact to form the MORULA
then these cells secrete fluid into cavity to become blastocyst
maternal RNA used until 2 cell stage
then zygotic transcription begins
(opposed to zebrafish where zygotic genome begins activating after the synchronous divisions enf and the asynchronous ones begin)
in compaction to form morula:
and morula polarisation
cells flatten
tight junctions and gap junctions form
cells polarise
-inside and outside distinction
-apical outisde w microvilli
-and basal on inside
-cells can wither divide symetrically in circumferential plane to give identical cells
-or divide asymmetriucally in radial plane to give non polarised cells in the middle
gives difference between the inside and outside of embryo
-these outer polarised cells become the trophectoderm
trophectoderm vs ICM lineage decision
E2.5 - Oct4, Cdx2 in all cells
E3.5 - Oct4 in ICM only, Cdx2 in trophectoderm only
how does Cdx2 become restricted to TE
Cdx2 restriction to future trophectoderm cells
Hippo signalling pathway regulates Cdx2 expression
-8 cell stage - Yap protein in all cells
-in late morula - big difference in Yap localisation between cells - outer have nuclear Yap, Inner have cytoplasmic Yap
(outer cells w nuclear Yap become TE
inner cells w cytoplasmic Yap become ICM)
This occurs via differential activation of Hippo signalling
-Hippo is active in inner cell:
>activates LATS kinase
>LATS phosphorylates Yap - Yap can no longer co-localise with Tead4 co-TF
>so doesnt go to nucleus - TEAD4 alone leaves Cdx2 OFF
>Cdx2 OFF in inner cells
-Hippo is inactive in outer cell
>hence Yap can co-localise with tead4 - activating Cdx2 in outer cells
consolidation of TE and ICM separation
mutual repression by Oct4 and Cdx2
consilidates difference between trophectoderm and pluripotent ICM lineage difference once it is set up
one more lineage segregation after TE and ICM before implantation:
preimplantation cell types:
depends on cells having slightly different FGF signalling levels (similar to the diff levels of notch and delta leading to lineage segregation)
Cells with lower FGF signalling
through Feedback loops secrete more FGF
but have low capability for FGF signalling
-Become epiblast
Some ICM cells have high levels of FGF signalling
-become the primitive endoderm (hypoblast?????????? - looks like that on diagram)
ICM vs primitive endoderm lineage segregation markers
nanog (ICM) and gata4 (PE) used as lineage markers
segregation of these factors is important for the lineage differentiation
Nanog important for epiblast formation
Gata4/6 for PE
Epiblast and primitive endoderm segregation mechanism
Future epiblast:
-Oct4+
-secrete FGF (due to Oct4)
-Oct4 promotes Nanog
-Nanog inhibits Gata6
future primitive endoderm:
-receive FGF signalling from future epiblast
-FGF4 signalling inhibits Nanog
-so Gata6 no longer inhibited by nanog so much (gata6 also inhibits Nanog - feedback)
-Gata6 activates Gata4 and FfgR2 (positive feedback by upregulating Fgf sensitivity)
thought that these two layers are different adhesively so separate out
pre implantation lineage segregations - mammal and amniote specific
ICM-TE segragation - mammal specific
Epiblast-Primitive endoderm - amniote specific
Totipotent blastomeres/ICM/Epiblast:
>totipotent
>give rise to embryo
trophoblast/primitive endoderm:
-make up extra-embryonic structures
trophoblast is mammal specific
extra-embryonic lineages importance:
for interactions
trophoblast important for interactions (placenta forms from it (in placental mammals) i think as well hence tropho)
Primitive endodewrm will be important for patterning the embryo
embryonic layers at implantation
has epiblast lineage now
with 2 extraembryonic lineages
polar trophectoderm goes on to differentiate into polar (at the top) trophectoderm
and mural (at the wall[of blastocoel ig]) trophectoderm
atm are just trophectoderm
blastocyst hatches
burrows into uterine lining
embryo 1 day after implantation
dramatic shape change
in mouse epiblast makes U shaped cup
cavity in the middle of the cup and around the outside
polar trophectoderm has divided a lot and has made a stock known as the egg cylinder
primitive endoderm has extended to cover inside (migrates all around the cavity) and outside of egg cylinder and lines the outside of the polar trophectoderm
cells derived from the mural trophectoderm - trophoblast giant cells on outside
now have eggcylinder - primitive endoderm - and epiblast cup
now to get epiblast ready for gastrulation
Mouse AP axis formation in the cylinder shape (as opposed to the disc one earlier)
epiblast and polar trophectoderm are lined by PE
2 diff cell types within the PE
formed because Ptrophectoderm signals to the epiblast - with BMP4 signalling it induces Nodal and Wnt3 expression at rim of cup (proximal end)
this causes the distal visceral endoderm (descendent of PE at distal end of egg cup)
this begins expressing nodal and wnt3 inhibitors (lefty1, Cer1, Dkk1)
meanwhile - BMP4 from polar ectoderm causes Nodal and wnt3 expression in the proximal epiblast
the distal visceral endoderm moves to one side - this will become the future anterior of the visceral endoderm
neighbouring visceral endoderm follows the distal visceral endoderm- forming the anterior visceral endoderm
formation of the AVE causes the nodal and wnt3 expression to move to posterior of the epiblast cup
somewhat forming an AP axis
anterior visceral endoderm action
reaches the embryo/extraembryonic junction
inhibiting nodal and wnt3 at the anterior
-this induces the anterior extoderm (future head and forebrain
nodal and wnt3 signalling at posterior of epiblast action
-the nodal and wnt3 signalling at the post end causes it to make mesoderm - forming the primitive streak (the structure from whicb mammals make mesoderm)
- cells here also make the definitive endoderm of the gut - which inserts between cells in the primitive endoderm and push them out of the way
-primitive streak initiates gastrulation in the epiblast posterior
gastrulation in cup shape mouse
cup shape
primitive streak induces at posterior
cells there make mesoderm
mesoderm cells then crawl to the anterior of the embryo - make the mesoderm wings and then line the whole cavity
meanwhile cells have been inserting into the primitive endoerm to make definitive gut endoferm
production of the 3 primary germ layers by gastrulation
xenopus gastrulates by takinf outer pluripotent cells and bringing them inside the embryo
MOUSE INSTEAD:
takes its inncer cells and pushes them outside to make the mesoderm and endoderm
can envisage the cup as the flat disc from earlier
chicks and humans also do this (flat disc w primitive streak at one end - mesoderm cells migrate forward and eventually make 3 germ layers)
fate choice in early embryo
after the first 2 lineage segregations from earlier
after these the epiblast makes the mesoderm endoderm and ectoderm
all 3 germ layer lineages will be source of more differentiated lineages
germ layer elaboration mouse
cup shape of embryo with beginnings of germ layers
-epiblast inside
-mesoderm middle
-endoderm on outer side
one day later see something more reminiscent of typical vertebrate shape
-head fold
-beginning of gut
-still has epiblast w primitve streak at back end of embryo
germ layer elaboration - cell movements in the primitive streak region
cells move from epiblast layer thru primitive streak where cells do epithelial to mesenchymal transition
then move out of streak as mesoderm
or if they dont move thru streak as mesoderm - will likely move away from primiitve streak and make ectodermal lineage
can have mesoderm and ectoderm lineages coming from same region - just depends whether they did the EMT at mid line
similarity of mouse to other vertebrates
differences seen as vertebrates approach gastrulation
sizes of embryos
-mouse timy
- chick embryo has v large yolk
then cells divide
and they do diff gastrulation strats:
moving cells inside (fish xenopus)
moving cells outside
all end up at phylotyic stage
vertebrate hourglass
vertebrates have distinct adult forms
and distinct early embryos
but converge on a phylotypic stage in embryo that looks similar
so hourglass of convergence and divergence to and past Phylotypic stage
frog/fish gastrulation strat
mouse pulls mesoderm from in to out
frogs pull mesoderm from outside to inside -(along that mesoderm band arround middle of blastula)
mouse is like an inside out frog
chick specification of AP axis from external signal
used gravity and egg rotation
yolk rotates in shell
causes primitive streak to appear on one side
Axis determinants in mouse
evidence against:
-absence of yolk, early genome activation
-huge capacity for regulation
regulation in preimplantation embryos - evidence against axial determinants
can take 2 morula embryos and sit next to each other in well
morulas stick together readilt and form chimeric embryo
survive just as efficientpy as non chimeric embryos and implant in foster mother
-suggests lack of obligate AP axis defines it yet as smashing two diff morula together is feasible
can also sort out inner or outer cells and make mouse out of each - also same survival rates as non chimera
also suggest lack of AP axis determines
evidence for cytoplasmic determinants in mouse zygote
biasing towards animal or vegetal cytoplasm by removing part of zygote - gives exact same embryos
so no evidence for determinants here
or could there be
as there was a low frequenct of liveborn mice (not jsut looking at embryi formation but survival post birth)
could just be due to a detrimental effect of general loss of cytoplasn - not necessarily due to axis determinants
