Mammalian Morphogenesis

Question 1

first steps

Answer 1

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

Question 2

when does zygotic gene transcritption begin

Answer 2

4 cell stage
one of first ones is E-cadherin
causes cells to stick together and compact

Question 3

compaction and trophectoderm formation

Answer 3

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

Question 4

blastocyst formation

Answer 4

trophectoderm forms
inner cell mass inside
trophectoderm begins letting fluid in - forming a blastocoel at the centre

Question 5

blastocyst hatching

Answer 5

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

Question 6

hypoblast/epiblast formation

Answer 6

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

Question 7

monozygotic twins cause

Answer 7

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)

Question 8

Cells separating inside the ZP

Answer 8

have two trophectoderms form
ICM forms hypoblast and epiblast in each
2 distinct systems with all the structures each

Question 9

two ICMs form inside same trophectoderm

Answer 9

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

Question 10

axes of embryo after epi/hypoblast formation

Answer 10

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

Question 11

solution for “marking 12 o clock” and generates the beginnnings of the axes

Answer 11

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

Question 12

3rd and rarest way of making MZ twins

Answer 12

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

Question 13

germ layer formation

Answer 13

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

Question 14

germ layer cell fates determinant

Answer 14

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)

Question 15

mouse cup peculiarity

Answer 15

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

Question 16

neurulation basic

Answer 16

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

Question 17

neurulation: when the cells at top meet

Answer 17

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

Question 18

failure of neural tube closure

Answer 18

failure to zip up properly can leave it open - vertebrae cannot form where this has happened

causes Spina Bifida

Question 19

Mesoderm development basic

Answer 19

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

Question 20

components of the mesoderm

Answer 20

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

Question 21

formation of structures from somites:

Answer 21

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)

Question 22

other mesoderm component diagram

Answer 22

-neural tube
-notochord below it (chorda mesoderm)
-paraxial mesoderm next (head and somites formation)
-intermediate mesoderm next to that
-lateral plate mesoderm next

Question 23

paraxial mesoderm give rise to

Answer 23

head

somites:
sclerotome, syndotome
myotome
dermatome, endothelial cells

Question 24

intermediate mesoderm

Answer 24

kidney
gonads

Question 25

lateral plate mesoderm

Answer 25

splanchnic somatic extra-embryonic

Question 26

the neural crest basic

Answer 26

small region of cells formed after neural tube pinches off some remains neural - others become different

Question 27

neural crest migration

Answer 27

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

Question 28

E9 twisting of mouse embryo

Answer 28

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

Question 29

phylotypic stage

Answer 29

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

Question 30

circulatory system basic

Answer 30

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

Question 31

E12 onwards

Answer 31

mostly organigenesis endoderm makes a tube forms: -gut -lungs -liver -pancreas branched from the gut bud off and form these organs

Question 32

bone formation

Answer 32

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

Question 33

intermediate mesoderm forms:

Answer 33

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

Question 34

foetus in fetu

Answer 34

where you get twinning a delayed embryo can become enveloped by the other twin end up with a delayed foetus growing within another animal

Question 35

generation of in vitro cell types needs understanding:

Answer 35

of fate choices so you can replicate them in culture understanding developmental decisions is important dor generating in vitro cell types from pluripotent cells

Question 36

understanding genetic defects:

Answer 36

the more like a mammal you are the more relevant to mammalian development genetic studies will be

Question 37

Pax6 mutant similar in mammals

Answer 37

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

Question 38

public health perspective of development study

Answer 38

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

Question 39

mouse as good model animal

Answer 39

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

Question 40

preimplantation development

Answer 40

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

Question 41

4 cell stage polar body

Answer 41

extra bump on 4 cell stage this is polar body extruded chromosomes not needed (from X inactivation ig??)

Question 42

8 cell to 9 cell stages

Answer 42

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

Question 43

mouse preimplantation embryos stages:

Answer 43

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)

Question 44

in compaction to form morula: and morula polarisation

Answer 44

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

Question 45

trophectoderm vs ICM lineage decision

Answer 45

E2.5 - Oct4, Cdx2 in all cells E3.5 - Oct4 in ICM only, Cdx2 in trophectoderm only how does Cdx2 become restricted to TE

Question 46

Cdx2 restriction to future trophectoderm cells

Answer 46

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

Question 47

consolidation of TE and ICM separation

Answer 47

mutual repression by Oct4 and Cdx2 consilidates difference between trophectoderm and pluripotent ICM lineage difference once it is set up

Question 48

one more lineage segregation after TE and ICM before implantation:

Answer 48

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)

Question 49

ICM vs primitive endoderm lineage segregation markers

Answer 49

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

Question 50

Epiblast and primitive endoderm segregation mechanism

Answer 50

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

Question 51

pre implantation lineage segregations - mammal and amniote specific

Answer 51

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

Question 52

extra-embryonic lineages importance:

Answer 52

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

Question 53

embryonic layers at implantation

Answer 53

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

Question 54

embryo 1 day after implantation

Answer 54

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

Question 55

Mouse AP axis formation in the cylinder shape (as opposed to the disc one earlier)

Answer 55

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

Question 56

anterior visceral endoderm action

Answer 56

reaches the embryo/extraembryonic junction inhibiting nodal and wnt3 at the anterior -this induces the anterior extoderm (future head and forebrain

Question 57

nodal and wnt3 signalling at posterior of epiblast action

Answer 57

-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

Question 58

gastrulation in cup shape mouse

Answer 58

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

Question 59

production of the 3 primary germ layers by gastrulation

Answer 59

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)

Question 60

fate choice in early embryo

Answer 60

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

Question 61

germ layer elaboration mouse

Answer 61

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

Question 62

germ layer elaboration - cell movements in the primitive streak region

Answer 62

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

Question 63

similarity of mouse to other vertebrates

Answer 63

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

Question 64

vertebrate hourglass

Answer 64

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

Question 65

frog/fish gastrulation strat

Answer 65

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

Question 66

chick specification of AP axis from external signal

Answer 66

used gravity and egg rotation yolk rotates in shell causes primitive streak to appear on one side

Question 67

Axis determinants in mouse

Answer 67

evidence against: -absence of yolk, early genome activation -huge capacity for regulation

Question 68

regulation in preimplantation embryos - evidence against axial determinants

Answer 68

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

Question 69

evidence for cytoplasmic determinants in mouse zygote

Answer 69

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