Les 1: Introduction + Fate and Potency
Understanding of development = mechanisms underlying development can only be studied by
disturbing the normal developmental processes in specific ways and then observe what happens. This
can be done by using model organisms, explants, or organoids.
Experiments to address sufficiency or necessity hypothesis
• Show-it/find-it experiments = shows correlaAve evidence, this forms the base for a hypothesis
Then we can test this hypothesis by disturbance:
• Block it/lose it experiment = LOF, this addresses necessity
• Move it experiment = GOF, this addresses sufficiency
Observa(ons in the earliest stages of development
1. Cell division: cells do not grow, the only divide and become smaller
2. Cell cleavage
3. FormaAon of blastomeres
4. Losing of the border
5. FormaAon of a cavity
Main ques:ons for this lecture:
• How to organize the development from a simple egg into an ordered mulAcellular body?
• How do cells become specified to a specific fate?
• What is the potency of cells?
Development starts at the ferAlizaAon of the egg. On top of that, development is a blend of different
processes, these processes oWen happen simultaneously:
• Cell division
• Cell differenAaAon
• Growth
• MigraAon
• PaYerning
• Morphogenesis
• Apoptosis
Development = zygote à blastula à gastrula (formaAon of the 3 germ layers) à neurula
Organiza:on of the embryo: at the top of the embryo, we have the animal pole (AP) and at the boYom
the vegetal pole (VP). At the AP the embryo will develop and at the VP the yolk develops.
1. Cell divisions
Cell division in early development is a lot shorter (~30 minutes) than in somaAc cell (~16 hours). Here,
it only has the S and the M faze. This makes that the cells don’t have Ame to grow, they just cleave.
Because the cells have no Ame to produces proteins, they get smaller. This division is especially high in
the stage from ferAlizaAon to gastrula.
It is also noted that in some species, like frogs, the embryonic genome is acAvated much later. This is
because in the early stages the embryo does not depend on the genome. These eggs are stuffed with
,maternal mRNA so that they can develop fast. This is necessary, because egg can be deposited in the
environment where they are exposed to danger.
In contrast, the embryonic genome of mammals is acAvated earlier since there is no pressure from the
outside.
So, to conclude: embryonic cleavage is way different than somaAc cleavage, it has no G1 and G2 faze.
Within cell division there are two forms:
• Asymmetric = one daughter cell will stay the same in the same fate as the mother, while the
other one changes
• Symmetric = two daughter cells get the same material and are of the same kind as the mother.
2. Pa0erns of embryonic cleavage
Cleavage of cells depends mainly on:
• The amount and distribuAon of egg yolk proteins in the cytoplasm. The yolk is heavy, no
division takes place. The embryo is pushed to one site
• Factors in the egg cytoplasm that influence the angle of the mitoAc spindle and Aming of its
formaAon
Cleavage can be categorized into two:
Holoblas:c = complete cleavage and isolecithal = with an even distributed yolk
1. Radial
2. Spiral
3. Bilateral
4. RotaAonal = in mammals
Holoblas:c and mesolecithal = moderate disposiAon of the yolk = radial (in amphibians)
Meroblas:c = incomplete cleavage and telocithal = dense yolk throughout most of the cell
1. Bilateral
2. Discoidal = in fish, repAles, and birds
Meroblas:c and centrolecithal = yolk in the center if of the egg = seen in most insects
Ac:va:on of the zygo:c genome
During oogenesis maternal mRNA is lead in the oocyte, aWer ferAlizaAon proteins are synthesized from
this. But when does the transcripAon of own embryonic mRNA start? In mammals this is in the 8-16
cell stage, which is earlier then in frogs (4096) cells. This is because the frog embryos need to develop
fast in the environment and for this use the mother’s mRNA.
3. Differen;a;on
= the generaAon of cellular diversity or specialized cell types
From a historical perspecAve to theories were formed:
1. RestricAon hypothesis = The cells either lost all genes except those relevant to their task
2. Potency hypothesis = The cells kept the enAre genome and selecAvely turned genes on or off
à true!
,Experiment by Hans Spemann
He found that cells in the early embryo are idenAcal and can give rise to a complete larva, the
experiment was as followed:
• With a hair he made a ligature in the 16-cell stage embryo, where one nucleus (blastomere)
was separated from the rest
• This one blastomere had the potency to become a whole embryo = toApotent cell
• When there was no nucleus in the separated embryo, no organism was formed
Waddington’s epigene(c landscape (WEL)
= the more cells cleave, the less potent they become. The more the cell rolls down the valley the more
restricted it becomes:
• Cells start at the top of the hill as a toApotent cell = can become anything
• Then it becomes a pluripotent cell/ESC = can become anything that an organism possesses
• At a bifurcaAon point, a progenitor state cells give rise to different descendants
• At the boYom we have a unipotent cells that is terminally differenAated
• A differenAated cell is very specific and will not change by itself
The first valley of differenAaAon is the determinaAon of the three germ layers:
• Ectoderm = epidermis and CNS
• Mesoderm = cardiovascular, UG, muscle, bones, and carAlage
• Endoderm = GI system, liver, pancreas, and the lungs
Withing these three germs layers the fate of the cells becomes progressively more restricted.
4. Potency
= a reflecAon of what a cell sAll can become
• ToApotent = zygote cell
• Pluripotent = ECS = blastocyst
• MulApotent = MSC = form an embryo or adult brain
• Limited differenAaAons potenAal = neuronal precursor = from the brain or spinal cord
• Limited division potenAal = differenAaAng neuronal precursor = from subareas of the brain
• FuncAonal non mioAc neuron = differenAated cells = from specific areas of the brain
Tes:ng the specifica:on of cells with the animal cap assay
SpecificaAon = what Assue would become if you explanted it an put it in another environment.
• When you take ectoderm Assue from the AP and put in in saline (without acAvin, a morphogen)
this becomes atypical epidermis
• When you add different concentraAons of acAvin on this, the Assue can become all sorts of
thing: blood, muscle cells, notochord cells and even hart cells, depending on the amount of
acAvin
à this means that the cells are not differenAated yet!
Specifica:on = a cell of Assue is specified to a given fate when it is capable of differenAaAng
autonomously when placed in isolaAon in a neutral environment, but the cell idenAty is sAll labile.
Determina:on
= a cell or Assue is determined when it is capable of differenAaAon autonomously even when placed in
another environment
• When taken a piece of Assue that will normally form eyes from a gastrula stage embryo and
implanAng it on a neurula host à the ‘eye’ cells become somaAc Assue à these cells are not
determined yet
, • When take a piece of Assue that will normally form eyes form a neurula embryo and implanAng
it on a neurula host à we get ectopic eyes à these cells are determined and are ‘deaf’ to cues
from the environment
Commitment = first specificaAon + determinaAon = differenAaAon
Cells that are terminally differenAated are usually post mitoAc
Gene expression altera:on
• By extracellular signals and cues
• Mechanical cues like stretch, flow, and pressure
• TF that are expressed in different combinaAons: the same TF can regulate expression of
different genes in a posiAve or negaAve fashion
So, to conclude this part, differenAal gene expression:
1. Every somaAc cell nucleus of an organism contains the complete genome established in the
ferAlized egg (genomic equivalence)
2. Unused genes of differenAated cells are neither destroyed nor mutated
3. A small porAon of the genome is expressed in each cell, and only a porAon thereof is specific
for that cell type
We also note that the characterisAc proteome a a cell is the result of:
1. DifferenAal gene expression
2. SelecAve pre-messenger RNA processing
3. SelecAve messenger RNA translaAon
4. DifferenAal posYranslaAonal protein modificaAon (PTM) regulates funcAon/localisaAon/turn-
over
5. Growth
• How do cells know that they have to stop dividing
• Different cells have different sizes
• Growth in the beginning is disproporAonal (babies have a big head)
6. Migra;on
In development we make a disAncAon of 5 types of cell migraAon:
1. Invagina:on = infolding of a sheet of cells (epithelium), much like a indenAon of a soW rubber
ball that is poked
2. Involu:on = inward movement of an expanding outer layer so that is spreads over the inner
surface of the remaining external layer
3. Ingression = migraAon of individual cells from the surface into the interior of the embryo, this
happens at EMT
4. Delamina:on = splinng of one cellular sheet into two ore more
5. Epiboly = movement and spreading of an epithelial sheet, usually over ectodermal cells to
cover the whole embryo
7. Pa0erning
= the process that establishes a well-ordered spaAal paYern of cells and Assue (for example a limb or
the neural tube), this includes:
• Establishment of a body plan: head anterior – tail posterior – an axial nervous system - limbs
lateral
• Asymmetric posiAoning of heart
• Stereotypic paYern of different neurons in the neural tube
, • Stripes of a zebra etc.
8. Morphogenesis
= the process of making changes in the form of the embryo, and involves coordinated cell growth, cell
migraAon and cell death. Morphogenesis usually co- occurs with paYerning.
Several morphogenic processes regulated by epithelial cells:
1. Dispersal = epithelium becomes mesenchyme (loose cells)
2. DelaminaAon = epithelium becomes mesenchyme, but only for a part of the structure
3. Shape change or growth = cell remain aYached, but the morphology is altered
4. Cell migraAon/intercalaAon = rows of epithelia merge to form fewer rows
5. Cell division
6. Matrix secreAon and degradaAon = synthesis or removal of ECM
7. MigraAon = formaAon of free edges
Several morphogenic processes regulated by mesenchymal cells:
1. CondensaAon = mesenchyme becomes epithelium
2. Cell division à hyperplasia
3. Cell death = cells die
4. MigraAon
5. Matrix secreAon and degradaAon
6. Growth = hypertrophy
9. Apoptosis
= programmed cell death
Apoptosis is criAcal in the physiological part of development because we generally have more
signatures before we are born.
From zygote to blastula
= a process of cleavage, the embryo does not grow it only divides.
Early development in mammals
• Oocytes of mammals are amongst the smallest oocytes in animal kingdom (~100 µm)
• Mammals only invest in a few oocytes per cycle
• Development inside another organism
• Need for supporAve extraembryonic Assues (placenta, umbilical cord, yolk sac, amnion)
Cleavage divisions: two rounds of differen(a(on
To go from a zygote to a blastomere the cells go through two rounds of differenAaAon, in this case we
use the mouse as a reference:
• From zygote (E0.5) up to an 8-cell stage (E2.5) cells are sAll toApotent (the stage in which twins
are formed)
• In the 8-cell stage we have toApotent blastomeres that undergo compacAon = there is going
to be a difference in inner and outer cells. The outer cells flaYen and become more polar, the
inner cells are apolar. Because of the toApotent blastomeres divide into the trophectoderm
(TE) that will form the placenta and chorion, and the inner cell mass (ICM), that is sAll
pluripotent.
• The ICM undergoes a second round and will form the primi:ve ectoderm (PrE) that will
become the supporAve Assue. It also becomes the epiblast (Epi), this is sAll pluripotent and
will form the embryo.
, • Later on, the cells from the PrE will cluster together and so will the Epi cells. Now a cavity is
with fluid that comes from the TE is formed, this pushes the Epi to the top and there the
embryo will develop
• In wave 1 and wave 2 we both have symmetrical and asymmetrical divisions
↑
• We end up with a blastocyst (E4.5) that will implant in the uterus
trophectoderm
totipotent
blasto
↑ rimitive endoder m-
ICM
epiblast =
pluripotent
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Wave 1 = cell division that results into inside (ICM) and outside cells (TE)
• Different blastomeres have a different spaAal posiAoning on the inside and outside
• Cells in the upper part of the cell are exposed to ECM
• Cells at the boYom are exposed to other cell types
• The inner nonpolar part is exposed to nothing
• Results in different signaling: Oct4 in ICM and Cdx2 in TE
TE = Aghtly adherend, is polarized along the apical basal axis. It has asymmetric cell-cell contact, zo the
apical side is not in contact with its neighbors.
TE cells also form a blastocoel cavity: this is iniAated by a diffusion of water across osmoAc gradient
and transport of water through aquaporins. The locaAon of this cavity determines the embryonic-
abembryonic axis. Because the epithelium of the TE is Aght, mainly salt results in the cavity expansion.
ICM = has symmetric cell-cell contact, and which prevents establishment of ab apical domain
Wave 2 = ICM segrega:on into the extraembryonic part (PE) and the epiblast cells (Epi)
• ICM in wave 1 make Fgf4
• Cells that end up in the ICM in the second wave have receptors for Fgf4 (Fgfr2)
• The ICM that is sAmulated in wave to differenAates into PE: these cells acAvate MAPK, this
inhibits NANOG. Since NANOG normally inhibits GATA6, GATA6 now is expressed.
• The EPI progenitor cells don’t have the Fgfr2, have no MAPK and thus sAll express NANOG and
no GATA6, hence they don’t become PE cells.
à EPI = high in NANOG and Oct4 (from the first wave)
à PE = high in GATA6, GATA4 and Oct 4
The gatekeepers of TE, EPI and PE fate are transcrip:on factors, these tell the cells what to become.
For example: Oct4 that is acAve in ICM cells, will promote TF important for PE or Epi cells and not TF
for the TE.
,Les 2: Methods in developmental biology
The ques(on we try to answer = how to can we organize the development from a simple fer(lized
egg into an exquisitely ordered mul(cellular body?
To understand this, we need to observe and describe developmental stages and discover what the
mechanisms are that govern this development.
Different approaches to look at embryos
Looking at embryos at different levels is the first observa(on we can do.
Histology
In the early days we looked at dead embryos, now we can use imaging and 3D reconstruc(on:
• Microscopy: using simple dissec(on microscopy to look at big embryos. We can also use light
and epifluorescent microscopy. With this we can look at sub embryonic structures, paJerns
and mRNA and protein distribu(on. A step further is confocal microscopy. By using this we
make sec(ons with light. This leads to a beJer resolu(on.
• In situ hybridiza9on: we stain the embryo with Ab. Nowadays we can look at even whole
embryos instead of just sec(ons.
• Live imaging: all the methods described before looking at sta(c embryos, but then a lot of
informa(on is lost, like signaling and migra(on. With live imaging we can add the factor of
(me. We can now look at, for example, the closure of the neural tube.
Fate mapping
= refers to the process of tracking the des(ny or fate of cells as they differen(ate and give rise to specific
(ssues and organs during embryonic development. It helps scien(sts understand how different cell
types are specified and organized within the developing organism. It goes as followed:
• The first experiment was done with Ascidians, because in the larvae stage it looks like a
vertebrate. The eggs are large and have a natural pigment at the vegetal pole.
• By following the cells and the pigment and by seeing where these ends up, we get an idea of
the fate of these cells à fate mapping
• We can also do this by placing a blue crystal on a specific loca(on at the blastula stage of the
embryo.
• The blue pigment of the crystal will deposit at this specific loca(on. Now we let the embryo
develop and we follow the color.
• In the past they did this all around the embryo to get the organ map.
Example of fate Mapping in Drosophila Embryo:
1. Marking Cells: Scien(sts inject a fluorescent dye into specific regions of the fruit fly embryo at
an early developmental stage.
2. Tracking Development: As the embryo develops, the labeled cells divide and differen(ate. By
carefully observing the labeled cells under a microscope, researchers can track which (ssues
and structures they contribute to as the embryo develops.
3. Analyzing Results: Through fate mapping, researchers can determine which cells give rise to
structures like the head, thorax, abdomen, legs, wings, etc. They can also iden(fy the lineage
rela(onships between different cell types.
4. Interpre(ng Results: Fate mapping experiments reveal lineage rela(onships and provide
insights into how cells become specialized during development. For example, they might show
, that certain groups of cells give rise to specific (ssues, such as the nervous system, while others
contribute to muscles or internal organs.
5. Applica(ons: Fate mapping is crucial for understanding normal development and can also
provide insights into developmental disorders or diseases. It helps in studying gene expression
paJerns, cell signaling pathways, and how muta(ons affect cell fate decisions.
A more advance method for fate mapping is by using gene9c encoded tracers, an example is by using
the Cre-ER, HSP90 LoxP system:
• Cre-ER is a fusion of Cre recombinase with the ER
• ER is a TF and needs to be in the nucleus for it to bind to DNA
• HSP90 is bound to Cre-ER and acts as a carrier
• When tamoxifen is added, HSP90 detaches, and Cre-ER migrates to the nucleus
• Cre can bind to the two LoxP sites that flank the desired DNA
• Cre can now remove the DNA that is between the LoxP sites
• Usually, this DNA is part of the LacZ gene repressor. When this repressor is cut out LacZ can be
expressed. This encodes for beta galactosidase that, with addi(on of X-gal gives a blue color.
• By using specific CreLox genes in different parts of the body can be expressed.
Manipula6on embryos
= a necessary method to understand the underlying mechanism of development.
1. Liga3on
An example of this is the experiment of Hans Spemann.
2. Transplanta3on
• For example, transplan(ng the blastopore lip from an embryo into an acceptor.
• Since the blastopore lip is an organizing structure, we get a secondary body axis and a larva
with two heads.
• This gives us cri(cal informa(on about the blastopore lip
3. Explants
For example, the animal cap assay: a piece of ectoderm from the blastopore was taken and put into
different concentra(ons of ac(vin. By adding ac(vin the animal cap could be pushed into different
direc(ons.
4. Abla3on
= killing cells or a specific layer and then let the embryo develop. We can see what structures we are
missing.
5. Microinjec3on
= the process of taken soma(c cells and puang them in another nucleus. An important ques(on in this
is: how important the nucleus in steering development is and what is the potence of the nuclei form
differen(a(on (ssue.
An example of this is the experiment of John Gurdon:
John Gurdon's experiment with frogs is a classic study that demonstrated the possibility of cell
reprogramming, which later became a key concept in the development of regenera(ve medicine and
the produc(on of induced pluripotent stem cells (iPSCs). He took the nucleus an adult frog's intes(nal
cell (soma(c cells) and implanted it into an egg cell from which its own nucleus had been removed due
to UV radia(on. This manipulated egg cell was then implanted into a tadpole from which the nucleus
,had been removed. The result was that the manipulated egg cell developed into an adult frog. This
meant that the nucleus of an adult intes(nal cell contained the instruc(ons to produce all types of cells
in a frog's body, including cells of the skin, muscles, nerves, and organs. This was a groundbreaking
discovery because it demonstrated that the nucleus of a specialized cell, such as an intes(nal cell, s(ll
contained all the gene(c informa(on needed to produce a complete organism. It showed that cells
could be "reprogrammed" to a pluripotent state, giving them the ability to develop into various cell
types rather than just one type.
6. Studying cell migra3on
This is done with labeled cells. You can inject cells from a donor into the acceptor and follow them. You
can do this with different kind of cells in different regions.
Molecules and embryos
What happens in development is an increasing complexity in a lot of processes. The increase in
complexity of the embryo happens gradually an involves: cell division, differen(a(on, growth,
migra(on, paJerning, morphogenesis, and apoptosis.
This whole process is coordinated by signal transduc(on and TF. TF especially are the controllers that
drive the expression of targeted genes that determine the phenotype of the cells.
What dis9nguished different cell types?
= the transcriptome (mRNA) and the proteome (en(re set of proteins expressed by a genome, cell,
(ssue, or organism at a certain (me under specific condi(ons. It encompasses the complete
complement of proteins, including their structures, func(ons, and interac(ons within a biological
system).
We can look at the transcript using in situ hybridiza9on:
• We start from RNA à cDNA à make an RNA probe
• The probe is complementary to the mRNA in the (ssue we want to visualize
• By this we can look a whole embryo
Immunohistochemistry = For looking at proteins. This is helpful, because some(me the mRNA can be
distributed in the whole cells, but the proteins are in a specific part. This is a important feature, because
the proteins are the site of ac(on.
• Direct method: Ab with label
• Indirect: with a secondary Ab
Gene, gene networks and embryos
What determines the difference between transcriptome (mRNA) and the proteome (whole picture)?
= gene regula(on, transcrip(on and transla(on
Differen(al gene expression is due because:
1. Every soma(c cell nucleus of an organism contains the complete genome established in the
fer(lized egg (genomic equivalence)
2. Unused genes of differen(ated cells are neither destroyed nor mutated
3. A small por(on of the genome is expressed in each cell, and only a por(on thereof is specific
for that cell type
The characteris(c proteome of a cell is the result of:
1. Differen(al gene expression
, 2. Selec(ve pre-messenger RNA processing
3. Selec(ve messenger RNA transla(on
4. Differen(al posJransla(onal protein modifica(on (PTM) that regulates func(on, localisa(on
and turn over
Gene regula3on with reporter genes
The ques(on is: Is SO regulated by EY and TOY? Is this the case and how does this work?
We here see the regula(on of 3 genes: We have the sine oculis gene (SO), the second gene is eyeless
(EY), and the third one is twin of eyeless (TOY)
• EY and TOY are closely related to the TXF PAX6
• All 3 genes are important for the development of the eye
• We have a piece of SO (part of the SO gene) hooked up to LacZ reporter, this turns blue if you
stain it
• SO DNA has all the informa(on to drive expression of a reporter gene in a developing eye
How did they get there:
• At the boJom we see the structure of the SO gene
• They took a piece exon (4), hooked it up in the structure and saw that this contains informa(on
the drive the expression of an eye
• Now, we can ask how small we can make piece 4 to s(ll have all the informa(on that we need
• They iden(fied fragment SO10 as the minimal sequence that is necessary to have all the
informa(on for the eye
Eventually we ended up with a DNA piece of 300 bp. To see whether EY and TOY s(ll bind to this part
of the SO gene, DNA footprin(ng was performed. When EY and TOY s(ll bind, there is no band seen,
because DNA is not cut.
In the end 5 boxes were found to be involved in binding of EY and TOY to SO:
• When there are TOY muta(ons, part of the expression is lost
• When all the boxes are mutated, there is no expression at all
Studying of gene networks by single cell approaches
This determines the transcriptome of a single cells, by this we can define individual cell types and
reconstruct gene expression paJerns
