The concepts and language of development

Question 1

Define Embryology

Answer 1

Embryology is the branch of medicine concerned with the study of embryos and their development

Question 2

What is the first stage in the development of a fetus?

Answer 2

The process of the egg and sperm coming together.

Question 3

Identify 4 key features of a fertilised egg

Answer 3

There are a few identifiable features of the fertilised oocyte:

1. Zona Pellucida (Bright ring) – this is glycoprotein material
2. Within the zona pellucida is the zygote with two pronuclei (with one haploid nucleus from the male and female respectively). They are yet to fuse.
3. The perivitelline space – a fluid filled space that surrounds the embryo
4. Polar bodies in the perivitelline space (either one or two bodies)

Question 4

Where do polar bodies come from?

Answer 4

They contain a portion of female genetic material. Females generate all of their germ cells prior to birth, where they form one oocyte.

1. A germ cell will duplicate its nuclear DNA, they briefly enter meiosis but then arrest after prophase 1 and will remain in this state into adult life.
2. When a single oocyte is chosen it will then finish meiosis.
3. Once meiosis continues the first meiotic division occurs to produce two cells that are unequal. Half of the genetic material is extruded into a polar body which will be found in the perivitelline body (the first polar body).
4. The selected oocytes will arrest in metaphase 2
5. In order for meiosis to now resume the oocyte must be fertilised.
6. Second meiotic division then occurs and another polar body will be extruded

Question 5

What is cleavage and describe the phases of it?

Answer 5

Cleavage stages (process of splitting into two cells without growth)

1. The pronuclei first fuse and the zygote will under mitosis.
2. Approximately thirty hours after fertilisation, the oocyte splits into two cells of equal size called blastomeres.
3. This will produce 2 cells and divide again to give 4 cells
4. All of this is contained in the zona pellucida, so the cells are getting smaller

Question 6

Describe how the blastocyst will form the morula and attachment to the endometrium of the uterus

Answer 6

1. After 3 more divisions, there are 12-16 cells. At this point, the group of cells is referred to as the morula. They have divided asyncrhonly and are all loosely attached to each other.
2. It will then undergo compaction where the cells on the outer part of the embryo will become flatter and form an epithelial barrier (with cell junctions between).
3. This allows for fluid to enter the central region of the embryo which is called the blastocoel and the embryo is now called a blastocyst. It consists of two cell types:
   a. Outer cell mass (trophoblast) – contacts with the endometrium of the uterus to facilitate implantation and the formation of the placenta. Inner cell mass (embryoblast) – responsible for the formation of the embryo itself.
4. N.B this is happening in the oviduct and anything after cleavage is called blastulation
5. This zona pellucida will then be dissolved by secretory enzymes from the embryo, the blastocyst will then hatch out around 6/7 days. This degrades the protein coat, the blastocyte then hatches out.
6. The trophoblasts have also differentiated to allow the blastocyst to implant into the uterine wall.
7. During the second week, the trophoblast and embryoblast divide into increasingly specialised cell types. The trophoblast divides into the syncytiotrophoblast and cytotrophoblast. The embryoblast divides into the epiblast and hypoblast, forming a two-layered structure; the bilaminar disk. The amniotic cavity forms within the epiblast.

Question 7

Describe the process of implantation

Answer 7

• After the initial rounds of cellular divisions, the embryo must implant into the endometrium of the uterus.
• During this process, the syncytiotrophoblast becomes continuous with the uterus – such that maternal blood vessels (known as sinusoids) invade the spaces within the syncytiotrophoblast (known as lacunae). At this point, uteroplacental circulation has begun; and further embryonic development can occur.

Question 8

What makes up the bilaminar germ disk?

Answer 8

The cells of the inner cell mass have also started to proliferate which have formed a bilaminar germ disk (a two layered embryonic disk). This consists of the epiblast cells and hypoblast cells.

Question 9

Summarise gastrulation

Answer 9

During the 3rd week of embryonic development, the cells of the bilaminar disk (epiblast and hypoblast) undergo a specialised process called gastrulation. During this time, the two cell layers become three germ layers and the bodily axes observed in the mature adult are created.

It is a process of cellular rearrangement involving migration, invagination and differentiation of the epiblast, largely orchestrated by the primitive streak. This is a groove in the midline of the epiblast which appears during the third week. Within the primitive streak lies a primitive node at the cranial end, and within the primitive node lies the primitive pit.

Question 10

Gastrulation – what happens to the bilaminar germ disk?

Answer 10

Cells of the epiblast layer break off and migrate toward the primitive pit. Here, they detach and penetrate through the epiblast layer to form three new germ cell layers:

1. Endoderm – formed by epiblast cells that migrate through the primitive pit and displace the hypoblast cells. (Lower)
2. Mesoderm – formed by epiblast cells that migrate through the primitive pit and lie between the epiblast layer and the newly created endoderm. (middle)
3. Ectoderm – formed by the epiblast cells that remain in position. (Top)

Question 11

What will the bilaminar germ disk form?

Answer 11

• The epiblast cells will then differentiate into endoderm cells which will then replace the hypoblast layer.
• Epiblast cells also forms the mesoderm. Epiblast cells will ingress and involute to form a 3 layered embryo
• On the top is ectoderm (cells from the epiblast that didn’t differentiate)
• Cells of the bilaminar germ disk will migrate anteriorly, posteriorly and laterally to form other tissues. These are shown by the arrows on the epiblast

Question 12

What is Hendsons node?

Answer 12

Hensons node is where gastrulation is being initiated and acts as a signalling centre by secreting growth factors that cause changes to the surrounding cells

Question 13

Summarise neurulation

Answer 13

• In the mesoderm we have a further differentiation of cells in the middle to form a chord known as notochord (beneath of the primitive streak). Its purpose is mainly in neurulation
• It induces a thickening change in the ectoderm above it called the neural plate
• The neural plate forms a tube and zips up it forms the neural tube, cells also invade the mesoderm from the ectoderm known as neural crest cells.

Question 14

Describe what can be seen at day 16 and 20

Answer 14

• Day 16 – the embryo will continue to divide and fold, lots of proliferation is going on but differs between tissues. This shows a sagittal section through the embryo: we have the cranial and caudal on the left and right. It is folding towards the midline from both ends.
• On the transverse section we can also see lateral folding.
• Folding brings the heart tissue inwards,
• Endoderm will form the lining of the gut
• Here we looking at a top-down view of the embryo with cranial towards the top and caudal towards the bottom.
• The pink structure is a developing nervous system, this sheet of cells will role to form the neural tube and then fuse.
• At day 22 the part of the neural tube has fused together to meet in the middle.
• In a cross section we have somite’s
• The grey is the notochord ventral to the neural tube
• Remember the anterior and posterior neuropore are exposed due to closure of the neural tube

Question 15

Describe the cloth purse model

What membranes close the cranial and caudal ends?

What forms the umbilical cord?

Where does the septum and the heart move?

Describe the planes

Answer 15

Folding to bring organs where they should be
Closed at the cranial end by the oral pharyngeal membrane and posterior by the cloacal membrane
• This process of folding is called embryonic folding
• It occurs in two planes, the horizontal plane and the median plane
• Folding in the horizontal plane results in two lateral body folds
• Folding the median plane results in the development of cranial and caudal fold
• The endoderm will move towards the midline and fuse to form the primitive gut tube which will differentiate into three main parts, the foregut, midgut and hindgut (caudal end)

Question 16

What are the results of folding?

Answer 16

Folding forms the endodermal gut tube

Formation of the umbilical cord

Question 17

• Changes in cell fate are accompanied by molecular changes

* How are these controlled?

Answer 17

• Option 1 – Mosaic development (cell autonomous specification) – information for what the cell will become is inherited and is contained in the cell
o Information inherited from parental cell
• Option 2 – Regulative development (conditional specification) – the cell can become a variety of cell types depending on the environment
o Cell influenced by its surroundings, or position, within the embryo

Question 18

What does Weismann’s nuclear determinants show?

Answer 18

• This image shows that after each cleavage each cell has inherited a different nuclear factor or determinant. This follows on from option 1 of mosaic development whereby factors are inherited.

Question 19

Describe Roux’s experiments on determination

Answer 19

• Roux’s attempt to show mosaic development. Destroying (but not removing) one cell of a 2-cell frog embryo results in the development of only one-half of the embryo.
• This is mosaic development – during stage two the embryo has inherited different factors forming a left and right side of a frog embryo. However this conclusion is wrong. This dead half is impeding with the regulation of the live right side.
• Tying a not round the 2-cell embryo and separating them results in the formation of two embryos each with a left and right embryo

It would seem like mosaic development – BUT THIS IS WRONG!

Question 20

What is robustness?

Answer 20

robustness is when a cell or particular group of cells are damaged during development and the cell is able to detect and compensate for it

Question 21

Why is C. elegans development mosaic?

Answer 21

This is mosaic development, it is early hatched and what cell types represent. Differentiation and specific cell fates can be traced back to their origins.

Question 22

Describe:

Driesch’s separation of sea urchin blastomeres demonstrates regulative development

Answer 22

Driesch’s demonstration of regulative development. (A) An intact 4-cell sea urchin embryo generates a normal pluteus larva. (B) When one removes the 4-cell embryo from its fertilization envelope and isolates each of the four cells, each cell can form a smaller, but normal, pluteus larva. (All larvae are drawn to the same scale.) Note that the four larvae derived in this way are not identical, despite their ability to generate all the necessary cell types. Such variations are also seen in adult sea urchins formed in this way (Marcus 1979).
Each cell is fully capable of forming all the cell types of the pluteus larva (shown in diagram above on right)

Question 23

Compare mosaic and regulative development

Answer 23

• Here we have an embryo that will give rise to specific cell types that will form specific parts of the body
• In mosaic development, removal of a cell type results in a loss of body part
• In regulative development, a cell type can compensate, but the body part is now derived from a different cell type
ht)

Question 24

Define fate

Answer 24

• Fate - what will normally happen to a cell during development

Question 25

What does commitment comprise off?

Answer 25

o Specification - what tissues will develop in an autonomous (“neutral”) environment
o Determination - an irreversible change in potential

Question 26

Define differentiation

Answer 26

• Differentiation - a restriction of potential with molecular/biochemical changes - term often used for mature cell types
• Differentiation = specification and determination

Question 27

Define potential or potency

Answer 27

• Potential or potency - the range of tissues which a cell can give rise to

Question 28

Define totipotent and pluripotent

Answer 28

• Potential or potency - the range of tissues which a cell can give rise to
o Totipotent - can give rise to all tissues
• Pluripotent - can give rise to many tissues

Question 29

What is orthotopic and heterotropic graft?

Answer 29

Orthotopic graft is the transplantation of a tissue or organ in its normal anatomic position. Heterotropic graft is the transplantation of a tissue or organ into a position it does not normally occupy

Question 30

During an experiment using a labelled donor:
What will happen if you graft the labelled donor cells onto an embryo via orthotopic graft?
What will happen if you isolate the tissue?
What will if you heterotropically graft the labelled donor cells onto an embryo at its early stage? at a later stage?

Answer 30

It will end up at its normal fate
you will find its speicification
there will be no determination as the early tissue are not committed
there will be determination as the early tissue are now committed

Question 31

What is induction?

Answer 31

• The introduction of tissue in a heterotopic way that results in the surrounding tissue responding to different factors that change the fate of the tissue. This is called induction.
• It relies on the receiving tissue to respond to the signals secreted by the new tissue
• This kind of induction where we can induce a whole second axis is called an organiser, such as Hensons node which also has organiser activity.
• Induction can change the body plan of an organism

Question 32

What does restriction in potential depend on?

Answer 32

Restriction in potential often depends on inductive interactions from neighbouring cells

Question 33

What is competence?

Answer 33

the ability to respond to an inductive signal

Question 34

What are the types of inductive interactions?

Answer 34

Permissive – this creates an environment where other factors can act to cause a tissue to be induced

Instructive appositional – where the instruction is passed by the close apposition or bringing together of two different tissue types to form a new tissue

Instructive morphogen gradient – here we have a localised signal of morphogen which is diffusible and is present at different concentrations along embryo and so different tissue types forms as a result of the different concentration of morphogens.

Question 35

Describe the french flag model of morphogens

Answer 35

• A morphogen is a diffusible molecule that triggers different cell fates at different concentrations. They provide a positional identify in the embryo
• Gradients need to be formed within the embryo and cell fate changed accordingly – how?
• Can provide positional information within the embryo

Question 36

What are Homeotic genes?

Answer 36

• Regulate the development of anatomical structures (identified in insects to control segment identity)
• eg theoretical animal with 4 segments, specified by morphogen gradient that sequentially activates genes 1-3 (a)
• Homeotic effects of altering morphogen gradient (b) or blocking morphogen (c)

Question 37

How can apparently simple diffusion result in complex patterns and structures?

Answer 37

• Turing’s reaction-diffusion model
• This proposes a diffusible activator that activates a diffusible inhibitor of itself which is diffusible in itself.

Turing patterns
Activator diffuses which activates an inhibitor that diffuses which inhibits the activator which then causes the inhibitor to leave.
Activator -> inhibitor -> activator

Question 38

Describe basic gene organisation

Answer 38

• The regulatory region is responsible for assemblying the RNA polymerase so it can generate RNA
• The RNA will then be transcribed to produce heterogenous nuclear RNA, containing both exons and intron sequeneces
• The RNA then becomes spliced to produce Mrna which then matures by a polyA tail and 5’ cap to produce a protein

Question 39

How are changes in cell fate reflected by changed in gene expression?

Answer 39

• Secreted factors and other signals allow cells to communicate
• Often results in regulation of gene expression
• Individual transcription factors don’t act alone, so effects can vary in different cell types (context – previous slide, for example). Transcription factors can be mediated
• Additional control at level of translation, post-translation, epigenetic changes leads to changes in cell fate

Question 40

What is the first and second cell fate in mammalian development?

Answer 40

• Inner Cell Mass (ICM) vs Trophectoderm (TE) in blastocyst (first cell fate)
o These cells become apparent morphologically at the blastocyst stage (blastula), but molecular changes occur before this
• ICM to Epiblast vs Hypoblast (second cell fate)

In mammalian embryos, two cell fate decisions are taken before implantation. These result in the setting apart of the extra-embryonic lineages (trophectoderm and primitive endoderm) from the pluripotent progenitors of the future body, the epiblast.

Question 41

What TFs are responsible for the first and second cell fate during embryonic mammalian development?

Answer 41

On image

Question 42

What genes are expressed in the ICM and TE?

Answer 42

• Oct3/4, Sox2, Sall4, Nanog expressed in ICM
• Cdx2, Gata4 expressed in TE
• Shown by immunofluorescence, staining of the mouse embryo
• DAPI stains all nucleus of cells

Question 43

What are the two possible mechanisms for the generation of multiple cell types and polarity?

Answer 43

A) Localized maternal determinant. In this model, a maternal determinant (pink) is unevenly distributed in the oocyte, and hence in the fertilized egg, and becomes inherited asymmetrically by daughter cells following cell division. The blastomere that receives this determinant can therefore adopt a cell fate that is different from a blastomere that does not receive the determinant. This can allow the generation of two cell types, as well as polarity, within the developing embryo.

Material sets asymmetrical, polarity -> cells

No signal, other external signals lead to polarity

(B) No localized maternal determinant. In the absence of such an asymmetrically localized maternal determinant, alternatives mechanisms must exist to allow the generation of multiples cell types and polarity within the embryo.

Question 44

How are the initial differences established between cells?

Answer 44

The combined model we propose here suggests that the core elements of each of the three models — early asymmetry, polarization and inside–outside — that have been proposed for the first cell fate decision identify concepts that are not exclusive.

The mouse egg has some asymmetry, possibly reflecting previous asymmetric meiotic divisions at the animal part of the egg, which leads to heterogeneity (being diverse) between the cells. The extent of this heterogeneity would depend on when cleavage divisions separate animal and vegetal parts of the embryo. Heterogeneity is revealed through asymmetry in epigenetic modifications at the 4-cell stage and through the expression levels of transcription factors such as CDX2 at the 8-cell stage. Such heterogeneity could generate differences in the timing or extent of blastomere polarization along the apical–basal axis that, in turn, would affect whether a cell divides symmetrically or asymmetrically.

Asymmetric divisions generate inherently different inside and outside cells that will occupy different positions in the embryo. Cell position further reinforces cell fate, possibly owing to the different environment of inside (yellow) and outside (green) cells. This combined model proposes that the development of polarity to affect cell fate occurs progressively. Feedback loops reinforcing cell fate decisions ensure that even a small initial bias is sufficient to break the symmetry.

Question 45

Describe how cdx2 mRNA induces different cell types in the early embryo

Answer 45

Cdx2 mRNA is not evenly distributed amongst cells of the embryo. At the 8 cell stage it is more concentrated in the outer polarised cells. These cells undergo symmetric divisions to produce daughter cells that contain cdx2 mRNA of equal amounts.

Cells that divide asymmetrically have different levels of cdx2 mRNA that produces different cell types

Question 46

Describe the inside-outside model

Answer 46

The inside–outside model proposes that cell fate is induced by the position of a cell during the morula stage rather than by cell-intrinsic differences, with cells on the inside forming the inner cell mass and cells on the outside taking up a trophectoderm fate

Question 47

Describe the cell polarity model

Answer 47

According to the cell polarity model4 , a trophectoderm fate is induced if a cell inherits the apical membrane domain (green) after cleavage of a polarized cell (for example, of the 8-cell embryo). Asymmetric divisions (resulting from a division plane (dashed line) that separates apical and basal domains) therefore produce an inner cell mass cell and a trophectoderm cell, whereas symmetric divisions dividing the apical domain produce two trophectoderm cells.

Question 48

What genes are expressed in the ICM and TE?

Answer 48

• Oct3/4, Sox2, Sall4, Nanog expressed in ICM

* Cdx2, Gata4 expressed in TE

Question 49

What is TEAD4 needed for?

What changes where Tead4 is expressed?

Answer 49

• Tead4 is a crucial regulator of TE formation
• Tead4 is required for the expression of Cdx2
• Where would you expect Tead4 to be expressed? – in the inner and outer cells but only activated in the outer cells of the embryo, this activation is controlled by the polarity of the cells through the hippo signaling pathway

Question 50

Describe the hippo signalling pathway

Answer 50

1. Outer cells are polarised which suppresses the Hippo pathway
2. Yap then moves into the nucleus where it interacts with Tead3 to turn on the TE genes such as cdx2
3. In the inner cells, they don’t have polarity. Hippo is activated which activates Lats which phosphorylates Yap so it cannot enter the nucleus, so Tead4 is not activated.

Question 51

As polarity increases what happens to Yap?

Answer 51

Yap gets localised to the nucleus in the TE

Question 52

Describe Oct4 kinetics

Answer 52

Differences in polarity at the 8 cell stage, these differences cause differences in gene expression.

However there are differences at the 8 cell stage

A fluorescent version of Oct4 is activated once it enters the nucleus of a cell at the 4 cell stage

Once the fluorescence is activated measure the decay of fluorescence in nucleus.

cells that shows a slow decay of Oct4 fluorescence , Oct4 is binding to more DNA sites. Cells that have a slow decay divide asymmetrically

Cells that show a fast decay of Oct4 fluorescence, Oct4 is binding to less DNA sites. Cells with fast Oct4 kinetics divide symmetrically

Question 53

How are Oct4 kinetics regulated by modifications of DNA?

Answer 53

• Oct4 kinetics are regulated by modifications of DNA, through methylation of a particular histone (Histone H3).
• This allows for more accessibility for Oct4 to bind to the DNA, so the cells are more likely to give rise to the inner cell mass whereas the lack of methylation leads to tighter winding of the DNA

As the embryo develops, DNA methylation occurs, particularly in the cells of the ICM. The promotor region of Elf5 is methylated in the ICM, but not in the TE. Therefore cdx2 is expressed in ICM cells to establish a feedback loop. Different TF’s can be inhibited in the same way

Question 54

Describe the 2 models of epiblast and primitive endoderm formation

Answer 54

• Formation of the epiblast and primitive endoderm (hypoblast)
• Epiblast and PE progenitors are initially distributed throughout the ICM
• Segregation of cell populations so that PE cells are adjacent to the blastocoel

On paper document

Question 55

Describe the segregation and relocation of epiblast and PrE cells

Answer 55

On document

Question 56

Gata 6 and Fgf4 are central in control of the second fate decision

Answer 56

• Sox17 here is a marker of Primitive Endoderm
• Nanog here is a marker of epiblast
• Sox17 not present in absence of Gata6
• Nanog not present when excess Fgf4 is added