Cleavage to implantation Flashcards
LO
- To understand cleavage - a special form of division
- To understand lineage development
- Follow the roles of Cdx2 and Oct 4
- The know the importance of the Zone Pelucida
What is the Zone Pelucida?
the thick transparent membrane surrounding a mammalian ovum before implantation.
Stages of development from zytogote to Late blastocyst
Tell me the stages of development in the early embryo from fertilisation to implantation
A crucial period- many events as an independent organism occur
- up to cleavage- mitosis begins (starts with activation – see last week) and organised cell division
- compaction – starting of cell polarisation (see 3)
- lineage formation – loss of totipotency and formation of Inner cell mass (pluripotent- ES cells derived from here: ES cells form the 3 germ layers which go on to form the tissues of our body) and trophoblasts
- Formation of a functional epithelium – needed for a blastocyst formation
- Hatching from zona pellucida
- Attachment and implantation to uterine wall
- For this many genes and embryo specific factors will need to be produced
- Genome reorganised – the methylation present in the material and paternal genome giving epigenetic control is largely removed and later reformed during development
What is the first stage of embryonic development?
Cleavage
Clevage in amphibian- scanning EM images
Tell me about the stages of embryonic cleavage and name the structres formed during the process
- The embryo divides repeatedly to convert the large cytoplasmic mass into a large cluster of small, cells called blastomeres.
- No growth occurs during this period (no growth during cleavage), only subdivision of mass, which continues until normal somatic cell size is attained.
- At the end of cleavage, the zygote has been divided into tens (mammals) or hundreds /thousands of cells (chick) and the blastula stage is formed (in mammals this is called the blastocyst)
What does the size of the zygote determine in embryonic cleavage?
The number of divisions and the positions of divisions
Embryonic cleavage examples
E.g., lots in a chicken, less in fish and frog and relatively few ~16 in mammals
This is governed by the amount nutrients held in the egg (yolk)
What is cleavage?
What forms due to it?
- Cleavage a rapid series of mitotic divisions that occur just after fertilization.
- Undifferentiated cells are formed which initially retain their totipotency
Tell me two reasons why cleavage is so important?
- Generation of a large number of cells that can undergo differentiation and gastrulation to form organs.
- Increase in the nucleus / cytoplasmic ratio.
Why are there embryonic proteins?
Eggs are cytoplasm rich (hold nutrients to support embryogenesis) but have only one nucleus to support a huge cytoplasm- limited numbers of mRNAs and hence embryonic proteins
A larger nucleus (gene) to cytoplasm ratio is optimal for what?
A larger nucleus (gene) to cytoplasmic ratio is optimal for cell function-for optimal RNA/ protein production.
Cell division occurs rapidly after fertilization to correct this problem.
Tell me a typical somatic cell cycle and what occurs in each stage
Also tell me how this can change during cleavage?
- During cleavage there is no need for cell growth – so G1 and G2 are reduced (mammals) or absent (most other animals)
- During the first cycles (cleavage divisions), the timing of the G1 phase is significantly reduced in mammalian embryos
- At the third cell cycle, the G1 phase takes only 1 h compared with 11 h of the normal cell cycle
- The length of the G1 and G2 phases is about 20% of normal somatic cells (on average)
What type of cleavage do mammals show and what is this?
Mammals show holoblastic cleavages- ie complete division of the whole cell this is typical with alterations for amphibians also (little yolk)
What type of cleavage do fish, reptiles or birds show?
But is not observed in fish, reptiles or birds where there is more yolk these show meroblastic or incomplete division
Holoblastic vs Meroblastic cleavage
In most animals, cleavage patterns are not random, explain?
- The plane of cleavage is always perpendicular to the spindle.
- Therefore, the orientation of the spindle determines the pattern of cleavage
Holoblastic (complete division) occurs in a few different forms, name and explain?
Where do mammalian eggs begin to cleave and continues to do so until it enters where?
Mammalian eggs have rotational cleavage that is holoblastic
The mammalian egg begins to cleave in the oviduct and continues until it enters the uterus (1 cleavage / 14 hr).
Tell me about mammalian asynchronous cleavage?
- Asynchronous cleavage: mammalian embryos are unusual Not all blastomeres divide at the same time- hence you may have odd numbers of cells
- The first cleavage is meridional, and the second cleavage is rotational. The 2 blastomeres divide in different planes (amphibians differ)
What remains present throughout cleavage?
Tell me why these embryos have limited mRNA/ ribosomes?
Maternal (pre-fertilisation) control molecules remain present through cleavage however gene activation (expression) occurs early in the mammal (unlike high yolk embryos)
These embryos have limited mRNA/ ribosomes
For embryonic gene activity, what must happen to the genome?
- For embryonic gene activity the genome must be remodelled
- new histones are placed on the DNA
- new DNA methylation patterns occur (generally unmethylated) gamete pattern is lost and by 8/16 cells pluripotent pattern seen
What does mammalian embryo compaction occur?
At the 8-cell stage
Tell me about the stages of compaction?
- Initial blastomeres of mammalian embryos have a loose arrangement, touch only at the basal surfaces.
- After compaction, blastomeres adhere tightly, maximizing the area of contact.
- During compaction, each blastomere undergoes polarization.
- This is initiated by E-cadherin expression forming adherence junctions
- Actin rearrangement (Cell shape alters)
- Tight junctions develop on peripheral contact surface, ZO proteins/ Occludins/ Claudins
- Apical and basolateral membranes specialize – see microvilli
- Gap junctions form on outer cells intercellular communication
E-cadherin goes from being spread everywhere to once compacted to be close to the cap junctions
What does cell polarity at compaction discriminate between?
Cell polarity at compaction discriminates outer and inner cells of the morula
No longer cleavage divisions as now have a polar outside and a non-polar inside (morula). Central cells are different to external cells
By the 16-cell Morula, what is present?
What has started at this stage?
1- Internal cells and external cells
2- the external cells seal off the inside of the sphere
The two cell groups are no longer the same- differential gene expression
Differentiation has started
In the Outer trophoblast OR inner cell mass the undifferentiated 8 cell embryo expresses what and what does this maintain?
The undifferentiated 8 cell embryo all cells express markers maintaining a totipotent state
In the Inner cell mass, what are the transcription factors and what can they become and repress?
The transcription factors Cdx2 and Oct4 are both present and mutually repress each other and can become either cell type when the levels of these TFs are balanced.
In the inner cell mass, at the 16 cell what is formed? What does this separate? What is altered?
At 16 cell - an outer layer is formed signals separating the fates of these layers are produced by altering the levels of Cdx2
What is the formation of the outer layer due to?
What does it drive?
- Due to a free outer membrane, we block Hippo signals here (this increases the TF – Yap in the nucleus)
- Drives Cdx2 expression
- And/or
What is the inner layer due to?
What is enhances/ blocked here?
Due to an increase cell – cell contact enhances Hippo signals (blocks nuclear Yap)
What is Yap?
Yap is a cofactor for the factor Tead4
What does Yap and Tead4 do together?
Together they cause the expression of Cdx2 to be upregulated in outer cells
Hence reducing Oct 4 here.
What does the phosphorylation of Yap in the inner cells prevent?
Phosphorylation of Yap in the inner cells prevents its entry into the nucleus
So Cdx4 levels reduce in inner cells
Hence increasing Oct 4 here
What is Hippo, Yap and Tead4?
Hippo – a protein kinase
Yap and Tead4 (TEF4) transcription factors
What drives the machinery for pluripotency in the inner layer?
In the inner cells – Oct4 is part of a mutual positive feedback loop maintaining the two other inner cell TFs Sox2 and Nanog
Together they drive the machinery for pluripotency
What is Oct4 expression lost due to?
What does this cause?
Where Oct 4 expression is lost (due to high Cdx2 in the outer cells) these cells loose pluripotency
What does Cdx2 drive expression of?
Cdx2 drives expression of Na+ channels (on the apical surface) and the Na/K ATPase pump (on the basal inner surface) of the outer cells
The presence of what type of junctions means that the Na+ channels and Na/K ATPase pumps are retained in different regions of the membrane?
Tight junctions
The presence of tight junctions in the inner layer means what?
Tell me the functions that can happen due to this?
Hence, we have a functioning polarised epithelium which differentially brings ions into the centre of the morula and with the 3/2 – Na/K bias (this ratio sets up an osmotic gradient)
This…
- Brings water as well via aquaporins (like the kidney tubule)
- The ionic gradient at the cell surface allows uptake of glucose /amino acids via Na co-transporters so nutrient transfer from the outside environment (like the gut and kidney)
The functioning polarised epithelium in the inner layer results in what?
Whats expressed now?
This results in cavitation – with a mass of Sox2 expressing pluripotent cells the Inner cell mass on one side of the cavity
After cavitation, the surrounding Cdx2 + cells form what?
Trophectoderm
These will become highly invasive once they interact with the Uterine wall
The cells covering the ICM also have an exposed surface and form what?
The central cells of the ICM is pluripotent, what is this called?
What do both of these form?
- The cells covering the ICM**** also have an exposed surface and form a second non- pluripotent epithelia-the hypoblast or the primitive endoderm (These cells express the TF GATA 4+)
- The central cell of the ICM is pluripotent (Sox2/Nanog/Oct4+) and called the epiblast or the primitive ectoderm
- THESE FORM THE EMBRYO
What is the blastocyst retained by?
The Zona Pellicida
Epi/ hypoblast
Tell me the chain of events that occurs when the Hippo is absent and when its present
What is Cdx2 important for?
What is the Zona pellucida?
Glycoprotein coat surrounding the oocyte- then later the blastocyst
What does the Zona pellucida protect?
developing oocyte
What is the Zona pellucida important for?
Interactions with granulosa cells
What does the Zona pellucida aid?
Spermatozoa binding before fertilisation
What does the Zona pellucida prevent?
- Prevents polyspermy after fertilisation
- Prevents ectopic implantation particularly occurring in the fallopian tube
Tell me about hatching from the zona pellucida?
Due to enzymes from the trophoblasts digesting a hole in the glycoprotein coat (?plasmin) pulsatile expansion of the blastocyst some minor role of uterine secretions…? Also, plasmin
LO lecture 2
- To hormonal priming of the uterus
- To understand how attachment and invasion is caused
- Describe the decidua and its importance
- The know the importance of the Zone Pelucida
Tell me the series of events of implantation
- Hormonal priming
- Attachment
- Entry / growth into uterine tissue (man) or engulfment by tissues (mouse)
- Vascular and secretory changes in uterine wall (decidua)
- Immunological tolerance
Tell me the layers of the blastocyst and what is considered the ICM
Name the TF associated with each of the layers
In implantation, tell me about the timings for the window of attachment
How does this differ between mice and humans?
- The window for attachment is short
- Mouse ~ 24 hrs (about E4- embryonic day 4, is 4 days after fertilisation)
- Human ~ 4 days (about E7)
During implantation attachment, what is the uterus primed to accept?
What is this priming due to?
Uterus is primed to accept the embryo at this point
– nb “implantation can occur in other tissues and is not time limited there!!- ie the permissive window is specific for the uterus)
The priming is due to the rise in E2- estradiol and P4 - progesterone
Where is the estradiol and progesterone from?
What do they help form?
Both from the ovary (P4- corpus luteum)
Are crucial forming the decidua
Whats Mifepristone, and what does it lead to?
Mifepristone – is an antagonist of progesterone leads to decidual degeneration
What hormones is ovulation driven by?
How does these hormones differ over a 28 day period?
Reminder – ovulation is driven by rises in LH and FSH in the Gonadotrophic axis driven by GnRH –> LH/FSH –> Estra/Prog cycle each is usually in a negative feedback loop (not at end of follicular phase)
If you provide high levels of either estradiol or progesterone, what does this block and therefore prevent?
If you provide high level of either Estradiol or Progesterone you will block GnRH through the feedback -ve loop and prevent ovulation (lower levels have other effects)
What does estradiol induce?
When does this occur in mice and humans?
estradiol (less degree progesterone) induce proliferation of uterine epithelium and stromal cells
The epithelial cells at ~E4 mouse/E7 human then differentiate in preparation to accept the embryo
What protein is highly expressed on the uterine epithelium?
What causes its loss and why?
Tell me the associated steps
- A mucin (MUC1) is expressed highly on the uterine Epithelium, this is inhibitory for attachment and is lost in the region where the Blastocyst is-? A paracrine event
- This loss of MUC1 allows the glycosylated transmembrane proteins/proteoglycans in the uterine epithelium to interact with lectins on the embryo trophectoderm
- Apposition— Adhesion—-Invasion
Tell me about trophoblast cells once attached
What do they release?
- The trophoblast cells once attached are extremely invasive and will spread out into the stroma of the uterus
- They release MMPs (MMP2) (matrix metalloproteinases)
What else may produce MMPs?
The epithelial uterine cells may also produce MMPs the uterine matrix is selectively degraded –compare to embryo
What is the decidua?
What is it maintenance driven by?
The decidua- a uterine wall thickening in the region of the blastocyst and begins active secretion
its maintenance is driven by progesterone
What does the decidua develop independently of?
The decidua develops independent of the embryo (as seen if foreign material is present)
What does the decidua interact with and then grow over?
The decidua interacts with the trophoblast and grows over the embryo on the luminal side, enclosing it in endometrium.
What does the decidua contain?
It contains enlarged endometrial stromal cells, which resemble epithelium, increased vascularity- sinusoids form (permeability)
What does the decidua produce?
oestrogen
progesterone
cortisol
CRH (corticosteroid releasing hormone.)
Tell me how the decidua helps with immune tolerance?
- The decidua produces oestrogen, progesterone, cortisol, CRH (corticosteroid releasing hormone.)
- All of which dampen the maternal immune system (immune suppression in the mother) to allow immune tolerance
- Further the trophoblasts do not express MHCI markers – so tissue mismatch responses do not occur from mother so the mother is then tolerant to the embryo
Label these layers seen at gd 6.5
As implantation occurs, changes between mice and man start at the inner cell mass. Tell me about these changes seen
In man -
- A second cavity (the amnion) appears in the inner cell mass gives a two layered disc– inner cells (primitive ectoderm) will form the Embryo outer cells (amnioblast/ amniotic ectoderm) the Amnion
- primitive endoderm line the embryonic disc and yolk sac
In mice-
The Inner cell mass grows down and forms a cylinder the primitive ectoderm –lined by (visceral) endoderm as above
Label the endoderms…
Differences between mice and men at 7.5 and 13 days…
Between the two layers of embryonic disc in humans, what does the primitive ectoderm (epiblast) do?
Between the two layers of the embryonic disc – the primitive ectoderm (epiblast) invaginates forming a groove (the primitive groove) and pushes the primitive endoderm (hypoblast) to one side and forms the embryonic (definitive) endoderm on the lower surface
Whats the Mesoderm and how is it formed?
When this is formed, what are the three germ layers of the embryo?
What does the formation of the germ layers depend on?
Give examples
Formation of these layers depends on changes in transcription factor expression
Expression of
- Cdx2 –>cells become trophectoderm
- GATA –> cells become primitive endoderm
During the formation of the late blastocyst, what happens to the ICM?
Tell me what happens to the TF here
- Inner cell mass–>primitive ectoderm (epiblast)- Oct4/ Nanog/Sox- these cells loose pluripotency (except for a group of posterior primitive ectoderm/ epiblast cells become PGCs)
- Loss of Nanog occurs first
- loss of Oct4 –> cells become embryonic ectoderm
- OR
- loss of Sox 2 –> cells become embryonic mesoderm and endoderm
What does the embryonic ectoderm form?
Embryonic ectoderm forms skin and nervous system
What does the embryonic mesoderm form?
Embryonic mesoderm makes muscle, bone, kidneys and gonads
What does the embryonic endoderm form?
Embryonic endoderm makes lung, gut, reproductive tract (not smooth muscle)
All of the previously mentioned are the crucial stages, in order for these to occur what stages have to occur?
Whats believed to have been lost over this period?
- initiate mitosis
- form the first epithelium with all junctions
- pump ions
- initiate energy formation
- induce hormonal changes
- attach – adhere- invade
- become (fertilisation) and loose totipotency (later morula)
- become and loose (most cells) pluripotency
- rearrange your genome
- differentiate into multiple cell types
Most embryonic loss is believed to occur over this period!
Unlike other vertebrates, where to mammalian embryos implant?
Into the uterine wall
So far from fertilisation to implantation these are the steps covered…
Do to implantation into the uterine wall, what do mammals spend extra time forming?
- Therefore, in early embryonic development, mammals spend time forming extra-embryonic tissues rather than establishing and organizing an embryonic body plan.
- Hence you might non placental vertebrate early development is simple….
- it’s not! - YOLK GETS IN THE WAY!!
How are eggs classified?
By how much yolk is present
What are the classifications of eggs? Tell me about each one?
What does the class effect?
- Isolecithal eggs (iso = equal) have a small amount of yolk, equally distributed in the cytoplasm (most mammals have isolecithal eggs).
- Mesolecithal eggs (meso = middle) have a moderate amount of yolk, and the yolk is present mainly in the vegetal hemisphere (amphibians have mesolecithal eggs).
- Telolecithal eggs (telo = end) have a large amount of yolk that fills the cytoplasm, except for a small area near the animal pole (fish, reptiles, and birds).
- Centrolecithal eggs have a lot of yolk that is concentrated within the center of the cell (insects and arthropods).
This effects how cleavage occurs…
Tell me about cleavage in amphibia?
Tell me about the name of each pole and what this means
we get uneven cleavage – the formation of cells in the vegetal pole is slower and fewer cells form here due to the presence of yolk
Top is animal pole= smaller and more cells
Bottom is Vegetal pole= larger and less cells
What class of eggs do amphibia have?
What type of cleavage do they undergo?
Tell me about each stage of cleavage and what is formed at the end
- Amphibia have mesolecithal eggs
- Amphibian eggs have a lot of yolk; however, they are still able to undergo holoblastic cleavage.
- The 1st cleavage is meridional
- The 2nd cleavage is meridional
- The 3rd cleavage is equatorial.
- (Small cells at the top and large cells at the bottom)
- The cleavage is displaced toward the animal pole due to the yolk. This results in 4 small animal blastomeres and 4 larges vegetal blastomeres.
Where is the blastocoel displaced to in amphibians?
The animal pole
From the 128-cell stage onward in amphibian embryos what is formed?
Blastula (hollow ball), from the 128-cell stage onward in amphibian embryo.
What is the outer surface of the amphibian blastula connected by?
What forms and why is this the case?
- The outer surface of the amphibian blastula has cells connected by Tight junctions and Desmosomes
- A blastocoele with multilayered cell wall forms – caused by ion pumps in the outer epithelial cells- see mammal!
What type of cleavage do birds, reptiles and fish undergo?
Tell me the type of eggs it forms and what this means
Discoidal meroblastic (incomplete) cleavage
This forms telolecithal eggs which is when there is an uneven distribution of yolk in the cytoplasm
much yoke that completely supports embryogenesis.
Unlike holoblastic cleavage, what does meroblastic cleavage leave?
Unlike holoblastic, meroblastic cleavage leaves a large portion of the zygote uncleaved into blastomeres.
What are the two types of meroblastic cleavage and what type of animals are they seen in?
Discoidal (birds, reptiles and fish)
and
Superficial (seen in insects).
Tell me the stages of cleavage of Discoidal cleavage…
Discoidal
- 1st cleavage is meridional. starts at the animal pole but does not progress.
- 2nd is meridional
- The 3rd cleavages is also meridional
- The 4th cleavage is equatorial, and it creates a layer of small cells on top of the huge uncleaved yolk.
Whats the blastoderm?
A layer of cells formed by cleavage of about 60,000 cells when the egg is laid
What is the next step in development after the formation of a blastoderm?
The next step in development of telolecithal eggs is formation two layers in the blastoderm.
Tell me the layers in the telolecithal egg and what lies between the layers
- The next step in development of telolecithal eggs (in birds, fish and reptiles) is formation two layers in the blastoderm.
- Epiblast: (epi = upon) the upper layer and it forms the embryo proper.
- Hypoblast: (hypo = under) the bottom layer that surrounds the yolk (extraembryonic endoderm).
- Blastocoel: lies between the 2 layers.
- Subgerminal space lies between the hypoblast and yolk.
In Mesolecithal (fish) and Telolecithal eggs (birds, fish and reptiles), what is there no need to wait for?
To develop specialisation
rather (see the mammal- which must implant) a need for large cell numbers Xenopus reaches 37,000 cells in 48 hrs, Chicken ~50,000 cells in 25hrs
(as mammals must soon implant cleavage only 3-4 divisions then differentiation must start)
What drives rapid division of cleavage (without G phases)?
maternal mitosis promoting factor
What are the maternal mitosis promoting factors?
Where is the mRNA stored and why is it located here?
maternal mitosis promoting factor
a dimer of cyclin B and cyclin dependent kinase 2
both needed for mitosis- cyclin B rapidly destroyed, its mRNA is stored in blastomere cytoplasm so S phase occurs while cyclin B reformed
Tell me four things needed for cleavage initiation
When does the normal form of cell cycle occur?
tell me the genes expressed here
The normal form of cell cycle occurs when embryo specific genes expressed- cyclin proteins (A, D and E) causing classical G phases… then cleavage stops
