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
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
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
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
Tell me the layers of the blastocyst and what is considered the ICM
Name the TF associated with each of the layers
Where is the estradiol and progesterone from?
What do they help form?
Both from the ovary (P4- corpus luteum)
Are crucial forming the decidua
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)
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
What else may produce MMPs?
The epithelial uterine cells may also produce MMPs the uterine matrix is selectively degraded –compare to 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…
Whats the Mesoderm and how is it formed?
When this is formed, what are the three germ layers of the embryo?
So far from fertilisation to implantation these are the steps covered…
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.
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 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
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.
Tell me four things needed for cleavage initiation
