Lecture 7 - Preimplantation development

Question 1

Cleavages: what are they, when do they occur, and what do they give rise to?

Answer 1

Asynchronous divisions of the larger embryo into smaller pieces, without net growth

Occurs every 10-12 hours while moving through the fallopian tubes

Blastomeres

Question 2

Compaction: what is it and what are cells like precompaction?

Answer 2

The name given to the cleavage of the embryo to form a compacted morula

Display totipotency - one singular cell from a blastomere can form a complete organisms, all cells have similar gene profiles precompaction

Question 3

Cavitation: what is it, when does it occur, how does it occur, and why is it necessary?

Answer 3

The formation of the blastocoel, a fluid-filled cavity that defines the blastocyst

After capacitation, at around day 5 after fertilisation

Na⁺/K⁺ ATPase pump results in the movement of Na⁺/K⁺ ions which causes water to flow into the embryo

Necessary step for embryonic development

Question 4

How is water retained and not lost from the embryo after cavitation?

Answer 4

Tight junctions between the cells of the mature trophectoderm epithelium

Question 5

ICM: what is it and where is it found?

Answer 5

Inner cell mass

Inside the blastocyst

Question 6

dpf: what does it stand for, what is the progress through the days,

Answer 6

days post fertilisation

1 - Fertilisation (zygote)
2 - 2-cell
3 - 4-cell
4 - 8-cell (EGA from 4-8 cells, between days 3-4)
5 - morula (compaction from 16-32 cells, between days 4-5)
6 - blastocysts
7-10 - implantation

Question 7

EGA: what is it and what does it do?

Answer 7

Zygotic/embryonic gene activation

Initial zygote development is controlled by stored maternal mRNA and proteins, but at this point, between 4-8 cells, the embryo takes over development - this step is required for capacitation and cavitation to occur

Question 8

Lineage specification: what are the main lineages of it and what is their function?

Answer 8

• Trophectoderm - required for proper implantation, formed from polar cells
• Epiblast - the actual foetus, formed from non-polar cells
• Hypoblast (primitive endoderm) - feeding epiblast, potential in signalling patterning

Question 9

Energy source during preimplantation development: what is the initial source and what is the later source?

Answer 9

Pyruvate until around the 8-cell stage

Glucose from this point, along with an increase in metabolic activity

Question 10

Trophectoderm: what is it and what does it do?

Answer 10

Epithelium surrounding the inner components of blastocysts

• Capable of pumping fluid to generate a blastocyst cavity
• Capable of interacting with the uterus for implantation and generating all placental lineages

Question 11

Epiblast: what is it and what does it do?

Answer 11

The ICM which contains the undifferentiated cells of the embryo, containing the genetic information capable of producing an entire organism

Question 12

Protein synthesis in the early mammalian embryo: what regulates the first cell cycles and what occurs before EGA?

Answer 12

First cell cycles - regulated at post-transcriptional level by oocyte derived mRNA

Before EGA the embryo carries abundant transcripts for genes that stabilise/control/degrade maternal mRNA

Question 13

EGA: what is it and what is the mechanism of the process?

Answer 13

Embryonic gene activation

• Release from transcriptionally oppressive environment with demethylation of paternal genome
• Opening of chromatin
• Protamine -> histone exchange
• Synthesis of transcription factor proteins from maternal mRNAs
• Post-translational modification of maternal transcription factors (disabling their function)
• Early translational initiator proteins
• Transcription activated even in arrested embryos (granted they’ve undergone the first mitotic division - unsure of exactly when the EGA switch is)

Question 14

EGA proteins: what is their activation caused by and what are some examples?

Answer 14

DUX4 expression is present at 4-cell stage of embryonic nucleus - results in EGA-associated gene activation (ZSCAN4 produced for example)

TPRX, OCT4, LEUTX (following minor EGA)

Question 15

DUX4: what is it, what does it do, when is it expressed, and what evidence is there of what it does?

Answer 15

Double homebox 4 - an EGA protein

Causes EGA-associated gene activation

4-cell stage of embryonic nucleus, though it has been reported to be transcribed at low levels after fertilisation

Force expression induces EGA-associated genes (ie ZSCAN4) in human-induced pluripotent stem cells and human embryonic stem cells

Question 16

Pioneering factors: what are they, what do they do, and what are some examples?

Answer 16

Factors that can interact with the inactivated chromatin and remodel it

Allows for other factors to access the inactive chromatin and conformationally change it to result in its activation

DUX4 and OCT4

Question 17

Compaction: what is it, when does it occur, what causes it to occur, and what also occurs at the same time?

Answer 17

The formation of a tight cluster of cells from the blastomeres

After EGA

Not exactly sure but PKC-α and β-catenin are theorised to be involved, Ca²⁺ is required

Polarisation - development of a polarised distribution of surface microvilli at one pole of the cell (apical pole)

Question 18

Cell-cell adhesion: what does it require and what does it result in during compaction?

Answer 18

Requires E-cadherin (and therefore Ca²⁺ as E-cadherin requires it to function)

Results in the usually contractile embryonic cells to adhere, it also later results in trophectoderm (TE) formation

Question 19

Embryonic polarity following compaction: what protein accumulates as a response and what does this cause to happen?

Answer 19

Ezrin at the outer membrane

Involved in cytoskeleton reorganisation

Question 20

Polarity and compaction: which happens first and why?

Answer 20

Either or, they are independent processes that happen at very similar times but do not directly depend on the other (they utilise similar mechanisms)

Question 21

Divisions during the 8-16 and 16-32 stages: what are the types, what do they cause to form, and why are they necessary?

Answer 21

Conservative division - symmetric division generate polarized outer cells

Differentiative division - asymmetric division, generates polar outer cells and apolar inner cells

the inheritance of the polarized state is influenced by the orientation of the cleavage plane in the blastomere

Question 22

Models for how TE and ICMs form: what are the types?

Answer 22

Inside-out model - dependent on cell contact, higher (inside) and lower (outside) levels result in differences in gene expression

Polarity model - presence or absence of an outer polar domain (ezrin-aPKC-Par6 complex) result in differences in gene expression

Cell contractility model - mechanical properties of cells affect their position, which is affected by polarity of the cells

Question 23

aPKC-Par6: what is it and what does it do?

Answer 23

Atypical protein kinase C (aPKC) and partitioning defective 6 (Par-6)

Proteins that work together to establish cell polarity in multicellular organisms

Question 24

Maintenance of ICM: what are the key proteins involved and what are their key features?

Answer 24

OCT4 - a pou domain (homeodomain) TF, unique to pluripotent cells

NANOG - divergent homeodomain protein, required for epiblast

SOX2 -Sry-related HMG box containing TF2, the first and most specific pluripotent factor in epiblast/ICM formation

Question 25

Totipotency

Answer 25

Any cell types

Question 26

Pluripotency

Answer 26

Any embryonic cell lineages but not extra-embryonic lineages (the lineages that form during embryonic development, etc)

Question 27

Cdx2: what is it, what does it do, why is it necessary, and what is its mutually antagonist gene?

Answer 27

Caudal-related homoeodomain transcription factor

Suppresses Oct4 in TE - necessary for blastocysts to not collapse and arrest

Oct4

Question 28

HIPPO pathway: what is it, how does it work

Answer 28

The pathway which causes the formation of the trophectoderm

HIPPO pathway kinases (LATS1/2) become active in unpolarused cells, causing the inhibition ofn

Hippo pathway active in adhering inner cells inhibits Yap and Taz so prevents nuclear localisation and activation of Tead 4 in inner cells
Nuclear Tead 4 only in outer TE up regulates CDx2
Gata 3 initiates some TE gene expression and needed for its proliferation
A number of these TE lineage-associated factors are also expressed in the
human embryo, though expression patterns are not necessarily always conserved
with the mouse. GATA3 is expressed in the human TE, but homologues
of key mouse TE factors such as ELF5 and EOMES are absent during
preimplantation development and absence of CDX2-regulated factors such as ELF5 suggests that Hippo signalling may not necessarily drive ICM-TE segregation or that Hippo signalling in humans regulates alternative TE factors upstream of CDX2.

Answer 29

The experiments revealed that the mother places two closely related proteins known as YAP1 and WWTR1 within each egg, which help to make placenta cells different from pluripotent cells.

Answer 30

The experiments revealed that the mother places two closely related proteins known as YAP1 and WWTR1 within each egg, which help to make placenta cells different from pluripotent cells.

Question 31

FGF/ERK inhibitors

Answer 31

Despite conservation of some lineage-specific genes between mouse and
human embryos, it is likely that distinct signalling pathways are required at
these early stages. However, the FGF receptor was not targeted
independently of inhibiting Erk, so it remains possible that FGF might function
via an alternative downstream pathway to regulate lineage specification.

in marmoset embryos, Erk or Wnt inhibition increased the proportion of NANOG expressing cells within the ICM and diminished, but did not abolish, GATA6-only PE cells

Components of the Nodal signalling pathway are enriched in the human Epi, and inhibiting TGFβ/Nodal
in the human embryo was recently shown to result in loss of NANOG expression, however, the results are controversial as other group reach an opposite conclusion.

Human embryos segregate putative hypoblast by day 7 of development. Human embryos were thawed and cultured in standard IVF medium until they formed cavitated blastocysts, upon which they were moved to N2B27 medium. Embryos were fixed and immunostained for Oct4 (white), Nanog (green) and Gata6 (red). At day 6 of in vitro development (A) Nanog is restricted to a few cells within the embryo, whilst Gata6 and Oct4 are broadly expressed. Confocal images of two representative embryos with a maximum projection of the 3D reconstruction of the blastocyst are shown. (B) A single slice in a z stack of each of the two embryos shown in (A), indicating that Nanog and Gata6 can both be expressed highly in the same cell (arrowheads) or that Gata6 can be low as Nanog is high (arrows). (C and D) Embryos were developed to day 7 in vitro and immunostained for Nanog (green), Oct4 (white) and Gata4 (red) (C) or Sox17 (red) (D). In contrast to the staining observed at day 6, Oct4 is restricted to the cells of the ICM. Gata4 and Sox17 are restricted to a subset of cells within the embryo, distinct from the Nanog positive cells: the putative hypoblast. In all embryos nuclei were counterstained with DAPI (blue). The total number of cells in each embryo is written in the top right hand corner of the panel

Question 32

Blastocyst components: what are their derivates as development occurs?

Answer 32

Trophectoderm:
* Chorionic ectoderm (cyto-/syncytio- trophoblast) - chorion/placenta

Inner cell mass:
* Extra-embryonic ectoderm - Amnion
* Extra embryonic mesoderm - allantois, yolk sac, chorion/placenta
* Extra-embryonic endoderm - yolk sac
* Embryonic ectoderm - skin, hair, mammary glands, and part of the nervous system
* Embryonic mesoderm - muscles, bones, heart, circulatory system, gonads, and urogenital system
* Embryonic endoderm - allantois, epithelium of the digestive tract, lungs, liver, pancreas, and thyroid

Question 33

Implantation: what must be separated from the blastocyst for it to occur, what causes it, and can this be done in vitro?

Answer 33

Hatching from the zona pellucida

• ZP thinning due to blastocyst cavity growth
• Stimulated by catechol-estrogen (a breakdown product of estradiol)
• Both uterine and embryonic serine proteases may be involved

Can be done locally - if not, IVF wouldn’t be possible

Question 34

Regulation of early embryo development: does it require specific circumstances to occur, what is it influenced by, and what are the effects of

Answer 34

Occurs autonomously in vitro in a simple salt solution

Nutritional and growth factors:
* Glucose/energy substrates
* Amino acids
* Growth factors - IGF/HB-EGF/FGF, etc
* Steroid hormones
* Cytokines
* Metabolic regulators

Potential short-term responses - developmental plasticity:
* Epigenetic modification
* Altered intracellular signalling
* Metabolic stress
* Gene expression changes
* Apoptosis
* Cell proliferation disturbed

Potential long-term consequences:
* Low female birth weight - low maternal protein diet (in rat models)
* High male blood pressure - low maternal protein diet (in rat models)
* Reduced implantation capacity
* Unbalanced fetal/placental allocations
* Abnormal fetal growth rate
* Altered setting of neuroendocrine axes
* Abnormal birth weight/postnatal growth
* Cardiovascular/metabolic syndromes

Question 35

Growth factors affecting early development? what are some examples and what do they do?

Answer 35

• IGF-1- survival factor for the human embryo and stimulates protein synthesis
• HB-EGF (Heparin-binding EGF-like growth factor) - stimulates murine and human TE formation
• FGF–4 - maintains the trophoblast stem cell population
• In mice LIF is necessary in the uterus for implantation

Question 36

Developmental programming: what is it and why is it important?

Answer 36

The ability of environmental factors to induce alterations during development to influence the health status of an individual

Prenatal epigenetic modifications are inherited during mitosis and can perpetuate specific phenotypes during early postnatal development and adulthood