Lecture 2 - Potency PDF

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Lecture on potency (ability of embryonic cells to develop into other cell types)...

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Potency

ANAT334 Lecture 2 13/7/18

Lecture Objectives: - Review cleavage and compaction o Loss of totipotency - Review blastocyst stage o Embryonic/extra-embryonic division o Hatching - Describe compaction as a physical process - Describe cell fate decisions o Intrinsic/extrinsic, bi-stable gene networks - Describe how to test developmental potency.

Cleavage, Compaction and Cavitation Cleavage up to the 8-cell stage involves cell division, not cell growth. At the 8-cell stage, around day 3, the e undergoes compaction. This is increased cell-to-cell adhesion, creating a truly multicellular organism. Comp is caused by altered surface tension, where the surface facing the outer edge of the embryo has increased (reduced area) and the surfaces facing each other have decreased tension (increased contact area). The ac cortex forming the cytoskeleton underneath the membrane is responsible for increasing the tension. On th to-cell surfaces there is E-cadherin, which disrupts the actin cortex and decreases tension. The actin cortex comprised of actin filaments, cross-linked by myosin motors and elastic proteins providing tension and elas Altering the contractility of these ‘actomyosin’ complexes alters surface tension. Surface tension (γ) of a ce be measured by placing a micropipette on a cell, maintaining a constant vacuum before and after compacti occurs, and then measuring the deformation that occurs. At the morula stage after compaction has occurred, around day 5, a cavity forms on the inside of the embryo. This is the point in time where it becomes a blastocyst. The first cell decisions are made, with the epiblast (inner cell mass) going on to make the embryo and the trophectoderm going on to make extraembryonic material. To cause cavitation, outer cells pump Na+ into the middle of the embryo, drawing water in by osmosis and causing swelling. Hatching refers to the blastocyst exiting the zona pellucida, around day 6-7, allowing implantation. This is an active process, where the trophectoderm cells secrete proteases that breakdown the zona pellucida and allow hatching. Thought Experiment – Why Delayed Hatching? Does the zona pellucida help keep cells stuck together, protect embryos from damage or help prevent ectopic pregnancy? In IVF, technicians often remove the zona pellucida right at the very start, hence neither of the first two options are correct. If the zona pellucida is intact, the embryo actually cannot implant, hence it is a measure to prevent ectopic pregnancy.

Potency Totipotency: ability of the cell to develop into all cell types of the organism, including the supporting structures for early development (e.g. placenta). Cells only exist in this state for a short time, losing totip the 2-8 cell stages. Pluripotency: ability of the cell to develop into all cell types of the body . The cells of the i pluripotent. Splitting cells at the 4-cell stage and implanting them into four individual cows produces four identical calves. This means that the cells retain full totipotency at least to the 4-cell stage. In humans, the first molecular decision is the division of the trophectoderm and inner cell mass, at which point these cells are pluripotent. The main extrinsic factor for this decision is cellular location, because the inner cells will be receiving cell signals from every direction but the outer cells will only be receiving cell signals from the cells underneath. There are also intrinsic factors in decision making: Bistable Gene Networks: cells have specific self-reinforcing gene expression and non-self repressive gene expression, often in transcription factors. This ensures that once a molecular decision is made it is not changed, by cells reinforcing their own cell type and repressing other cell types. A key transcription factor network in the early embryo

involves CDX2 (trophectoderm), OCT4, KLF4 and SOX2 (inner cell mass/pluripotency) genes. produced by the inner cell mass bind to the OCT4 region of the genome and support its exp reinforcement’). CDX4 produced by the trophectoderm represses the OCT4 region (‘non-sel versa. Cellular location is likely to be one of the key factors in which cells express which of th Chromatin-immunoprecipitation (ChIP) is one technique scientists can use to see where the factors bind. Demethylation: once a molecular decision is made, TET proteins demethylate DNA in promoter regions of pluripotency genes, leading to increased gene expression and self-reinforcement. Pluripotency factors also enhance the expression of these proteins, making the whole system self-regulating. Remember that DNA methylation levels remain low until pluripotency is established and then increases greatly, because the genes for totipotency are silenced.

Culturing Pluripotent Cells We can culture the cells of the ICM to create embryonic stem cells , which are self-renewing and retain developmental potency in vivo. There are multiple ways of testing pluripotency, from least to most reliable: - Differentiating the ESCs in specific cultures and observing what cell types they can derive. - Injecting ESCs underneath the skin of an immune-compromised mouse, forming a teratoma containing all different cell types of the body. - Injecting ESCs into a host blastocyst to create a chimera. - Injecting ESCs into a tetraploid (4n) blastocyst should create an organism only with cells derived from the donor, because a normal tetraploid blastocyst should not be able to form an embryo (only a placenta). This is the most stringent method of testing pluripotency....