Developmental Biology Lecture 5 PDF

Lecture 5 Early development Cleavages and cell-fate decisions Cell-fate decisions are being made in the morula stage (16-cell) because the in the blastocyst, the cells have now been restricted in their potential Morula stage:...

Lecture 5 Early development Cleavages and cell-fate decisions Cell-fate decisions are being made in the morula stage (16-cell) because the in the blastocyst, the cells have now been restricted in their potential Morula stage: At the 8-cell stage ⇒ Cells undergo compaction due to the upregulation of a cell adhesion molecule called E-cadherin By the time the embryo reaches the morula stage ⇒ Cells are tightly associated with each other → makes it hard to discern individual cells Blastocyst stage: The embryo consists of 2 distinct cell populations ICM and trophoblasts/trophectoderm Cell Potential restrictions: By the blastocyst stage ⇒ Cells have been restricted in their potential Implies that at some point prior to the blastocyst stage, the cells were more flexible in their developmental potential and that the ICM/trophoblast decision was not yet made Relationship between the maternal vs. zygotic transcripts in early mouse embryo The oocyte is loaded with maternal transcripts by the surrounding follicles before fertilization the maternal transcripts are RNA molecules provided by the mother to the egg these transcripts are eventually translated into proteins that are necessary for early embryonic development the zygote needs this until it can begin to produce its own transcripts Lecture 5 1  they provide a “head start” for the developing embryo zygote transcription after fertilization ⇒ the zygote’s own genome begins to be transcribed → production of zygotic transcripts the transition represents of the embryo’s own developmental program the embryo relies entirely on maternal transcripts for protein synthesis until the zygotic transcripts increase, leading to the maternal transcripts decreasing zygotic transcripts are predominantly from male pronucleus DNA Methylation PGCs migrating through the hindgut, they are highly methylated in the early embryo, but then lose methylation once they enter the primitive gonad because cells are still dividing and haven’t decided a cell fate methylation is lost but later acquired during late stages of gamete maturation methylation rates drastically go down once the PGCs differentiate into the sperm or egg once the gametes mature their methylation goes up post-fertilization ⇒ methylation remains high in imprinted genes but DNA in male pronucleus undergoes rapid de-methylation in the zygote done enzymatically de-methylation in the female chromosomes occur more slowly these changes account for greater levels of transcription in paternal genome during very early development blastocyst stage = equal high methylation levels returned demethylation represents the ability of gene expression in the eggs or sperm Lecture 5 2  methylation high = low expression vice versa Parental Imprinting definition imprinted genes are a subset of genes where only one allele is expressed and the other is silenced through DNA methylation either copy from mother is inherited or copy from father is inherited, but not both established during gametogenesis mammalian specific about 150 genes have been identified in mammals parent-specific expression ⇒ imprinted genes show parent-specific expression patterns, meaning that they are exclusively expressed from maternal or paternal allele consequences non-viable embryos ⇒ if both maternal and paternal nuclei are replaced in an embryo with pronuclei from the same sex, the resulting embryo will not be viable. this is because the imprinted genes will be expressed at too high of a level or not expressed at all depends on if they are normally expressed from paternal or maternal allele paternal nuclei = 2 paternal pronuclei Embryo = stunted (small and not properly developed) and nearly normal placenta maternal pronuclei = 2 maternal pronuclei Embryo is mostly normal but the placenta will be very small and unable to sustain embryo during development Lecture 5 3  reason that same-sex couples cannot contribute to the embryo implications ICM vs. Trophoblast decision: 16-cell stage (morula) Cell Potency and Commitment At the 16-cell stage, all the cells (blastomeres), are still considered pluripotent, pluripotent = stem cells meaning that they are able to develop into any cell type in the embryo or the extra-embryonic tissue These cells are not yet committed to becoming either ICM or trophoblast fate influenced by position in the morula Morula Compaction: at the eight cell stage cells undergo compaction Positional Influence on Cell Fate: Experiments have demonstrated that a cell's position in the morula dictates its future fate Inner cells are more likely to become part of the ICM Outer cells are more likely to become part of the trophoblast Transplantation Experiments: When cells from the inside of a morula are moved to the outside, they will become trophoblast cells, and vice versa Plasticity: This indicates that the cells at the 16-cell stage are still plastic and their fate is not yet fixed. At the 32-cell stage, the fate of the cells becomes irreversible and fixed Lecture 5 4  Ex: if a cell is taken from the outside of a 32 cell morula and transplanted into the centre of a 16 cell morula, it will still become a trophoblast cell4. Molecular Mechanisms: The decision between ICM and trophoblast fate is regulated by the expression of key transcription factors: CDX2 ⇒ trophoblast cells It is upregulated in the outer cells If CDX2 is removed, the embryo cannot form a proper blastocyst and the cells will not downregulate the expression of the ICM-specific transcription factor Oct4 Oct4 ⇒ ICM Initially, Oct4 is expressed in all the cells of the morula It is upregulated in the inner cells of the morula, while it is downregulated in the outer cells that will become trophoblast4. Blastomeres intially expressed both Cdx2 and Oct4 and when they choose their fate, they will upregulate and downregulate their respective transcription factors Cross-repression: Oct4 and CDX2 have a cross-repressive relationship⇒ If one is being expressed, the other is not This cross-repression ensures that the cells commit to a particular cell fate and does not adopt a mixed cell fate Hippo Signalling Pathway This signaling pathway plays a crucial role in determining whether a cell becomes ICM or trophoblast Cell-Cell Communication: The Hippo pathway involves cell-cell communication, such that when Hippo receptors on two neighbouring cells bind to each other, the Hippo pathway is Lecture 5 5  activated LATS Kinase Activation: When the Hippo pathway is activated, the LATS kinase will phosphorylate and degrade the YAP protein YAP and CDX2 Expression: YAP is a transcription factor that interacts with TEAD4 (T to turn on expression of CDX2. When YAP is degraded, CDX2 will not be expressed. Spatial Regulation: Cells in the centre of the morula have more neighbors and therefore receive more hippo signaling This results in the degradation of YAP, and consequently the expression of CDX2 is reduced. Conversely, cells on the periphery receive less hippo signaling, which means that YAP is not degraded, which in turn leads to the expression of CDX2. Consequences of Dissociation: If the cells of a 16-cell morula are dissociated and spread out on a petri dish, they will receive little or no hippo signalling. As a result, these cells are predicted to become trophoblast cells, expressing CDX2 Transcriptional Circuitry of ES cells pluripotency transcriptional circuitry = maintains the pluripotency of embryonic stem (ES) cells drawing from the mechanisms involved in the inner cell mass (ICM) formation during early embryonic development Oct4 and Sox2: Lecture 5 6  These are two key transcription factors that are initially expressed in the ICM Co-activation: Oct4 and Sox2 work together to activate the expression of other genes involved in maintaining pluripotency, including Nanog Core Regulatory Circuitry: Together, Oct4, Sox2 and Nanog form the core of a transcriptional circuit that is essential for maintaining the pluripotent state of ES cells Nanog = transcription factor that is expressed in pluripotent cells of the ICM Positive Feedback Loop: Nanog works with Oct4 and Sox2 to activate its own expression creating a positive feedback loop that further reinforces the pluripotent state example of auto-activation Amplification: The auto-activation of genes like Nanog and Oct4 is important leads to the expression of many other genes involved in pluripotency. ES specific transcription factors repress the expression of Cdx2 this activation loop ensures pluripotency state is maintained and that the cells don’t differentiate prematurely When Oct4, Sox2 and Nanog are activated, they will turn on many other genes that are required for the ICM Interdependence If one of the key transcription factors is missing or not expressed, the entire pluripotency circuit collapses and the cells are unable to maintain their undifferentiated state. Figure b Lecture 5 7  (B) The interconnected regulatory circuit whereby Oct4, Sox2, and Nanog each activate themselves and each other’s synthesis. 2nd week of Development Formation of the embryonic Bilaminar disk Embryonic shield = Bilaminar disk Following implantation ⇒ Inner cell mass (ICM) undergoes a critical transformation → formation of the bilaminar disc Disc consists of two distinct layers ⇒ Epiblast ⇒ destined to form the embryo proper becomes 2-layered Hypoblast ⇒ contributes to extra-embryonic tissues such as the yolk sac Anything that isn't part of the embryo proper is considered extra- embryonic primitive endoderm Epiblast or Hypoblast cell fate decision is not well understood, but two key genes involved in this process Nanog = epiblast Gata6 = hypoblast Initially, the expression of Nanog and Gata6 appears random within the ICM2. However, the cells sort themselves out so that all the Gata6-expressing cells are in the hypoblast layer and all the Nanog-expressing cells are in the epiblast layer Lecture 5 8  Tissue and germ layer formation in early human embryo epiblast → gastrulation → 3 germ layers Human Monozygotic twinning Monozygotic twinning in humans arises from the splitting of cells during early embryonic development the timing of split influences the structures that the twins will share fraternal = separate fertilization events where two oocytes are fertilized by two sperm no more genetically similar as siblings These cells can compensate for any reduction in cell numbers caused by the split identical twins = monozygotic twins originate from a single fertilization event the cells separate during the blastocyst stage, or even when they are part of the inner cell mass or epiblast, two separate embryos can form The timing of the splitting event determines the relationship of the twins to the amniotic sac and uterus in turn determines the degree of sharing of extraembryonic structures 1. Early Split: Split occurs very early, before the cells have committed to becoming either the inner cell mass or the trophoblast ⇒ two separate blastocysts can form a. Each blastocyst then develops its own inner cell mass and its own trophoblast. b. These result in identical twins that each have their own amnion and chorion. Lecture 5 9  2. Intermediate Split: a. If the splitting occurs after the inner cell mass has formed but before the epiblast and hypoblast layers are determined ⇒ two clumps of inner cell mass cells exist within the same blastocyst b. Each of these clumps then goes on to form its own epiblast and hypoblast c. This results in twins sharing a chorion but having separate amniotic sacs 3. Late Split: a. Splitting that occurs at the epiblast stage results in embryos that share both the chorion and the amnion Lecture 5 10