Embryonic Development: Key Processes & Cell Fate

• Early development begins

Cleavages and Cell-Fate Decisions

• Cell-fate decisions happen at the morula stage (16-cell) because blastocyst cells have restricted potential.
• At the 8-cell stage, cells compact due to E-cadherin upregulation.
• By the morula stage, cells are tightly associated, making individual cells hard to discern.
• The embryo consists of two cell populations in the blastocyst stage.
• ICM and trophoblasts/trophectoderm make up the two populations.
• By the blastocyst stage, cells' potential has been restricted.
• Prior to the blastocyst stage, cells were more flexible in their developmental potential, and the ICM/trophoblast decision was not yet made.

Relationship Between Maternal vs. Zygotic Transcripts in Early Mouse Embryo

• The oocyte is loaded with maternal transcripts by surrounding follicles before fertilization.
• Maternal transcripts are RNA molecules from the mother's egg.
• These transcripts are translated into proteins for early embryonic development.
• The zygote needs maternal transcripts until it can produce its own.
• They give the developing embryo a "head start".
• After fertilization, the zygote's genome is transcribed, producing zygotic transcripts.
• This transition represents the embryo's developmental program.
• The embryo relies entirely on maternal transcripts until zygotic transcripts increase, decreasing maternal transcripts.
• Zygotic transcripts are predominantly from the male pronucleus.

DNA Methylation

• PGCs migrating through the hindgut are highly methylated in the early embryo but lose methylation upon entering the primitive gonad.
• This is because cells are dividing but haven't decided on a cell fate.
• Methylation is lost and later acquired during late stages of gamete maturation.
• Methylation rates decrease when PGCs differentiate into sperm or egg.
• Gametes' methylation goes up once they mature.
• After fertilization, methylation remains high in imprinted genes, but DNA in the male pronucleus undergoes rapid demethylation in the zygote.
• Demethylation is done enzymatically.
• Demethylation is slower in female chromosomes.
• These changes cause greater transcription in the paternal genome during early development.
• The blastocyst stage has equal high methylation levels returned
• Demethylation allows gene expression in eggs/sperm.
• High methylation means low expression, vice versa.

Parental Imprinting

• Imprinted genes constitute a subset where only one allele is expressed, and the other is silenced through DNA methylation.
• Either the mother's or father's copy is inherited, but not both.
• Imprinting is established during gametogenesis.
• Imprinting is mammalian-specific.
• About 150 genes have been identified in mammals as imprinted.
• Parent-specific expression means imprinted genes show expression patterns that are exclusively expressed from maternal or paternal alleles.
• If both maternal and paternal nuclei are replaced in an embryo with pronuclei from the same sex, the resulting embryo will not be viable.
• Non-viable embryos occur because the imprinted genes will be expressed at too high a level or not expressed at all.
• This depends on whether they are normally expressed from the paternal or maternal allele.
• With 2 paternal pronuclei, the embryo is stunted but the placenta is nearly normal.
• With 2 maternal pronuclei, the embryo is mostly normal, but the placenta is small and cannot sustain embryo development.
• Same-sex couples cannot contribute to the embryo because of imprinting.

ICM vs. Trophoblast Decision: 16-Cell Stage (Morula)

• At the 16-cell stage, all cells (blastomeres) are considered pluripotent.
• Pluripotent cells can develop into any cell type in the embryo or extra-embryonic tissue.
• Cells are not yet committed to becoming either ICM or trophoblast.
• Fate is influenced by position in the morula.
• Compaction starts at the eight-cell stage.
• Cells undergo compaction.
• 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.
• When cells from inside a morula are is moved to the outside, they will become trophoblast cells, and vice versa.
• At the 16-cell stage cells are still plastic.
• At the 32-cell stage, cell fate becomes irreversible.
• If a cell is taken from outside of a 32 cell morula and transplanted into a 16 cell morula, it will become a trophoblast cell.
• The decision between ICM and trophoblast fate is regulated by key transcription factors involved.
• CDX2 leads to trophoblast cells.
• It is upregulated in outer cells.
• Without CDX2, the embryo cannot form a proper blastocyst.
• Without CDX2, cells will not downregulate Oct4 expression.
• Oct4 leads to ICM.
• Initially, Oct4 is expressed in all morula cells.
• It is upregulated in the morula's inner cells and downregulated in the outer cells that will become trophoblast.
• Blastomeres initially expressed both Cdx2 and Oct4
• Blastomeres will upregulate and downregulate their respective transcription factors.
• Oct4 and CDX2 have a cross-repressive relationship.
• Cross-repression ensures cells commit to a particular fate and does not adopt a mixed cell fate.

Hippo Signaling Pathway

• Hippo signaling determines whether a cell becomes ICM or trophoblast.
• The Hippo pathway involves cell-cell communication; Hippo receptors on two neighboring cells bind to each other.
• After Hippo pathway activation, the LATS kinase phosphorylates and degrades YAP protein.
• YAP is a transcription factor interacting with TEAD4 to turn on CDX2 expression.
• If YAP is degraded, CDX2 will not be expressed.
• Cells in the morula center have more neighbors, receiving more Hippo signaling.
• This results in YAP degradation, lowering CDX2 expression.
• Cells on the periphery receive less hippo signaling, and YAP is not degraded, leading to CDX2 expression.
• If a 16-cell morula's cells are dissociated and spread on a petri dish, they receive little or no hippo signaling.
• As a result, these cells are predicted to express CDX2 and become trophoblast cells

Transcriptional Circuitry of ES Cells Pluripotency

• Transcriptional circuitry maintains the pluripotency of embryonic stem (ES) cells.
• This circuitry uses mechanisms involved in inner cell mass (ICM) formation.
• Oct4 and Sox2 are two key transcription factors initially expressed in the ICM.
• Oct4 and Sox2 work together to activate other genes involved in maintaining pluripotency, including Nanog.
• Oct4, Sox2, and Nanog form the core of a transcriptional circuit essential for maintaining the pluripotent state of ES cells.
• Nanog = transcription factor expressed in pluripotent cells of the ICM.
• Nanog works with Oct4 and Sox2 to activate its own expression.
• This creates a positive feedback loop that further reinforces the pluripotent state.
• Auto-activation of genes, like Nanog and Oct4, is important.
• This leads to the expression of many other genes involved in pluripotency.
• ES-specific transcription factors repress Cdx2 expression.
• This activation loop ensures that the pluripotency state is maintained and that the cells don't differentiate prematurely.
• When Oct4, Sox2, and Nanog are activated, they turn on many other genes required for the ICM.
• If one of the key transcription factors is missing, the entire pluripotency circuit collapses, and the cells are unable to maintain their undifferentiated state.
• Oct4, Sox2, and Nanog each activate themselves and each other's synthesis in the regulatory circuit.

2nd Week of Development Formation of the Embryonic Bilaminar Disk

• Embryonic shield = Bilaminar disk.
• After implantation, the inner cell mass (ICM) undergoes a critical transformation, forming the bilaminar disc.
• The epiblast is destined to form the embryo proper.
• The epiblast becomes 2-layered.
• The hypoblast contributes to extra-embryonic tissues such as the yolk sac.
• The primitive endoderm is anything that isn't part of the embryo proper.
• Nanog = epiblast.
• Gata6 = hypoblast.
• The expression of Nanog and Gata6 appears random within the ICM.
• Cells sort themselves out so that all Gata6-expressing are in the hypoblast layer and vice versa for Nanog.

Tissue and Germ Layer Formation in Early Human Embryo

• epiblast turns to gastrulation turns to 3 germ layers

Human Monozygotic Twinning

• Human monozygotic twinning arises in early embryonic development when the cells split
• Timing of the split affects what structures will be shared by the twins
• Fraternal twins come from separate fertilization events; two oocytes are fertilized by two sperm and are no more genetically similar; they are siblings
• After the split, cells are able to compensate for cell reduction
• Identical twins = monozygotic twins, originating from a single fertilization
• Identicals split can occur during the blastocyst or when part of the inner cell mass, allowing formation of two separate embryos
• Timing of the splitting event determines relationship between the twins to the amnionic sac and uterus
• Timing then determines the degree of extraembryonic structure sharing amongst the twins

Human Monozygotic Twinning - Early Split

• Early Split comes before cells commit to inner cell mass and or trophoblast, allowing separate blastocysts to form
• Each blastocyst develops its own inner cell mass and its own trophoblast
• The result are twins with their own amnion and chorion.

Human Monozygotic Twinning - Intermediate Split

• Intermediate comes after inner cell mass forms and then becomes distinct epiblast and hypoblast layers determined, forming two inner cell mass clumps within the same blastocyst
• Each clump forms its own epiblast and hypoblast
• Results in twins sharing chorion, and separate amniotic sacs

Human Monozygotic Twinning - Late Split

• Late split at epiblast, results in sharing for both the chorion and the amnion

Explore the roles of Oct4, Sox2, and Nanog in embryonic stem cells. Learn about the ICM's transformation post-implantation and the derivatives of the epiblast. Understand Nanog's and Gata6's roles in cell fate decisions. Also, examine monozygotic vs. fraternal twins.