lecture 2: early development

Question 1

What occurs between fertilisation and implantation in the development of the mammalian embryo?

Answer 1

• the egg is ovulated (shed from its layer of cells sitting in a follicle) into the fallopian tube/oviduct
• gap between oviduct and ovary can lead to ectopic pregnancy
• egg is fertilised in the oviduct
• finishes meiosis at fertilisation
• travels down the oviduct
• changes:
  
  • spindle formation
  • cell division
  • 2 cells, 4 cells, 8 cells etc
  • morula
  • blastocyst
• implants into the uterine wall
• endometrium and myometrium
• embryo implants into the glandular portion of the uterus
• trophoblast will invade the uterine tissue to start creating placenta etc
• pre implantation and post implantion: important concept because different things happen at different times

mouse pre-implantation development

• zona pellucida: protein coat that protects the embryo as it is travelling down the oviduct and confers species specificity
• hardens once egg is fertlised to restrict the ability of other sperm to bind to that zona pellucida
• when it reaches the uterus it must hatch out of that zona pellucida
• believed that ectopic pregnancy might occur if hatching process happens too early
• size of the embryo is not changing - cleavage occurs without cell growth untl the blastocyst change
• blastocyst starts to grow and expand
• creates blastocoel cavity
• inside and outside cells
• trophectoderm (outside cells)
  
  • before implantation
  • after implantation = trophoblast
• inside cells become embryo proper
• 64 cells
  
  • 8 - 10 cells make up ICM
  • rest became placenta
• 8 cell stage cells start to compact → morula

1. fertilised egg with two nuclei
2. division to 8 cells with minimal contact
3. totipotent up to this stage
4. compaction → sides of cells closer together → polarised
   
   • when they divide some are on the outside and some on the inside required to form the embryo
5. 16 cell morula
6. 32 cell morula
7. cells getting smaller, embryo not getting bigger
8. at the 16 cell stage the outer cells are still somewhat similar to the inner cells
9. at 32 cell the outer cells have separate identity
10. cavitation = formation of blastocoel cavity
11. 64 cell stage = early blastocyst
    
    • two cell lineages
    • trophectoderm and inner cell mass
12. > 100 cell late blastocyst
    
    • three cell lines
    • trophectoderm, epiblast and hypoblast
    • from these two stages that we can select ICM cells and create ES cells
    • late blastocyst is the best stage for creating ES cells

Question 2

What occurs post-implantation in mammalian embryonic development?

Answer 2

• trophectoderm (trophoblast) expands and invade into uterine wall to create the placenta, anchor the embryo, similar to the invasive process of metastatic cancer
• rest of the embryo is changing shape
• outer layer of hypoblast, inner epiblast - form egg cylinder
• forms cavity
• perietal endoderm
• embryo starts to undergo gastrulation

Question 3

What is gastrulation?

Answer 3

• embryo undergoes a radical rearrangement of its cells
• sets aside its germ cells and forms:
  
  • primary germ layers (ectoderm, mesoderm, endoderm)
  • axes (anterior-posterior, dorsal-ventral, left-right)
• gastrulation is critical for development
• at the egg cylinder stage ICM/epiblast are still pluripotent
• during gastrulation cells travel across the epiblast to the primitive streak and then travel around the embryo
• travelling ‘down’ (according to the picture) the primitive streak they form the head region, end is the tail (A-P axis), L-R also established and front and back

Question 4

What are the derivatives of the three primary germ layers?

Answer 4

• ectoderm
  
  • skin cells of epidermis
  • neuron of brain
  • pigment cell
• endoderm
  
  • pancreatic cell
  • thyroid cell
  • lung cell (alveolar cell)
• mesoderm
  
  • cardiac muscle
  • skeletal muscle cells
  • tubule cell of the kidney
  • red blood cells
  • smooth muscle (in gut)

Question 5

During gastrulation, where are the germ cells?

Answer 5

• primordial germ cells are a derivative of the epiblast and they avoid signals that direct them to adopt a particular cell fate, like becoming ectoderm for example, by being sent outside the gastrulating embryo
• region just outside of the embryo, posterior end of the primitive streak

Question 6

What are the germ layer origins?

Answer 6

Question 7

Where and what types of stem cells are available from the early embryo?

Answer 7

• ES cells from ICM
  
  • day 3.5 and 4.5
• Epiblast like SC, from pre and post implantation
  
  • 4.5 and 5.5
• hypoblast (PE) (XEN SC)
• 4.5
• trophoblast stem cells (TS SC)
• 4.5, 5.5
• studied in various labs for different reasons
• ability to derive these stem cell types confirms pluripotent potential of early embryo

Question 8

What occurs during compaction?

Answer 8

• E-cadherin, tight junction and gap junction protein expression initiated
• compaction critically important for formation of first two cell types
• outer cells form an epithelial layer with apical-basal polarity
• formation of external epithelial layer:
  
  • cell can divide asymmetrically (2) or symmetrically (1)
  • symmetrical division leads to two out cells (apical-basal divisino)
  • asymmetric division leads to an oter and an inner cell (at right angles to A-B plane)

Question 9

How do we get segregation of TE and ICM?

Answer 9

• outer cells of morula stages are polarised (have A-B polarity)
• from 8 cells, Cdx2 and Oct4 co-expressed
• 16 cell stage they are still fluid, not confirmed in their identity, can change (expressing both transcription factors)
• around 32 cell stage (and Key transcription factors differentially expressed):
  
  • Inner cells become ICM (Oct 4: downregulates Cdx2)
  • outer cells become TE (Cdx2: downregulates Oct4)

Question 10

How do we get segregation of ICM into Epiblast and Hypoblast?

Answer 10

• in morula stages, Nanog and Gata 6 co-expressed in inner cells
• by E3.5, due to mutual inhibition of transcription factors:
  
  • nanog expressed in epiblast and downregulates Gata 6
  • Gata 6 expressed in hypoblast and downregulates nanog
• current model suggests that ICM cells are randomly specified as epiblast or hypoblast, then sort into respective layers

Question 11

What is a model of first lineage formation and role of key transcription factors (in vivo)?

Answer 11

• once we form the morula we are capable of forming inner and outer cells by mutual inhibition of Cdx2 and Oct4
• ICM: mutual inhibition of gata6 and nanog allow formation of hypoblast and epiblast
• so mutual inhibition of key transcription factors lead to formation of first three lineages

Question 12

What is the expression of Oct 4 during pre-implantation development in the mouse?

Answer 12

• Oct4: maternal mRNA – zygote
• Oct4: zygotic mRNA ~ 2 cell
• 8 cell stage – all cells presumed totipotent
• post-gastrulation Oct4 expressed only in germ cells of foetus and adult
• all of early preimplantation and post implantation right through to epiblast
• then only expressed in primordial germ cells
• pre morula it is a marker of totipotency, post morula a marker of pluripotency