Chick - gastrulation

Question 1

Explain the story of gastrulation in the chick in terms of gene expression and its relation to cell lineages and movements.

Answer 1

• Cell movements through organiser must be accompanied by changes in gene expression
  
  • We know this as gene expression of goosecoid remains in the node but cells are moving in and out.
  • therefore at least in early embryos, gene expression patterns are not indications of cell lineage history but rather of the current position of the cells
• Are the early goosecoid expressing cells (in the blastoderm) precursors for late goosecoid expressing cells?
  
  • Labelled early goosecoid expressing with DiI. Then incubate the embryo to later stage. The fix the embryos and photoconvert the dye and do ISH for gsc. ​
  • There is one population of cells (brown from DiI and blue from ISH) therefore they are precursors.
• Do goosecoid expressing cells induce other cells to express goosecoid?
  
  • Transplant of goosecoid expressing cells from quail donor. Then ISH with chick-specific gsc probe.
  • Was shown to induce goosecoid in neighbouring chick cells
• Cells from two different sources contribute to Hensen’s node.
  
  • It was shown that overlying epiblast cells are induced to express goosecoid. “Polonnaise movements”

Question 2

Explain the story of gastrulation in terms of cell movements and what drives them.

Answer 2

• (Cells from the epiblast at the primitive streak undergo an epithelial to mesenchymal transition and ingress at the primitive streak to form the germ layers. The primitive streak is formed at the beginning of gastrulation and is found at the junction between the extraembryonic tissue and the epiblast on the posterior side of the embryo and the site of ingression)
• By using 2 photo microcopy a 3D image of the cell movements has been mapped.
• The combination of posterior midpoint convergence and midline extension from the epiblast resembles a Polish dance
  *

Question 3

What argues that the default model is too simplistic? ERNI, BERT AND GEMININ

Answer 3

• Missexpression of BMP antagonists do not form a complete neural axis
• When organiser is grafted for 5 hours (not sufficient time to induce a nervous system) and then remove and replaced by Chordin-secreating cells the cells maintain an early neural marker, Sox3.
  
  • This finding suggests that signals from the organiser other than BMP antagonists are requird for cells to become responsive to BMP antagonists.
  • However, even more steps must be involved because even after carrying out this process late neural markers, Sox2, are not expressed.
• What happens at the 5 hour time point?
• Did a differential screen using cDNA libraries from single cells exposed and not exposed to grafted organiser for 5 hours
  
  • ERNI and Churchill were identified
• ERNI transiently expressed in cells destined to become nerual plate (<2hrs)
  
  • FGF8 induces ERNI and is expressed in precursors of hensens node and hensens node itself
  • ERNI mutant induces sox2
  • Mammalian 2-hybrid system found ERNI to dimerises
    
    • ERNI inhibits Sox2 expression by binding to Geminin
• Cytotrap 2-hybrid system (a modified version - with increased efficacy)
  
  • Identified BERT
  • BERT induces Sox2
• Bimolecular fluroescence complementation
  
  • ​(It is based on the association of fluorescent protein fragments that are attached to components of the same macromolecular complex.)
  • Showed that all three proteins interact with each other
• Interactions between three coiled-coil domains, ERNI, Gemini and BERT control the N2 enhancer
  
  • N2 enhancer is the earliest enhancer of Sox2
  • Competitive interactions between these three proteins modulate repressors and regulate Sox2 expression
• Conclusions:
  
  • Timing of gene/protein activity is important
  • A function of ERNI may be to delay the induction of Sox2 while gastrulation takes place: cells expressing ERNI are multipotent
    *

Question 4

What are argues the default model is too simplistic? CHURCHILL

Answer 4

• But FGFs have many functions in development. Even at this early stage, they are involved in mesoderm induction as well as in the initiation of neural induction, events that happen almost simultaneously at the beginning of gastrulation yet their outcomes are incompatible with one another (a cell cannot become both mesoderm and neural). How do cells decide how to respond to these signals? Answer churchill…
• Churchill was another protein found in the differential screen
  
  • It is a zinc-finger transcriptional activator
  • Expressed in late stages of gastrulation
  • Target Sip1 than in turns inhibits brachury that is required for mesoderm formation
  • It in inhibits ingression through the primitive streak
  • Also induced by FGF8 but later
  • Churchill seems to act as a delaying mechanism, separating two functions of FGF: an early requirement for mesoderm induction and a slightly later role in initiating neural plate development

Question 5

Summary of neural induction chick

Answer 5

• We propose that ERNI functions as an inhibitor of premature Sox2 expression during early gastrulation: cells expressing ERNI are multipotent and can generate any cell type. Cells that remain in the epiblast at the end of gastrulation and acquire expression of BERT to activate Sox2, which, most likely together with other genes involved in neural specification, assigns a neural plate fate.

1. Early pre-neuronal state:
   
   1. FGF induces Gem and ERNI
   2. N2<–Brm–>Gem<–ERNI–>HP1-gamma–|Sox2
2. Commitment to neuronal plate:
   
   1. BERT is induced (unknown what induces)
   2. BERT bind ERNI and Geminin allowing for Sox2 expression

Question 6

Gastrulation.

Answer 6

1. Prior to gastrulation future “dorsal” tissue are found just central to and adjacent Koller’s sickle
2. The terrioties move towards the centre of the blastoderm and gradually become replaced by more lateral regions of epiblast
3. These movements comprise convergence of epiblast towards the posterior mid-point and extension along the midline
4. but do not seem to occur by the process normally called “convergent extension” in that it is not accompanied by significant cell shape changes.
5. The combination of posterior midpoint convergence and midline extension resembles a Polish dance (“Polonaise”)
6. Intercalating movement of cells have been attributed to Non-canonical Wnt signalling (PCP)
7. Computer models have been generated to explain the epiblast movements,
   
   1. due to tension under tight epithelium, cells have a preferred direction of intercalation in the streak forming region

• Spratt chopped embryo into 4 sections - each one made an embryonic axis. Therefore, gravity and localized maternal components can, at best, only bias polarity but do not act as definitive determinants.
• The fact that isolated fragments of blastodiscs can spontaneously initiate axis formation indicates that cell interactions, rather than definitive determinants, must be involved. What are the signals, and where do they come from? Three main sources have been proposed: the hypoblast and/or endoblast, Koller’s sickle and the posterior marginal zone (PMZ).
• it was shown that complete removal of the hypoblast leads to the formation of multiple streaks at random positions, suggesting that the hypoblast emits an antagonist of axis formation.
• Analysis of expression patterns and misexpression experiments then suggested that Cerberus, a Nodal antagonist, is responsible.
• Cerberus is expressed in the hypoblast but not in the endoblast, which is consistent with the fact that the primitive streak starts to form precisely at the time when the hypoblast is displaced away from the posterior edge of the area pellucida by the incoming endoblatst
• PMZ
  
  • cVg1 is expressed in PMZ
  • misexpression of cVg1 in marginal zone induces new axis
  • Only in the marginal zone can a new axis be formed. why?
  • Wnt8c is expressed in marginal zone
    
    • inhibiting Wnt stops axis fomation
    • therefore, Wnt activity defines Marginal Zone
  • Vg1 and Wnt induce Nodal
• Hypoblast
  
  • Expresses cerebrus and crescent
  • Cerebrus inhibits nodal, bmp and wnt
  • crescent inhibits wnt
  • removal of hypoblast generates many streaks

Question 7

Conclusion of gastrulation

Answer 7

In conclusion, all three tissues (hypoblast/endoblast, Koller’s sickle and PMZ) proposed to have axis inducing activity do indeed have the ability to influence primitive streak formation. However, their mechanisms of action and relative importance differ. The sickle has inducing ability but this is probably only because it contains some of the cells fated to form Hensen’s node (the avian organizer – see below). The earliest influences appear to come from the PMZ, where Vg1 and Wnt activities overlap. Vg1+Wnt induce expression of Nodal in the neighboring area pellucida epiblast, but Nodal can only act (presumably to induce mesendoderm) when the hypoblast has been displaced by the incoming endoblast.