Chick - General

Question 1

Why is chick superior for temporal based experiments?

Answer 1

• Best staging system of any of the model systems described by Hamilton and Hamburger
• Classically could not control timing of genetic manipulation in frog and fish

Question 2

What are the techniques used in classic chick embryology?

Answer 2

• Embryological techniques
  
  • Structure/ ultrastructure (optical and EM level)
  • Natural mutants
  • Gene expression studies (ISH and antibody)
  • Fate mapping/ lineage analysis with
    
    • fluorescent dyes (single cell and groups of cells)
    • Chick-quail chimeras
  • Time-lapse filming to look at cell movement
  • Transplantation to study cell interactions
• Molecular techniques
  
  • Beads with chemical or protein factors
  • Retrovirus mediated gene transfer (for lineage analysis and misexpression)

Question 3

What are the new techniques used in the chick?

Answer 3

• Electorporation and Sonoporation
  
  • DNA, morpholinos, siRNA (gain and loss of function)
• Lentivirus mediated transgenic chicks
• Embryonic stem cells
  
  • (including homologous recombination)
• Culture Primordial Germ Cells (PGcs)
  
  • (including transgenesis)
• Chick genome now available
• Promoter studies
  
  • (identification of conserved studies and electoporation of reporters)
• Positional cloning of genes underlying “old” mutants
• FISH for gene mapping

Question 4

Describe the limb bud

Answer 4

• Consists of mesenchyme surrounded by ectodermal cells
• Transplantation of mesenchymal cells from posterior (ZPA) to anterior
  
  • duplication of limb/limb digits
  • earlier transplantation gave rise to more proximal duplication whereas later transplantation lead to distal duplication (i.e forelimb of digits)

Question 5

What is a morphogen?

Answer 5

A morphogen is a compound whose CONCENTRATION determines cell fates. It must have have at least 2 thresholds (i.e specify at least 3 fates). The French flag model represents the activity of a morphogen.

(if there is just one threshold then it is simply an inducer)

Question 6

What is the ZPA? And what found by altering the distance of the second ZPA graft?

Answer 6

1. The zone of polarising activity established the A-P axis of the limb bud. It acts as if emitting a morphogen.
2. When the distance from the second ZPA is closer to the first then the morphogen concentration is increased and only one threshold is achieved (Only the most posterior digit is specified)

Question 7

What is the ZPA morphogen?

Question 8

What is the AER?

Answer 8

• Distal apical ectodermal ridge of the limb bud
• When removed surgically removed:
  
  • ​Limb formation is defective
  • Time of removal determines where the defective will occur (i.e Limb, forelimb or digits)
• A slow release FGF bead implanted where the AER is removed
  
  • ​Compensates for the loss of the AER

Question 9

What happens when a FGF bead is implanted in the flank?

Answer 9

• A limb will be formed. Forelimb or hindlimb depending on the anterior-posteior postioning of the bead.
• Therefore, FGF also controls limb bud formation (initation)

Question 10

What

Question 11

Explain proximal-distal patterning of the limb bud.

Answer 11

• It is determined by the progress zone
• A group of dividing stem cells below the AER are continually proliferating and move distally
• Cell that are left behind differentiate

Question 12

Explain non-equivalence in the limb bud.

Answer 12

• Transplantation of proximal mesenchymal cells from the hindlimb to the forelimb just under the AER
  
  • ​Hindlimb tissue is formed but it is of distal nature.
  • This showed that distal proximal commitment occurs after forelimb or hindlimb commitment.

Question 13

Explain dorsal-ventral patterning of the limb bud

Answer 13

Barely goes into detail. Papers in your reading list…

Question 14

Describe the story of the somites and PNS development. Cellular and molecular.

Answer 14

• Somites formation from anterior to posterior
• Chick makes roughly 65 pairs of somites
• Cellular
  
  • Antibody staining found that motor nerves (and sensory) only in rostral half of somite
    
    • Is this property of the somites of the NS?
  • Transplantation of rotated neural tube into host
    
    • ​The paterns of axons was normal
  • Transplantation of roated somites
    
    • ​The patterns of axons was defective. This showed that segmentation of the nervous system is down to the somites and not the NS.
• Molecular
  
  • Peanut agglutin binding to caudal half of somite. What protein is the PNA binding to?
  • collapse assay. Homoegenise somites and then apply homegenate to axons causes collapse
  • PNA chromatography regonised 2 protein bands
  • Stain somites form embryos at various stages and found three expression patterns of hairy-1 (a member of the Notch signalling pathway)
    
    • It was found that there was a 90min expression pattern.
    • A mathematical model, the segmentation clock, was proposed describing the wave of expression
  • A transgenic mouse with a luminescent reporter confirmed the expression pattern of the segmentation clock
  • As cell enters the posterior end of the embryo it experiences 10-12 waves of expression before forming a somite
  • A more complicated “clock and wavefront model” was proposed.
  • HOWEVER, transplantation of the node and posterior primitive streak to the lateral plate mesoderm
    
    • ​the node can dorsalise posterior primitive streak. Transfroms lateral plate mesoderm into somites
  • Transplant of posterior primitive streak with Noggin-releasing cells
    
    • ​This mimiced the effects of the node
  • The graft somites looked morphological the same as normal somites
  • Transplantation of noggin-secreating-cell-somites into host somites
    
    • ​graft somites were fully incorporated and could from all derivatives.
  • A GFP transgenic chick doner of somites
    
    • ​Showed that somites did not form sequentially
  • Grafted somites were different from normal somites in one way - they lacked the subdivision between caudal and rostral halves. ISH showed they lacked the caudal somite markers, hairy1 and hair2.
    
    • This suggested that the clockwave mechanism does not determine size or shape of somites but the rostral caudal division.
  • Taken together, the hypothesis for what causes somite size and shape is local cell-cell interactions, e.g. constraints to cell packing

Question 15

Describe the development of Left-right asymmetry in the chick.

Answer 15

• An earlier hypothesis of what determined left-right asymmetry was the description of an F shaped molecule that was found in every cell in the early embryo
• Serendipitous discovery of asymmetric (left) SHH discovery in Hensons node
  
  • It was also found that the Activin receptor IIA is expressed on the right
  • These were the first asymmetric genes discovered!
• Then the discovery of left asymmetric expression of Nodal in the lateral plate mesoderm and node.
• Activin soaked bead implanted on the left
  
  • ​made expression of its receptor double and inhibited SHH expression
• SHH soacked bead implanted on the right
  
  • ​causes over expression of Nodal
• Epistatic analysis of other asymmetrically expressed genes
  
  • ​Pitx2, Cerebrus, Fgf8
• inversus viscerum mutant mouse
  
  • mutated gene encodes a cytoskeletal motor protein involved in monocilia
  • The mouse node contains monocilia with a similiar structure to originally propsed F shaped molecule!
• Forcing fluid flow from left to right in WT mice
  
  • reverses asymmetry
• Mutant mice placed in an artifical flow
  
  • ​Rescue asymmetric phenotype
• Other theories a proton-potassium pump that is asymmetrically distrubuted
• There is a lot of differences between the model systems. However they all converge on Nodal and Pitx2.
• This field of research is in its infancy - ongoing!