Vertebrate limb Flashcards
Vertebrate limbs
Use tetrapod limbs as model - chick and mouse
New experimental biology
Molecular biology and genetics, signalling pathways - SHH FGF
Thumb is anterior and the chick is the best model to see this
Key concepts
Patterning - embryos plan for development
Axis determination
Default state
Morphogen gradient
Tetrapod limbs
All share homologous skeletal features along proximal distal axis
Evolved from common ancestor but change for required purpose e.g. flying/swimming
Limb bud
First sign of limb growth
Form on either side embryo with forelimbs (closest to head) and hindlimbs (closest to tail)
While bud grows begins differentiating and growing skeleton
The distinct regions of the limb bud
Ectoderm
- Apical ectodermal ridge (AER) (epithelium) thickens along tip of ectoderm
Mesoderm
- Progress zone - Mesenchymal cells that below AER, as cells push out of AER they realise need to differentiate and do
- Differentiating tissues
Early experimental biologist examined AER in 1960’s
Removal AER truncates limb
Are you remove AER causes limb to truncate and stop growing
Later you take out AER, the more development you have before it stops
The progress zone requires something secreted by AER
Older AER can substitute younger AER, signal not instructing progress zone on what to make its just permissive to growth
When put older AER in place of younger AER in younger limb buds growth would return as normal
Research to find what activates AER
Researchers look for genes expressed right time place to be AER activity
Found signal molecules - FGFs (Fibroblast growth factors)
Are 22 FGFs in mice but only FGF4 and * expressed strongly at right time in overlapping domains (the area where it is having its effect) so are most likely factors activate AER
Gene knockout - mouse genetics
If knockout FGF8 then mouse has mild limb defect
If knockout FGF4 no limb defect
FGF8 and FGF4 redundancy (will need to update when gone back over lecture)
Double mutation (knockout of both FGFs) phenocopies AER ablation (lack of AER)
Genetic redundancy common in vertebrates
Redundancy - inclusion extra components not strictly necessary to functioning
FGF signalling required for limb development
Would say that FGF4 is redundant (just check this)
Experimentation FGF8 soaked beads
FGF8 soaked beads substitute for AER in dose dependent manner
More FGF8 = more limb growth
FGF8 beads rescue AER ablation but dependent on amount added
This argues isn’t something else missing that also making the AER
Ectopic expression FGF
Ectopic expression FGF in flank leads to ectopic limbs, using beads or genetic method
Experiment shows FGF8 signalling sufficient for limb development, if in right context and in flag region
Anterior to Posterior
Found if took most posterior section of donor wing bud and transplanted to anterior most section host wing bud, caused interesting digit pattern of 43234 to form
Named zone ZPA - zone of polarising activity
Another experiment with ZPA (need to update)
When ZPA removed toes developed distally but had no A/P axis - this is default state
Then repeated this but at end added APR from donor to anterior most region of bud, created normal limb with reverse A/P polarity
Could hypothesis that signal secreted by … (Need to finish)
Model - secreted morphogen (check back over this and maybe summarise diagram)
Diffusion of morphogen to establish gradient
Think it’s primarily concentration that determines if limb will or wont form
Mapping ZPA over time
Sonic hedgehog (Shh) expression correlates to ZPA
Shh is secreted ligand
Hedgehog signals first identified in Drosophila
Vertebrate hedgehog ligands given different names like Sonic
