vertebrate limbs Flashcards
is your thumb anterior or posterior
anterior
is your palm dorsal or ventral
ventral
what do bodies have and what does this give us
-axis
-gives us anterior and posterior, and ventral and dorsal
what do tetrapod limbs all share
-homologous skeletal features along the proximal distal axis and
therefor probably evolved from a common ancestor
-or limb developed 2x during evolution called convolution
-tetrapod = 3 fingers (digits)
what does homologous mean
-same shape and function between different animals compared to humans (in this case)
what does early cleavage lead to
-early cleavage -> gastrulation -> somitogenesis/neurulation -> limb development
-The first sign of the limbs is the limb buds that form on either side of the embryo.
-forelimbs and hindlimb
what are the Three distinct regions in the limb bud
-Ectoderm:
=apical ectodermal ridge (epithelium)(AER)- runs along anterior to posterior axis
-Mesoderm:
=Progress zone (mesenchymal cells that are below the AER)
=Differentiating tissues
-Early experimental biologists examined the AER using surgical methods.
-distal is always furthest away from limbs
what occurs in the Removal of the AER truncates the limb
-removal of AER leads to a larger ridge and random epithelia isn’t going to work
-Model: the progress zone requires something that is secreted by the AER.
-Older AER (looks perfectly fine and works fine) can substitute for younger AER (AER telling part of the limb to be made by the progression zone):
The signal is not instructing the progress zone on what to make, rather it is permissive to growth
what are FGFs
-signal molecules - genes that are expressed at the right time and place to be the AER activity
-Fgf8 expression (RNA in situ hybridisation) - good candidate for proliferation signal
-There are 22 Fgfs (many different versions) in mice, some have been shown to direct cells to proliferate. Fgf8 and Fgf4 are expressed strongly, at the right time and in overlapping expression domains
what does FGF stand for
fibroblast growth factor
Mouse genetics – gene Knock-outs
-Fgf8 mutation has a mild limb defect
-And, the Fgf4 mutant has a no limb defect
what happens when Fgf8 and Fgf4 are redundant
-FGF signalling is required for limb development.
The double mutant phenocopies (looks like) AER ablation.
Genetic redundancy is common in vertebrates.
-FGF I signalling therefore required for limb development
what does redundancy mean
-the inclusion of extra components which are not strictly necessary to functioning
what can FGF8 soaked beads (protein) substitute for
-the the AER in a dose dependent manner
-This suggests that the AER is only required as a source of FGF.
FGF8 soaked beads rescue AER ablation.
-implant the beads into embryo. AER can be removed therefore you get truncated limb
-more beads= more respiration of the limbs
what does Ectopic expression of FGF in the flank lead to
-ectopic limbs (using beads or genetic methods) (expressing something in the wrong place
-FGF signalling is sufficient for limb development
-(Note FGF signalling has many different roles – not just in limb development)
whats ZPA
-Zone of Polarizing Activity
-(patterning the identities)
-look at slide 14
-donor wing bud and host wing bud
-donor embryo moved to recipient
-taking tissues and moving it around to polarising tissue
whats the hypothesis for ZPA signalling
-a signal is secreted by the ZPA to to give the A/P axis polarity
-investigated ZPA - is it part of the ectoderm or underlying mesoderm
-cut off limb bud from embryo
-removed mesoderm tissue and separated cells
-mix them up
-centrifuge them
-put them back into embryo
-loose ability to polarise
new donor put into anterior part of the limb
-slide 15
whats secreted morphogen model
-Diffusion of the morphogen to establish a gradient
-lowest concentration of morphogen gives us 2 digits (limb)
-additional polarising region grafted to anterior margin gives us a mirror image of gradient regarding concentration of morphogen
-small number of polarising region cells grafted to anterior margin= curve decreases then slightly increases (conc of morhphogen)
what does Mapping the ZPA over time look like
-Sonic Hedgehog (Shh) expression correlates to the ZPA
-Shh is a secreted ligand. Hedgehog signals were first identified in Drosophila. When vertebrate Hedgehog ligands were identified, they were given different names – like Sonic Hedgehog.
-ZPA moves along distal part of limb
whats Shh required for
-A/P axis
-Shh-/- mouse mutant phenocopies (looks like) removal of ZPA, no redundancy for Shh-/- and formed a truncated limb
-Also: Shh soaked beads induce posterior cell fates in a dosage dependent manner
-(Note SHH signalling has many different roles – not just in limb development)
Proximal to Distal Axis
-AER is required for outgrowth and
=surgical removal results in loss of distal structures.
-FGF’s mimic the effect of the AER and are a limb organizer
=expressed in the right place
=beads rescue the loss of the AER
=double mutants loose limbs
=beads can induce ectopic limbs (sufficient)
Anterior to posterior patterning
-ZPA is required for posterior fates and polarizes the limb.
=surgical removal results in loss of posterior structures.
-Shh mimics the effect of the ZPA
=expressed in the right place
=beads rescue the loss of the ZPA
=beads can induce reversed polarity in limbs
=mutant loses posterior limb structures
-Shh forms a gradient that acts as a morphogen
