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Development of the Tetrapod
Limb - Placement on the Axis,
Forelimb Vs. Hindlimb
Gilbert - Chapter 16
Today’s Goals
• Become familiar with several aspects of limb
formation in the tetrapod
– Limb initiation
• Forelimb vs. hindlimb
• Where to make a limb?
• Examine molecules involved in specification of the above
– Limb patterning
• Dorsal/ventral
• Anterior/Posterior
• Proximal/distal
Position of the
tetrapod limb is
conserved:
Forelimb always
at cervical to
thoracic
transition
Hindlimb always
at lumbosacral
transition
Evidence that Limb field is
Specified
• If remove presumptive limb field, no
limb will form
• If move limb field tissue to new axial
level (flank), a limb will form in the flank
• Fate mapping presumptive limb field
shows those cells participate in limb
formation
Hox Genes and Limb Field
Specification
• Different Hox genes are expressed at
different points along the A-P axis
– “Hox code”
• Expression of certain Hox genes maps to the
level of limb formation
• Conserved throughout the tetrapod
organisms
• Thought to be important for establishing limb
field, level of limb field
What about snakes?
In snakes
• Radical change to the body plan
• No Limbs!
– Lost them through evolution
– First the forelimb and then the hindlimb!
– Fossils have been found of snakes w/ hindlimb,
but no forelimb!
– Primitive snakes develop a rudimentary femur
(pythons)
• Don’t continue to form (no SHH to extend limb - we’ll see
more later. . . )
– Can be explained by Hox expression
• Hox C6 and C8 expressed through most of body -> ribs
throughout
From Specification to
Induction
• Once limb field is set-up, how does the embryo
begin to form a limb?
– Mesenchyme cells in somatopleure LP proliferate
– Myotome from somite proliferates
– These 2 cell types form a bulge or limb bud
• Cells in limb field were specified from A-P
signals
– Now must signal locally to form this swelling
Primaxial
Abaxial
Limb Bud Formation
• Lateral plate mesoderm cells
– Secrete FGF-10 (fibroblast growth factor)
– Appears to be important for limb bud
formation
– HOW CAN WE TEST THIS??
• Is it in the right place?
• Is it sufficient to form a limb? (Over-express)
• Is it necessary to form a limb? (Knockout)
FGF-10 and Limb bud
formation
• FGF-10 is expressed in the lateral plate
mesoderm at the presumptive limb field
regions (correlative evidence)
• Ectopic expression of FGF-10 results in
ectopic limb formation (gain-of-function
evidence)
• FGF-10 null mutant mice (Knockouts) do not
form limb outgrowths (loss-of-function
evidence)
Forelimb or Hindlimb?
• Depending on placement of the ectopic
FGF-10 protein in the flank either
forelimb or hindlimb will form
– If placed closest to forelimb, ectopic
forelimb
– If placed closest to hindlimb, ectopic
hindlimb
– If place directly at midpoint, chimeric
fore/hindlimb forms
Specifying Forelimbs and
Hindlimbs
• Members of a family of transcription factors
related to T(Brachyury) called T-box genes
have been implicated
– Tbx-4, Tbx-5
– Transcription factors
• Initially, expression data showed Tbx-5
expressed only in mouse forelimb, Tbx-4 only
in mouse hindlimb
– CORRELATIVE evidence
Tbx-5 Loss of Function
• Humans with heterozygous for TBX5
have upper limb abnormalities
– Not completely show support for upper
limb specification
– Not turn upper limb into a lower limb
– Good enough evidence to continue to
pursue these genes
Tbx genes: Gain-of-Function
evidence
• FGF-bead experiments. If FGF placed:
– Closest to forelimb
• Ectopic forelimb formed
• Tbx-5 expression induced in new bud
– Closest to hindlimb
• Ectopic hindlimb formed
• Tbx-4 expression induced in new bud
– Between fore and hindlimb
• Ectopic chimeric fore/hindlimb
• Anterior 1/2 expresses Tbx-5, posterior 1/2
expression Tbx-4
Tbx-5
Tbx-4
Viral Misexpression of Chick
Proteins
• In Chick embryos, avian viruses can be
engineered to express a gene of interest
• The embryo can then be injected with the
virus at an early stage
• As virus spreads through the developing
tissue, gene expression is turned on
• In this way we can express genes ectopically
in chick tissue
– Like a gain of function mutation in a mouse, but
limited to a specific cell population
Viral misexpression of Tbx
proteins in chick
• Tbx-4 misexpression throughout the flank of
the embryo
– Then an FGF bead is placed in flank
• Results in formation of mostly ectopic hindlimbs
• This further supports Tbx genes in hindlimb
vs. forelimb identity
– NOT YET PROVEN!
• BUT: it is necessary to establish the
expression of Tbx genes
– This occurs further upstream: ??HOX??