1. Development of the Tetrapod Limb - Placement on the Axis, Forelimb Vs
Development of the Tetrapod Limb - Placement on the Axis, Forelimb Vs. Hindlimb
Gilbert - Chapter 16

2. 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

3. Position of the tetrapod limb is conserved:
Forelimb always at cervical to thoracic transition
Hindlimb always at lumbosacral transition

4. 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

6. 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

8. What about snakes?

9. 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

11. Retinoic Acid and Limb Field Specification
Vitamin A derivative
Similar to Accutane (Isotretoin)
Affects Hox gene expression
Not just a teratogen!
Normally expressed in an anterior to posterior gradient along embryo
Blocking normal retinoic acid function in embryo
Prevents limb bud initiation

12. 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

13. Primaxial
Abaxial

14. 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)

15. 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)

17. 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

20. 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

21. 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

22. 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

23. Tbx-5
Tbx-4

25. 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

26. 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??