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??