Developmental Biology 9

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Embryonic Chick Limb Development

Back 1

Limb bud: small protrusions from the body wall that eventually form the limbs
Appear 3 days after egg is laid

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Embryonic Chick Wing

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Limbs well developed 10 days after egg laid
Cartilage at this stage: later ossified to form bone
Feather buds are also visible

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Limb Bud Development

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Limb bud is composed of Loose mesenchymal cells derived from the lateral plate mesoderm: form the skeletal and connective tissue elements, outer layer of ectodermal epithelial cells
Apical epidermal ridge (AER)
Progress zone
Condensation

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Apical Ectodermal Ridge (AER)

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Closely packed columnar epithelial cells
Allows flattened shape of the bud to be maintained
Its length determines the width of the bud

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Limb Development

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Progression from proximal to distal
AER disapears after all basic elements in place
Followed by a growth phase

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Limb Location is Determined Early

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The body plan is determined at the neurula stage
Occurs before any visible sign of differentiation
Limb regions may be specified by Hox genes
Pitx1 specifies hind limbs
Considerable capacity for regulation (pre-limb bud and limb bud)

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Two Signaling Regions in the Limb Bud

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The zone of polarizing activity (ZPA) is located as the posterior margin: Specifies position along the A/P axis
The AER at the tip of the limb bud: Required for limb outgrowth

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The AER is required for Limb Outgrowth

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Essential for out growth and proximal-distal patterning
Due to its role in inducing and maintaining the progress zone
Cells from the progress zone produce the initial outgrowth of the limb bud
The progress zone may be the location where cells acquire their positional information

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The AER is Required for P/D Development

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Removal of the AER leads to truncation of the limb
The limb develops in a proximal to distal sequence
Following removal cell proliferation in the progress zone is greatly reduced and cell death is observed

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AER Signaling

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Grafting experiment: AER causes ectopic outgrowth
FGFs are the AER signaling molecules: FGF-8 is throughout the AER, FGF-4 is in the posterior AER, Both FGFs can mimic the action of the AER

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Signaling b/w the Limb Bud Centers

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THe AER, ZPA and progress zone are mutually dependent on one another
Sonic hedgehog (shh) is expressed in the ZPA
Part of a positive feedback loop b/w the AER and ZPA

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FGF-4 Application Induces a New Limb

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FGF-4 application initiates the formation of a new AER and FGF-8 from the ectoderm
FGFs stimulate Shh production
Shh in turn stimulates host embryo's FGF-4 expression

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ZPA

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Specifies position along the A/P Axis

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Shh is a Candidate Morphogen from the ZPA

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Shh is produced by the ZPA cells Fibroblasts producing Shh can act like ZPA cells
Beads coated with Shh also mimic ZPA cells: the concentration of Shh determines the pattern of digits that form

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Hx is a Mouse Shh Mutant

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Hx is a single point mutation in the Shh enhancer region
Mutation results in ectopic expression of Shh
This results in the formation of extra digits: polydactyly

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Mutations Similar to Hx Occur in Humans

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Human mutations occur in a similar Shh enhancer region
Also results in the formation of extra digits: polydactyly

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Shh and Digit Number

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Shh signaling is involved
GII3 is the transcription factor
Shh inactivated: GII3 repressor does not allow digit formation
Shh + GII3 inactivated: many digits form, but there is no A/P polarity
There must be some underlying mechanism that sets up digit formation

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Shh and Digit Identity

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Shh may be acting as a morphogen
BMPs are also involved (downstream of Shh?))
The BMPs appear to act in the regions between the digits
Digit identities can be altered by: removal of this interdigital region, altering BMP concentrations in this region

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Proximo-Distal Axis Specification

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P/D axis specification may be determined by a timing mechanism
As the limb grows cells leave the progress zone (PZ)
Longer they remain in the PZ the more "distal" they become
Cells given a positional value

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The D/V Axis is Controlled by the Ectoderm

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Limbs have obvious D/V patterning
Reconstituted chick limb bud experiments: Rotate the ectoderm 180 so dorsal and ventral are switched relative to the internal mesenchyme
Resulting limb has distal regions inverted (proximal normal), Indicates ectoderm specifies dorso-ventral patterning

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The D/V axis is Controlled by the Ectoderm: Mouse genes

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WNt-7a is expressed in the dorsal ectoderm: Inactivation causes double ventral limb, the two halves are mirror images
Engrailed is expressed in the ventral ectoderm: Inactivation causes double dorsal limb

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Limb Cells Interpret Positional Signals

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Positional signals in the wing and leg bud appear to be the same
This is revealed by grafting experiments: ZPA from wing bud onto a leg bud: extra toes not wing digits
Positional signals are conserved between vertebrates: Mouse AER grafted in place of chick AER: almost normal development
Signals are interpreted according t othe origin of the responding cells

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Limb Cells Interpret Positional Signals

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Take proximal cells from the leg bud that normally would develop into thigh
Graft them to the distal region of the wing bud
Result is toes instead of wing digits in the developed limb

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Hox Genes in Limb Patterning

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Thought to record positional values with the limbs
Hoxa and Hoxd cluster genes are both expressed in forelimbs and hind limbs
Expressed in a nested pattern in the A/P axis
Pattern corresponds to the three main limb regions

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Hox Genes in Limb Patterning: PZA

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Deletion of all genes in the Hoxa and Hoxd clusters: Severe limb truncations: likely due to the absence of Shh expression
PZA graft experiment causes a change in Hox gene expression

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More 5' Hox genes Control More Distal Regions of the Limb

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Triple Mutants made for each paralogous group of Hox genes
The more 5' genes (hox13) affect the distal portion of the limb
The more 3" genes (Hox9) affect the proximal portion of the limb

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Mutation in the Human Hoxd13 Gene

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Results in fusions of some of the fingers
Mutations in other Hox genes can result in polydactyly
currently no explanation for these particular phenotypes