Limb Patterning: Proximal-Distal Axis

Gilbert - Chapter 16

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Today’s Goals

• Become familiar with several aspects of limb formation in the tetrapod– Limb initiation

• Forelimb vs. hindlimb • Where to make a limb?

– Limb patterning• Dorsal/ventral• Anterior/Posterior• Proximal/distal

Generating Limb Axes

• Classical embryonic manipulations from the dating from the 1940’s– John Saunders– Series of surgical rearrangements that later

became the basis for what we know about molecular signals in limb formation

– He identified specialized areas in the limb that were necessary for development of the different axes

John Saunders

Proximal Distal Axis

• Growth along P-D axis made possible by interaction between 2 tissues

• Apical ectodermal ridge (AER)– Thickening of ectoderm at distal tip of limb bud– Very important for several aspects of limb

formation

• Progress zone (PZ)– Mesenchyme directly underneath AER– Proliferates to lengthen the limb bud

PZ

Experimental Manipulations

• Remove AER at any time– Distal limb development ceases

• Graft extra AER to existing limb– Extra distal structures form

• CONCLUDE: – AER is necessary and sufficient for distal

limb formation

Removal of AER at different times: loss of various structures

More Experimental Manipulations

• Remove PZ from wing, replace it with leg PZ– Toes form at distal tip of wing!

• Remove PZ from wing, replace with mesenchyme from non-limb– Limb development ceases, AER disappears

• CONCLUDE:– PZ involved in specifying limb type– AER dependent on PZ to continue developing

Overall conclusions: AER, PZ

• AER required for distal limb development

• AER keeps PZ in a proliferative state, and PZ sustains AER (feedback loop)

• What molecules are involved in this interaction??

FGFs and the AER

• FGFs, including FGF10, FGF8– Important in formation, sustaining the AER

• FGF10 expression in the mesenchyme of the forming limb bud induces formation of AER in the overlying ectoderm– This occurs at the distal tip of the limb– FGF10 induces expression of FGF8 in AER– FGF8 is secreted from AER, signals to PZ to keep

dividing– FGF8 acts in feedback loop to instruct PZ to

maintain FGF10 expression

FGF8 expression

FGFs and The AER

• If the AER is removed from the developing limb– Normally, development of distal limb

ceases– BUT - if remove AER and put in a bead

coated in FGF• NORMAL DEVELOPMENT OF LIMB!

How are cells specified with regard to P-D axis?

• The AER and PZ allow for limb outgrowth, but how do proximal cells know to form humerus, distal cells know to form digits?

• 2 Models– Progress zone model – Early allocation and progenitor expansion

model

P-D axis specification: Guess who?

• Regardless of which model for the P-D axis wins out . . .

• Hox genes appear to be involved!– These genes are expressed in a nested

pattern that changes during limb development

– When mutations occur, changes can occur in the P-D axis

Specific Hox Paralog groups are expressed in specific regions of the limb

A= Wild Type Mouse; B = Hox a-11, Hox d11 double mutant mouse; C = Human Polysyndactyly (HomozygousHoxD13 mutation)