Limb Patterning: Proximal-Distal Axis

1. Limb Patterning: Proximal-Distal Axis Gilbert - Chapter 16 Do you want to hear a talk from an ASTRONAUT 4/17??

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

4. 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

5. John Saunders

6. 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

7. PZ

8. 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

10. Removal of AER at different times: loss of various structures

11. 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

13. 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??

14. 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

15. FGF8 expression

16. 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!

18. 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

19. 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

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

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