1. Vertebrate Embryonic Patterning 6 Limb Formation

2. Tetrapod Limb anterior posterior proximal distal ventral dorsal

3. Limb Bud Formation

4. Establishment of Limb Fields

5. Hox Gene Expression in Trunk During Limb Bud Development Fig 23.21 Analysis of Biological Development 2 nd ed, Kalthoff

6. FGF10 Induces FGF8 supernumerary limbs from implanting FGF10 beads

7. Positioning of the Limb Field Forelimb position lies near anterior expression boundary of hox b8, b9, c6, c9, & d9 FGF8 expression in intermediate meso + hox genes induces FGF10 in the lateral plate meso Wnts in the lateral plate maintain FGF expression in regions of hoxd9 expression Between the fore & hindlimb buds, the developing kidney (mesonephros) inhibits FGF expression Exogenous FGF can induce supernumerary limbs

8. Forelimb vs Hindlimb

9. Forelimb vs Hindlimb Correlates with Tbx5 & Tbx4 Expression Respectively Tbx5 Tbx4

10. Tbx4 Gain of Function Study Tbx4 expressing retrovirus infected embryo

11. Proximal Distal Limb Axis AER –overlying ectoderm –responsible for maintaining outgrowth of limb Progress zone –underlying mesoderm –region of cell division –maintained in active mitotic state by AER

12. AER – Apical Ectodermal Ridge

13. AER Necessary for Limb Bud Outgrowth Removal of AER at various times prematurely truncates limb

14. Reimplantation of older AER into younger bud causes loss of proximal structures AER Necessary for Limb Bud Outgrowth Reimplantation of younger AER into older bud causes duplication of distal structures

15. Interactions of AER and PZ

16. FGF8 in the AER FGF in ectodermAER 3 day embryo limb buds

17. Hox Code of the Limb ProximalDistal Hoxa & d = forelimb; Hoxc & d = hindlimb

18. Mammalian Hox Genes

19. Hox Genes Required for Proper Limb Development Hoxa-11/Hoxd-11 double knockout wild-type human with synpolydactyly resulting from homozygous HOXD-13 null mutation

20. Proximal-Distal Structures

21. Anterior-Posterior Limb Axis Zone of Polarizing Activity ZPA

22. Classic Experiments: Retinoic Acid Induced Limb Duplication Implanting RA soaked beads caused duplication of zeugopod/autopod structures Depletion of RA reduces the normal structures

23. Shh is Expressed in ZPA

24. Demonstration that Sonic Hedgehog is Primary ZPA Component

25. Dorsal-Ventral Gene Patterning in Limb Bud Fig 23.23 Analysis of Biological Development 2 nd ed, Kalthoff

26. Dorsal-Ventral Limb Axis Lmx 1.Fgf-8 induces Shh 2.Shh & Fgf-8 induce Fgf-4 3.All these induce Lmx-1 4.Wnt-7a in the ectoderm represses Lmx-1 and induces Shh BMPs

27. Molecular Interactions in Limb Bud

28. Dorsal-to-Ventral Transformations WT mouse autopod Wnt7a -/- mouse autopod

29. Homology of Process: A-P Axis

30. Homology of Process: D-V