VERTEBRATE LIMB DEVELOPMENT

1. VERTEBRATE LIMB DEVELOPMENT Differential Response of Shh Expression Between Chick Forelimb and Hindlimb Buds By FGF-4 (Wada and Nohno, Dev. Dyn., 221(4): 402-411) Robert Herrington

2. Why Study Limb Development? Fundamental Understanding of the process Clinical relevance Some statistics: Frequency of abnormality depends on severity Duplication of digits is fairly common at ~1 in 300 births Major failures of formation is less common, at ~ 1 in 100,000 births (Indiana Hand Center)

3. What are these abnormalities?

4. How do these abnormalities arise? THROUGH IMPROPER DEVELOPMENT!!!

5. Limb Development is A Part of Organogenesis The process of organogenesis involves using the basic body plan (organized embryo) to develop specific organs (limbs, heart, eyes) in specific regions that leads to the fully functional organism, capable of independent survival Involves: pattern formation, specification of positional information, induction, morphogenesis, and cellular differentiation

6. How are these processes involved? Pattern formation ? directs cell identity and leads to ordered spatial pattern of cell activity Positional information ? directs where organs will form Induction ? direction of specific cell fate Morphogenesis ? changing the form of cells Differentiation ? acquire functional and structural identity distinct from their surrounding cells

7. Model System � Development of the Chick Limb I Chick limbs begin to develop on the 3rd day after laying The limb develop from limb buds on the body wall of the embryo

8. Development of the Chick Limb II

9. Development of the Chick Limb III

10. Embryonic Chick Wing (day 10)

11. What Directs Limb Development? The APICAL ECTODERMAL RIDGE (AER) is responsible for the proximo-distal patterning of the limb, which occurs over time through the progress zone Signals are sent both from the AER to the progress zone, and from the progress zone to the AER

12. What Directs Limb Development?(cont�d) The ZONE OF POLARIZING ACTIVITY (ZPA) is responsible for antero-posterior patterning of the limb The ECTODERM controls patterning along the dorso-ventral axis Differences in the activation of genes that control the interpretation of positional information is responsible for development of different limb structures (i.e.. wing or leg)

13. What Molecules Direct Limb Development? Sonic Hedgehog (Shh) The Shh gene encodes inductive signaling molecules that function during embryogenesis Involved in establishing cell fates Key signal in patterning of the antero-posterior limb axis Alx-4 represses Shh expression Fibroblast Growth Factor 4 (FGF-4) Member of FGF family Mesodermal cell stimulator and directs developmental signaling FGF-4 -/- die on E 5.0 Transcriptionally inactive in adult tissues

14. Differential response of Shh expression between chick forelimb and hindlimb buds by FGF-4 Wada and Nohno, Dev. Dyn., 221(4): 402-411

15. Hypothesis Since differences in limb morphology result from distinct gene expressions and subsequent signal transduction pathways in the limb bud, then� The cells constituting the fore- and hindlimb bud of chick embryo are likely to have distinct responsiveness to signaling molecules

16. Objectives To determine if there are different responses between the fore- and hindlimb buds to signaling molecules Can FGF-4 induce Shh in the different limb buds? Can Shh change the positional identity of cells? Does FGF-4 repress a Shh repressor in limb buds? Do cells migrating to the limb buds express Shh? Can other FGFs induce Shh expression in limb buds?

17. Experimental Procedures Embryo Manipulations Beads containing FGF solution were grafted into a slit under the anterior margin of the AER in fore- and hindlimb buds Whole Mount In Situ Hybridization Probes used were antisense cRNA Staining Skeletal patterns were analyzed after staining of cartilage elements

23. Summary of Results FGF-4 can induce Shh expression in the chick hindlimb, but not the forelimb Induction of Shh expression can lead to digit duplication and respecification of positional information FGF-4 does not appear to repress Alx-4 Migrating mesechymal cells do not contribute to induction of Shh expression FGF-2 and FGF-8 do not induce Shh in the chick limb bud

24. Discussion What do these results mean? There is a difference between the fore- and hindlimb buds in the cell competence of Shh induction in response to FGF-4 These results suggest that signal transduction pathways in fore- and hindlimb buds may differ, with the hindlimb having molecular cascades through which FGF-4 could activate hindlimb specific Shh inducers, leading to Shh expression

25. Relevance Why are these results important? It is important to compare the responsiveness of fore- and hindlimb buds to signaling molecules, because the results will provide useful information for clarifying morphologic differences in the limbs

26. Future Directions TO DETERMINE DIFFERENCES IN LIMB DEVELOPMENT: Look at other signaling molecules that may that may show a differential response between fore- and hindlimb ( i.e., Hox genes) Look at cascade pathways/molecules (PKC) that may be activated by signaling molecules such as FGFs or Shh proteins

27. THANK YOU� ANY QUESTIONS???