Vertebrate Limb Development

1. Vertebrate Limb Development Carolyn Hedley March 25, 2002

2. Introduction develop from limb buds 3 developmental axes: proximo-distal, antero-posterior, and dorso-ventral early limb bud: progress zone, apical ectodermal ridge, polarizing region

3. Apical Ectodermal Ridge (AER) 1 of 2 organizing regions. length of ridge controls width of bud. essential for both outgrowth and proximo-distal patterning; truncated limbs result after removal.

4. AER continued positioning determined by gene Radical Fringe (r-Fng): develops at boundary of r-Fng-expressing and non-expressing cells. AER signals to underlying mesenchyme. signals: include proteins of fibroblast growth factor (Fgf) gene family FGF8: expressed throughout ridge FGF4: in posterior region - can act as a functional substitute for apical ridge

5. Progress Zone (PZ) lies beneath AER. produces initial outgrowth of limb bud: rapidly proliferating mesenchymal cells. cells begin to differentiate only after leaving the progress zone. differentiation proceeds distally as limb extends - cell fate determined by time spent in progress zone.

6. Zone of Polarizing Activity (ZPA) second organizing region. diffusible morphogen specifies position of cells along antero-posterior (A/P) axis; by concentration of morphogen, or short-range signals. Sonic Hedgehog (SHH) protein key component.

7. ZPA, PZ and AER signals ZPA and PZ signals are necessary for survival and function of AER; FGFs from AER maintain ZPA and PZ. positive feedback loop: between SHH protein in mesoderm and FGF4 expression in ridge, via bone morphogenetic proteins (BMPs).

8. Dorso-ventral (D/V) axis specified by ectoderm Wnt-7a gene identified in mice - if mutated, dorsal tissues adopt ventral fates.

9. Forelimbs vs Hindlimbs same signals interpreted differently. 23 different Hox genes expressed: Hoxa-d gene clusters Hoxa & d - expressed in both forelimbs and hindlimbs Hoxb & c - restricted to either forelimb or hindlimb bud

10. Cartilage elements can develop in absence of polarizing region. reaction-diffusion mechanism - establishes a prepattern by periodic peaks in a morphogen. acquires positional information in PZ.

11. Muscle muscle cells in early embryo are all equivalent; migrate into the limb bud from somites. dorsal and ventral blocks of muscle initially. Divisions give rise to individual muscles; pattern determined by connective tissue.

12. Apotosis programmed cell death key role in formation of digits - separation depends on death of cells between cartilaginous elements. mesoderm determines cell fate

13. Normal limb development in conditional mutants of Fgf4 Moon, A. M., Boulet, A. M., and M. R. Capecchi. 2000. Development. 127: 989-996.

14. Objective To produce a conditional mutant of Fgf4 for assessing the role of FGF4 during limb development in living murine embryos.

15. Fibroblast Growth Factors expression strongest in the posterior AER at E10.5-11.0. hypothesized to mediate AER activity in vivo. FGF4 - may provide a proliferative signal to the posterior PZ to support limb outgrowth. evidence for SHH-FGF4 feedback loop hypothesis: lack of Fgf4 expression is associated with i) failure to maintain Shh expression and, ii) abnormal patterning of distal limb elements.

16. Methods: Conditional mutagenesis technique used to conditionally inactivate Fgf4 in a developmentally regulated, tissue-restricted manner. used the Cre/loxP system - two mouse lines required: 1) vital Fgf4 coding sequences are flanked by loxP sites 2) conventional transgenic mouse line with Cre targeted to a specific tissue or cell type

17. Methods cont�d 3 components of Fgf4 conditional mutagenesis: 1) mice carrying either a conditional or a null allele of Fgf4 were created by gene targeting in embryonic stem cells ? transgenic �switch� generated to inactivate the conditional allele 2) Southern analysis used for identification of offspring with Fgf4 mutant alleles 3) animals genotyped using PCR technique

18. Evaluation of Cre-mediated combination the human alkaline phosphatase gene (AP) is expressed in the conditional allele following Cre-mediated inactivation of Fgf4. stained for AP; AP activity labels cells in which recombination has occurred

19. Results: phenotypic appearance of AER and limb bud initiation, location, or size were indistinguishable between embryonic conditional mutants and littermates at E10.0-11.5. patterning and outgrowth of forelimbs of Ffg4 conditional mutants were normal in E15.5 embryos, newborns, and adults. Normal external appearance and function in living animals.

21. Results: Shh, Bmp2 and Fgf8 expression no detected changes in Shh expression in forelimbs of Fgf4 conditional mutants. normal expression of Fgf8, Bmp2.

22. Discussion may be other in vivo AER factors involved compensation of another FGF for absence of FGF4. FGF1, FGF2 and FGF8 can each support limb outgrowth in absence of AER. FGF8 can induce and maintain SHH expression in the absence of the AER.

23. Discussion the normal skeletal and molecular phenotypes of conditional mutants are unlikely a function of early or persistent Fgf4 gene function no detection of Fgf4 transcripts in forelimb AER at E 10.0-11.5.

24. Summary: Implications Fgf4 conditional mutants had normal limb outgrowth development. in vivo results indicate that FGF4 is not required for normal limb development or Shh expression in the ZPA. some of the functions currently attributed to FGF4 may be performed by other AER factors.

25. Future directions determine role of Fgf8 in limb development by conditional mutagenesis. most likely possible to disrupt multiple genes simultaneously in conditional mutagenesis eliminate more than one FGF in the AER

26. Additional research Sun et al. (May 2000) - upregulation of Fgf8 and Fgf9 did not occur in Fgf4 conditional mutants. Moon & Capecchi (Dec. 2000) - conditional mutagenesis of FGF8. Fgf8 absolutely required in the anterior AER, and to maintain Shh expression. Fgf4 expression increased at E11.5 in Fgf8 conditional mutants; possible compensation.

27. References Cohn, M. J., and P. E. Bright. 1999. Molecular control of vertebrate limb development, evolution and congenital malformations. Cell Tissue Research. 296: 3-17. Moon, A. M., Boulet, A. M., and M. R. Capecchi. 2000. Normal limb development in conditional mutants of Fgf4. Development. 127: 989- 996. Moon, A. M., and M. R. Capecchi. 2000. Fgf8 is required for outgrowth and patterning of the limbs. Nature Genetics. 26: 455-459. Sun, X., Lewandoski, M., Meyers, E. N., Liu, Y., Maxson, R. E., and G. R. Martin. 2000. Conditional inactivation of Fgf4 reveals complexity of signaling during limb bud development. Nature Genetics. 25: 83-86. Wolpert, L. 1998. Principles of Development. Oxford University Press, New York, NY.