Joint formation in chick limb bud CAM grafts

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Discussion

Areas of limbs where no staining occurred indicate areas of fully formed non-cartiliginous bone or gaps in cartilage or bone. Joints are, by definition, gaps formed by apoptosis in limbs (Pacifici et al., 2002). Importantly, joints do not form as interacting bones, such as the metatarsals&emdash;the foot region, individually form and grow together, rather parts of continuous rods of bone die off creating joints and separate interacting bones (Pacifici et al., 2002). Gaps in staining, then, most likely indicate joints. Our control limbs showed normal joint formation, including clean digit, elbow, and knee joint formation. The staining gaps in our limb grafts closely matched those of the controls, and presumably indicate similar joint formation. Digital joints were the most clearly formed in all but two grafts. Interestingly, other joints such as the elbow and knee joints were not as clearly or cleanly formed (as indicated by haziness in gaps or incomplete staining gaps) in all grafts.

CAM grafts receive calcium and other nutrients important for growth from the chorioallantoic membrane's rich blood supply (Hamburger, 1960). Signals for proper limb growth, however, hypothetically do not pass across the CAM from the host embryo (Judy Cebra-Thomas, personal communication). As grafts are limbs which have been removed from the rest of the embryo, they offer a chance to examine endogenous signaling within the limb. Within our limb graft group, however, joint formation was not regular, and in fact, 12.5% (two of sixteen) grafts did not form joints. Interestingly, the grafts with less regular or no joint formation were exclusively wing grafts. Perhaps hind limbs contain a joint formation signal at eight days that fore limbs do not contain or contain sometime after day eight. More experimentation should be done to determine if different endogenous joint formation signals reside in fore and hind limbs. Additionally, our experimental sample size was not large enough to determine the accuracy of our results. Further, irregularities seen in limb graft patterning could be due to imprecise limb bud extraction where too little of the bud was used as a graft. We propose that CAM grafting with eight day old grafts cannot yet be determined as an effective method for joint formation experimentation. Further experimentation with larger sample sizes and more precise grafts should be undertaken.

Acknowledgements

We would like to thank Professor Judy Cebra-Thomas for her guidance and advice throughout this project, Bill Gresh for his skilled assistance during the project, and Fraser Tan for her advice and assistance during laboratory periods. Thank you to Katy Lewis, Katie Crawford, and Erin Betters for donating the limb buds from their experiment for our grafts. We would also like to extend our gratitude to Andy Nicol at F&M for his write-up "Chorioallantoic Membrane Grafting with Chick Embryo Limb Buds" which we referenced for this project.

Works Cited

Gilbert, S.F. 1997. Developmental Biology, 5th ed. Sunderland: Sinauer Associates, Inc., 76, 359-361.

Hamburger, V. 1960. A Manual of Experimental Embryology. Chicago: The University of Chicago Press, pp. 79-81.

Nichol, Andy. Chorioallantoic membrane grafting with chick embryo limb buds. <http://www.swarthmore.edu/NatSci/sgilber1/DB_lab/Chick/CAM.html>. 2000.

Pacifici, Maurizio et. al. 2002. "Joint Formation: new findings shed more light on this critical process in skeletogenesis." Philadelphia: Lippincott, Williams and Wilkins, p.339-343.

Pitsillides AA, Archer CW, Prehm P, Bayliss MT, Edwards JC. "Alterations in hyaluronan synthesis during developing joint cavitation." J Histochem Cytochem. 1995 Mar; 43(3):263-73.

Ribatti, Domenico. "The Chick Embryo Chorioallantoic Membrane as a model for in vivo Research on Anti-Angiogenesis." <http://www.bentham.org/cpb1-1/D.%20Rabbati/D.Ribatti.htm>. 2001.

Tuan, R. 1987. "Mechanisms and regulation of calcium transport by the chick embryonic chorioallantoic membrane." Journal of Experimental Zoology. [Suppl.] 1: 1-13

Nakada K, Mishima T, Miyazaki JI, Hirabayashi T. "Novel tissue culture method: skeletal muscle implantation under gizzard serous membrane of a chick." J Exp Zool. 2000 May 1; 286(6):615-24.

J. N. White 5/13/04

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Last Modified: 13 May 2004

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		Discussion Areas of limbs where no staining occurred indicate areas of fully formed non-cartiliginous bone or gaps in cartilage or bone. Joints are, by definition, gaps formed by apoptosis in limbs (Pacifici et al., 2002). Importantly, joints do not form as interacting bones, such as the metatarsals&emdash;the foot region, individually form and grow together, rather parts of continuous rods of bone die off creating joints and separate interacting bones (Pacifici et al., 2002). Gaps in staining, then, most likely indicate joints. Our control limbs showed normal joint formation, including clean digit, elbow, and knee joint formation. The staining gaps in our limb grafts closely matched those of the controls, and presumably indicate similar joint formation. Digital joints were the most clearly formed in all but two grafts. Interestingly, other joints such as the elbow and knee joints were not as clearly or cleanly formed (as indicated by haziness in gaps or incomplete staining gaps) in all grafts. CAM grafts receive calcium and other nutrients important for growth from the chorioallantoic membrane's rich blood supply (Hamburger, 1960). Signals for proper limb growth, however, hypothetically do not pass across the CAM from the host embryo (Judy Cebra-Thomas, personal communication). As grafts are limbs which have been removed from the rest of the embryo, they offer a chance to examine endogenous signaling within the limb. Within our limb graft group, however, joint formation was not regular, and in fact, 12.5% (two of sixteen) grafts did not form joints. Interestingly, the grafts with less regular or no joint formation were exclusively wing grafts. Perhaps hind limbs contain a joint formation signal at eight days that fore limbs do not contain or contain sometime after day eight. More experimentation should be done to determine if different endogenous joint formation signals reside in fore and hind limbs. Additionally, our experimental sample size was not large enough to determine the accuracy of our results. Further, irregularities seen in limb graft patterning could be due to imprecise limb bud extraction where too little of the bud was used as a graft. We propose that CAM grafting with eight day old grafts cannot yet be determined as an effective method for joint formation experimentation. Further experimentation with larger sample sizes and more precise grafts should be undertaken. Acknowledgements We would like to thank Professor Judy Cebra-Thomas for her guidance and advice throughout this project, Bill Gresh for his skilled assistance during the project, and Fraser Tan for her advice and assistance during laboratory periods. Thank you to Katy Lewis, Katie Crawford, and Erin Betters for donating the limb buds from their experiment for our grafts. We would also like to extend our gratitude to Andy Nicol at F&M for his write-up "Chorioallantoic Membrane Grafting with Chick Embryo Limb Buds" which we referenced for this project. Works Cited Gilbert, S.F. 1997. Developmental Biology, 5th ed. Sunderland: Sinauer Associates, Inc., 76, 359-361. Hamburger, V. 1960. A Manual of Experimental Embryology. Chicago: The University of Chicago Press, pp. 79-81. Nichol, Andy. Chorioallantoic membrane grafting with chick embryo limb buds. <http://www.swarthmore.edu/NatSci/sgilber1/DB_lab/Chick/CAM.html>. 2000. Pacifici, Maurizio et. al. 2002. "Joint Formation: new findings shed more light on this critical process in skeletogenesis." Philadelphia: Lippincott, Williams and Wilkins, p.339-343. Pitsillides AA, Archer CW, Prehm P, Bayliss MT, Edwards JC. "Alterations in hyaluronan synthesis during developing joint cavitation." J Histochem Cytochem. 1995 Mar; 43(3):263-73. Ribatti, Domenico. "The Chick Embryo Chorioallantoic Membrane as a model for in vivo Research on Anti-Angiogenesis." <http://www.bentham.org/cpb1-1/D.%20Rabbati/D.Ribatti.htm>. 2001. Tuan, R. 1987. "Mechanisms and regulation of calcium transport by the chick embryonic chorioallantoic membrane." Journal of Experimental Zoology. [Suppl.] 1: 1-13 Nakada K, Mishima T, Miyazaki JI, Hirabayashi T. "Novel tissue culture method: skeletal muscle implantation under gizzard serous membrane of a chick." J Exp Zool. 2000 May 1; 286(6):615-24.
		J. N. White 5/13/04