Joint formation in chick limb bud CAM grafts

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Introduction

CAM grafting

The chorioallantoic membrane (CAM) is formed on day four after fertilization of a viable chick egg embryo, fusing together the chorion and allantois into a semi- permeable membrane that facilitates gas exchange through direct contact with the overlying shell. From days four to eleven, vasculature augments as capillaries begin to proliferate throughout the membrane (Ribatti, 2001). This allows for exchange of materials (i.e. gas, calcium, and other nutrients) between the developing embryo and the membrane (Nichols, 2000). Previous experiments have shown that CAM-grafted limbs can form well-grouped muscle fibers and that these muscle fibers can regenerate from the existing tissue and remain in place for at least sixty days (Nakada et. al., 1999). This regenerated muscle tissue is reportedly free from exogenous tissues and motor neuron innervation. This ability to isolate and culture tissues outside the original organism and apart from influencing factors such as migrating somites and other simultaneously-differentiating tissues exogenous to myocyte development suggests that CAM grafting could function as a means of isolating the inductive events that lead to myocyte differentiation and regeneration. Similarly, the results of the Nakada experiment demonstrate that osteogenesis and feather development can occur in grafted limbs (Nakada et. al., 1999). It is evident from these previous studies that tissue formation can continue to occur in grafted limbs since both oxygen and nutrients from the host circulatory system can cycle through the grafted tissues, but there are also transcription factors and other signaling molecules that are necessary for proper joint development to take place which might be altered or absent in the grafted tissue.

Joint Formation

Cavitation between regions of presumptive cartilage formation precludes normal limb development. Events prior to cavitation include the expression of N-Cadherins concurrent to mesenchyme condensation, forming cartilage nodules that preface proliferation of osteoblasts throughout the developing limb (Gilbert, 2003). Signaling molecules that have been linked to joint development include GDF-5, Wnt-14, BMPs, and FGFs. In a study conducted by Maurizio Pacifici and colleagues, signaling molecules such as Wnt-14, BMPs, GDF-5, and several FGFs were found at the site on joint formation.

The function of these molecules was further analyzed through a retroviral vector which contained a mutated form of the particular signaling molecule to be assayed. In order for joint formation to occur, the chondrocytes (mature cartilage cells) in the region of the presumptive joint must de-differentiate to allow for gaps to form between the future bones of the limb. Wnt 14 and Cux 1 have been implicated in the de-differentiation of cartilage cells while GDF-5 and BMP expression promotes cartilage growth. FGF-2 and FGF-3 have been shown to block the ectopic expression of GDF-5 outside the region of the articular chondrocytes. Cux1 transcription factor is believed to be a downstream effector of Wnt-14 and GDF-5 because it does not directly induce their transcription. C-1-1 is another transcription factor thought to induce expression of Tenascin-C, a protein localized in the synovial capsule and thought to determine the location of the interzone (Pacifici et. al., 2002). Histochemical mapping of regions of presumptive joint formation reveal the presence of hyaluronan (HA), which may be a key factor in the differentiation of cells adjacent to the gap regions between bones of the joint that will become a part of the synovial capsule, the fluid-containing cavity surrounding the fully-formed joint (Pitsillides, 1995).

These studies indicate that there are at least several key molecular mechanisms involved in joint formation of normal chicks that may or may not be present in order to allow for proper cartilage and joint formation in limb bud CAM grafts. Therefore, the purpose of this experiment is to determine whether or not normal joint formation can occur in CAM-grafted chick limb buds.

J. N. White 5/13/04

@ Cebra-Thomas, 2000
Last Modified: 13 May 2004

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		Introduction *CAM grafting* The chorioallantoic membrane (CAM) is formed on day four after fertilization of a viable chick egg embryo, fusing together the chorion and allantois into a semi- permeable membrane that facilitates gas exchange through direct contact with the overlying shell. From days four to eleven, vasculature augments as capillaries begin to proliferate throughout the membrane (Ribatti, 2001). This allows for exchange of materials (i.e. gas, calcium, and other nutrients) between the developing embryo and the membrane (Nichols, 2000). Previous experiments have shown that CAM-grafted limbs can form well-grouped muscle fibers and that these muscle fibers can regenerate from the existing tissue and remain in place for at least sixty days (Nakada et. al., 1999). This regenerated muscle tissue is reportedly free from exogenous tissues and motor neuron innervation. This ability to isolate and culture tissues outside the original organism and apart from influencing factors such as migrating somites and other simultaneously-differentiating tissues exogenous to myocyte development suggests that CAM grafting could function as a means of isolating the inductive events that lead to myocyte differentiation and regeneration. Similarly, the results of the Nakada experiment demonstrate that osteogenesis and feather development can occur in grafted limbs (Nakada et. al., 1999). It is evident from these previous studies that tissue formation can continue to occur in grafted limbs since both oxygen and nutrients from the host circulatory system can cycle through the grafted tissues, but there are also transcription factors and other signaling molecules that are necessary for proper joint development to take place which might be altered or absent in the grafted tissue. *Joint Formation* Cavitation between regions of presumptive cartilage formation precludes normal limb development. Events prior to cavitation include the expression of N-Cadherins concurrent to mesenchyme condensation, forming cartilage nodules that preface proliferation of osteoblasts throughout the developing limb (Gilbert, 2003). Signaling molecules that have been linked to joint development include GDF-5, Wnt-14, BMPs, and FGFs. In a study conducted by Maurizio Pacifici and colleagues, signaling molecules such as Wnt-14, BMPs, GDF-5, and several FGFs were found at the site on joint formation. The function of these molecules was further analyzed through a retroviral vector which contained a mutated form of the particular signaling molecule to be assayed. In order for joint formation to occur, the chondrocytes (mature cartilage cells) in the region of the presumptive joint must de-differentiate to allow for gaps to form between the future bones of the limb. Wnt 14 and Cux 1 have been implicated in the de-differentiation of cartilage cells while GDF-5 and BMP expression promotes cartilage growth. FGF-2 and FGF-3 have been shown to block the ectopic expression of GDF-5 outside the region of the articular chondrocytes. Cux1 transcription factor is believed to be a downstream effector of Wnt-14 and GDF-5 because it does not directly induce their transcription. C-1-1 is another transcription factor thought to induce expression of Tenascin-C, a protein localized in the synovial capsule and thought to determine the location of the interzone (Pacifici et. al., 2002). Histochemical mapping of regions of presumptive joint formation reveal the presence of hyaluronan (HA), which may be a key factor in the differentiation of cells adjacent to the gap regions between bones of the joint that will become a part of the synovial capsule, the fluid-containing cavity surrounding the fully-formed joint (Pitsillides, 1995). These studies indicate that there are at least several key molecular mechanisms involved in joint formation of normal chicks that may or may not be present in order to allow for proper cartilage and joint formation in limb bud CAM grafts. Therefore, the purpose of this experiment is to determine whether or not normal joint formation can occur in CAM-grafted chick limb buds.
		J. N. White 5/13/04