Joint formation in chick limb bud CAM grafts

Abstract
        

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.

Joint Lab Protocol

Objective: To determine whether normal joint formation occurs in chick limb buds that have already begun cartilage patterning

Materials

• 8 day old donor eggs

• 10 day old host eggs

• fine forceps

• Scotch tape

• gentle agitating device (we used Nutator TM )

• 5% Trichloroacetic acid

• PBS

• 100% ethanol

• methyl salicylate

• dH2O

• Alcian green stain

• ethanol

Procedure

Adapted from Developmental Biology website:

I. Chorioallantoic Membrane Grafting (Hamburger, 1960)

For each host embryo*, complete the following procedure:

1. Sterilize a 10-day old donor egg with 70% ethanol.

2. Poke a hole into the blunt end and peel away the shell with forceps. Open a hole in the shell approximately an inch in diameter, or large enough to insert the graft and isolate veins. There will be a whitish membrane on the surface inside the opened shell. Carefully remove this white membrane without disturbing the clear CAM membrane immediately underneath. The CAM should remain intact in order to preserve the circulatory system of the host embryo so that it can distribute nutrients and oxygen to and from the graft.

3. Look for a large Y- shaped junction of blood vessels in the clear chorioallaintoic membrane. This is where the graft will be placed once the donor limbs are prepared. Peel back the white membrane around the Y-shaped junction to prepare the area for grafting. Cover the hole with scotch tape and store the egg in the incubator at 37 degrees Celcius.

4. Sterilize an 8-day old donor egg with 70% ethanol, and then transfer to a dish with Ringer's solution.

5. Remove all membranes surrounding the embryo.

6. Excise limbs (2 wing buds and 2 hind limbs) with a tungsten knife, and include an extra flap of cells from the flank to help move the graft into the proper position.

7. Transfer one limb graft to each host egg.

8. Position transferred limb over the large Y- junction.

9. Re-tape the eggs to prevent infection and desication. Incubate for 7-10 days (but not more than 10 or the host chick will hatch! ) before removal of the graft for staining.

10. Carefully remove the tape covering the shell opening above the CAM graft (you may want to candle the embryos in a dimly lit room so as to avoid opening already-dead host chicks. The dead hosts will show little vasculature, a sallow yellow coloring on the inside of the egg, and no sign of a dense mass surrounded by extensive vasculature indicating the presence of the living host embryo).

11. Gently use forceps to excise the limb graft, which should appear as a dense mass of tissue often darker around the edges that the host tissue, with possible feather bud formation. In order to excise the limbs, you may need to clip the CAM and the surrounding vasculature as it often has a tendency to adhere to the limb bud.

*36 host embryos were used in this experiment, each containing a single limb graft. The average survival rate for host chicks was 11/16, with the successful retrieval of about 18 limbs. Therefore, have a large population of host chicks to ensure a sufficient enough sample of limbs in order to more accurately assess the occurrence of joint formation.

II. Preparing the control embryo

1. Retain one 8-day old embryo to use later as a control.

2. Excise the chick from its shell after the incubation period, and remove

Limb buds

3. Stain using Alcian green

III. Alcian green staining for cartilage formation

1. Wash 3X with PBS.

2. Fix limbs or embryos in 5% TCA for 1 hour at room temperature.

(Eviscerate embryos before fixation).

3. Immerse in Alcian green stain for 4 hours (or preferably overnight*) with gentle agitation.

*Note: If limbs are immersed in the stain overnight, the following ethanol treatments should also be conducted overnight to effectively dehydrate the limbs before the methyl salicylate wash.

4. Wash 3X with 70% ethanol. Each wash should be at least two hours long, but it can be left overnight.

5. Wash with 85%ethanol, 95% ethanol, 100% ethanol, several hours to overnight each.

6. Clear by washing 1-3X with methyl salicylate. The limb bud grafts will begin to become transparent after immersion in the methyl salicylate. The grafts can remain in this solution for an indeterminate amount of time. Photograph limb buds and record observations. 

Materials and Methods

Grafting

The limbs used in this experiment were excised from 8 day old chick embryos, and the host embryos that received the grafted limbs were 10 days old. The grafted limbs were placed in Howard Ringer's solution (DB Lab, 2003) until they could be moved to the CAM of the host embryo. Thirty six host embryos were used in this experiment, each containing a single limb graft. Egg shells were demarcated in lead pencil to differentiate between limbs and wings, as it can be difficult to distinguish between the two during later development. The average survival rate for host chicks was 11/16, with the successful retrieval of about 18 limbs. Therefore, it helps to have a large population of host chicks to ensure a large enough sample of limbs in order to more accurately assess the occurrence of joint formation. The limb bud grafts were removed from the host eggs seven days later and placed in embryo ringers solution.

Alcian Green Staining and Dehydration

Excised limbs were then taken out of embryo ringers solution and fixed in 5% TCA for one hour at room temperature and rinsed twice with PBS solution. The limbs were then immersed in alcian green stain overnight. The alcian green stain was prepared according to the protocol listed on Developmental Biology Lab Website (DB Lab, 2003). In order to dehydrate the limbs after staining, limbs were induced over a period of four days with successively greater concentrations of lithium chloride at 70, 85, 95 and 100 per cent, immersing in each concentration overnight. After the dehydration process, limb buds were placed in methyl salicylate and stored in glass containers, as methyl salicylate will dissolve plastic. Embryos were photographed after twenty three hours.

Results

Figure 1. Control limbs were detached from fifteen day old chick embryos and stained for cartilage with alcian green. Arrows point to examples of fully formed joints, spaces created by cell death along rods of cartilage. A wing showed digit and elbow joints (A). The joints of the toe digits were clear in a chick foot (B1). The knee joint and toe joints were easily seen in a chick leg (B2).

A	B

C	D

Figure 2. Fifteen day old limb grafts (eight day old limbs grafted for seven days) were extracted from seventeen day old hosts and stained for cartilage with alcian green. Arrows point to examples of fully formed joints, spaces created by cell death along rods of cartilage. Leg grafts showed somewhat regular patterning and discernable toe and knee joints (A and B). A leg graft showed an irregular patterning, but discernable joints (C). Another leg graft showed an irregular patterning (possibly a duplication of limbs) and discernable joints (D).

A	B

Figure 3. Fifteen day old limb grafts (eight day old limbs grafted for seven days) were extracted from seventeen day old hosts and stained for cartilage with alcian green. Arrows point to examples of fully formed joints, spaces created by cell death along rods of cartilage. A wing had a somewhat regular patterning and discernable digit and elbow joints (A). Another wing had a somewhat regular patterning and somewhat discernable elbow joint (B).

A	B

Figure 4. Fifteen day old limb grafts (eight day old limbs grafted for seven days) were extracted from seventeen day old hosts and stained for cartilage with alcian green. Both wing grafts showed irregular patterning and cartilage/bones were completely intact with no discernable breaks in cartilage/bone formation (A and B).

All limbs were extracted at day seven of experimentation when chick embryos and grafts were approximately fifteen days old. Limbs were stained for cartilage with alcian green and observed and photographed under a microscope. Discernable gaps along rods of cartilage/bone were noted. Green staining indicated cartilage, blue staining cartilage and bone, and no staining either gaps in cartilage or bone or fully formed bone.

Control limbs detached from fifteen day old chick embryos were normally patterned, relatively fully formed wings and legs (Figure 1 A, B1, B2). No staining occurred in upper regions of the leg, and in discrete spaces between green or green-blue stained regions (Figure 1).

Patterning and staining varied among grafts. Three of seven (43%) hind limb or leg grafts were patterned somewhat normally, that is, they looked like the control leg (Figure 2 A, B). The remaining four (57%) hind limb grafts were irregularly patterned, that is, did not look like the control leg (Figure 2 C, D). No staining occurred in discrete spaces between green or green-blue stained regions similar to the hind limb control (Figure 2 A, B, C, D) in all hind limb grafts.

Most fore limb or wing grafts (seven of nine&emdash;78%) were patterned like the control wing (Figure 3 A, B and Figure 4 A, B). No staining occurred in discrete spaces between green or green-blue stained regions (like the fore limb control) in six of nine (66%) of our wing grafts (Figure 3 A, B). In one fore limb (11%) green-blue staining appeared hazy where discrete areas of no staining occurred in the control (Figure 4 A). Green-blue staining occurred in continuous rods with no discernable spaces along those rods in two of nine (22%) fore limb or wing grafts (Figure 4 B). These two fore limb grafts were among the seven grafts that were patterned like the control (Figure 4 B).

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.