Optimized Ex-ovo Culturing of Chick Embryos to Advanced Stages of Development

Podsumowanie

Viewing and accessing the chicken embryo during development can be challenging. We have developed an ex ovo method that is simple, cost effective, and can easily be used in a classroom or research setting. This method provides access to the embryo into late stages of embryonic development (HH 40).

Streszczenie

Research in anatomy, embryology, and developmental biology has largely relied on the use of model organisms. In order to study development in live embryos model organisms, such as the chicken, are often used. The chicken is an excellent model organism due to its low cost and minimal maintenance, however they present observational challenges because they are enclosed in an opaque eggshell. In order to properly view the embryo as it develops, the shell must be windowed or removed. Both windowing and ex ovo techniques have been developed to assist researchers in the study of embryonic development. However, each of the methods has limitations and challenges. Here, we present a simple, optimized ex ovo culture technique for chicken embryos that enables the observation of embryonic development from stage HH 19 into late stages of development (HH 40), when many organs have developed. This technique is easy to adopt in both undergraduate classes and more advanced research laboratories where embryo manipulations are conducted.

Wprowadzenie

Ex ovo culturing has played an important role in the study of development of the chicken^{1, 2}. This culturing method has been used to study neurological diseases, limb development, craniofacial development, and as a model to investigate malformations associated with diabetes ^{3, 4, 5}.

There are many variations to the ex ovo technique. The most common approach is to use a Styrofoam cup^6,7,8 or a glass bowl⁵. In these methods, the cup or bowl is lined with plastic wrap to cradle the embryo, a lid is placed on the cup, and the embryo is then placed in an incubator with appropriate humidity⁶. This set up however, can be technically challenging. The first challenge is the plastic wrap that is used to cradle the embryo. This wrap is difficult to work with and often does not adhere to the cup very well. To solve this problem, an elastic band is placed around the cup to hold the wrap in place. Despite this, the wrap can still slip, which is fatal to the embryo. The plastic wrap has the potential to tear or get punctured by forceps or needles that may be used during embryo manipulations and observations. Finally, this set-up is not very stable and students can easily knock the cups over. The height of the cups also makes it very difficult to place the embryo under a stereomicroscope, which has a limited objective to stage height. These challenges make it difficult for undergraduate students to work with live chick embryos in teaching labs, such as advanced developmental biology courses.

The above challenges in the ex ovo method has meant that researchers turn to the windowing method ^9,10 to view embryonic chick development. In this technique, a hole or “window” is made in the eggshell overlying the embryo. The hole can be re-sealed with tape or wax⁹ to allow for further embryonic development. Although the windowing method has some advantages, such as the ability to view embryonic development and easy maintenance, this method also has several limitations. The first is that the window needs to be fairly large in order to view the entire embryo (especially at late stages). Secondly, large windows are difficult to seal; an improper seal will lead to sterility and survivability problems. Using molten wax as a sealant adds another inconvenient and messy step to the protocol. Therefore, although the windowing method may be ideal for chick embryos at young stages (HH 11 – HH 27), viewing the entire embryo at late stages is not easily accomplished.

Here, we describe an improved and simple ex ovo culturing technique¹¹ that avoids the need for high tech equipment, is easy to handle under a stereomicroscope, gives the embryo enough support to perform microscopic manipulations, and enables researchers to view the growth of the embryo in its entirety well into the later stages of development (up to HH 40-41). With these advances in the ex ovo technique, individuals gain access to a more complete understanding of embryonic development. For instance, growth into later stages allows individuals to observe developmental processes that do not occur until this time point, such as ossification, feather development, and advanced limb and eye development. The entire embryo and extraembryonic membranes and vasculature are clearly visible. More advanced research can also be performed, such as, embryonic manipulations (i.e., implanting beadssoaked in inhibitors or inserting barriers between tissue layers), and researchers are then able to observe the effect of the manipulations in later stage embryos.

Protokół

Note: All supplies are listed in Table 1.

1. Storing the Chicken Embryos

1. Incubate chicken eggs of the strain Gallus gallus horizontally at 37^oC with approximately 40% humidity and turn eggs once or twice daily. Turning eggs is important to prevent the embryo from adhering to the eggshell.
2. Do not to turn the egg in the 24 hr prior to setting up the culture as otherwise the embryo will be located ventral to the yolk mass and will be damaged on opening the egg in step 3. In addition, keep the eggs at 4^oC degrees for no more than one week before incubation to “halt” development but this is not ideal.

2. Staging Chicken Embryos

1. Stage the chicken embryos using the Hamburger and Hamilton¹² staging table. The ideal stage for setting up the ex-ovo culturing is HH stage 19-20 (around 3- days of incubation) because this is shortly after the head turns at 53 hpf.
   Note: HH stage 19-20 is characterized by the following morphological traits: somites are extended into the majority of the tail, however the very end of the tail remains unsegmented, the tail bud is curled, the allantois is small and has limited vasculature, the leg-buds are larger than the wing-buds and the eyes are unpigmented or have a grayish hue.

3. Removing the Embryo from the Shell

1. Before removing the embryo from the shell, spray the shell with 70% ethanol and allow it to dry. Do not turn the egg rapidly as this will damage the embryo.
2. Being careful not to alter the orientation of the egg, carefully crack the egg on the lower side (i.e., the side that was ventral during incubation) and release the embryo into a sterile weigh boat (88 x 88 x 23 mm). Wipe down weigh boats with 70% ethanol. Inspect the embryo to ensure that the yolk sack is not damaged. If the yolk has broken, discard the embryo, as it will not survive.
3. Observe the embryo to ensure that it is viable; the heart is beating, vasculature appears normal, and no obvious abnormalities are present. Ensure that the edges of the weigh boat are dry.
4. Using a pipette, drop 40 μl of penicillin/streptomycin (5,000 units penicillin, 5 mg streptomycin per ml) on top of the albumin to help prevent infection.

4. Preparing the Humidity Chamber

1. Place a small stack of kim-wipes and/or an absorbent pad made of cotton in the bottom of a sterile plastic container (12 x 12 x 6 cm) wiped with 70% ethanol.
2. Add sterile, distilled water to moisten the kim-wipes and/or cotton (approximately 150 ml of water).

5. Assembling the Ex Ovo Culture

1. Place the weigh boat containing the embryo on top of the moist padding. Then, place half of a square sterile petri-dish (9.5 x 9.5 cm) on top of the weigh boat to form a loose lid.
2. Cover the plastic container with its lid. Press down two corners of the lid, ensuring a partial seal that still allows for good airflow inside the chamber.
3. Carefully place the setup into the 37^oC incubator until desired stage.
4. Place containers of water in the incubator to help control the humidity, if a controlled humidity incubator is unavailable. Sterilize all water in the humidity chambers and in the incubator and replenish as needed during the incubation period. 40% humidity is ideal.

Wyniki

This ex ovo method allows for the observation of embryos from early stages of development (HH 19/20) to late stages of development (HH 40-41) (Figure 1A and 1B). Setting up the culture at HH 19-20 increases survivability of the embryos in the culture. Prior to the head turning (before 53 hpf) survivability is very low in culture and after stage 21, the embryo tends to stick more to the shell on removal so fewer intact embryos are obtained. In general, survivability of the embryo...

Dyskusje

Ex ovo culturing and windowing both have advantages and challenges. Here we compare the advantages and challenges of the Styrofoam cup ex ovo method and the windowing method to our optimized ex ovo method shown here. Our method enables manipulation and easy observation of the chick embryo at late stages of development and our refinements to the traditional ex ovo method^{1, 2, 3} make it additionally very easy to use in undergraduate teaching laboratory classes.

Materiały

Name	Company	Catalog Number	Comments
Penicillin/Streptomycin	Sigma	P4458	Make small aliquots to avoid freeze/thaw events
Square Petri Dish			9.5 cm x 9.5 cm
Weigh Boat	Fischer Scientific	8732113	88 x 88 x 23 mm
Ziplock container	Ziplock	N/A	12 cm x 12 cm x 6 cm