LAB 10 

            CHICK HEART DEVELOPMENT 

I. Introduction 

In the chick, early development (cleavage) occurs before the egg is laid, but for matters of convenience the age of the embryo is usually described as so many hours of incubation at 37.5^oC. The heart of the chick embryo develops from the fusion of paired precardiac mesodermal tubes between 24 and 29 hours of incubation. The cells that compose these tubes have migrated over long distances to the heart-forming area.  

As the single heart tube fuses, and before the actual circulation of blood commences, the fused ventricular portion begins to pulsate (at about 29 hours of incubation). As fusion continues the atrial portion of the heart is incorporated, and the rate of the heart beat increases because of the controlling cells of the atrium (33 hours). Further fusion of the tubes continues as the heart becomes twisted in an S shape, and by 44 hours the sinus venosus is incorporated, and again the heart-beat rate changes.  

Following completion of fusion further changes in shape (morphogenesis) occur, transforming the simple S-shaped tube into a four chambered heart. Development of cells, tissues or organs in the living organism is called in vivo development, while that in culture is called in vitro development. In an in vitro situation one can study the influence of chemicals and other living tissue on one particular tissue or organ when it is isolated from the influence of the body as a whole.  

In explanting the whole organ (heart) primordia and dissociating the heart cells of the chick we are using the methods of organ and cell culture respectively. It has taken many years of experimentation with growth requirements to establish the type of medium that will supply a growing portion of an organism with all the requirements so that it can grow independently of the whole organism. The objectives of today's lab are as follows: 

1.         To become familiar with the structures visible at different stages of chick development. 

2.         To determine the heart structures, direction of blood flow, and heart rate of a 48, 72  and 96 hour old chick embryo (rotate eggs daily). 

3.         To use sterile techniques for cell and organ culture procedures. 

4.         To answer the developmental questions posed in the exercise. 

 

II. Procedure 

A. Review of Heart Structure  

Examine your whole mounts of the 24, 33, 48, 72 and 96-hour chick embryos using a dissecting microscope (which is what you will use to observe living embryos). The living chick you will be observing is at about the 55, 72 and 96-hour stage. Study the 48-hour whole mount carefully. Notice that the embryo is growing on top of the yolk. The yolk sac which is composed of all three germ layers; ectoderm, mesoderm and endoderm (now only a small disc).  It seems like living embryos of the classical commercial stages are only obtained after longer incubation periods, for example:

                              Commercial Stage                            Incubation Time at 38C

                                     33 hours                                               38-42 hours

                                    48 hours                                               55-60 hours

                                    72 hours                                               75-82 hours

 

The developing embryo is now turned on its side. It is made of a tubule of endoderm (gut), surrounded by mesoderm, which is in turn surrounded by ectoderm. The nervous system has already begun to develop from folding of the neural plate. It is composed basically of a long tube with the brain anterior and the spinal cord posterior. The developing heart (mesoderm) is prominent. It has developed from a tube and now takes on an S-shape configuration.

 Blood flows in living embryos from the yolk sac to the sinus venosus, atrium, ventricle and aortic arches to the whole body and out to the yolk again, thereby transporting food and oxygen and disposing of waste material. Blood flow to and from the yolk sac can be observed in the arteries and veins of the yolk. The amnion, chorion and allantois have not yet formed. Notice the developing somites lateral to the spinal cord. Identify the main features of the 48-hour chick so that you will be familiar with them in the living specimen.

 B. Sterile Techniques 

Aseptic or sterile technique is the execution of microsurgery or tissue culture procedures without introducing contaminating microorganisms from the environment. In doing tissue culture work, 70% of the problems are due to a lack of good sterile technique.  Microor­ganisms causing the contamination problems exist everywhere, on the surface of all objects and in the air. A conscious effort must be made to keep them out of a sterile environment. Because many and sometimes awkward manipulations are required for various techniques, tissue culture media used are often supple­mented with antibiotics. 

 Antibiotics do not eliminate problems of gross contamination, which result from poor sterile technique or antibiotic-resistant mutants.  Autoclaving renders pipettes, glass­ware, and solutions sterile. Nutrient medium cannot be autoclaved.  The compounds in nutrient medium are destroyed by the heat of autoclaving.  Medium must therefore be sterilized by passing it through a sterile filter small enough in pore size to hold back bacteria and mycoplasmas (Millipore Sterivex - GS 0.22u disposable filter units). Here are some rules of thumb to follow to keep your medium, cultures, and glassware from becoming contaminated:

 1.         Wipe your work area and hands with 70% ethanol before starting. Ideally this work is done in one of the department’s two laminar flow hoods. Because of the number of students in lab, work often must be done on the open lab benches, much as the early experimental embryology was done.

 2.         Never uncover a sterile flask, bottle, petri dish, etc., until the instant you are ready to use it.  Return the cover as soon as you are finished.  Never leave it open to the environment.

 3.         Sterile pipettes should never be taken from the cylinder or wrapping until they are to be used.  Keep your pipettes at your work area.  Sterile pipettes do not have to be flamed.  Pipetting your cells with a hot pipette will kill them.

 4.         When removing the cap from a bottle, flask, etc., hold it down, grasping it with the little finger of your right hand is convenient once you get the hang of it -- do not place the cap on the lab bench. Do not hold the opening straight up into the air.  If possible, tilt the container so that any falling microorganisms fall onto the lip.

 5.         Be careful not to talk, or shake your head allowing cells, spores, exfoliating epidermis to contaminate your sample when you are per­forming these sterile procedures.

 6.         Do not draw from a bottle more than once with the same pipette.  Because such a pipette has been exposed; the chance for contamination is too great; use sterile pipette each time -- especially when pipetting medium.

 7.         Techniques should be performed as rapidly as possible to minimize contamina­tion. 

 

You may find yourself involved in a procedure, which these sterile technique "rules-of-thumb" do not cover.  Therefore, you must constantly be aware that microorganisms are everywhere and take proper steps to keep them out of your cultures.  When first developing your aseptic technique you must always be thinking of sterility.   

Eventually it will become second nature to you.  Mastering good aseptic technique will save you considerable frustration in the labs to follow.  Furthermore, the same princi­ples for good aseptic technique also minimize biohazard risk to the investigator when infec­tious organisms or dangerous chemicals are used.

 

C. Observation of the Living Embryos 

Before you obtain your chick embryo, check all your equipment to be sure you have everything in readiness. Each group of students will be provided with four eggs at three different stages of development. The first, for gross observation and the practice heart explantation. The second will be used for the experimental heart explantation. Each pair of students should have sterile finger bowls, sterile petri dishes, watchmaker’s forceps, fine scissors, and 3 sharp glass or cactus needles in a Coplin jar with alcohol.  

Optional - During this period you should prepare your surgical glass needles. Break off the thin glass bridge at a point that is very fine but that still has rigidity. Another option is to prepare cactus needles as per directions of the instructor. Rinse your instruments in the alcohol to prevent contamination of your chick embryo. Take care not to touch the embryo with alcohol or get the alcohol near any open flames.  

Obtain a 48 and 72 hour old egg. Hold it in exactly the same orientation as it had in the tray. In subsequent experimental labs we will be making windows in the eggshell, in today's experiment we will completely remove the contents of the egg from the shell.  

To do this you will pour about 1cm of warm sterile Howard's Ringer (HR) solution, a physiological saline solution, into a fingerbowl. You can use sterile gloves or wipe your hands and the surface of the egg with EtOH, then crack the egg on the side of the bowl. Carefully lower the egg into the Ringer's solution.  

The  object is to not break the yolk. If the yolk breaks at this point, try to remove the vasculated area, that is the area with the blood vessels to a large petri dish filled with warm HR. If you did not break the yolk you should be able to see the heart beating using the dissecting microscope. If no embryo is visible, use your finger to try to flip or roll the egg over to expose the embryo. On your embryo:    

 

                        1. Identify the sinus venosus, atrium, and ventricle; 

                        2. Determine the place of origin of the heart beat; 

                        3. Determine the direction of the blood flow.

It is essential that you do not allow the embryo and yolk to dry out. Apply several drops of warm sterile HR as necessary. The area of the yolk covered with blood vessels is called the area vasculosa. At this stage of development the blood in the vessels picks up oxygen and food for the embryo and disposes of embryonic waste. As the embryo grows this area increases. Refer to your atlas for a diagram of the 48-hour chick. Use your light to keep the embryo warm, and continue to keep it moist with HR. Identify other prominent structures. Is the heart rate slowing down? If so, why?  

 

D. Explantation of the Heart Rudiments/Cell and Organ Culture 

Obtain a 35 mm culture dish with sterile culture medium. In most tissue culture work the scientist is greatly concerned about maintaining sterile conditions to eliminate contamination of her cultures. Prepare to remove the embryo including the area vasculosa to a sterile petri dish containing warm, sterile HR. This procedure should be practiced 2-3 times before actually performing the experiment.  

Carefully cut around the outside of the area vasculosa, gently supporting the embryo with a pair of forceps, or hold the embryo with a forceps placed across the body below the heart. Do not let the embryo drop down into the  yolk. When the circular incision is completed remove the embryo and the area vasculosa to the sterile HR in the petri dish. Try to carry over as little yolk as possible. Pull the embryo through the solution to free it of excess yolk. Turn the embryo dorsal side down, yolk side up. The heart should then be uppermost.   

An alternative method for removing the embryo is to cut out a life-saver shaped disk from filter paper and lay this disk on the area vasculosa. The hole in the middle should be approximately the size of the embryo and the entire disk should be slightly smaller than the area vasculosa. Make sure the filter paper is wet with sterile ringers. Hold on to an edge of the paper and cut along the outside. The embryo can be moved to a petri dish by transferring the entire filter paper disk.  

Dissect out the heart from above (anterior to) the truncus arteriosus (the main trunk vessel leaving the ventricle) and below (posterior to) the sinus venosus. This is best performed with glass or cactus needles and/or fine scissors and forceps. The easiest way to do this is to first sever the head portion of the embryo from the heart region. Then sever the posterior portion of the whole embryo from the heart region. Remove the heart region from the rest of the embryo*.  

Usually you will remove the heart and digestive tract. Then the heart can finally be freed from all other tissue. Place the heart (with a pipette) into the 35 mm culture dish. After careful observation (see below) cover and label your dish as to name, date and time. Place the culture dish in a moist environment in a 37.5^oC incubator.  

Optional - Repeat this procedure, excising the whole heart, and then cut it into three regions. The anterior region can be distinguished from the posterior if the heart is beating. If it does not beat at first, allow time for it to warm up. The beat in the whole heart begins at the sinus--thus the posterior end of the heart can be determined. Try to cut the heart into the three regions corresponding to the sinus, atrium, and the ventricle. Place the three regions of the heart into a 35 mm culture dish. You may find it helpful to mark off three pie-shaped regions on the underside of the dish and label A,V,S before you put in the pieces of the heart.                               

 

If the media and explants are warmed sufficiently by the light, and if you have not injured the heart tissue, the whole heart and heart fragments will begin to beat. Observe. Record the rate of beating. Do all the fragments beat at the same rate? Do they beat at the same rate as the whole heart in vivo?

If your techniques have been good, it is quite possible to keep the heart alive and beating for hours, days, and if fed, weeks. Make observations at 12 hour intervals. Using your experimental evidence from the organ culture of the heart, answer the questions at the end of this section.

For the cell culture procedure, remove 2-3 hearts using the procedures outlined above*. Place them in sterile ringers in a 35 mm petri dish. Then transfer the hearts into 2 ml of trypsin-EDTA and cut-up into small pieces. Observe under the inverted microscope. Now allow the trypsin to digest the heart fragments for up to 30 minutes in a 37.5C incubator.  Use a 27 gauge syringe needle to dissociate the embryonic cells. Disperse the cells with a sterile pipette, then add 6 ml of culture media to inactivate the trypsin and feed the cells. Incubate in a 37.5C incubator and observe at 12 hour intervals under the inverted microscope and record your results.  

* This procedure can be carried out for the whole embryo in addition to just for the heart.  

 

III. Data Analysis 

1.         Is the heart beat an intrinsic property of the cardiac muscle, or is it a result of nervous or hormonal signals? 

2.         (OPTIONAL - Does each section of the heart tube still have its own separate rate?)

 3.         OPTIONAL - Are the new heart-beat rates that appear during development the result of all the heart cells changing their rates, or is the new rate merely imposed by the new section of the heart that is being incorporated?

 4.         Is the further morphogenesis of the heart programmed into the tissue itself, or does the heart develop its form in conjunction with other structures of the body and the fluid dynamics of the   functioning circulatory system?  

 

VI. Materials 

1. Water bath set at 40^oC for Howard's Ringers

2. 6 l of Sterile Howard's Ringers in the water bath

3. 37^oC incubator with an open tray of water to maintain a high humidity

4.  sharpie marking pens

5. Sterile 100 and 35 mm petri dishes

6. 3 battery operated electric pipettes and thumb operated pipettes

7. Watchmakers forceps, regular forceps, and fine scissors

8. Coplin jar filled with 70% alcohol and cotton on the bottom - DANGER - KEEP

   ALCOHOL AWAY FROM OPEN FLAMES!

9. Glass, cactus or tungsten dissecting needles, small (000⁺) insect pins

10. 48, 72 and 96 hour old live chick embryos

11. Sterile filter paper and pasture pipettes and bulbs

12. 1% solution of Neutral Red

13. T-25 tissue culture flasks

14. Enzyme: Trypsin-EDTA (1X) (Sigma T5775)

15. Several tins of Sterile 1 and 10 ml glass pipettes

16. Antibiotics: Pen Strep Concentrate (Sigma P0906)

17. Small insect pins, wooden sticks and dental wax to mount needles

18. Inverted phase-contrast microscopes