Embryonic Anatomy and Physiology

Practical organiser - Prof Keith Brennan (email: [email protected])

 To investigate the effects of temperature on heart rate

 To observe embryonic structures, particularly the somites

Learning outcomes

By the end of the practical, students will hopefully:

1. Appreciate the utility of embryos for the study of anatomy and physiology

2. Recognise the advantage of having an accessible embryo in an egg for studies of development

3. Appreciate the dynamic nature of development

4. Understand how heart rate is regulated and how external factors can affect it

Risks Associated with Practical

Eggs have the potential to be contaminated with Salmonella. Wear gloves throughout and students should wash their hands before leaving the lab.

Dissection implements are sharp, so students should take care not to cut themselves or other students.

Students should wear a labcoat to protect themselves from egg splatter. 1. INTRODUCTION

Adult anatomy and physiology, in all their complexity, are derived from a single cell through the process of development. Development is largely a matter of self organization for embryonic cells, which must decide where to make a heart, a brain, two eyes and so forth, with little or no help from outside.

The embryos of different vertebrate species appear very similar to one another at early stages of development, and then diverge as development proceeds to make a flamingo or a python or a gorilla. This early stage at which embryos are similar to one another is termed the ‘phylotypic stage’ and it indicates that early events in embryo formation are similar between different vertebrate species, including humans. This is very useful for researchers as it means that a variety of organisms can be used as models of embryonic development – they illustrate general principles of vertebrate development, not merely the development of their own species. At the phylotypic stage, the embryo lays out the basic vertebrate body plan: heart, head, eyes, vertebrae and spinal chord; and leaves the species-specific details, such as scales, feathers, hairs, glands, beaks, lips and teeth, until later on. This early similarity between embryos was recognized in the early 19th Century (e.g. von Baer’s law, 1828; ‘The general anatomical characters of the group to which an embryo belongs appear in development earlier than the special characters’), and recent molecular studies have confirmed that the same genes act to regulate this stage of development in different species.

Chicken eggs as a developmental model A particularly convenient system in which to study vertebrate embryonic development is the chicken egg. Studying embryos in eggs does not require removing them from a mother, in contrast to the study of mammalian embryos, and chicken eggs are plentiful. In addition, it is possible to open a ‘window’ in an eggshell, observe and manipulate the embryo, and then allow incubation resume to see what the effects of the experimental manipulation were.

While many interesting experiments can and have been done using chicken embryos, the time constraints of a short practical mean that students will only be able to focus on a couple of processes: embryonic morphology and heart rate. For this practical, 3 day old chick embryos will be used. Somites are forming at this age and the heart is beating and is at about the same stage of development as a human embryo 5 weeks after conception.

Somites Many animals have body plans that are organized around repeating segments along their length. These segments are most obvious in creatures that have exoskeletons, such as insects, though vertebrates have repeating segment units too. Our most obvious segments are the stack of vertebrae that make up the spinal column and that give the ‘vertebrates’ their name. The vertebrae are derived from temporary embryonic structures called somites. Somites form in pairs along the length of an animal from the head to the tail. They are easily seen as blocks of tissue, and go on to produce not only the vertebrae, but also make muscles of the trunk and limbs and the dermis of the skin. Figure 1. Stained chick embryo

Heart Unlike manufactured objects, such as microscopes or computers, an embryo has to function before it is fully made. While the embryo is very small, simple diffusion is sufficient to carry gasses and nutrients to and from all of its cells. As it grows, however, it reaches a size at which diffusion alone is no longer enough, and so circulation of blood is needed. This requires a functional heart and a reasonable network of blood vessels. Thus, the heart starts working early in development, and is usually the first organ to function – beating and pumping blood even before it is fully formed. The importance of the heart during development is highlighted by the fact that cardiac malformations are estimated to lead to the death of 5-10% of human embryos, and heart rate is an important diagnostic indicator during pre-natal screening.

The embryonic heart is quite easy to see in fertilized eggs at early stages of development, with no requirement for specialised equipment. This allowed chicken heart development to be studied even by the Ancients (“the heart appears, like a speck of blood, in the white of the egg. This point beats and moves as though endowed with life”, Aristotle, Historia Animalium ~340 B.C.). Heart cells (cardiomyocytes) will beat ‘as though endowed with life’ even without being a part of an embryo. Stem cells grown in vitro that are persuaded by various treatments to become heart cells are found to beat of their own accord. This makes sense – as the heart is the first organ to function, it cannot rely on instructions from other organs for its regulation.

Biological material Students will be supplied with fertile eggs, most of which will contain chicken embryos that have been developing in an incubator for 3 days. The only functional organs in these embryos are the heart and circulatory system; the brain and nervous system are several days away from being able to sense anything, including pain. In recognition of this UK law (ASPA 1986) protects vertebrates after their nervous system has formed. Practically, this happens around half way through their incubation or gestation. Consequently, as chickens hatch after a 21 day incubation, the specimens used in this practical are not capable of perceiving pain based on their neuroanatomy and are not protected by UK law.

Purpose of the practical The overall aim of this practical is to determine how the external environment regulates embryonic heart rate and to examine embryonic development. In the first part of the practical, the effect temperature has on heart rate will be examined. In the second half of the practical, Indian ink will be used to reveal the morphology of the early chick embryo. 2. EXPERIMENTAL PROCEDURES

A. Opening an egg and determining heart rate

1. Incubate eggs at 38.5°C for 3 days to allow development to proceed until the heart and somites form.

2. Split the eggs into three groups and transfer to 20°C (room temperature), 30°C and 40°C at least two hours before the practical to equilibrate to the new temperature

3. Collect an egg and a heavy and a fine forceps.

4. Put the egg into a small beaker with the blunt end up (and so the pointed end is down).

5. Use the heavy forceps to tap a hole in the top of the egg, be careful not to push the forceps too far into the egg.

6. Use the fine forceps to pick bits of shell out. You will see an air space below the hole and the yolk below that.

7. If you do not see the embryo (a ring of blood vessels should be visible) then gently swirl the egg so that it floats up to the top of the yolk. If this doesn’t work you will need to take another egg.

8. Use the fine forceps to break off pieces of shell down to the yolk so that the embryo (visible as a ring of blood vessels) is exposed and the rim of the shell is just above the surface of the egg white or albumen.

9. The inside of the shell is covered by a translucent vitelline membrane which you should peel off.

10. You may be able to see the heart beating without magnification, if not then put the egg under a magnifying glass and heart rate can be determined by counting the beats over a 15 second period and multiplying by 4 (giving beats per minute).

B. Viewing embryonic morphology, including the somites

1. Make a 1:10 dilution of Indian ink in PBS (conc) and draw the solution up into a 1 ml syringe fitted with a 23G syringe needle.

2. Open an egg as described above. Once the shell has been removed down to the level of the yolk and the vitelline membrane has been removed, slide the syringe needle under the embryo. It is easiest to insert the syringe needle vertically at the edge of the egg initially and then rotate the needle until it is almost horizontal using the edge of the egg shell as a support. The tip of the needle should end up just below the embryo in the centre of the egg. 3. Slowly inject the Indian ink solution to reveal the embryo and view under a dissecting microscope.

4. It should be possible to identify the eye, heart and forming somites, along with vitelline artery and vein. The somites are regular blocks of tissue lined up in pairs along most of the body posterior to the head.