Learning Objectives:
- Recognize the parts of an amniote egg.
- Recognize and know the function of the four extra embryonic membranes.
- Understand the similarities and differences between cleavage in the chick embryo and the amphibian embryo.
- Understand the similarities and differences between cleavage in the chick embryo and the amphibian embryo.
- Understand the process of cleavage and gastrulation in a chick embryo.
- Recognize the structures present in the 24 hour chick embryo.
- Understand how the central nervous system beings to develop in vertebrates using the chick as an example.
- Understand how the cardiovascular system beings to develop in vertebrates using the chick as an example.
- Understand how the gut beings to develop in vertebrates using the chick as an example.
- Understand what structures arise from the endoderm, ectoderm and mesoderm in vertebrates using the chick as an example.
- Record and interpret results of an experiment.
• In the this lab we will look at early stages of vertebrate development using the Chicken as a model genetic organism.
• We will start be familiarizing ourselves with the chicken egg.
• After we are familiar with the egg we will look at the early stages of development up to 24 hours after the egg has been laid.
• By 33 hours of development in the chick (or 4 weeks of human development) there are two major organ systems that have obviously begun to develop; The central nervous system (CNS) and the cardiovascular (CV) system.
• We will look at the development of the CNS from the ectoderm.
• Then we will discuss the development of the CV system in the chick embryo.
• We will discuss additionally structures, such as the gut, obvious in the 33-hour chick embryo along with what they will give rise to in the adult.
• Finally, we will look and interpret the results of experiments applying a variety of chemicals to the newly developed heart.
• Chickens, like all birds, reptiles and mammals have embryos that are amniotes. This means they have an extra-embryonic layer (the amnion) that protects the egg from dehydration.
• All amniotes have 4 extra-embryonic layers called the chorion, amnion, allantois and yolk sac.
• Evolutionary these 4 layers first appeared in early reptiles. This freed vertebrates from developing in water (like the amphibian and brine shrimp eggs we looked at in the last two labs) allowing then to lay eggs on land.
• These eggs are classified as macrolecithal meaning they have a large amount of yolk.
• The yellow yolk is surrounded by a thin yolk cell membrane.
• Outside the yolk cell membrane is the vitelline envelope which protects and gives shape to the egg yolk and separates it from the albumen (egg white).
• The egg also contains a with watery albumen (aka - egg whites).
• The yolk provide enough nutrients until hatching in about 21 days.
• Notice that the chicken eggs in Figure 6.1 have one blunt end that marks an airspace. Look for this the next time you see an egg. When you crack an egg open look for the air space at the blunt end.
• When eggs are first produced in the reproductive tract of the hen (female chicken) they do not have a shell and are no where near the size they are when they are laid. Look at Figure 6.2 to see how the egg enlarges before it is laid.
• Let's first look closer at the shell which is added to the developing egg just before it is laid.
• The shell has been carefully crafted during evolution to provide protect the embryo from physical damage while still allowing for gas exchange.If you look close you will see the shell is porous to allow for gas exchange.
• The shell itself is composed of 3 layers. We will start from the outside and work our way in to the center of the egg (FIGURE 6.3).
• The outermost layer of the shell is the cuticular membrane. Before the egg is laid this layer is wet and slippery and helps with oviposition (egg laying). Once the egg is laid it appears thin and shiny. It consists of glycoproteins and serves to protect the egg from drying out and against invading microbes.
• The next two layers of the shell contain calcite (calcium carbonate). 98% of the egg shell is calcium. The hen gets the calcium to make the shell from her diet. If there is not enough calcium in her diet she will get it from her bones (your mom did this for you, not to make an egg shell, but so you could make your bones).
• The outer calcite containing layer is the spongy layer (aka the crystalline layer).
• The inner most calcite containing layer is the mammillary layer.
• Both of these layers give the egg its strength.
• Just inside the shell are is the shell membrane. In the chicken egg there are two shell membranes, an inner and an outer shell membrane. These provide protection from drying out and from invasion by microbes.
• Now let's look more inside the egg.
• As we work our way towards the center of the egg we next encounter the albumen (egg white)
• Notice various viscosities of albumen
• Around the yolk is a narrow band of thick albumen and external layer of thin albumen.
• Albumen is 88% water . This is major source of water for developing chick.
• The rest of the albumen is mostly glycoproteins, mostly ovalbumin.
• There are also some proteins are antimicrobial. These include:
1. Lysozyme which kills bacteria by disrupting bacterial cell walls.
2. Ovotransferrin which binds iron preventing bacteria from using it.
3. Avidin which binds the vitamin biotin. These cause these nutrients (needed for bacterial growth) to be unavailable to bacteria
4. Ovomucin and 5. Crystatin which are antiviral.
• Additionally the high pH of albumen also inhibits bacterial growth
• Notice that part of the albumen is very dense and coiled into 2 cordlike calalaza that are tightly attached to the yolk membrane.
• These suspend the yolk in the middle of albumen and allow it to rotate so that it always orienting to gravity so animal pole is up.
• Now look at the yolk itself. That is one giant cell! It is surrounded only by a delicate cell membrane and a vitelline envelope.
• It appears yellow because it contains carotenoids (a precursor of vitamin A). The carotenoids come from the hens diet and are added by the hen before the egg is laid. Because more carotenoids are added during the day, while she is eating than at night while she is sleeping, the yolk appears as concentric rings of dark and light yolk.
• The yolk is rich in nutrients that the embryo will need to survive until it hatches in 21 days.
• There is only a small puddle of cytoplasm in the yolk. This is called the blastodisc. This tiny puddle will become the future chick.
• Only the central region forms the body of the embryo.
• Other regions form the extra embryonic membranes (Figure 6.4)
• The Amnion protects embryo from drying out and cushions against mechanical damage.
• The Chorion protects and nurtures the embryo. Where the chorionic is attached to the allantois it allow gas exchange
•The Allantois stores nitrogenous waste and, where it attached to chorion, provides a respiratory surface. It can also absorb Calcium from the egg shell
• The Yolk sac absorbs nutrient rich yolk and transports it to embryo via the vitelline blood vessels.
• Unlike the sea urchin and frog, chickens have internal fertilization.
• This means it is not easy to see the early stages of development in the chick. The early stages occur while the egg is still in oviduct.
• Experiments have shown that cleavage divisions are meroblastic meaning that cleavage is partial due to the presence of the thick yolk (Figure 6.5). Most of the bulk of the chicken ovum is not cytoplasm. Instead it is spheres of yolk lipids in a sea of yolk proteins,
• Yolk is so disproportionately abundant compared to the cytoplasm.
• The cytoplasm is displaced to the animal pole and sits in a puddle on top of the yolk.
• This tiny area, called the blastodisc, will become the chick.
• The fertilized egg already contains in its cytoplasm all the germ-layer components.
• Cleavage divides the single cell into many smaller building blocks that can be rearranged and molded into a multicellular organism (Figure 6.5).
• The yolk is so dense that it completely impedes cleavage furrow (recall yolk slowed it down in the frogs)
• Cleavage is partial (meroblastic) and the cleavage pattern is discoidal (restricted to a circular disc of cytoplasm, the blastodisc)
• The cleavage furrow cuts down the animal pole toward the yolk and stop at the yolk.
• The cells are called blastomeres (just like in all the other embryos we have looked at).
• The center of blastoderm cells become separated from yolk by a space called the subgerminal cavity (Figure 6.7)
• This space makes this central area appear lighter and more translucent so it is called the area pellucida.
• The surrounding area of blastoderm, still connected to yolk looks darker and opaque and is called the area opaca.
• The movement of the cells here is very different from the movements we saw in the amphibian and sea urchin embryos. In those embryos we had a ball of cells (A blastula).
• Here we have a flat sheet of cells called a blastoderm.
• Regardless of how the cells move, at the end we will once again have 3 germ layers with an outer ectoderm (will give rise to the epidermis and nervous system), a middle mesoderm (will give rise to muscle, the skeleton, the dermis , circulatory system and kidneys) and an inner endoderm (will form the lining of the gut, pancreas and liver).
• Gastrulation in the chick starts with the lower layer of cells in the blastodisc losing their affinity for the cells above them. This is accomplished by a change in their cell surface adhesion molecules. This process is called delamination.
• As a result of delamination we have two sheets of cells. There is an upper epiblast and a lower hypoblast. Between them is a space called the blastocoel (Figure 6.7)
• The upper epiblast at this point contains presumptive ectoderm and mesoderm and still some endoderm.
• The hypoblast contains both extra embryonic endoderm that will participate in forming extra embryonic membranes and presumptive endoderm which will become part of the embryo.
• Presumptive endoderm still remaining in the epiblast will also contribute to the embryo but must first join the hypoblast before doing so.
• Embryonic endodermal cells still in the epiblast move to the hypoblast by moving to the center and then down to join the hypoblast.
• The line along which cells are moving is called the primitive streak.
• The presumptive mesodermal cells enter primitive streak, next but they don’t move to the hypoblast. Instead they make a U-turn and move between epiblast and hypoblast.
• The Primitive streak is similar the blastopore in frogs embryos. At the the top of the primitive streak, where the notochordal cells are ingressing and migrating forward is Hensen's node.
• Hensen’s node is analogous to the dorsal lip where the notochordal cells converge and ingress and migrate forward rather than laterally to form a streak of notochordal cells down the midline.
• Note that gastrulation begins in anterior region first and moves progressively posteriorly.
Anterior regions may have finished gastrulation and gone on to organ formation while the posterior region is still gastrulating.
• This anterior- to- posterior wave of progression results in the primitive streak becoming confined to ever more posterior regions and is often referred to as regression of the primitive streak.
• This means embryo has been incubating for 24 hours after being laid.
• It has been developing longer than this. (You looked very much like this at ~3 weeks of development)
• Look at whole mount in Figure 6.9. The cellular region has been removed from yolk and mounted on a slide.
• The embryos are transparent so all levels can be seen by focusing up and down through the embryo.
• By now the anterior half is undergoing neurulation, while posterior half is still gastrulating
• The Area opaca has greatly expanded and now occupies too large an area to be mounted on your slide and much has been trimmed away
• 2 subdivisions of area opaca can be seen; the area vitelline where cells contain many yolk granules (vitelline = yolk) and the Inner area vasulasa where blood islands are forming. Masses of blood-forming cells that will condense to form capillary network that will bring yolk nutrients to embryo
• The Area pellucida looks like a footprint with the Proamnion just in front of embryos head. This particularly clear area (nothing to do with amnion)
• It appears more translucent because it consists of only ectoderm and endoderm. The spreading mesoderm has not yet reached this area.
• Notice the folding and pinching at anterior end. These are these are the head folds where the anterior fold has undercut developing head and raised it above the levels of the blasotoderm.
• The neural folds can be seen medial to the head folds. The neural folds converge and fuse at the midline to form the neural tube.
• Notice that head fold has caused the endoderm to fold into a closed tube to from the foregut.
• A very noticeable feature is the invaginating neural ectoderm which was previously an open neural plate, but now, the anterior region has folded upward to form the neural tube.
• At the anterior tip of the embryo notice neural plate does not close completely, but leaved an open channel – anterior neuropore which will close by 36 hours.
• Sometimes neural folds in spinal region fail to close this is called spina bifida.
• Look at open midgut region and notice the mesoderm has segmented into blocks of somites on either side of the neural tube.
• As the embryo develops more somites form posteriorly from unsegmented somite mesoderm.
• Somites will form axial skeleton (vertebra and ribs), muscles of axial skeleton and limbs and dermis.
• Between the two rows of somites in the midline underneath the neural ectoderm notice a streak of condensed tissue. • This is the notochord.
• At the posterior end notice a primitive streak, a linear band of thickened epiblast.
• Anterior to the primitive streak is Hensen’s node, a group of cells that act as the organizer in the chick embryo and through which gastrulating cells migrate anteriorly to form tissues if the future head and neck.
• The posterior location of Hensen's node and the primitive in the 24-hour chick embryo shows that ingression is still occurring in this region.
· The ectoderm above the notochord rises up to form the neural plate, which folds inwards to form the neural folds.
· The neural folds fuse at the midline forming the neural tube.
· The neural tube will become the CNS (brain and spinal cord).
· Once the neural tube forms anteriorly (it is not completely closed yet, anteriorly it is still open at the anterior neuropore) it begins to swell to form three primary vesicles (Figure 6.9).
· From anterior to posterior the primary vesicles are the; prosencephalon (forebrain), metencephalon (midbrain), and the rhombencephalon (hindbrain).
· Two of the primary vesicles then subdivide. One remains undivided to form five secondary vesicles.
The prosencephalon divides to for the telencephalon and diencephalon.
· The rhombencephalon divides to form the metencephalon and myelencephalon.
· The mesencephalon remains undivided
· That is a lot of encephalons (which means brains) that start with the letter “m”. Fortunately, the order is easy to remember since they appear in alphabetical order!
· The telencephalon (at this point any area of the brain anterior to the optic vesicles) will later become the bilobed cerebrum.
· In the chick this area will contain olfactory receptors for the sense of smell. In humans it is an area for higher thinking.
· The diencephalon is complex. It evaginates later in development to form a variety of structures.
· The lateral walls of the diencephalon evaginate to form both the thalamus (a region of sensory integration) and the optic vesicles which will later invaginate to form the optic cups (Figure 6.10).
o The inner surface of the optic cups give rise to the neural retina.
o The outer surface of the optic cups give rise to the pigmented retina.
· The roof of the diencephalon evaginates to form both the epiphysis which later becomes the Pineal gland. This gland sets diurnal rhythms by secreting melatonin. The roof also forms the anterior choroid plexus; a highly vascular area that secreted cerebral spinal fluid into CNS.
· The floor of the diencephalon evaginates to form both the infundibulum (which later becomes the posterior pituitary gland (or neurohypophysis) and the hypothalamus.
· The hypothalamus sends hormones to the posterior pituitary gland including antidiuretic hormone (ADH) and oxytocin.
· The Mesencephalon (Midbrain) is an oval shaped structure that serves to process data from the eyes and the ears. The Mesencephalon appears as a primary vesicle and remains undivided as a secondary vesicle.
· The Metencephalon (formed by the subdivision of the Rhombencephalon) forms two structures in the adult brain. Dorsally it forms the Cerebellum (coordinates stimuli about body position. Ventrally the Metencephalon forms the Pons (shunts information between the Cerebellum and the Cerebrum).
· The Myelencephalon, also called the medulla oblongata, contains a series of enlargements, called neuromeres, which are associated with specific motor and sensory neurons.
· In the epidermis, in the area of the myelencephalon, Otic placodes form on either side of the head. These will later become the inner ear.
· The remainder of the neural tube will become the spinal cord.
· The heat arises from an area in the mesoderm appropriately called the cardiac mesoderm.
· When the heart first forms it appears as a tube with two swellings (Figure 6.11). Your heart once looked like this too.
· Later this tube will undergo a series of movements and will fold on itself. The 2 sides will fuse to form a heart with four chambers.
· At this point the heart consists of two chambers; an anterior ventricle and a posterior atrium.
· If you know your adult heart anatomy you will know this is upside down! After the morphogenic movements the atria will be located anteriorly, and the ventricles will move to the posterior.
· The heart will start beating at about 48 hours of development once the vascular system is complete.
· Once the heart starts beating blood, rich in nutrients from the yolk, will be carried by the Vitelline veins from the extraembryonic regions to the Sinus venosus.
· The sinus venosus later shrinks to because the sinoatrial (SA) node and acts as the pacemaker for the heart.
· Blood flows from the sinus venosus into a single, still undivided atria and then into the undivided ventricle.
· From the undivided ventricle blood enters into the Bulbus cordis.
· The Bulbus cordis later forms the base of the 2 largest arteries in the body; the aorta and the pulmonary trunk.
· Blood leaves the Bulbus cordis via paired ventral aortas and then into paired dorsal aortas.
· The gut will form from a combination of the endoderm and the mesoderm.
· The endoderm forms only epithelial tissue lining the inner portion of the gut organs.
· The mesoderm surrounding the endodermal tube forms the muscle and connective tissue layer of these organs.
· Let’s start by looking at the endoderm.
· The gut tube forms as the head folds and lateral body folds lift up the embryo and then meet in the foregut and hindgut regions and fuse at the dorsal midline
. At the anterior end of the tube the foregut forms the pharynx.
· The pharynx gives rise to the esophagus and a number of outpocketings and invaginations.
· In this region two ventral evaginations form. The anterior of these two evaginations gives rise to the thyroid gland while the posterior evaginations forms the lungs.
· A lateral evagination becomes the pharyngeal pouches that later form the estuarian (auditory) tube and middle ear cavities well as the epithelium lining the tonsils, thymus, parathyroid, and ultimobranchial body.
· The posterior region of the foregut forms the lining of the stomach.
· Between the foregut and the midgut lies the anterior intestinal portal.
· The midgut is still open and attached to the yolk sac via the vitellointestinal duct.
· The midgut will later for the small intestine.
· Between the open midgut and the hindgut lies the posterior intestinal portal.
· The hindgut will give rise to the Colon (large intestine) in the adult.
· Now let’s turn our attention to the more complex mesoderm.
· As previously mentioned, the heart will arise from the mesoderm from a region called the cardiac mesoderm.
· Located ventral to the neural tube at the midline of the embryo is a dark streak called the notochord. This structure is found in all chordates.
· The notochord acts as a primary organizer. It induces and initiates an axial organization in the organism.
· The notochord provides axial support for the embryo until the vertebra form and take over this function.
· In the adult the notochord will become the gelatinous center of the intervertebral discs called the nucleus pulposus.
· Lateral to the notochord are the somites.
· Somites appear as blocks of tissue on either side of the notochord. They appear in pairs and are often used to help determine the age of the embryo.
· The somites are subdivided into three parts; a sclerotome, a myotome and a dermatome.
· The sclerotome gives rise to the ribs and the vertebra.
· The myotome gives rise to the skeletal muscle of the back and the limbs.
· The dermatome gives rise to the dermis.
· Lateral to the somatic mesoderm lies a streak of nephrogenic mesoderm.
· The nephrogenic mesoderm gives rise to the kidneys, urogenital ducts and the gonads.
· Next to the nephrogenic mesoderm, on either side of the embryo lies the Lateral Plate Mesoderm which delaminates into two layers; The somatic and splanchnic layers. A cavity, called the coelom, forms between these two layers (Figure 7.5).
· The Somatic Lateral Plate Mesoderm associates with the epidermal ectoderm above it. Together they are referred to as the somatopleure which will later form the body wall and in the extra-embryonic region the amnion and chorion.
· The Splanchnic Lateral PlateMesoderm associated with the underlying endoderm and together they are called the splanchnopleure.
· The splanchnopleure gives rise to the gut wall (smooth muscle and connective tissue) and in the extra-embryonic region the allantois and yolk sac.
· This unique set of cells appear only in vertebrates.
· They first appear on the crest of the neural tube as it closes and soon migrate to form a wide range of cell types including melanocytes, membranous bones of the head, the adrenal medulla, sympathetic ganglia of the autonomic nervous system and smooth muscle.
· In vertebrates that have teeth (not birds) they also produce the dentin secreting cells of the teeth.