Lab 6
Phylum Chordata and Embryonic Development

    The animals belonging to phylum Chordata, although a very diverse group, share certain essential characteristics.  Although these characteristics are often not apparent in adult forms, they are easily observable in larval forms and/or in various stages of embryological development.  The four basic characteristics are: 1) a dorsal tubular nerve cord, 2) a notochord, 3) pharyngeal pouches, 4) and a post-anal tail.  All chordates show these characteristics at some point between the zygote stage of development and the adult animal.

    Phylum Chordata contains the most structurally advanced bilaterally symmetrical  animals in the kingdom Animalia.  The phylum is divided into three subphyla.  In order of evolutionary and structural importance these subphyla are:

Subphyla:
        Urochordata - a name meaning "tail chord" indicating that the notochord does not extend into the head region.  Examples include little know animals such as tunicates and sea squirts.

        Cephalochordata - "head chord", indicating that the notocord does extend into the head region.  Animals classified in this subphylum are know as lancelets (Amphioxus).

        Vertebrata - "dorsal vertebrate column". 

Phylum Vertebrata has two primary division":
         Superclass Agnatha - "jawless fishes", the hagfish and lampry.

         Superclass Gnathostamata - jawed fish and all tetrapods.

Superclass Gnathostamata has 6 primary divisions:
            Class Chondrichthyes - "cartilaginious fish"; sharks, skates, and rays.
            Class Osteichthyes - "boney fish"; all other fishes.
            Class Amphibia - terrestrial animals but still dependant on water; frogs, toads, salamanders.
            Class Reptilia - the first true land vertebrates; snakes, turtles, lizards, alligators, crocodiles.
            Class Aves - "birds"; feathered, homeothermic vertebrastes.
            Class Mammalia - "mammary glands"; homeothermic vertebrates with hair and mammary glands.

    Vertebrates are characterized by an endoskeleton that includes a backbone composed of a series of vertebrae.  The vertebrae develop around a notochord (usually present in the embryo only).  The symmetry of this group is described as being bilateral.  The body cavity is lined completely by mesoderm (represented by the peritoneum), and is referred to as a coelom.  The circulatory system is closed, and the digestive tract is complete with a mouth and an anus.  Segmentation is exhibited in the arrangement of the vertebrae and organization of the muscles.  The vertebrae kidney is closely associated with the closed circulatory system, and it functions in accordance with the animal's environment.  The nervous pathways consist of a central and peripheral system.  Gas exchange occurs by diffusion across cell membranes, gills, and lungs.

Question 1:  Name and describe the four chordate characteristics.

Question 2:  List the three subphyla of chordates and give an example of each.

Question 3:  What are the factors that unify these three groups of animals?

    The most frequent points of observance of the chordate characteristics is during embryological development.  Early chordate development also shows the linkage between the chordates and lower animal forms.  In fact, the patterns of early chordate development are most easily seen and exemplified by early development of the Echinoderms, as chordate developmental patterns have been modified by evolution to accommodate a large mass of yolk (stored food).  In moving toward land, animals had to develop ways of feeding developing embryos in a hostile, unnatural environment, and this caused the basic patterns of early development to be somewhat obscured. 

Question 4:  In what group of animals is early chordate developmental patterns most easily observed?

LAB OBJECTIVES
    1.  To observe the chordate characteristics and through them learn the relationships between the three chordate subgroups.
    2.  To observe and learn the patterns of early chordate development.
    3.  To learn characteristics and representatives of superclass Agnatha and classes of superclass Gnathostamata.

PROCEDURE
    Observe the indicated slides, preserved specimens, and filmstrip for the chordate characteristics and patterns of embryological development as indicated.

I.  The Chordate Characteristics

    A.  Subphylum Urochordata
        1.  Observe the preserved specimens of adult tunicates.  Be able to identify their phylum and subphylum.
        2.  Obtain the slide labeled "Tunicate larvum".  Using figure 1, note the four chordate characteristics.

    B.  Subphylum Cephalochordata
    The representative of this subphylum, Amphioxus (also known as lancelets), is thought to have evolved as a result of neoteny.  Neotony is a phenomenon wherein a larval form fails to progress into adult stages, becomes sexually reproductive, and produces descendents that exhibit the larval traits.  Theoretically Amphioxus evolved from larval tunicates, and modern adults exhibit many characteristics of the tunicate larval stage.       
       
        1.  Observe the preserved specimens of Amphioxus.
        2.  Obtain the slide labeled "Amphioxus w.m".  Using figure 2, locate the labeled structures, especially the four chordate characteristics.  Note the similarities to the tunicate larvum.

    C.  Subphylum Vertebrata
    This subphylum is divided into two superclasses, Agnatha and Gnathostamata, and the latter has six diverse classes. 
       
        a. Superclass Agnatha, jawless fishes, is the most primitive vertebrates.  The sea lamprey belongs to this class.  The larval form of the sea lamprey, ammocoetes, closely resembles Amphioxus establishing a link between the vertebrates and the cephalochordates.
       
        1.  Obtain the slide labeled "ammocoetes".  Using figure 3, observe the four chordate characteristics.  Note the similarity to Amphioxus.
        2.  Observe the adult sea lamprey.

        b. Superclass Gnathostamata consists of jawed fish and all tetrapods, and has six diverse classes (see above)
        1. Your instructor will show you representatives and tell you characteristics of each class.

Question 5:  What is the phylum and subphylum for tunicates?

Question 6:  What is neoteny?

Question 7:  What is the phylum and subphylum for Amphioxus?

Question 8:  What is the phylum, subphylum, and superclass for the sea lamprey?

Question 9:  From your observations, how are the three subphyla of chordates visibly linked together?

Question 10:  List the 6 classes of superclass Gnathostamata and write the characteristics your instructor

 illustrated.

II.  Embryology and Development

    The union of sperm and egg with subsequent fusion of egg and sperm nuclei (amphimixis) and activation of the egg cytoplasm constitutes fertilization.  This initiates changes that result in the formation of a new individual.  Fertilization produces a single-celled zygote which undergoes cleavage (rapid mitotic divisions) to produce a solid ball of cells known as the morula (mulberry).  The morula undergoes further development becoming a hollow ball of cells, the blastula.  By invagination (turning in) of surface cells of the blastula a third developmental stage is achieved, the gastrula.  Additional development results in organ formation and the establishment of an embryo.

    A.  Human Sperm Smear
        1.  Observe the slide marked "Human Sperm Smear" (your instructor may show you this slide as a demonstration).  The sperm are male reproductive cells produced by the testes of the male.

    A sperm cell consists of (1) a head, which contains 23 chromosomes, the anterior tip of which is differentiated into an acrosome which functions in egg penetration; (2) a middle piece, which houses many mitochondria (sites of ATP production which provides energy for the propulsion of the sperm) and contains the base of the flagellum; and (3) a long tail called a flagellum which is used for locomotion.

Question 11:  Where are sperm cells produced?

Question 12:  Draw and label a sperm cell.  Include the function of the three primary structures.

    B.  Development of the Sea star
        1.  Unfertilized Egg:
Obtain the slide marked "sea star, early cleavage".  (Note: step 1 through step 5 below may be found on this slide.)  Several shapes will be found on the slide.  Find an unfertilized egg.  The unfertilized ovum of a sea star is a simple, spherical cell showing cytoplasm, a nucleus, and a nucleolus within the nucleus.  Unfertilized eggs possess an extracellular membrane, the vitelline membrane, which adheres tightly to the plasma membrane of the egg and thus cannot be seen.  Try to locate an egg that appears to have only an outer plasma membrane.

        2.  Fertilization Membrane (One-Cell Stage):
When a sperm enters the sea star egg, the vitelline membrane is lifted from the egg surface as the fertilization membrane preventing further sperm penetration.  Find a fertilized egg by looking for the fertilization membrane.

        3.  Two-Cell Stage:
With the development of the fertilization membrane, mitotic divisions begin.  Look for an egg that has divided into two cells.  (Note that the cells remain in contact with each other.) 

        4.  Four-Cell Stage:
The first plane of cleavage (above) is vertical, passing through the main axis of the egg, and therefore at right angles to the equatorial plane of the cell.  The plane of the second division is at right angles to the plane of the first division.  Note that the cells appear to be about half the size of those in the two-cell stage.

        5.  Eight-Cell Stage: 
The third cleavage plane is at right angles to the first two planes.  Thus, it is horizontal or parallel to the equator of the egg.  Care must be taken in choosing the eight-celled stage and not a more advanced one.

        6.  Later Cleavage Stages (16 to 32 cells):
Obtain the slide marked "Sea star, late cleavage".  The cells continue to divide until a large number of cells have been produced.  The stage of development reached when there is a ball of sixteen or more cells with no large cavity in its center is called the morula.

        7.  The Blastula:
Obtain the slide marked "Sea star, blastula".  This stage is a hollow sphere containing a fluid-filled cavity known as the blastocoel.  On a whole mount slide the blastula will appear as a solid ball, more transparent in the hollow center and with a thicker rim around the outside.  Locate the side where the cells are the thickest.  It is this slide that will subsequently push in by the process of gastrulation to produce the next stage, the gastrula.

        8.  The Gastrula:
Obtain the slide marked "Sea star gastrula".  The gastrula stage appears as an in-pushing of the cells on one side of the blastula forming two cell-layers; an inner endoderm and an outer ectoderm.  As a result of this movement, two cavities are now evident; the old blastocoel and the new cavity, the archenteron or primitive gut.  This cavity later becomes the digestive tract of the adult animal.  The opening to the archenteron is the blastopore, which is destined to become the future anus in deuterostomes.  (The blastopore becomes the mouth in protostomes.)

If your gastrula is a late one, find the two mesodermal pockets forming as out-pockets at the blind end of the archenteron.  The mesodermal or middle germ layer forms primarily by the budding off into the blastocoel of mesenchyme cells from these pockets.

Question 13:  What is the vitellin membrane?

Question 14:  What is the fertilization membrane?

Question 15:  What is the morula?

Question 16:  What is the blastula?

Question 17:  How does the size of the blastula compare to that of the one-celled stage?

Question 18:  How does the sizes of the individual cells compare to the one-cell stage?

Question 19:  What two layers are created by gastrulation?

Question 20:  What is the archenteron?

Question 21:  What is the blastopore?

    C.  Human Development
Time permitting, your lab instructor may show a video on human development.  Watch for the chordate characteristics.

Question 22:  List all the similarities you observed between early human development and the sea star.

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