MITOSIS AND DEVELOPMENT IN MULTICELLULAR ORGANISMS Student Learning Outcomes 1

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MITOSIS AND DEVELOPMENT IN MULTICELLULAR ORGANISMS Student Learning Outcomes 1. Be able to list and identify the phases of a cell’s life cycle known as Prophase, Metaphase, Anaphase, Telophase and Interphase. 2. Be able to draw a pie diagram indicating the relative proportions of time a plant, like Allium (onion), spends in the different mitotic phases. 3. Be able to diagram and describe the stages of embryonic development common to all animals (zygote, morula, blastula, and gastrula). 4. Be able to diagram and describe the function of a fertilization membrane surrounding a fertilized Sand dollar egg. 5. Be able to contrast the end results of the 2 kinds of cell division observed in Eukaryotic cells: Mitosis and Meiosis. Introduction ? The tremendous diversity of structure and function that eukaryotic cells assume is remarkable when you consider that most multicellular organisms begin life as a single fertilized egg, the zygote. Through repeated divisions, this cell gives rise to all the cells that make up the organism. The series of events experienced during cell division by actively reproducing cells is termed mitosis. Mitosis also serves as the basic mechanism of reproduction in unicellular organisms. In this lab, you will examine several essential aspects of mitosis, and of the role of mitosis in the early development of multicellular organisms. ? Mitotic processes are rather easily observed in situations where rapid increases in cell numbers are occurring. Two cell types that will be used in this lab are 1) the cells produced as a consequence of fertilization in the sand dollar, Dendraster, and 2) cells in the growing root tips of the onion, Allium. I. Mitosis in Plants ? At no time other than cell division can a cell's chromosomes be observed. Although it is also possible to see chromosomes during meiosis, today we will examine mitosis in a rapidly growing tissue, the root tip of the onion Allium. ? Although animal and plant cells differ somewhat in structure as well as in some of the fine points of mitosis, the mitotic phases are essentially the same in all eukaryotic cells. While the process is continuous and there is some gradation among the various steps, four general phases can be identified: Prophase, Metaphase, Anaphase, and Telophase. Mitosis is followed by cytokinesis, the division of the cell's cytoplasm, which also takes the two new nuclei into separate daughter cells. When a cell in a growing tissue phase is not in mitosis, we say it is in Interphase (this phase is not regarded as one of the phases of mitosis). During this relatively long period, the DNA is replicated in the nucleus and extra amounts of other cellular components are synthesized, in preparation for distribution to future daughter cells. No evidence of this activity can be seen until the next mitosis, when the nuclear membrane dissolves and the chromosomes condense into the short, thick, stainable bodies which are visible through the light microscope. ? For any given species, there is usually one characteristic number of chromosomes (n) found in the gametes and double that number (2n) found in cells of the sexually reproducing adult. There is wide variation in the number of chromosomes characteristic of species, and the n does not necessarily indicate how much hereditary material is present. In some species, there is a large n but the chromosomes are very small, while other species have fewer, larger chromosomes. The chromosomes of onion cells are large and relatively few (2n=14), making them easier to study than the cells of many other organisms. A. Observation of Mitotic Phases in Onion (Allium) Root Tip Cells The growing onion root tip is one of the most widely used materials for the study of mitosis, since it is easily cultivated in quantity and preparations of the dividing cells are easily made. There are regions of rapid cell division in root tips; therefore, the chances are good that within such tissues one can identify every stage in mitosis. The following study will involve an examination of preserved and stained thin sections of onion root tips, with the stains making the chromosomes of the cells quite visible. You will need a microscope for this portion. Be sure to use two hands. Make sure that the cord is not dangling to prevent a tripping hazard. Before using the microscope, check for the condition the microscope was left in from the prior class. 1. 2. 3. 4. Was the microscope placed back in its assigned compartment with the arm facing out toward you? Was the cord wrapped between the stage and objectives with the plug tucked inside the cord? Was the cord relatively untangled? Was the ocular lens clean? (If not, clean the dirty lens with the appropriate lens cleaner and lens paper (not a paper towel). 5. Was the light turned off? 6. Check that the Condenser is at its highest point, directly below the stage. There is a knob connected to the Condenser that allows it to be moved up and down. 7. Was the mechanical stage centered so that the stage clips don’t hang over the edge of the stage? 8. Was the scanning objective lens (4x) (not another objective) placed over the stage? If not, rotate the nosepiece until the scanning objective is facing the stage. 9. Was the stage lowered to the lowest setting possible position? If not, use the coarse focus knob to do so, not the fine focus knob. 10. Were there any slides remaining on the stage? If so, remove the slide and notify your instructor. It is important that the slide is placed in the correct box, or it may get lost. 11. If any of these conditions were problematic, please let your instructor know. Procedure: 1. Plug in the cord and turn up the light intensity.to its maximum value and adjust the iris diaphragm to its most closed setting. As you proceed you can increase the light passing through the specimen by gradually opening the iris diaphragm. 2. Adjust the distance between the oculars: Without placing the prepared slide on the stage yet, look through the oculars. You are likely going to see two circles of white light. Do not try to focus your eyes on any one thing, as nothing is in focus yet. Slowly, move the two oculars together and/or further apart until you see the single Field of View. 3. Lower the stage using the coarse focus knob, and make sure the (shortest) scanning objective is facing the stage, so that there is no chance of the slide scratching any objective lenses. 4. Obtain a slide of Allium root tips. Hold the slide above a sheet of white paper, and note the series of dark streaks on the slide. Each streak is a very thin longitudinal section through an onion root tip. 5. Before looking through the eyepiece (ocular), open the stage clip, and place the slide on the stage of the microscope beneath the objective, with the coverslip visible on the upper side. The stage clip should be holding the slide in place, not pressing the slide under it. Center the object below the objective without looking through the oculars. Nothing is in focus yet. 6. Coarse adjustment with scanning lens: With the scanning lens in place, move the stage up to its highest point without looking through the oculars. Nothing is in focus yet. Looking through the ocular with your right eye only (squint or cover your left eye), bring the specimen into focus by turning the coarse focus adjustment knob slowly until the specimen is generally in focus. Then turning the fine adjustment knob will bring the specimen into sharper focus. 7. Focus your left eye: Viewing the specimen with both eyes through both oculars, turn the left ocular diopter until the specimen is clear in both eyes. 8. Adjusting on low and high power objectives - use fine adjustment knobs only: If you wish to view the specimen using higher magnification, center the specimen in the field, and carefully rotate the revolving nosepiece to bring the next higher power objective into place beneath the body tube. The specimen will no longer be in focus. In order to sharpen the image of the specimen, adjust the focus using only the fine focus adjustment knob. (Again, the light may have to be adjusted with the iris diaphragm.) Each time you move to the next higher power objective, be sure you center the specimen beforehand. 9. Locate one of the root longitudinal sections under scanning power and then move to low power, to determine whether it shows clear mitotic stages. Since each section is very thin, not all will be equally good for study. A good section will have many cells showing the dark, strap-like, chromosomes. Notice that the "best" area for mitosis is not at the very tip end of the root or high up the root where it was cut, but above the root cap (See Figure 1). 10. After preliminary examination under low power, change to high-dry power, being very careful not to break the slide. Keep in mind the sequence in which the different stages occur, but do not try to find them in sequence. Thus, if you happen to find an Anaphase first, study it before proceeding to another stage. Chances are that most of the cells will be in interphase. (You may even see cells which appear to have no nucleus. Remember that this is a very thin section and the slice you see captured the edge of some cells, missing the nucleus.) Keep this slide for the rest of the lab exercise. 11. Obtain one of the folders entitled Plant Mitosis (set #55), view it through the mini-scope, and use it to become familiar with the stages of mitosis. Figure 4 shows these stages also. Refer to the plant mitosis model sequence on the side lab bench to help you visualize the process. 12. Scan the prepared slide of the Allium root tip until you can reliably identify the four mitotic phases. Be able to describe the chromosomal changes which occur during each phase. (It is not necessary to be able to count every chromosome; just describe their positions, shapes, and orientations.) For your information, descriptions of these phases follow. Mitotic Phases Prophase During Prophase the chromosomes become distinguishable in the nucleus. The nuclear membrane breaks down and the chromosomes become distributed haphazardly through the cytoplasm. At this stage in the onion root tip the chromosomes often appear as a partially coiled mass. These elongated chromosomes later become condensed into shorter chromosomes and the nuclear membrane disappears. Even at this early stage each chromosome has doubled, although this will be difficult to see on the slide. Metaphase During Metaphase the chromosomes become arranged near the center of the cell, with the ends of the chromosomes generally pointing away from the midline. This stage is apparently a preparation for the equal division of chromosomes between the daughter cells, a process that begins in the next phase. During or somewhat before Metaphase, small, threadlike structures (spindle fibers), form in the dividing cell. Some of them appear to be attached to the chromosomes and seem to provide the machinery for the movement of the chromosomes, although the way in which this is accomplished is not yet understood. These fibers usually appear most clearly in Anaphase. The composition of the spindle fibers is not known. It has been suggested that they form by the aggregation of protein molecules. Under the electron microscope these fibers appear as fine, straight, hollow tubules. Although they lengthen and shorten during Mitosis, they do not appear to get thicker or thinner. This suggests that they do not stretch or contract but that new material is added to the fiber or removed from it as the spindle changes shape. Anaphase At the beginning of Anaphase, the two members of each of the previously doubled chromosomes separate and move toward opposite ends of the cell. This stage can be recognized in the onion by the two groups of V-shaped chromosomes on opposite sides of the cell. The sharp end of the V is pointed away from the center toward the cell wall. An onion cell has 14 chromosomes; hence it is seldom possible to see all of them at one time. Reduce the light coming through the objective of the microscope and see if you can find any spindle fibers near the center of the cell. They will appear as very fine lines between the two groups of chromosomes, but they are not often visible in a study of this kind. Telophase Cell division is completed during Telophase, and reorganization of the cell contents of the two daughter cells begins. It is often difficult to distinguish between late Anaphase and early Telophase in the cells of the onion root tip. During Telophase, however, a cell plate starts to form as a fine line across the center of the cell. When complete, the cell plate will divide the original cell into two daughter cells. In some cells it will be indistinct. As Telophase progresses, the nuclei begin to reorganize, and the chromosomes become indistinct. In both plant and animal cells, the daughter cells resulting from mitotic division have the same number and kinds of chromosomes as the original cell from which they came. Thus, in the onion each daughter cell has 14 chromosomes, just as the original cell had. Figure 1. A simplified drawing of a longitudinal section through the root tip of an onion (Allium). Refer to the laboratory models and charts, your slides, or your textbook's diagrams of the mitotic phases in plants. In the spaces below, make realistic rough sketches (not cartoons) of each stage of the cell cycle (including interphase) as one onion root tip cell divides into two daughter cells. Take a picture of your drawing and insert it in the appropropriate location below. Insert diagrams here: Prophase: Metaphase: Anaphase Telophase: Interphase: Double click here and replace this text with a Double click drawing. here and replace this text with a Double click drawing. here and replace this text with a Double click drawing. here and replace this text with a Double click drawing. here and replace this text with a drawing. List the main actions that happen during this phase here. Use arrows and words to label important parts: Figure 2. Diagrams Showing the Different Phases an Onion Cell Moves Through during a Mitotic Cell Division. B. Durations of Phases in the Cell Cycle The cell cycle is the name for the entire sequence of events from one division to the next, including all the steps of mitosis, plus cytokinesis and interphase. The time it takes for the cell cycle varies widely, with cells of developing embryos dividing the fastest. (You may have observed the first cleavage of the sand dollar less than an hour after fertilization.) As you will observe, some of the mitotic phases take longer than others. Now that you can identify the various stages of mitosis, you are ready to determine the relative percent of time spent in each phase during the life of a cell. We do not have enough lab time to sit at the microscope and watch one live root tip cell go through its entire cycle, since that might take many hours. Therefore, we will examine a group of cells in a root tip at one point in time and deduce the relative time spent on the different phases of the cell cycle. In order to make this determination, some assumptions are necessary: a. In the growing region of a root tip, all of the cells are assumed to have the same overall cell cycle time (including interphase). b. The phase of the cycle which takes the most time (i.e., Interphase) will be that phase which is seen in the greatest number of cells. c. Since cells quickly pass through phases which require less time, the phase which takes the least time will be seen in the fewest cells. d. If we census a large block of cells in the growing area of a root tip, identifying the phase of each one, the relative numbers will tell us which phases require more time and which require less time. Procedure: 1. A pair of students should work together observing the Allium root tip cross-section through the microscope. 2. Plug in the cord and turn up the light intensity.to its maximum value and adjust the iris diaphragm to its most closed setting. As you proceed you can increase the light passing through the specimen by gradually opening the iris diaphragm. 3. Adjust the distance between the oculars: Without placing the prepared slide on the stage yet, look through the oculars. You are likely going to see two circles of white light. Do not try to focus your eyes on any one thing, as nothing is in focus yet. Slowly, move the two oculars together and/or further apart until the two circles of white light become one circle of light. This circular field of light is the Field of View. 4. Prepared slide: Hold the prepared slide up to the light and examine it. You will see two or three tube shaped images which have been permanently mounted, using resin or some similar material, between the surfaces of the slide and the coverslip. These are two or three root tips that would normally cut off the tips of green onion, Allium 5. With other items, often a stain is used in preparation to allow the observer to clearly see the specimen if it is transparent. By placing the slide on the white paper next to this text, you will see the color of the stain which will help you locate where the specimen is. Generally, it is in the middle of the coverslip, but in some preparations, it is off slightly to one side. 6. Place the slide on the stage: Lower the stage using the coarse focus knob, and make sure the (shortest) scanning objective is facing the stage, so that there is no chance of the slide scratching any objective lenses. Before looking through the eyepiece (ocular), open the stage clip, and place the slide on the stage of the microscope beneath the objective, with the label and coverslip visible on the upper side. The stage clip should be holding the slide in place, not pressing the slide under it. Place the slide so that you can easily read the words on the label. They should not be upside down on the lower side or facing away from you. Center the object below the objective. A specimen should always be viewed first using the scanning objective. 7. Coarse adjustment with scanning lens: With the scanning lens in place, move the stage up to its highest point without looking through the oculars. Nothing is in focus yet. 8. Looking through the ocular with your right eye only (squint or cover your left eye), bring the specimen into focus by turning the coarse focus adjustment knob slowly until the specimen is generally in focus. Then turning the fine adjustment knob will bring the specimen into sharper focus. 9. Focus your left eye: Viewing the specimen with both eyes through both oculars, turn the left ocular diopter until the specimen is clear in both eyes. 10. Iris diaphragm: The light coming through the microscope may be either too bright or too dim. If the amount of light is not satisfactory, it can be adjusted by carefully regulating the size of the opening of the iris diaphragm by moving the lever beneath the stage. The iris diaphragm is part of the condenser which concentrates the light coming from the light source. 11. Once again, locate the growing region of the root tip, where cells in mitotic phases are evident. This is above the root cap which comprises cells that are long. Above the root cap the cells are very small. Then you will see cells become much more organized in lines or rows of cells. Some of the cells in this region have clearly delineated nuclei, whereas the nuclei of others look more like little spiders or dark squiggles. If you continue up further, the cells are much more rectangular and elongated, and the nuclei become more pale or even appear to be lacking altogether. You may not be able to see all of this detail yet, but you need to be in the area above the root cap. Once you go to the next step, review looking at these three areas: the root cap, the area above the root cap and the elongated part. 12. Adjusting on low and high power objectives - use fine adjustment knobs only: If you wish to view the specimen using higher magnification, center the specimen in the field, and carefully rotate the revolving nosepiece to bring the next higher power objective into place beneath the body tube. The specimen will no longer be in focus. Only research grade, precision microscopes are parfocal, keeping specimens in focus as the objective lens is changed. Adjust the focus using only the fine focus adjustment knob. (Again, the light may have to be adjusted with the iris diaphragm.) Each time you move to the next higher power objective, be sure you center the specimen beforehand. Use the objective which you can identify the phases most easily. 13. While looking at the root tip under the microscope, one member should identify the mitotic phase (or Interphase) for each cell observed, moving longitudinally up and down the root tip. The other member of the team will keep a tally (Table 1) of the number of cells in each phase. Skip those cells with no visible nucleus (what happened to these cells?), but count all of the others, including those in Interphase. (Try to avoid counting the same cell twice.) 14. The cell phases should be tallied until 100 cells have been observed and counted. You should trade places after 50 cells so that both of you get the practice of identifying the mitotic phases.) Record your tallies in Table 1 below. Table 1. Number tally of onion root tip cells in different phases of the cell cycle Group Tally Other Group Counts Class Total Percent of Time Class Average Interphase Prophase Metaphase Anaphase Telophase Total Cells 15. Calculate the percent of the cells' total cycle time spent in each phase using the following formula: percent of time spent in phase = ???????????????????????? ???????? ???????????????????? ???????? ????????????????? ???????????????????? ???????????????????????? ???????? ???????????????????? ???????????????????????????? 16. Record the values in Table 1 and in the class data table on the board. = Question 1. Based on your calculations which phase plant cells spend the most time. Be able to explain why. Replace this text with your answer. Question 2. Why is a class average more likely (than your group's data alone) to reflect the true percentage of time spent in each phase? Replace this text with your answer. Question 3. Using the class averages for each phase (Table 1, previous page), use google docs, excel, or this online chart tool to draw a "pie chart" that graphically demonstrates the relative amount of time an average onion root tip cell spends in each phase of mitosis. Remember to give your chart a title. Replace this text with your answer. Question 4. Compare your chart with a general pie chart for plant mitosis found in your textbook or some other reference. Describe the similarities and differences. Replace this text with your answer. Question 5. Speculate on why our class' pie chart percentages may not agree exactly with those seen in your reference. Replace this text with your answer. Clean up 1. Removing slides: When you need to remove a slide, be sure to rotate the nose piece to the scanning objective. Then using the coarse adjustment knob, lower the stage to its lowest position. Then open the stage clip and remove the slide. Be sure the slide goes back to the correct slide box. Keep your “e” slide for now, since you still need it. 2. Return the prepared slides to their appropriate slide boxes. II. Fertilization and Early Embryonic Development in Sand Dollars The development of the zygote into a complex of interdependent cells, tissues, and organs that make up an adult animal is one of the more fascinating processes in biology. Using the following procedure, you will examine the process of fertilization and the resulting mitotic divisions which quickly follow. The complete developmental sequence from zygote to adult cannot be accomplished within a one-lab time frame. However, several increasingly complex larval stages could be observable over the next few days (depending, of course, on larval mortality rates in the lab). The most abundant sand dollar found along the San Diego coastline is a small organism (up to 3 1/2 inches in diameter) living in sandy areas below the surf zone. A member of the Phylum Echinodermata, the sand dollar is covered with a very short blanket of movable spines (used for locomotion), among which are short, moving hair-like projections called cilia. Sand dollars filter small nutrient particles and plankton from the sea water. They have provided many generations of biologists with a ready source of gametes for reproductive and developmental studies. Each female will produce millions of eggs and each male will eject even more sperm. A. Observation of Adult Sand Dollar Procedure: Take a few moments to study an adult sand dollar. The focus knobs are found on the arm of the dissecting microscope. The magnification can be changed by rotating the knobs on the head of the microscope. Then turn the Top light on. Place a few paper towels on the stage of a dissecting microscope, and then place a sand dollar on the paper towels. Question 6. Are the aboral (top side) and oral (under side) surfaces similar? If not, how do they differ? Replace this text with your answer. Question 7. Closely observe the flat oral surface. Locate the main opening in the center, surrounded by the five-pointed star-like structure. The star-like structure is often difficult to see in a sand dollar as it is covered by many of the moveable spines. However, this structure is much more easily seen in a relative of the sand dollar, an urchin. View the following video to observe this structure, Urchin teeth. What is this structure’s function? Replace this text with your answer. Clean up 1. Be sure to return the sand dollar to its water as soon as you are done observing it to prevent it from overheating and drying out. 2. Throw the paper towels in the trash, and turn off the microscope light. B. The Gametes: Eggs and Sperm The external anatomy of sand dollars will not reveal their sex, but by injecting them with a few milliliters of an isotonic potassium chloride solution, they can be induced to shed their gametes. Due to the decreasing numbers of echinoderms, such as sand dollars, in general, we will observe the gametes using videos. Question 8. Observe the following short video: Video “Fertilization of Sand Dollar Eggs” (1:07) fertilization of sand dollar eggs. Draw an egg and a few sperm cells, including all the subcellular detail of the egg that is apparent. Take a picture of your drawing and paste it here. Replace this text with your drawing. Question 9. Describe the sperm movement. Does it appear to be directed or random? Replace this text with your answer. Question 10. In the spaces below, briefly describe the form (shape) of an egg and a sperm. Then describe the mobility (movement), if any, of the two kinds of cells. Now compare the gametes' sizes (make sure you are viewing them both at the same magnification). Sperm Egg Form Mobility Size Suggest a possible explanation for the observed differences. Question 11. Surrounding the egg cells, you will notice a protective gelatinous material containing colored granules. This material is outside the cell membrane of the egg. (Label this material in the image labeled Unfertilized Egg in your drawing.) C. Fertilization Question 12. It is usually not possible to observe fertilization (nuclear union) but you can tell when it has occurred, for a fertilization membrane will develop around each fertilized egg, inside the egg’s gelatinous cover. (Label this on the image of the Fertilized Egg in Figure 4.) This halo-appearing membrane emerges from the surface of the cell and signals the formation of the zygote. Draw a fertilized egg showing the fertilization membrane. Take a picture of it and paste below. What might be the function of the fertilization membrane? Replace this text with your drawing. D. Mitosis and Cleavage Cleavage is a form of mitotic cell division which is not accompanied by cellular growth. In fact, each successive cleavage will increase the number of cells while their volume is halved. Thus, the total mass of the embryo remains the same while the cells proliferate. The cleavage sequence includes all the stages from zygote through blastula. However, even though all the mitotic stages (Prophase, Metaphase, Anaphase, etc.) are occurring with each cleavage stage, we would not be able to actually see the sand dollar chromosome. This is due to their small size, the large quantity of opaque cytoplasmic material in the cells, and the need for a specialized staining technique to make them visible. Chromosomes will be more easily observed in the stained onion root tip cells found on prepared slides used in Part I of today's experiment. In mitosis the nucleus divides in a process known as karyokinesis, which apportions identical chromosomal material to each of the two daughter nuclei. This is soon followed by the more obvious cytokinesis, or division of the cell body. This first mitotic cell division requires only a few minutes and should occur within an hour after fertilization. Subsequent divisions will probably occur at longer intervals. For your reference, data are available for the time frame of cleavage in the sea urchin, a relative of the sand dollar (see Table 2). Question 13. Draw arrows between each of the stages in Figures 1 and 2. Then on the top of the arrow, label what process is happening (formation of fertilization membrane, first cleavage, etc.) based on what is listed in Table 2 below. On the bottom of the arrow, label how long the process takes (e.g. 2-5 minutes, etc.). See the example on Figure 1. Table 2. Approximate Time Sequence for the Development of Fertilized Sea Urchin Eggs (Note: cell division is temperature dependent) formation of fertilization membrane 2-5 minutes first cleavage 50-70 minutes second cleavage 78-107 minutes third cleavage 103-145 minutes blastula 6 hours hatching of blastula 7-10 hours gastrula 12-20 hours pluteus larva 24-48 hours Question 14. On entering the egg, the sperm nucleus carrying the male chromosomes unites with the nucleus of the egg to produce a zygote with the chromosomes of both egg and sperm. What is the relative chromosome number of the zygote compared to the gametes? THINK! Replace this text with your answer. E. Early Cleavage Stages (See also Figure 4) Procedure: 1. Obtain a mini-scope and one of the folders entitled Cleavage (set #60) from the lab prep table. 2. Observe the first three frames and read the text paragraph in the folder. Question 15. Were the photographs of all the stages pictured in frames one through six taken at the same power magnification? a. Within individual frames in the early cleavage stages, how do the individual cell sizes compare? Replace this text with your answer. b. How does the size of the entire cell mass compare at these different stages? Replace this text with your answer. 3. Obtain a glass prepared slide of starfish early development stages and observe it under the compound microscope. (The starfish's developmental stages are similar to those of the sand dollar since they are close relatives.) Identify an unfertilized egg, fertilized egg, two cell stage, four cell stage, eight cell stage, 32 cell stage, morula, blastula, gastrula, and larval stages. Question 16. How does the size of the entire cell mass (such as the 8 cell stage, 32 cell stage, etc.) compare at these different stages? Do you see the same or different trend in starfish, as you did in sand dollars? Replace this text with your answer. F. Late Cleavage Stages (See also Figure 5) Procedure: 1. Observe Frame 5 ("mass of cells") through the mini-scope. Question 17. What stage (cell number) appears to be pictured? The mass of cells you see is called a morula. Draw the morula. Be sure to label it as a solid sphere of cells. Replace this text with your answer. 2. After the sixth or seventh division the cells undergo their first morphogenic migration, meaning that cells move quite a distance from one area of the embryo to another. The cells in the center begin to move to the outside, leaving a hollow center. The hollow ball of cells is known as a blastula. Question 18. Draw a blastula. Be sure to label it as a hollow, fluid-filled sphere. Replace this text with your drawing. Question 19. Compare the size of a blastula to that of an unfertilized egg. Has the embryo actually grown? Estimate the number of cells the blastula contains: Replace this text with your answer. 3. The cells of the blastula continue to divide and sometime between 12 and 24 hours, depending on temperature, the blastula will hatch, leaving the fertilization membrane. Use the mini-scope and the sea star development slide to observe the various stages of cleavage development. Question 20. Describe how you tell if an embryo is a simple mass of cells ("morula") or actually a blastula (fluid-filled sphere of cells). (Hint: remember how "depth of field" is perceived through the microscope.) Replace this text with your answer. 4. To appreciate the three-dimensional shapes of the developmental stages, examine the sequence of animal cleavage models on the side lab bench in the laboratory. G. Gastrula In the late blastula phase, the cells undergo their second morphogenic migration. At one point on the blastula surface the cells migrate inward toward the other side of the sphere and results in the formation of an embryo composed of two primary cell layers. The outer cell layer is the ectoderm and the inner one the endoderm. Within a short time the mesoderm will begin to form between these layers and by midgastrulation these three germ layers will have developed. All subsequent tissues and organs will develop from these three primary germ layers. Procedure: Refer to Frames 7 and 8 of the mini-scope Cleavage folder and also observe gastrulas on the prepared slide of starfish development. Question 21. Draw a gastrula. Label the ectoderm and endoderm (using arrows to point to parts). Replace this text with your drawing. Question 22. A gastrula will develop into free-swimming "pluteus" larvae in a day or two. In contrast a seastar forms a bipinnaria larva. What does gastro- (as in gastrointestinal tract) mean? Replace this text with your answer. Questions 23. At what point in development would you logically expect the larvae to be capable of growth (growing in size)? Explain your answer. Replace this text with your answer. When the sand dollar embryos reach the larval stage, they have a complete digestive system and are capable of feeding. They consume phytoplankton and become significant members of the zooplankton trophic level. The free-swimming larvae will, in time, metamorphose into tiny sand dollars and settle to the bottom of their marine environment. Those which settle in an appropriate sandy area will become the new generation and they will compete with their parents for the available habitat. As a review, observe the following short video of a sea biscuit, another echinoderm relative of a sand dollar. “Sea Biscuit’s Life” (3:36) A Sea Biscuit's Life Identify the unfertilized egg, fertilized egg, two cell stage, four cell stage, eight cell stage, 32 cell stage, (skipped in the video: morula, blastula), gastrula, and larval stages. Clean up. Returning your Compound Microscope to the Cabinet: 1. Rotate the nose with the objectives so that the scanning objective is facing the stage. 2. Lower the stage to the lowest setting possible setting, using the coarse focus knob, not the fine focus knob. 3. Remove your slides. 4. Center the mechanical stage so that the stage clips don’t hang over the edge of the stage? 5. Turn the light intensity knob down to “1.” 6. Turn off the light. 7. Check to see if the ocular and objective lenses are clean. (If not, clean the dirty lens with the appropriate lens cleaner and lens paper (not a paper towel).) 8. Wrap the cord between the stage and objectives with the plug tucked inside the cord. 9. Place your microscope back in its assigned (numbered) compartment with the arm facing out toward you. Clean up of the remainder of the lab materials. 1. Return any prepared slides to their appropriate boxes. 2. Remove the coverslip from your wet mount slides. Then rinse off the slides. 3. Place any wet mount slides (not prepared slides!) in the container marked “Used slides.” Place the coverslips in the trash. 4. Wipe down your lab bench with lab bench cleaner and paper towels. 5. Wipe down side lab benches with lab bench cleaner and paper towels. 6. Push in your chair. Formation of a fertilization membrane, 2 - 5 min. Figure 4. Early Stages in the Developing Sand Dollar Figure 5. Later Stages in the Developing Sand Dollar I. Review of Sand Dollar Development Answer the following questions with reference to the letter designations in the chart below. Processes Stages a. fertilization f. larva b. morphogenic migration g. gametes c. hatching h. blastula d. cleavage i. zygote e. metamorphosis j. gastrula Question 24. Which of the following represents the sequence of occurrence for the developmental processes? Use the highlighter tool ( 1. 2. 3. 4. 5. ) to highlight the correct answer. a, b, c, d, e a, d, b, c, e b, e, d, c, a e, d, b, a, c a, d, e, c, b Question 25 . Which of the following represents the sequence of occurrence for the developmental stages? Use the highlighter tool above ( 1. 2. 3. 4. 5. f, g, h, i, j j, h, i, f, g g, h, i, j, f g, i, h, j, f i, g, f, j, h ) to highlight the correct answer.