Vanessa Bishop Seabreeze High School Daytona Beach, Florida	Phil Talbot  Skyline High School  Salt Lake City, Utah

 

 
Introduction  to top
    In the sequence of mitotic divisions which result in the single-celled zygote becoming a complete organism consisting of trillions of cells, three main processes are involved. The three components of embryonic development are cell division, differentiation, and morphogenesis.
     After the zygote is formed, growth and development occurs through mitotic divisions. Differentiation allows cells with identical chromosomes to express different genes, thereby giving rise to specialized cells responsible for an organism's development.
     Morphogenesis involves the movement, arrangement and differentiation of cells during early development  to produce a 3D body shape. In embryonic development, a sequence of events takes place that establishes the basic body plan of the animal.
   An important premise about cell differentiation during development is that at fertilization the egg is not uniform in its chemical makeup. The side opposite from where the sperm penetrates has a high concentration of Ca++. The distribution of mRNA's within the cell is also uneven. Hence, when the cell divides the two new cells have different chemical/ molecular concentrations that affect the expression of their genes in a unique manner.  The molecular difference between cells may be great enough to suppress or activate the expression of genes in one cell and  inhibit them in another.
     How cells differentiate has been a major question among developmental biologists. The discovery of  morphogens, often described as growth factors, has helped to answer some of these questions.  The first morphogens were isolated around 20 years ago. The most studied in the Drosophila appear to be the bicoid, nanos, and hunchback, Dpp, hedgehog. These are responsible for anterior, posterior, and the banding pattern in the midsection. Cells determine their location in the embryo by detecting concentration gradients of the different morphogens.
    Cells in the center of the embryo, for example, determine their position because the anterior and posterior morphogens are of equal concentration at the midsection. If the concentration of the first was slightly higher than that of the second, then the cells determine their location in the larva as anterior to the midsection. In order to fine tune their location as development progresses, more tissues produce such molecules and the concentration of these morphogens helps to give each tissue a precise reference to its position in the body. Morphogens may also help in inducing genes that help the cells differentiate. This activity focuses on the role morphogens have on development, even tough other factors are involved.
     The purpose of this exercise is to help students understand the concept of how varying concentration gradients aid the cells in locating their position in the embryo.
    The effect of morphogens is based on their concentration in the cells, which is regulated by: (1) The rate of diffusion, which involves how fast the morphogen can get to the target cells (a function of distance) , (2) The rate at which it decays. The stability of the molecule and presence of chemicals that may degrade it are important factors that affect this rate. (3) The reaction. The morphogen may have to be over a threshold level concentration to trigger an effect on gene expression in a given cell.
       During early gastrulation, bicoid expresses two genes that cause the striping of the larva. Bicoid protein arises from mRNA at the anterior end pole of the egg. Following fertilization these mRNA's are translated and the proteins diffuse posteriorly to form a gradient beyond the midpoint of the larva. This gradient helps to organize the body's segmentation. It appears that each cell nucleus measures the exact concentration of bicoid protein and directly reads the slope of the gradient. Bicoid drives high levels of hunchback protein transcription and then binds it to specific regulatory genes in target zones. The protein products of the regulatory genes form local concentrations gradients, which control the transcription of genes further down the hierarchy.  Each cell layer has a definite anterior-posterior orientation. Following this event,  the dorsal-ventral orientation divides the embryo like a checkerboard. Each would be a parasegment responsible for generating a precise region of the larva and adult. Cells from neighboring compartments will have different affinities, and tend to minimize their mutual contact to limit cell movement between them. Thus, when a signal does cross over the border into the next square, all the cells within range are affected.
    Evidence suggests that Dpp is a long range morphogen produced in the anterior end of each compartment. Dpp is triggered by the release of the hedgehog which is produced in the posterior region of the previous compartment. Hedgehog appears to be a short-range inducer to elicit a long-range morphogen (Dpp).
    The concentration landscape of the morphogen gradients contains three types of information. The scalar concentration provides positional information about how far a given cell is from the peak. The direction of maximal change provides information about orientation with respect to the source. The slope of the gradient relates to the size of the field.
    Although we discussed the Drosophila model exclusively, there are examples of vertebrate morphogens, such as activin. This protein can determine the cell state in mesodermal cells.
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• Determine rates of diffusion.
• Analyze and graph data.
• Describe the role of morphogens in the development of the larva.
• Explain the chemical dynamics of a cell, and its influence on the cell's genome.
• Understand the significance of embryo polarity during development.

• Grade level: 6-12
• Biology, Anatomy and Physiology, Genetics

Notes to the Teacher: to top
  Before the lab, the teacher must prepare the models of the larva:

• Roll out the modeling clay so that it is 1/4" thick.
• Using the fuel tank in a kit from an inexpensive model airplane kit ( 2"-3"), make as many molds as you have teams (you may want to use this mold over the years, so make as many molds as you need for large classes). A battery (AA) works in making a mold.
• Pour 2% agar into the molds to make a model of the Drosophila larva.
• After the larva models have solidified, place them in a zip lock bag and keep refrigerated until lab time. Be careful not to break them as they are fragile.

• Modeling clay
• Fuel tank from a model plane, or a AA battery
• 2% agar
• Food coloring (red, yellow, and blue)
• Metric ruler
• Graph paper
• Petri dishes
• Zip lock bags
• Razor
• Forceps
• 3 pieces of filter paper (size: 1/4" x 3/4")

• The significance of embryo polarity during cleavage.
• The early stages of embryogenesis (from the unequal distribution of the chemicals found in the cytoplasm of the egg to the formation of the larvae).
• The concept of chemical gradient.
• The process of diffusion.

    After 20 minutes, when the dye has been absorbed, remove the filter papers.

• What is the rate of diffusion of the anterior morphogen (represented by red dye)?
• Graph the rate of migration of the individual dyes overtime.
• Identify the dependent and independent variable in the above graph.
• Identify the anterior, posterior, and midsection morphogens that have been identified in Drosophila larvae (from the reading)
• How might a cell determine its position in the embryo by use of a morphogen concentration?
• What morphogen (in the Drosophila larvae) is represented by (1) red, (2) blue, and (3) yellow?
• What structures do you think will develop from: (1) red , (2) orange, (3) yellow, (4) green, and (5) blue.
• What happened to the concentration of the morphogen as it moved away from the point of origin? Explain.
• Do all the cells respond to the same concentration of a given morphogen equally?
• Do all cells respond to the same concentration of a given morphogen equally?
• What kind of biological molecule is a morphogen? Hypothesize about the the characteristics of the morphogen molecule in terms of (1) size, (2) solubility, and (3) other structural characteristics that allow the morphogen to perform its function. (ESSAY)
• Cells identify their relative position by use of anterior and posterior morphogens in the embryo. How can cells more accurately fine tune their embryological position? (ESSAY)
• Design an experiment that demonstrates the role of morphogen in differentiation in the Drosophila larvae.

• Have students make a colored drawing of the Drosophila larvae showing the banding pattern. It may be helpful to have them look at photographs of such pictures first. Pictures are found in the magazine "Cell" vol. 85., 1996
• Suggest that students research anomalies found in Drosophila due to errors in development caused by the lack of morphogens, timing of morphogen release, etc.

• The Biology Project, University of Arizona, November 8, 1996. URL address:                                                    http://www.biology.arizona.edu/dev.../Developmental_Mechanisms/09a.html
• Reaction-Diffusion,   Ingo K. Hunsinger, 1996.      URL address: http://www.informatik.uni-bremen.de/~ingo/Publications/tos/node3.html
• Toward an Evolvable Model of Development Autonomous Agent Synthesis, Dellaert and Beer,  1996. URL address: ftp://alpha.ces.cwru.edu/pub/agents/dellaert/alife/alife.html
• Manipulating the anteroposterior pattern of the Drosophila embryo, Frohnhofer, Lehmann, Nusslein-Volhard, 1986. Journal of Embryology, 97 Supplement, 169-179.
• Synergy between the Hunchback and Bicoid Morphogens is Required for Anterior Patterning in Drosophila, Simpson-Brose, Treisman, Despain, 1994. Cell, Vol.78, 855-865, September 9, 1994.