Abstract:
Effectively communicating how three-dimensional tissues change shape over time is significant challenge to understanding developmental biology and embryology (Slish, 2000). Both my developmental biology laboratory course and my independent research provide students with opportunities to use the tadpole visual system as an accessible experimental to study molecules that influence neuronal development. Specifically, students study retinal ganglion cell axons, neurons that provide the sole physiological connection between the eye and the brain. Thus, retinal axon wiring must be executed with extreme precision. Both the timing of retinal axon extension and anatomical trajectory of the axons from the eye to the target region of the brain have been well-documented (Chien and Harris, 1994; Lom and Hauptman, 2000). All current documentation of these anatomical and developmental events, however, is limited to two-dimensional, static representations that obviously do not thoroughly convey how developing tissues in time and space. I propose to use animation software to transform static anatomical brain images into interactive animations that not only present the tadpole brain in three dimensions, but also include time as the fourth dimension to demonstrate the developmental progression of growing retinal axons. By visualizing axons growing within the 4D brain, students will visualize and understand a dynamic developmental process that is a difficult, but very fundamental concept in neurobiology and developmental biology.

Specific objective:
To use Flash and/or LiveMotion animation software to build a virtual four dimensional tadpole brain that dynamically demonstrates the developmental processes of axon growth and guidance.

Pedagogical implementations of a four-dimensional tadpole brain animation:

1. as a laboratory demonstration in Biology 306 (Developmental Biology - taught annually) where students will be performing investigative experiments on the development of retinal axons in the tadpole
2. as a lecture demonstration of axonal navigation, growth, and guidance in Biology 362 (Seminar in Developmental Neurobiology - taught annually)
3. as a demonstration to biology 371/372 students (Independent/Honors research) of the model experimental system that my laboratory employs to study neuronal development
4. as a presentation tool in research seminars presented by myself or my students
5. as a web published demonstration freely available for students and instructors engaged in developmental biology and neurobiology courses at other institutions

Technical requirements, acquisition of skills, and institutional support:

To execute this project during the summer of 2001 I will need access to quality tadpole brain images, computer software, hardware, and support, and time to learn to use the animation software to produce vector-based animations that can be downloaded quickly and easily via the web. My postdoctoral research generated a body of images sufficient to build an accurate model of the tadpole central nervous system. Davidson College currently provides me with access to recent Macintosh G4s in my office and laboratory, space on the biology department server to publish the resulting animations, and IT support. Thus, I already have access to the necessary raw material, technology, and support. The only limit to creating the 4D tadpole brain is that I do not yet have the technical skills to create animations. During a brief introduction to Flash, I was able to create a simple animation and found the software intuitive and well supported. Since Flash and LiveMotion are industry-standards for animation that have been used successfully for creating pedagogical animations, numerous resources are available for learning and using the software including local colleagues, workshops, tutorials, and books. Further, I have extensive, self-taught experience in digital image processing and a keen desire to produce biological animations. Thus, I have little doubt that I can develop the technical skills necessary to implement this proposal.

Evaluation plan:
Assessment of student learning with regard to the major concepts of retinal axonal navigation can be accomplished in two objective fashions. First, the amount of class time required to explain retinal axon development during class in 2001 using static 2D images will be compared to the amount of class time required to explain the concept in 2002 using the 4D model. Second, student scores on written lab reports will be compared with and without the benefit of the 4D model. In addition, student feedback will be solicited after viewing the 4D brain to obtain subjective evaluation of its effectiveness as a learning tool. It is anticipated that the 4D tadpole brain model will provide students with a more accurate and rapid understanding of the dynamic means by which axons extend in a growing brain.

Future applications:
Obviously teaching embryology and developmental biology involves describing numerous four-dimensional developmental anatomical events such as cleavage, gastrulation, and neurulation. By acquiring the skills to create a 4D animation of retinal axon development, I hope to be able to expand these skills to create animations of other developing tissues for use in my developmental and introductory biology courses.

Brief methodological outline:
The major events of retinal axon guidance occur over the second, third, and fourth day after fertilization in Xenopus laevis development. By placing a dye in the lens of tadpoles at relevant stages of development (Cornel and Holt 1992), I can obtain clear snapshots of retinal axons as they extend through the brain (figure 1a). Camera lucida tracings of these images are then used to generate an accurate representation of the salient features, namely brain outlines and retinal axonal trajectories (figure 1b). Camera lucida representations of frontal and transverse views of brains from early axon extension to termination at the target (stages 32 - 42 Nieuwkoop and Faber, 1956) will be compiled (figure 2) and the brains translated into three-dimensional outlines. I will then use the animation packages to connect these static three-dimensional brains in a time-lapse fashion, adding the fourth dimension of developmental time and revealing the dynamic process of how the brain wires itself during embryonic development.

References:

a b

Figure 1. Photomicrograph (a) and camera lucida (b) representations of dye-labeled retinal axons in the developing Xenopus brain. Images from Lom and Hauptman (2000).

Figure 2. Lateral and transverse views of tadpole brains illustrate RGC development. RGC axons (green) follow a predictable route in the midbrain, while dendrites (yellow) arborize near their cell bodies (blue) within the retina. Images adapted from Nieuwkoop and Faber (1956), Chien and Harris (1994), and Sakaguichi et al. (1984).