Robert A. Boomsma, Ph.D.

Professor of Biology

Epidermal Growth Factor Receptor During Early Development

in the Japanese Medaka

Boomsma, R.A., Scott, H. and Walters, K., 2001.

Immunocytochemical localization of epidermal growth factor-receptor in early embryos of the Japanese medaka fish (Oryzias latipes).

The Histochemical Journal 33(1):37-42.

Introduction

Growth factors are secreted by cells which can then control the development of neighboring cells. Growth factor receptors are found on the cell surface and are required in order for a cell to respond to that growth factor.
Therefore, identifying cells that contain a receptor to a particular growth factor will give insight into those cells that may be affected by that growth factor.
Epidermal Growth Factor (EGF) is important in the development of various systems such as the epidermis, brain, lung, retina and kidney.
EGF acts through the epidermal growth factor receptor; this receptor also mediates the action of other ligands as well, most notably transforming growth factor-a (TGF-a).
The purpose of this study was to identify cells that contain the EGF-receptor in early embryos of the Japanese medaka in order to identify cells that might respond to EGF or TGF-a.
This study was done with the help of two students: Kendra (VanderWildt) Walters ('97) and Heather (Verstraete) Scott ('99).

Methods

Japanese medakas were bred in a standard fish tank and the embryos were maintained in petri dishes containing appropriate rearing medium.
Embryos at various stages (1-26) were prepared for microscopy.
Embryos were stained for EGF-receptor by immunocytochemistry using a specific antibody to EGF-receptor.

Immunocytochemistry involves treating the slide with a primary antibody that is specific for the molecule (antigen) you want to observe, in this case EGF-receptor.
The slides are then treated with a second antibody labeled with biotin, then with an avidin-biotin complex that sticks to the second antibody via the biotin.
The avidin-biotin complex has a peroxidase enzyme associated with it. The peroxidase is allowed to react with the substrate, DAB, which places a brown precipitate on the slide.
Therefore, the location of the antigen is marked by the presence of the precipitate.

Results

Japanese medakas begin development as a small disc of cells at the top of the yolk and then grow around the yolk as shown in the figures below. It takes the embyro approximately 56 hours to reach stage 26 at room temperature. (from http://biog-101-104.bio.cornell.edu/BioG101_104/tutorials/Medaka_stills.html)


Stage 3: 2 cells	Stage 26: retinal pigmentation

After sectioning and immunocytochemical staining, cells containing the EGF-receptor stain brown.

Stage 3: 2 cells Both cells of the embryo stain for EGF-receptor equally.
Stage 9: 64 cells All cells still contain equal amounts of EGF-receptor.
Stage 9: 64 cells This slide was treated with control antibody. The lack of staining proves that our antibody is specific for EGF-receptor.
Stage 19: Beginning Neuralation The future neural tube (spinal cord) is forming. Notice that the surface ectoderm which will form the epidermis of the skin and the cells surrounding the sides of the yolk contain a lot of EGF-receptor. Other cells do not contain much, if any receptor.
Stage 20 Note that many cells lack EGF-receptor while other have it.
Stage 22 The surface epidermis and selected cells of the neural tube contain EGF-receptor.
Stage 26 The epidermis, aorta, kidney and intestines contain EGF-receptor

Conclusions

EGF-receptor is present in early Japanese medaka embryos.
All cells start out with EGF-receptor, but later the receptor is localized in a limited number of cells types.
EGF must be important in the development of the epidermis, aorta, kidney and intestines.