Sheffield Lab

The major interests in our laboratory concern the development of the retina in the chick.  Our efforts have been directed towards an understanding of those factors which lead to the formation of the distinctive cell and fiber layers that are characteristic of this tissue.  

1.  Do the cells in the retina move during development of the tissue?  Time-lapse microscopy of living retinal slices, or of aggregates of cells, shows that cells do indeed move within the tissue.  Most of the movement occurs in the radial dimension.  Examples of this movement can be seen in the following mpeg sequences. (These sequences are best viewed with the program VMPEG by Steve Ekhart, which allows one to control the framing rate of the playback. You can get more information about it here).

sequence 1,  1 MBytes  This sequence shows the cellular activity in a living slice of a 14 day embryonic chick retina.  Cells can be seen moving within the inner nuclear layer.

sequence 2, 700 Kbytes  This sequence shows the cellular activity in a living slice of a 5 day embryonic retina. There is very active cellular blebbing throughout the tissue in addition to slow nuclear movement.

sequence 3,  900 Kbytes  This sequence shows the  cellular activity in an aggregate of 6 day embryonic retina cells which were cultured for 1 day.  Cells can be seen moving radially within the tissue.

2.  Are these movements accompanied by the expression of an extracellular protease?  Several lines of evidence suggest that this is the case.

a.  Isolated retinal neurons in vitro remove fluorescent gelatin from the substrate on which they are growing.  In the following image of cells grown on fluorescent gelatin, a phase contrast image is superimposed on a fluorescence image of the same cells.  The dark spaces in the fluorescence image represent regions from which the gelatin has been removed. Click on the thumbnail for a larger image.

Click for larger image.

b.  Inhibitors of Metalloproteinases alter the behavior of retinal growth cones in vitro.  Click on the images for a fuller discussion and time-lapse views.

Effect of HS-LFA

c.  Western blots and immunohistochemistry demonstrate that the developing retina does contain material which reacts with antibodies to the Matrix Metalloproteinase 2 (Gelatinase A).

d.  The MMP-2 is not easily detectable by zymography because of the presence of a significant amount of the Tissue Inhibitor of Metalloproteinases 3 (TIMP-3).

3. What is the fate of mitochondria during layer formation?  Since the mature chick retina lacks a vascular system, the retina changes from a tissue that relies primarily on aerobic metabolism to one that is mostly anaerobic. Click on the image for more information.

Mitochondrial Stain

 

4.  Is there a role for apoptosis in layer formation?  Initial studies indicate that there are waves of apoptosis, as measured by TUNEL stain, that pass through each of the cellular layers at characteristic times during development.