What do we know? -Differentiated cells in flatworms do not divide -Thus, neoblasts are the only dividing cells in flatworms (Egger et al 2007)
-Some Catenulids possess neoblasts in their epidermis, while no known Rhabditophora do -Is this a feature of Catenulids, or rather a primitive condition of flatworms? -S. virginianum along with other groups possess this feature, thus indicating a primitive condition of Platyhelminthes
Study of the cell-cycle could answer whether or not the cells in the epidermis are proliferating cells
Antibody “double-labeling technique” or the “sandwich” technique
The antibodies of the primary antibody host attach to the antigen of interest -Which is then rinsed -Placed into a solution of secondary antibody with dyes attached -The secondary anti-bodies are what contain the dye and allow fluorescence
The primary anti-bodies used were anti-PCNA and anti-phosphoH3
Fluorescent dyes that attached to the primary anti-bodies were Alexa flour 488/Cy2/Cy3/Cy5
1° Ab binding: (mouse anti-PCNA / rabbit anti-phosphoH3) 2° Ab binding: (Dye-labeled donkey anti-rabbit / donkey anti-mouse) Wash away excess Ab
Goal I: Osmium vs. Acetone/formaldehyde vs. Methacarn fixation methods -In a standard fixation the worms are placed into a vial filled with acetone after freezing on a LN2-cooled block - After an overnight storage in the freezer, warmed to room temperature and fixed with formaldehyde -In an osmium fixation the worms are placed into a vial filled with both acetone and 0.04% osmium - After an overnight storage in the freezer, warmed to room temperature, osmium worms already fixed -Test of Methacarn fixation, recommended DNA/PCNA protocol -Which one of these methods gives the best antibody staining and nuclear morphology? -I predict that the acetone/formaldehyde only fixations will be the more effective and optimal method of fixation for Stenostomum virginianum
Goal II: Optimization of primary antibody dilutions -Too high of a primary antibody concentration gives overstaining and high background; too low of a primary antibody concentration gives weak staining -I tested the effect of a 1:200 vs. 1:500 dilution of the primary antibodies -I predict that the 1-200 dilution will yield more positive proliferating cells -5uL of primary placed into 1mL buffer in 1:200 vs. 2.5mL in 1:500
Goal III: Overall distribution of PCNA/phosphoH3 positive cells -With the methods used above, what is the overall distribution of proliferating cells? -Where are they located? -Do the dyes exhibit non-specific binding? -S-phase lasts much longer than M-phase in the cell cycle -Therefore, I predict there will be more positive PCNA cells than phosphoH3 positive cells
Goal I: -In the Osmium fixations the mucus granules was highly fluorescent and could disrupt the ability to detect proliferating cells in the epithelium -The acetone fixations proved to have a significantly less non-specific labeling of the mucus granules allowing better distinction between the parenchyma and epithelial cells -Methacarn fixation proved to have poor immunostaining and imaging -The traditional method of acetone/formaldehyde is the optimal fixation method when using Stenostomum virginainum
Goal II: -The 1:200 vs. 1:500 dilutions showed no difference in immunolabeling between the samples -PhosphoH3 positive cells were found to be present in both dilutions, while there were no signs of any PCNA positive cells -From the samples collected there did appear however to be a sharper contrast in imaging from the 1:500 dilutions -Higher dilutions, such as 1:1000 seem plausible -Cost efficient
Goal III: -Distribution of cells are found to be highly concentrated along the formation of division planes, and all along body cavity -Some positive PhosphoH3 cells were found in a majority if the samples, however there was not a significant number found in the epidermis -A cell spends much longer (7.5hr-10hr) in S-phase than M-phase (1hr) -Expected to see lots of S-phase cells, but saw none -Does lack of PCNA detection indicate lack of PCNA in S. virginianum? - PCNA primary anti-body was raised in mouse anti-human, could be the result of ineffective binding -Cy3 will bind to the mucus granules as well as to some positive phosphoH3 cells
Egger, Bernard, and S. Ishida. "Chromosome fission or duplication in Macrostomum ligano (Macrostomorpha, Plathelminthes) - remarks on chromosome numbers in archoophoron turbellarians." (2004): 127-32.
Smith III, Julian P.S., and Sara Merlie. Stem Cells in Stenostomum virginianum. Department of Biology, Winthrop University.
Smith III, Julian P.S., Bernard Egger, Seth Tyler, Peter Ladurner, Johannes Achatz, and Sara Merlie. Neoblasts in Nemertodermatida.
Smith III, Julian P.S., Kevin Ryan, and Sara Merlie. Neoblasts in Catenulida. Department of Biology, Winthrop University.