Thesis

Effects of α-mannosidase in sea urchin embryo gastrulation

The sea urchin embryo is a NIH designated model system to study mechanisms that may be involved in human health and disease. In order to determine if mannose residues are involved in sea urchin embryo cellular interactions at gastrulation, 6324 Lytechinus pictus embryos were incubated with varying concentrations of independently characterized (assayed for purity and activity) α-mannosidase for 24 hours using a quantitative microplate assay. The results showed a variety of morphological deformations even for α-mannosidase activity of as low as 0.06 U/ml. These effects did not occur using heat treated (that we call denatured) α-mannosidase. Thousands of replicates were statistically evaluated and the results suggest that α1-2-, α1-3-, and/or α1-6-linked mannose residues may play an important role during sea urchin gastrulation. Additionally, formaldehyde fixed, 48 hour old Lytechinus pictus embryos were microdissected to attempt to identify a specific site of α-mannosidase action within the embryo. The procedure involved separation of archenterons from the blastocoel roofs. Microdissected control groups maintained the adhering properties of the cells located on the tip of the archenteron to the roof of the blastocoel when the dissected pieces were placed back together. But, in these small sample preliminary experiments embryo pieces that were incubated with α-mannosidase lost their adhesive properties suggesting the tip of the archenteron and roof of the blastocoel are possible sites for the effect of alpha-mannosidase. Both the microplate and microdissection approaches used here independently suggest a role for D-(+)-mannose residues in sea urchin gastrula adhesive interactions.

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