Thesis

The effects of lectins in sea urchin Lytechinus pictus during gastrulation in low calcium sea water

In order to help learn about the possible role of carbohydrate – containing molecules in gastrulation in the model sea urchin embryo (Lytechinus pictus), 24 hr sea urchin embryos were incubated with 3 lectins (carbohydrate binding proteins) Triticum vulgaris (wheat germ agglutinin), Artocarpus integrifolia agglutinin and Phaseolus vulgaris PHA-L agglutinin at 0.1-0.00001 mg/ml. Triticum is a specific binder of N-acetyl-D-glucosamine- like residues, as is Phaseolus, while Artocarpus is a specific binder of D-galactose-like residues. The embryos were treated with and without these lectins at all 5 concentrations for an additional 24 hrs at 15 ⁰C in lower calcium artificial seawater (that speeds entry of molecules into the interior of the embryos) in 96 wells well flat bottom microplates. The wells were treated with 10% formaldehyde to fix the embryos at the 48 hr stage (late gastrula). All embryos in each well were scored as to their morphologies: complete archenteron, incomplete archenteron, non-invaginated, exogastrulated or dead. Thousands of embryos were scored. Means of percentages of each morphology for each concentration of each lectin was plotted with standard error bars and a t-test was used to determine if any differences in the experimentals vs. controls were statistically significant (P<0.05). The results indicated statistically significant concentration dependent effects of all 3 lectins on altering the morphologies of the embryos. These preliminary results suggest that N-acetyl –D- glucosamine and D-galactose groups maybe involved in archenteron elongation and organization. The microplate assay is an effective means of quantitatively determining the precise effects of reagents on sea urchin morphologies, an NIH designated model for higher organisms including humans.

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