Because of its transparency, accessibility, simplicity and availability, the sea urchin embryo has been widely used to study cellular interactions that also occur in human development and disease. That is why the sea urchin embryo nas been designated a NiH model system and isthe rationale for its use in this study. Our long-term goal is to understand the molecular basis of one fuhctipnaliy important cellularInteraction, namely the attachment of the developing gut (archenteron) to the roof of the blastoeoel. Our working hypothesis, based on our published preliminary results, is that secondary mesehchyme cells at the tip of the arehenteron bind to cells of the blastoeoel roof via ligands containing mannose/glucose moieties associatirig with lectin-like receptors for these ligands.
These specific aims will test this hypothesis: We will determine: (1) if microdissected components of thes cellular interaction bind together via mannose/gluoose moieties and their receptors;(2) if molecules that we isolated from living embryos, that block the cellular interaction in live embryos, can be purified and characterized;(3) if the active components of these molecules are specific sugars and receptors;(4) if the purified molecules block the cellular interaction in living embryos;(5) if the molecules are localized on the right cell types arid at the right developmental time;(6) if masking the molecules in live embryos blocks the cellular interaction;(7) if the findings are similar in two species of sea urchins. This study is innovative and novel because the system is totally accessible to probes in living embryos and allows microdissection to isolate and access the components of the cellular interaction for direct experimentation. Of importance in this revision is that we isolated molecules by anion-exchange HPLC that meet criteria for mediating the cell interaction. Two active HPLC fractions contain 1 (35 kD) and 2 (35 kD and 150 kD) bands on PAGE. Milligram quantities of the pufifi.ed molecules are easily Obtainable and will be sequeheed and compared with known proteins;frorri various genome databases. Relevance to Public Health. This study will identify molecular mechanisms that control a specific cellular interaction using the exquisitely accessible sea urchin erhbryo, a NIH designated model. These mechanisms will be of major importance in the elucidation of similar mechanisms that control human development and cancer spread, in human systems that are not very accessible to experimentation.
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