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	...	The Effects of NiCl2on Spicule Formation Heather Stallman & Matt Listner, Millersville University Jessica Ann Billet, Franklin and Marshall College Background Sea urchins exhibit radial holoblastic cleavage, eventually forming a blastula. Shortly after the blastula hatches from the fertilization membrance, the embryo begins gastrulation. Gastrulation begins when the vegetal side of the blastula begins to thicken and flatten. This flat sheet of cells is called the vegetal plate. In the center of the vegetal plate a small group of cells begins to change. These cells extend and contract long, thin filopodia. These cells then break off from the epithelium and migrate into the blastocoel. These migrating cells are known as the primary mesenchyme cells (PMCS). Before the PMCs migrate, there are equatorial ring patterns that are formed in certain regions of the embryo for the PMCs to navigate to where skeletogenesis can occur. The ring formed by the PMCs during gastrulation is composed of two aggregates of cells: the ventrolateral clusters and the ventral and dorsal chains (Guss, 1997). Eventually the migrating cells localize within the ventrolateral region of the blastocoel. It is in this area that the PMCs fuse together to form syncytial cables. Syncytial cables will eventually form the axis of the calcium carbonate spicules of the larval skeleton. Spicules are the rods of the sea urchin larval skeleton that are formed from skeletogenic mesenchyme cells in the early gastrula stage and are composed of calcium carbonate (Gilbert, 2010). In normal sea urchin development during gastrulation, the spicules and skeleton morphogenesis is influenced by the interaction between the underlying ectoderm, the PMCs, and the rest of the embryo. There are several PMC-species gene products that participate in the synthesis of the skeleton, but the four proteins that are part of the spicule matrix are msp130, SM50, SM30, and PM27 (Guss, 1997). The purpose of this lab is to explore the development of spicules in a sea urchin's larval skeleton. NiCl2 is one of the few chemicals that can interfere with the dorsoventral axis and the development of the spicule and skeletal formation. Nickel chloride interferes with spicules development by altering the interaction between the PMCs and the rest of the embryo. Nickel chloride also disrupts the location and amount of dorsal and ventral tissues (Hardin et. al., 1992). Nickel chloride influences the ectoderm of the sea urchin blastula wall (Armstrong et. al., 1993). By introducing half of the embryos into a solution of NiCl2, we hope to observe the effects of blocking skeleton formation. We will stain all of the embryos with an Ig8 immunofluorescent antibody. Ig8 will stain or 'tag' the primary mesenchyme cells of the developing embryo. Another way is to observe spicule foramtion by exposing the sea urchin embryos to polarizing light under a compound microscope because the calcium carbonate from the spicules bends polarized light and the spicules become visible. Objective: To observe the developmental effects on the formation of spicules on the larval skeleton in sea urchin embryos. To observe the spicules in the gastrula stage and the larval skeleton using polarized light. The hypothesis is the nickel chloride will affect skeleton formation by the primary mesenchyme cells during gastrulation.