Look Ma, No Archenteron! Sulfate role in sea urchin early development
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Objective
To observe the role sulfate plays in sea urchin gastrulation, and to replicate the findings of Karp and Solursh, that sea urchin embryos fail to gastrulate without sulfate. To test whether endoderm differentiation can occur in the absense of the movements of gastrulation.
Abstract
Sea urchin development requires not only internally produced signals, but also materials incorporated from the environment. Karp and Solursh have hypothesized that secondary mesenchyme cells, which form the filopodia of the developing archenteron (primary gut) require sulfate (to form sulfated proteoglycans which act as something like an adhesive) in order to migrate along the acid mucopolysaccharide of the extracellular matrix within the blastocoel of a developing sea urchin (1974). Developing sea urchin embryos were raised in either artificial sea water or sulfate-free sea water, fixed, and stained for alkaline phosphotase enzyme activity and with immunofluorescent antibodies to vegetal archenteron cells. Embryos raised in a sulfate-free environment, unlike controls, did not develop archenterons. Staining indicated, however, that these cells did differentiate to become gut cells and had invaginated, but the cells could not migrate up the blastocoel wall. This data supports the findings and hypothesis of Karp and Solursh (1974).
Sea urchin development progresses in a predictable and easily observable way (Gilbert, 2000). The vegetal plate thickens and primary mesenchyme cells ingress and form spicules, the urchin skeleton (Figure 1). The vegetal plate then invaginates, forming the archenteron, or primitive gut. The archenteron migrates up the sea urchin's blastocoel wall with the help of secondary mesenchyme cells (Figure 1).
The migration of the archenteron depends not only on signals and proteins already present in the egg, but also on extracellular materials that have been incorporated into the organism. Karp and Solursh have hypothesized that secondary mesenchyme cells, which form the filopodia of the developing archenteron (primary gut) require sulfate (as something like an adhesive) in order to migrate along the extracellular matrix within the blastocoel of a developing sea urchin (1974).
Presumably, sea urchin embryos incorporate sulfate from the environment into their extracellular matrixes. The extracellular matrix contains acid mucopolysaccharide, which when bound to sulfate, is rough in appearance (Karp and Solursh, 1974). This roughness is akin to velcro's roughness, allowing secondary mesenchyme cells to pull the archenteron up along the blastocoel cavity. If sulfate is not present, it has been observed that an archenteron does not form (Karp and Solursh, 1974). Fixed and stained embryos will indicate if the gut endoderm cells have differentiated (and have simply failed to migrate).
Figure 1. A schematic drawing of a gastrulating sea urchin embryo. Note the secondary mesenchyme cells, in the form of a filopodia, attaching to the blastocoel wall to pull the archenteron up through the blastocoel to form the gut cavity.
Materials and Methods
Lytchinus pitus or Strongylocentrotus purpuratus (from the Pacific coast), and Arbacia punctulata or Lytchinus variegates (from the Atlantic and Gulf of Mexico) can be used for this experiment. Lytchinus variegatus was used for our experiment. Sea urchin gamete release was induced by injection of KCl and eggs and sperm were obtained in separate beakers as described in basic protocol "sea urchin gamete collection" .
