Incubate embryos at 18-20ûC and score perturbations at early gastrula.

Rinse incubated embryos and transfer to petri dish with 10% Steinberg's solution.Ħ. Incubate embryos at 18-20ûC for 10 minutes.ĥ. Transfer the remaining embryos to a control petri dish with 10% Steinberg's solution.Ĥ. Rinse healthy embryos and transfer 3/4 to the experimental petri dish containing 0.3M lithium chloride in 0.1x MBS. Dejelly early blastula embryos before the 32 cell stage.ġ) Select and separate embryos below the 32 cell stageĢ) Remove jelly membrane using sharpened forceps.Ĥ) Place dejellied embryos in 10% Steinberg's solution.ĥ) Select embryos that remain below or at the 32 cell stageĢ. As mentioned, lithium has the ability to inhibit glycogen synthase kinase-ß(beta), which is a substance which initites the wnt pathway in xenopus embryos that leads to dorsal axis formation (Klein and Melton 1996).ġ. 2000) This event occurs at the same time in development that the dorsal blastopore lip would form if there had been no lithium chloride treatment administered. The most extreme results of this treatment are embryos with radial symmetric heads, a cylindrical heart, and bands of eye pigment. The result is an embryo that has a continous doral side. Dorsalization is when the dorsal blastopore lip forms around the entire embryo at the same time. When early embryos are treated with lithium chloride before the 64 cell stage, it has been shown that the embryos undergo dorsalization (Kao and Elison 1988, 1989 Slack 1988). The dorsal blastopore lip is not only the point of entry of cells, but also sets the dorsal-ventral axis and marks the posterior of the embryo. The cells which are migrating into the embryo travel in through the dorsal blastopore lip. This region has rotated, the cortical(outer) cytoplasm rotates 30û, so that its location is between the animal and vegetal pole. The gray crescent is the region which is opposite from the location of sperm entry. Gastrulation initiates on the future dorsal side of the organism which is marked by the gray crescent. After the blastula has formed and the blastocoel, the hollow cavity inside the blastula, is present, then gastrulation begins. The earliest stages of Xenopus development after fertilization of the egg is characterized by the rapid cleavage of cells, which is holoblastic(uneven) due to the large concentration of yolk at the vegetal pole. Lithium is believed to be the cause these perturbations due to its ability to inhibit glycogen synthase kinase-ß(beta) which initiates developmental pathways. INTRODUCTION: When the developing embryos of many organisms, such as the lytechinus variegatus (sea urchin) and xenopus (frog), are treated with lithium chloride, they experience alterations or perturbations in development. PURPOSE: The objective of this experiment is to study the effect of lithium chloride treatment on early blastula xenopus embryos.

Justin A Spenillo, Franklin & Marshall College DEVELOPMENTAL EFFECTS OF LITHIUM CHLORIDE ON XENOPUS EMBRYOS

Definition of blastopore