Pattern formation during development is widely thought to involve specification of cells' positions in the embryo by gradients of informational molecules collectively known as morphogens. This is in keeping with much embryological data, but little success has been made in identifying such morphogens. In some lower organisms cyclic AMP, intracellular free calcium, or pH have been implicated as diffusible morphogens, but the involvement of these agents in vertebrate pattern formation has not been carefully investigated. Teratogens are often employed in the study of pattern formation, in particular the lithium ion, but its pathological effects on whole embryos are difficult to interpret. The recent demonstration that microinjection of Li+ into a specific blastomere of the early frog embryo induces formation of a second complete head now provides a powerful tool for such studies. Li+ has two well-established biochemical effects: inhibition of the phosphatidylinositol cycle (PI cycle) and of adenylate cyclase. Since the former's products, inositol trisphosphate and diacylglycerol, are involved in intracellular free calcium and pH regulation and the latter enzyme produces cyclic AMP, these agents may be involved in vertebrate pattern formation. The long-term goal of this study is to employ the teratogen, Li+, to test the involvement of pH, free Ca2+, cAMP, and inositol phosphates in pattern formation.
Specific aims are to: (1) determine the distribution of PI cycle intermediates, cAMP levels, and intracellular free Ca2+ and pH values in normal frog embryos; (2) determine the effects of Li+ microinjection on these distributions and attempt to correlate these with head formation; (3) determine whether other inhibitors of the PI cycle and cAMP action mimic lithium's head-inducing ability; and (4) test whether direct manipulations of these distributions can mimic or prevent lithium's effects. Methodologies employed include measurement of intracellular free Ca2+ and pH with fluorescent probes and digital image analysis, chromatographic assay of PI cycle intermediates, radioimmunoassay of cAMP, microinjection, and microelectrodes. Health relatedness: These studies may contribute to our understanding of the mechanisms of teratogens such as Li+ (which is widely employed clinically), and of some birth defects of the neural tube and head, such as anencephaly. They may also fundamentally contribute to our understanding of vertebrate pattern formation.
