Lithium chloride inhibits development along the animal vegetal axis and anterior midline of the squid embryo.

When squid embryos, Loligo pealeii, are cultured in vitro (1) they may be individually manipulated with classic and molecular techniques, providing insights into the conservation of developmental pathways. Dorsoventral polarity in many embryos is associated with precise gene transduction cascades involving the Wnt signaling pathway (2). Results from experiments with frog (3), fish (4), and mouse (5) embryos suggest that a component of this cascade, -catenin, plays a major role in axis formation. Additional support for the role of -catenin in the early development of many embryos comes from studies using lithium chloride (LiCl). LiCl is a known vegetalizing agent for sea urchins (6), and in echinoderms generally, it enhances and expands levels and regions of nuclear -catenin localization coincident with increases in endoderm and mesoderm (7, 8). In contrast, its effect on amphibian embryos is speciesand stage-specific. For example, when gastrulating embryos are treated with LiCl, they develop reduced notochords and enhanced vegetal structures, but when treatments are given at earlier or later cleavage stages, dorsalizing and anteriorizing effects, respectively, are observed (3, 9). In this study, embryos cultured in vitro were treated with lithium chloride to determine its effect on development; this is the first step towards understanding the molecular mechanisms of patterning in squid. Embryos were fertilized in vitro (1) and cultured at 17 °C in 60-mm plastic petri dishes (Falcon) that were lined with 0.2% agarose (Type II-A, Sigma), filled with Millipore (0.22 M) filtered seawater (MFSW), and supplemented with bovine serum albumin (BSA) (0.5%). Each LiCl treatment dish also contained 20, 40, or 60 mM LiCl (Sigma). Three trials of 15 embryos per treatment were performed. Dishes and solutions were changed every other day. Embryos were treated with LiCl for the first 6 d of development, by which time epiboly of the outer yolk cell was complete. Development was observed until the control embryos began to hatch from their chorions, at 19–20 d after fertilization. The classical stages of J. M. Arnold (10) were used to describe embryonic development. Embryos cultured in the presence of LiCl exhibited a dosage-dependent inhibition of development that was evident by 6 d in culture (Fig. 1a, b, 6 d post-fertilization (dpf), stage 18), but it was more easily detected later, during organogenesis (Fig. 1d, e, f, 17 dpf, stage 27). Development was inhibited in many structures normally associated with ectodermal tissues, such as tentacles, eyes, mantle, fins, and funnel. Moreover, convergence and inhibition of anterior midline structures was observed in embryos treated with 40 and 60 mM LiCl. In one trial, for example, 8 of 16 embryos cultured in the presence of 60 mM LiCl had anterior midline structures that were inhibited. As a result, the eyes were abnormally placed: either converged (5/16, Fig. 1f), fused (2/16), or cyclopic (1/16). In contrast, structures that normally form on the posterior body wall such as the funnel or paired statocysts, although reduced, were always present in these embryos. These observations suggest that various regions in the embryo are differentially sensitive to LiCl treatment. Lithium chloride treatment inhibits development along the animal-vegetal axis in the squid embryo and causes convergence and fusion of anterior cephalic structures. This result is consistent with the notion that LiCl treatment induces vegetalization of the squid embryo and thereby enhances mesodermal and endodermal structures at the expense of ectodermal derivatives, as it does in other invertebrate and vertebrate embryos (3, 6, 7, 8, 9). That LiCl treatment may induce convergence, fusion, and cyclopia in squid embryos strengthens this interpretation (see 11 for a survey of the early literature on cyclopia produced by experimental means) and provides insights into the possible conservation of developmental mechanisms in cephalopods. This work was made possible by support from a Faculty Development Grant and the Aldom-Plansoen Distinguished Endowed Professorship in Contemporary Studies to K.C. from St. Mary’s College of Maryland. K.C. is most grateful to Bill Eckberg, Howard University, who graciously provided laboratory space, collaborative guidance, and digital imaging assistance.