Essential and opposing roles of zebrafish beta-catenins in the formation of dorsal axial structures and neurectoderm.

In Xenopus, Wnt signals and their transcriptional effector beta-catenin are required for the development of dorsal axial structures. In zebrafish, previous loss-of-function studies have not identified an essential role for beta-catenin in dorsal axis formation, but the maternal-effect mutation ichabod disrupts beta-catenin accumulation in dorsal nuclei and leads to a reduction of dorsoanterior derivatives. We have identified and characterized a second zebrafish beta-catenin gene, beta-catenin-2, located on a different linkage group from the previously studied beta-catenin-1, but situated close to the ichabod mutation on LG19. Although the ichabod mutation does not functionally alter the beta-catenin-2 reading frame, the level of maternal beta-catenin-2, but not beta-catenin-1, transcript is substantially lower in ichabod, compared with wild-type, embryos. Reduction of beta-catenin-2 function in wild-type embryos by injection of morpholino antisense oligonucleotides (MOs) specific for this gene (MO2) results in the same ventralized phenotypes as seen in ichabod embryos, and administration of MO2 to ichabod embryos increases the extent of ventralization. MOs directed against beta-catenin-1 (MO1), by contrast, had no ventralizing effect on wild-type embryos. beta-catenin-2 is thus specifically required for organizer formation and this function is apparently required maternally, because the ichabod mutation causes a reduction in maternal transcription of the gene and a reduced level of beta-catenin-2 protein in the early embryo. A redundant role of beta-catenins in suppressing formation of neurectoderm is revealed when both beta-catenin genes are inhibited. Using a combination of MO1 and MO2 in wild-type embryos, or by injecting solely MO1 in ichabod embryos, we obtain expression of a wide spectrum of neural markers in apparently appropriate anteroposterior pattern. We propose that the early, dorsal-promoting function of beta-catenin-2 is essential to counteract a later, dorsal- and neurectoderm-repressing function that is shared by both beta-catenin genes.

Characterization of mouse Rsk4 as an inhibitor of fibroblast growth factor-RAS-extracellular signal-regulated kinase signaling.

Receptor tyrosine kinase (RTK) signals regulate the specification of a varied array of tissue types by utilizing distinct modules of proteins to elicit diverse effects. The RSK proteins are part of the RTK signal transduction pathway and are thought to relay these signals by acting downstream of extracellular signal-regulated kinase (ERK). In this study we report the identification of ribosomal S6 kinase 4 (Rsk4) as an inhibitor of RTK signals. Among the RSK proteins, RTK inhibition is specific to RSK4 and, in accordance, is dependent upon a region of the RSK4 protein that is divergent from other RSK family members. We demonstrate that Rsk4 inhibits the transcriptional activation of specific targets of RTK signaling as well as the activation of ERK. Developmentally, Rsk4 is expressed in extraembryonic tissue, where RTK signals are known to have critical roles. Further examination of Rsk4 expression in the extraembryonic tissues demonstrates that its expression is inversely correlated with the presence of activated ERK 1/2. These studies demonstrate a new and divergent function for RSK4 and support a role for RSK proteins in the specification of RTK signals during early mouse development.