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The Wnt family of secreted proteins has been proposed to play a conserved role in early specification of the bilaterian anteroposterior (A/P) axis. This hypothesis is based predominantly on data from vertebrate embryogenesis as well as planarian regeneration and homeostasis, indicating that canonical Wnt (cWnt) signaling endows cells with positional information along the A/P axis. Outside of these phyla, there is strong support for a conserved role of cWnt signaling in the repression of anterior fates, but little comparative support for a conserved role in promotion of posterior fates. We further test the hypothesis by investigating the role of cWnt signaling during early patterning along the A/P axis of the hemichordate Saccoglossus kowalevskii. We have cloned and investigated the expression of the complete Wnt ligand and Frizzled receptor complement of S. kowalevskii during early development along with many secreted Wnt modifiers. Eleven of the 13 Wnt ligands are ectodermally expressed in overlapping domains, predominantly in the posterior, and Wnt antagonists are localized predominantly to the anterior ectoderm in a pattern reminiscent of their distribution in vertebrate embryos. Overexpression and knockdown experiments, in combination with embryological manipulations, establish the importance of cWnt signaling for repression of anterior fates and activation of mid-axial ectodermal fates during the early development of S. kowalevskii. However, surprisingly, terminal posterior fates, defined by posterior Hox genes, are unresponsive to manipulation of cWnt levels during the early establishment of the A/P axis at late blastula and early gastrula. We establish experimental support for a conserved role of Wnt signaling in the early specification of the A/P axis during deuterostome body plan diversification, and further build support for an ancestral role of this pathway in early evolution of the bilaterian A/P axis. We find strong support for a role of cWnt in suppression of anterior fates and promotion of mid-axial fates, but we find no evidence that cWnt signaling plays a role in the early specification of the most posterior axial fates in S. kowalevskii. This posterior autonomy may be a conserved feature of early deuterostome axis specification.

In the 150 years since Darwin, the field of evolutionary biology has left a glaring gap in understanding how animals developed their astounding variety and complexity. The standard answer has been that small genetic mutations accumulate over time to produce wondrous innovations such as eyes and wings. Drawing on cutting-edge research across the spectrum of modern biology, Marc Kirschner and John Gerhart demonstrate how this stock answer is woefully inadequate. Rather they offer an original solution to the longstanding puzzle of how small random genetic change can be converted into complex, useful innovations.In a new theory they call \"facilitated variation,\" Kirschner and Gerhart elevate the individual organism from a passive target of natural selection to a central player in the 3-billion-year history of evolution. In clear, accessible language, the authors invite every reader to contemplate daring new ideas about evolution. By closing the major gap in Darwin's theory Kirschner and Gerhart also provide a timely scientific rebuttal to modern critics of evolution who champion \"intelligent design.\"

The dorsal--ventral axis in frog embryos is specified during the first cell cycle, when the cortex rotates relative to the cytoplasmic core along parallel microtubules associated with the core. Cytoplasmic transfer experiments suggest that dorsal determinants are transported 90 degrees from the vegetal pole to the dorsal equator, even though the cortex rotates only 30 degrees. Here we show that, during rotation, small endogenous organelles are rapidly propelled along the subcortical microtubules toward the future dorsal side and that fluorescent carboxylated beads injected into the vegetal pole are transported at least 60 degrees toward the equator. We also show that deuterium oxide, which broadens the zone of dorsalization even though it reduces the extent of rotation and is known to randomize the microtubules, also randomizes the direction of organelle transport. Moreover, β -catenin, a component of the Wnt signaling pathway that possesses dorsalizing activity in Xenopus, colocalizes with subcortical microtubules at the dorsal side of the egg at the end of rotation. We propose that cortical rotation functions to align subcortical microtubules, which then mediate the transport of dorsal determinants toward their plus ends on one side of the egg.

A large library of phage-displayed human single-chain Fv antibodies (scFv), containing 6.7 × 109members, was generated by improving the steps of library construction. Fourteen different protein antigens were used to affinity select antibodies from this library. A panel of specific antibodies was isolated with each antigen, and each panel contained an average of 8.7 different scFv. Measurements of antibody-antigen interactions revealed several affinities below 1 nM, comparable to affinities observed during the secondary murine immune response. In particular, four different scFv recognizing the ErbB2 protein had affinities ranging from 220 pM to 4 nM. Antibodies derived from the library proved to be useful reagents for immunoassays. For example, antibodies generated to the Chlamydia trachomatis elementary bodies stained Chlamydia-infected cells, but not uninfected cells. These results demonstrate that phage antibody libraries are ideally suited for the rapid production of panels of high-affinity mAbs to a wide variety of protein antigens. Such libraries should prove especially useful for generating reagents to study the function of gene products identified by genome projects.

It has long been thought that anteroposterior (A-P) pattern in the vertebrate central nervous system is induced in the embryo's dorsal ectoderm exclusively by signals passing vertically from underlying, patterned dorsal mesoderm. Explants from early gastrulae of the frog Xenopus laevis were prepared in which vertical contact between dorsal ectoderm and mesoderm was prevented but planar contact was maintained. In these, four position-specific neural markers (engrailed-2, Krox-20, XIHbox 1, and XIHbox 6) were expressed in the ectoderm in the same A-P order as in the embryo. Thus, planar signals alone, following a path available in the normal embryo, can induce A-P neural pattern.

It has long been held that the parathyroid glands and parathyroid hormone evolved with the emergence of the tetrapods, reflecting a need for new controls on calcium homeostasis in terrestrial, rather than aquatic, environments. Developmentally, the parathyroid gland is derived from the pharyngeal pouch endoderm, and studies in mice have shown that its formation is under the control of a key regulatory gene, Gcm-2. We have used a phylogenetic analysis of Gcm-2 to probe the evolutionary origins of the parathyroid gland. We show that in chicks, as in mice, Gcm-2 is expressed in the pharyngeal pouches and the forming parathyroid gland. We find that Gcm-2 is present not only in tetrapods but also in teleosts and chondrichthyans, and that in these species, Gcm-2 is expressed within the pharyngeal pouches and internal gill buds that derive from them in zebrafish (Danio rerio), a teleost, and dogfish (Scyliorhinus canicula), a chondrichthyan. We further demonstrate that Gcm-2 is required for the formation of the internal gill buds in zebrafish. We also have identified parathyroid hormone 1/2-encoding genes in fish and show that these genes are expressed by the gills. We further show that the gills express the calcium-sensing receptor, which is used in tetrapods to monitor serum calcium levels. These results indicate that the tetrapod parathyroid gland and the gills of fish are evolutionarily related structures, and that the parathyroid likely came into being as a result of the transformation of the gills during tetrapod evolution.

In and evaginations of 2D cell sheets are major shape generating processes in animal development. They result from directed movement and intercalation of polarized cells associated with cell shape changes. Work on several bilaterian model organisms has emphasized the role of noncanonical Wnt signaling in cell polarization and movement. However, the molecular processes responsible for generating tissue and body shape in ancestral, prebilaterian animals are unknown. We show that noncanonical Wnt signaling acts in mass tissue movements during bud and tentacle evagination and regeneration in the cnidarian polyp HYDRA: The wnt5, wnt8, frizzled2 (fz2), and dishevelled-expressing cell clusters define the positions, where bud and tentacle evaginations are initiated; wnt8, fz2, and dishevelled remain up-regulated in those epithelial cells, undergoing cell shape changes during the entire evagination process. Downstream of wnt and dsh expression, JNK activity is required for the evagination process. Multiple ectopic wnt5, wnt8, fz2, and dishevelled-expressing centers and the subsequent evagination of ectopic tentacles are induced throughout the body column by activation of Wnt/β-Catenin signaling. Our results indicate that integration of axial patterning and tissue morphogenesis by the coordinated action of canonical and noncanonical Wnt pathways was crucial for the evolution of eumetazoan body plans.

Qualitative and quantitative information are crucial to a detailed understanding of the function of protein phosphorylation. MS is now becoming a quantitative approach to analyze protein phosphorylation. All methods that have been described either require the elaborate/expensive use of stable isotopes to compare a limited number of samples or do not provide phosphorylation stoichiometries. Here, we present stable isotope-free MS strategies that allow relative and absolute quantitation of phosphorylation stoichiometries. By using the developed methods, we can normalize to robustly account for run-to-run variations and variations in amounts of starting material. This procedure monitors the unmodified proteolytic peptides derived from the protein of interest and identifies peptides that are suitable for normalization purposes. Also, we can determine changes in phosphorylation stoichiometry by monitoring the changes in the normalized ion currents of the phosphopeptide(s) of interest. Absolute phosphorylation stoichiometry are measured by monitoring the ion currents of a phosphopeptide and its unmodified cognate as the signal intensity changes of both peptide species are correlated. The method is applicable to multiply phosphorylated species (for which one more sample with varying phosphorylation stoichiometry than number of phosphorylation sites is required to correct for the differences in the ionization/detection efficiencies of the phosphopeptide, its partially phosphorylated and unphosphorylated cognates). Last, we can quantitate species with ragged ends resulting from incomplete proteolysis and measure phosphorylation stoichiometries of single samples by controlled dephosphorylation. These approaches were validated and subsequently applied to the phosphorylation of the yeast transcription factor Pho4.

Transgenic Xenopus laevis embryos were produced by transplantation of transfected cultured cell nuclei into unfertilized eggs. A Xenopus cell line, X-C, was stably transfected with plasmids containing a hygromycin-resistance gene and genes for either β-galactosidase with a heat shock promoter or chloramphenicol acetyltransferase (CAT) with a muscle-specific actin promoter. Nuclei transplanted from these cells into unfertilized eggs directed development of embryos containing stably integrated copies of the plasmids in each cell. Transgenic embryos showed somite-specific expression of CAT and uniform expression of β-galactosidase. Transgenic embryos produced by nuclear transplantation should be useful for testing the function of cloned genes in amphibian development.