Electron microscopic visualization of tRNA genes with ferritin-avidin: biotin labels

A method is described for indirect electron microscopic visualization and mapping of tRNA and other short transcripts hybridized to DNA. This method depends upon the attachment of the electron-dense protein ferritin to the RNA, the binding being mediated by the remarkably strong association of the egg white protein avidin with biotin. Biotin is covalently attached to the 3' end of tRNA using an NH2 (CH2) 5NH2 bridge. The tRNA-biotin adduct is hybridized to complementcrry DNA sequences present in a single stranded nonhomology loop of a DNA:DNA heteroduplex. Avidin, covalently crosslinked to ferritin is mixed with the heteroduplex and becomes bound to the hybridized tRNA-biotin. Observation of the DNA:RNA-biotin:avidin-ferritin complex by electron microsdopy specifically and accurately reveals the position of the tRNA gene, with a frequency of labeling of approximately 50%

Fez function is required to maintain the size of the animal plate in the sea urchin embryo

Partitioning ectoderm precisely into neurogenic and non-neurogenic regions is an essential step for neurogenesis of almost all bilaterian embryos. Although it is widely accepted that antagonism between BMP and its inhibitors primarily sets up the border between these two types of ectoderm, it is unclear how such extracellular, diffusible molecules create a sharp and precise border at the single-cell level. Here, we show that Fez, a zinc finger protein, functions as an intracellular factor attenuating BMP signaling specifically within the neurogenic region at the anterior end of sea urchin embryos, termed the animal plate. When Fez function is blocked, the size of this neurogenic ectoderm becomes smaller than normal. However, this reduction is rescued in Fez morphants simply by blocking BMP2/4 translation, indicating that Fez maintains the size of the animal plate by attenuating BMP2/4 function. Consistent with this, the gradient of BMP activity along the aboral side of the animal plate, as measured by pSmad1/5/8 levels, drops significantly in cells expressing Fez and this steep decline requires Fez function. Our data reveal that this neurogenic ectoderm produces an intrinsic system that attenuates BMP signaling to ensure the establishment of a stable, well-defined neural territory, the animal plate

Gene Regulatory Network Interactions in Sea Urchin Endomesoderm Induction

A major goal of contemporary studies of embryonic development is to understand large sets of regulatory changes that accompany the phenomenon of embryonic induction. The highly resolved sea urchin pregastrular endomesoderm–gene regulatory network (EM-GRN) provides a unique framework to study the global regulatory interactions underlying endomesoderm induction. Vegetal micromeres of the sea urchin embryo constitute a classic endomesoderm signaling center, whose potential to induce archenteron formation from presumptive ectoderm was demonstrated almost a century ago. In this work, we ectopically activate the primary mesenchyme cell–GRN (PMC-GRN) that operates in micromere progeny by misexpressing the micromere determinant Pmar1 and identify the responding EM-GRN that is induced in animal blastomeres. Using localized loss-of -function analyses in conjunction with expression of endo16, the molecular definition of micromere-dependent endomesoderm specification, we show that the TGFβ cytokine, ActivinB, is an essential component of this induction in blastomeres that emit this signal, as well as in cells that respond to it. We report that normal pregastrular endomesoderm specification requires activation of the Pmar1-inducible subset of the EM-GRN by the same cytokine, strongly suggesting that early micromere-mediated endomesoderm specification, which regulates timely gastrulation in the sea urchin embryo, is also ActivinB dependent. This study unexpectedly uncovers the existence of an additional uncharacterized micromere signal to endomesoderm progenitors, significantly revising existing models. In one of the first network-level characterizations of an intercellular inductive phenomenon, we describe an important in vivo model of the requirement of ActivinB signaling in the earliest steps of embryonic endomesoderm progenitor specification

Sea Urchin FGFR Muscle-Specific Expression: Posttranscriptional Regulation in Embryos and Adults

AbstractWe have shown previously byin situhybridization that a gene encoding a fibroblast growth factor receptor (SpFGFR) is transcribed in many cell types during the initial phases of sea urchin embryogenesis (Strongylocentrotus purpuratus) (McCoonet al., J. Biol. Chem. 271,20119–20195, 1996). Here we demonstrate by immunostaining with affinity-purified antibody that SpFGFR protein is detectable only in muscle cells of the embryo and appears at a time suggesting that its function is not in commitment to a muscle fate, but instead may be required to support the proliferation, migration, and/or differentiation of myoblasts. Surprisingly, we find thatSpFGFRtranscripts are enriched in embryo nuclei, suggesting that lack of processing and/or cytoplasmic transport in nonmuscle cells is at least part of the posttranscriptional regulatory mechanism. Western blots show that SpFGFR is also specifically expressed in adult lantern muscle, but is not detectable in other smooth muscle-containing tissues, including tube foot and intestine, or in coelomocytes, despite the presence ofSpFGFRtranscripts at similar concentrations in all these tissues. We conclude that in both embryos and adults, muscle-specific SpFGF receptor synthesis is controlled primarily at a posttranscriptional level. We show by RNase protection assays that transcripts encoding the IgS variant of the ligand binding domain of the receptor, previously shown to be enriched in embryo endomesoderm fractions, are the predominant, if not exclusive,SpFGFRtranscripts in lantern muscle. Together, these results suggest that only a minority ofSpFGFRtranscripts are processed, exported, and translated in both adult and embryonic muscle cells and these contain predominantly, if not exclusively, IgS ligand binding domain sequences

Integration of Canonical and Noncanonical Wnt Signaling Pathways Patterns the Neuroectoderm Along the Anterior–Posterior Axis of Sea Urchin Embryos

<div><p>Patterning the neuroectoderm along the anterior–posterior (AP) axis is a critical event in the early development of deuterostome embryos. However, the mechanisms that regulate the specification and patterning of the neuroectoderm are incompletely understood. Remarkably, the anterior neuroectoderm (ANE) of the deuterostome sea urchin embryo expresses many of the same transcription factors and secreted modulators of Wnt signaling, as does the early vertebrate ANE (forebrain/eye field). Moreover, as is the case in vertebrate embryos, confining the ANE to the anterior end of the embryo requires a Wnt/β-catenin-dependent signaling mechanism. Here we use morpholino- or dominant negative-mediated interference to demonstrate that the early sea urchin embryo integrates information not only from Wnt/β-catenin but also from Wnt/Fzl5/8-JNK and Fzl1/2/7-PKC pathways to provide precise spatiotemporal control of neuroectoderm patterning along its AP axis. Together, through the Wnt1 and Wnt8 ligands, they orchestrate a progressive posterior-to-anterior wave of re-specification that restricts the initial, ubiquitous, maternally specified, ANE regulatory state to the most anterior blastomeres. There, the Wnt receptor antagonist, Dkk1, protects this state through a negative feedback mechanism. Because these different Wnt pathways converge on the same cell fate specification process, our data suggest they may function as integrated components of an interactive Wnt signaling network. Our findings provide strong support for the idea that the sea urchin ANE regulatory state and the mechanisms that position and define its borders represent an ancient regulatory patterning system that was present in the common echinoderm/vertebrate ancestor.</p> </div

Preventing Wnt/β-catenin signaling allows activation of presumptive ANE specification throughout the embryo.

<p>(A) Diagram showing the regulatory factors shared between the sea urchin ANE and the vertebrate forebrain/eye field. Both territories are restricted by mechanisms dependent on posterior Wnt signaling. (B, D, F, H) <i>foxq2</i> expression in glycerol control (B, D) or Axin mRNA-injected embryos (F, H) at the 32-cell stage (B, F) and late blastula stage (D, H). <i>six3</i> expression in glycerol control (C) and TCF-Eng mRNA-injected early blastulae (G). (E) A normal 3.5-d pluteus stage embryo. (I) A 3.5-d embryo misexpressing Axin mRNA. White box outlines the ANE. Serotonergic neurons (green), DAPI (nuclei, blue). (J) Schematic showing the stages of ANE restriction in 32-cell stage (6 hpf) (a), early blastula stage (7–15 hpf) (b), and mesenchyme blastula stage (24 hpf) embryos (c). ANE (blue), nβ-catenin (orange nuclei at 32-cell stage), endomesoderm territory (orange at blastula stages), posterior ectoderm (gray at blastula stages), and unknown restriction mechanism activated by posterior nβ-catenin (orange arrows).</p