ECB-ART-43089Evodevo 2013 Nov 04;41:31. doi: 10.1186/2041-9139-4-31.
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Nuclearization of β-catenin in ectodermal precursors confers organizer-like ability to induce endomesoderm and pattern a pluteus larva.
BACKGROUND: In many bilaterians, asymmetric activation of canonical Wnt (cWnt) signaling at the posterior pole is critical for anterior-posterior (AP) body axis formation. In 16-cell stage sea urchins, nuclearization of β-catenin in micromeres activates a gene regulatory network that defines body axes and induces endomesoderm. Transplanting micromeres to the animal pole of a host embryo induces ectopic endomesoderm in the mesomeres (ectoderm precursors) whereas inhibiting cWnt signaling blocks their endomesoderm-inducing activity and the micromeres become ectoderm-like. We have tested whether ectopic activation of cWnt signaling in mesomeres is sufficient to impart the cells with organizer-like abilities, allowing them to pattern normal embryonic body axes when recombined with a field of mesomeres. RESULTS: Fertilized eggs were microinjected with constitutively active Xenopus β-catenin (actβ-cat) mRNA and allowed to develop until the 16-cell stage. Two mesomeres from injected embryos were then recombined with isolated animal halves (AH) from uninjected 16-cell stage embryos. Control chimeras produced animalized phenotypes (hollow balls of ectoderm) and rarely formed skeletogenic mesoderm (SM)-derived spicules, endoderm or pigment cells, a type of non-skeletogenic mesoderm (NSM). In contrast, over half of the 0.5 pg/pL actβ-cat mesomere/AH chimeras formed a partial or complete gut (exhibiting AP polarity), contained mesenchyme-like cells similar to SM, and produced pigment cells. At three days, chimeras formed plutei with normal embryonic body axes. When fates of the actβ-cat mRNA-injected mesomeres were tracked, we found that injected mesomeres formed mesenchyme-like and pigment cells, but endoderm was induced. Higher concentrations of actβ-cat mRNA were less likely to induce endoderm or pigment cells, but had similar mesenchyme-like cell production to 0.5 pg/pL actβ-cat mesomere/AH chimeras. CONCLUSIONS: Our results show that nuclear β-catenin is sufficient to endow naïve cells with the ability to act as an organizing center and that β-catenin has both cell-autonomous and non-autonomous effects on cell fate specification in a concentration-dependent manner. These results are consistent with the hypothesis that a shift in the site of early cWnt signaling in cleaving embryos could have modified polarity of the main body axes during metazoan evolution.
PubMed ID: 24180614
PMC ID: PMC3835408
Article link: Evodevo
Genes referenced: LOC100887844 LOC100888042 LOC115919910 LOC590297 LOC594353 pole
Article Images: [+] show captions
|Figure 1. Summary of the controls used in experiments. (A-D) Controls, bright field images. (C’, D’) Controls, fluorescent images. (A) Uninjected embryo at 27 hours post fertilization (hpf). (B) The uninjected animal half (AH) forms a hollow ciliated ball of ectoderm lacking endomesoderm and patterning along the oral-aboral axis (dauerblastula), 24 hpf. (C, C’) A chimera was made at the 16-cell stage in which micromeres removed from an embryo injected with rhodamine-dextran (RDX) were recombined with the uninjected AH. This produces a nearly normal prism stage embryo, although few pigment cells were present in many individuals. The RDX-labeled micromeres contributed to mesenchyme-like cells and occasionally to pigment cells (not shown). (D, D’) When RDX-labeled mesomeres from a 16-cell embryo were recombined with an AH, the chimera formed a dauerblastula and most of the labeled cells remained in the ectoderm. Note the expanded apical plate at the animal pole (B and D, D’). This is a typical feature of the dauerblastula. Orientations of the embryos are as follows: (A) Lateral view, oral side up. (B, C, C’, D, D’) All other images are frontal views with the animal pole oriented towards the top. Scale bars = 100 μm.|
|Figure 2. Summary of cell transplantation experiments and results at 23 to 27 hours post fertilization. (A) A mesomere pair from a 16-cell embryo injected with actβ-cat mRNA (shaded embryo) was recombined with the eight mesomeres of an uninjected animal half (AH). (B-G) Bright field images of chimeras in which a labeled mesomere pair injected with 0.5 pg/pL actβ-cat mRNA was recombined with an uninjected AH. (B’-G’) Corresponding fluorescent images. Labeled/injected mesomeres formed mesenchyme-like cells and pigment cells. Absence of label in the gut (gut indicated with arrowheads in B’, C’, D’) suggests that this tissue is induced in mesomeres from the uninjected AH. Some embryos were similar to uninjected controls (B, B’, C, C’, F, F’, G, G’), forming a complete gut, mesenchyme-like cells (indicated with arrows in B’) that produced spicules, and pigment cells (development in the injected cells and transplants is delayed compared to that in uninjected embryos). Others (D, D’) only formed a partial gut or lacked a gut (E, E’). (F, F’-G, G’) In some, the labeled cells were observed at the tip of the archenteron (arrowheads in F’ and G’). In this Figure, labeled mesenchymal cells are also clearly evident in the blastocoel. These are likely skeletogenic mesoderm and/or non-skeletogenic mesoderm cells such as pigment cells, but it is interesting that, unlike other chimeras, many of the labeled cells in these individuals remain concentrated at the tip of the archenteron. Orientation: (B, B’) vegetal view, (C-E, C’-E’) lateral views. (F-G, F’-G’) Frontal views with the animal pole at the top of the photograph.|
|Figure 3. Results of cell transplantation experiments at 3 days post fertilization. (A) Uninjected control. (B) Animal half (AH) control. (C) Bright field image of a 3-day chimera formed from a labeled mesomere pair injected with 0.5 pg/pL actβ-cat mRNA recombined with an uninjected AH. Note that features such the spicules, a tripartite gut, and pigment cells are all present. Larvae appear to be normal. (C’) Fluorescent image shows that the actβ-cat mRNA-injected mesomeres gave rise to mesenchyme-like cells and pigment cells (arrows in C, C’, D, and D’ indicate pigment cells). (D, D’) Different focal plane in the same embryo, showing that cells in the gut are not derived from the actβ-cat mRNA-injected mesomeres. Arrowheads in C, C’, D, and D’ indicate the induced, unlabeled gut.|
|Figure 4. Chimeras generated using higher levels of actβ-cat (1.0 pg/pL). (A) Some embryos appeared to be nearly normal, forming spicules, a gut, and pigment cells. (B) In other cases, the gut was absent, but many pigment cells and some skeletal elements (arrowhead) are present, or (C) few pigment cells formed. All images are lateral views.|
References [+] :
Angerer, The evolution of nervous system patterning: insights from sea urchin development. 2011, Pubmed, Echinobase