Forming and interpreting gradients in the early Xenopus embryo

Abstract

The amphibian embryo provides a powerful model system to study morphogen gradients because of the ease with which it is possible to manipulate the early embryo. In particular, it is possible to introduce exogenous sources of morphogen, to follow the progression of the signal, to monitor the cellular response to induction, and to up- or down-regulate molecules that are involved in all aspects of long-range signaling. In this article, I discuss the evidence that gradients exist in the early amphibian embryo, the way in which morphogens might traverse a field of cells, and the way in which different concentrations of morphogens might be interpreted to activate the expression of different genes.

Figure 1.

Mesoderm induction. (A) Drawing of a Xenopus embryo at the midblastula stage (Nieuwkoop 1956). The animal hemisphere is to the top and the vegetal hemisphere to the bottom. The mesoderm forms in the equatorial region of the embryo between the two. (B) Mesoderm induction. A signal from the vegetal hemisphere of the embryo (white arrows) causes equatorial cells to form mesoderm. (C) Demonstration of mesoderm induction. Animal pole tissue derived from an embryo uniformly labeled with a fluorescent lineage label is juxtaposed with vegetal pole tissue from an unlabeled embryo and the resulting conjugate is cultured for 3 days. (D) The result of such an experiment. The fluorescently labeled tissue differentiates as notochord and muscle rather than epidermis. Photograph courtesy of Les Dale and Jonathon Slack.

Figure 2.

Threshold formation. (A,B) Normal expression of Xbra. (A) Sectioned in situ hybridization showing expression of Xbra in red. Animal pole is to the top and vegetal pole to the bottom. Photograph courtesy of Linda Essex and Michael Sargent. (B) Whole-mount in situ hybridization viewed from the vegetal pole. Notice expression of Xbra throughout the marginal zone. (C) Whole-mount in situ hybridization showing expression of goosecoid in the dorsal region of the embryo. Photograph by Susie Zoltewitz. (http://www.xenbase.org/xenbase/original/WWW/Marker_pages/organizer/goosecoid.html). (D) Differential expression of Xbra in response to different concentrations of activin. Disaggregated animal pole cells were exposed to the indicated concentrations of activin, reaggregated, and cultured until the equivalent of early gastrula stage 10. They were then assayed for expression of Xbra and EF-1α by RNase protection. Note that Xbra is only activated by intermediate concentrations of activin. (E) Activation of Xbra by intermediate concentrations of activin. An activin-soaked bead was placed between two animal pole regions and cultured for 5 hours before being assayed for expression of Xbra. Note that Xbra is not activated close to the bead but at a distance, where the concentration of activin is at the appropriate level (Papin and Smith 2000). (F) Activation of goosecoid by high levels of activin. An identical experiment to that illustrated in (E), but assaying for expression of goosecoid (Papin and Smith 2000). (G,H) An attempt to understand threshold formation. (G) Left-hand side illustrates that activin activates both goosecoid and Xbra, and the two gene products repress each other's expression. The same relationshiop with activin represented as M (morphogen) and goosecoid and Xbra as “A” and “B”, respectively. (H) Equations describing the rates of synthesis of A and B in response to M. M, A, and B represent the concentrations of each component, k_a and k_b are the synthesis rates of A and B, respectively, kd_a and kd_b are the decay rates, α and μ are the cooperativities of repression of A by B and B by A, respectively, and μ is the cooperativity of induction by M (Saka and Smith 2007).

Figure 3.

Following long-range signaling in the Xenopus embryo. (A) Animal pole tissue expressing enhanced green fluorescent protein (EGFP)-tagged Xnr2 (cells with red membranes) was juxtaposed with unlabeled tissue and cultured for 2.5 hours. Note the presence of EGFP fluorescence (green) in the unlabeled tissue. Photograph courtesy of Hugh Williams. (B) Visualising activation of the activin signal transduction pathway by Smad2/4 bimolecular fluorescence complementation (BiFC). Animal pole regions derived from embryos previously injected with 25 ng (top) or 50 ng (bottom) mRNA encoding activin (left-hand side) were juxtaposed with tissue derived from embryos expressing GPI-CFP as a membrane-bound lineage marker (right-hand side). Both tissues were also expressing Smad2/4 BiFC constructs. Note nuclear fluorescence in cells at a distance from the source of activin, and that the range of signaling is greater when cells are expressing greater levels of activin. Photograph courtesy of Anja Hagemann.