Plouhinec JL et al. (2013), Chordin forms a self-organizing morphogen gradi...

XB-ART-47690

Proc Natl Acad Sci U S A December 17, 2013; 110 (51): 20372-9.

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Chordin forms a self-organizing morphogen gradient in the extracellular space between ectoderm and mesoderm in the Xenopus embryo.

Plouhinec JL , Zakin L , Moriyama Y , De Robertis EM .

Abstract
The vertebrate body plan follows stereotypical dorsal-ventral (D-V) tissue differentiation controlled by bone morphogenetic proteins (BMPs) and secreted BMP antagonists, such as Chordin. The three germ layers--ectoderm, mesoderm, and endoderm--are affected coordinately by the Chordin-BMP morphogen system. However, extracellular morphogen gradients of endogenous proteins have not been directly visualized in vertebrate embryos to date. In this study, we improved immunolocalization methods in Xenopus embryos and analyzed the distribution of endogenous Chordin using a specific antibody. Chordin protein secreted by the dorsal Spemann organizer was found to diffuse along a narrow region that separates the ectoderm from the anterior endoderm and mesoderm. This Fibronectin-rich extracellular matrix is called "Brachet''s cleft" in the Xenopus gastrula and is present in all vertebrate embryos. Chordin protein formed a smooth gradient that encircled the embryo, reaching the ventral-most Brachet cleft. Depletion with morpholino oligos showed that this extracellular gradient was regulated by the Chordin protease Tolloid and its inhibitor Sizzled. The Chordin gradient, as well as the BMP signaling gradient, was self-regulating and, importantly, was able to rescale in dorsal half-embryos. Transplantation of Spemann organizer tissue showed that Chordin diffused over long distances along this signaling highway between the ectoderm and mesoderm. Chordin protein must reach very high concentrations in this narrow region. We suggest that as ectoderm and mesoderm undergo morphogenetic movements during gastrulation, cells in both germ layers read their positional information coordinately from a single morphogen gradient located in Brachet''s cleft.

PubMed ID: 24284174
PMC ID: PMC3870759
Article link: Proc Natl Acad Sci U S A
Grant support: [+]

Species referenced: Xenopus laevis
Genes referenced: admp bmp1 bmp2 chrd.1 fn1 szl
Antibodies: Smad1 Ab7

Article Images: [+] show captions

	Fig. 1. A gradient of BMP activity patterns the D-V axis of the Xenopus gastrula. (A) BMP activity along the D-V axis results from a series of direct protein–protein interactions between Chordin and other partners (black arrows), transcriptional regulation (blue arrows), and protein flux (red arrows). The entire embryo participates in forming the BMP gradient, which results from the dueling activities of the dorsal and ventral signaling centers. (B) Transverse optical section at gastrula (stage 11) showing a ventral- (V) to-dorsal (D) gradient of BMP activity using anti-pSmad1/5/8 antibody as readout. (C) Gastrula (stage 11) embryo sectioned sagittally, showing higher BMP activity in the ventral animal cap and marginal zone nuclei as assessed by pSmad1/5/8 immunostaining.
	Fig. 2. Overexpressed BMP-GFP fusion proteins diffuse in Brachet’s cleft. (A and B) Dorsally expressed zADMP-GFP and xBMP2-GFP fusion proteins diffuse within the narrow confines of Brachet’s cleft (marked by arrowheads) away from the point of mRNA injection in ectodermal cells (indicated by a red dotted line) (n = 7 and n = 6, respectively). GFP fusion proteins were detected by GFP immunostainings of transverse optical sections of stage-12 embryos through the animal–vegetal axis. (C) Diagram of stage-12 sagittal section of a Xenopus embryo (after P. Nieuwkoop, ref. 38). Brachet’s cleft is the narrow cavity that separates the mesodermal and anterior endodermal layers from the ectoderm and encircles the entire D-V axis.
	Fig. 3. Endogenous Chordin protein diffusion within Brachet’s cleft compared with the expression of chordin mRNA. (A and B) Immunostainings of Chordin protein in late gastrula (stage 12) embryos in transverse (n = 16) or sagittal (n = 15) sections, respectively, using an affinity-purified Chordin antibody. Chordin protein is detected throughout the entire length of Brachet’s cleft (arrowheads) forming a gradient from dorsal toward ventral. Chd, Chordin. (C) Imaging of the Chordin gradient following the entire circumference in a clockwise manner (circular arrow) or in a radial manner (numbered arrows). (D) Measurement of the Chordin gradient of the embryo seen in C. Note that the gradient forms over a very long distance of almost 2 mm; similar profiles were obtained in the four embryos analyzed. (E) Intensity of fluorescence plotted along five radial lines from central to peripheral. Line 1 is dorsal and shows Chordin protein peaks in organizer mesoderm and in Brachet’s cleft. In the other lines a peak is seen in Brachet’s cleft, even in the ventral-most region. (F and G) In situ hybridization of stage-12 embryo in transverse or sagittal section showing that chordin mRNA is transcribed only in the dorsal side (arrowheads); compare with the panels (A and B) showing Chordin protein localization at the same stage.
	Fig. 4. Analysis of BMP signaling and Chordin protein localization in embryos depleted of Tolloid (Tld MO) or Sizzled (Szl MO). All embryos were siblings allowed to develop for the same period; all images were processed identically. (A–F) pSmad1/5/8 (Upper) and Chordin (Lower) immunostainings of sagittal optical sections. The gradient of BMP activity was complementary to that of Chordin localization in WT embryos (Left, n = 6 and n = 4, respectively). When translation of the Chordin-degrading enzyme Tolloid was inhibited in Tld MO-injected embryos (Center, n = 5 and n = 3, respectively), BMP activity was decreased on the dorsal side and increased accumulation of Chordin was observed in Brachet’s cleft (arrowheads). Conversely, when Sizzled was depleted (Szl MO), BMP activity (nuclear pSmad1/5/8) was greatly increased in the embryo, and Chordin failed to accumulate in the Brachet’s cleft (Right, n = 3). Diffuse accumulation of Chordin in dorsal ectoderm is indicated by the arrow. (G–L) pSmad1/5/8 (Upper) and Chordin (Lower) immunostainings of embryos sectioned transversely through the animal–vegetal axis at late gastrula (stage 12). pSmad1/5/8 staining was decreased in Tld MO embryos (H, n = 6) and increased in Szl MO embryos (I, n = 4) compared with WT (G, n = 6). Tld MO increased (K, n = 7) and Szl MO decreased (L, n = 5) Chordin staining in Brachet’s cleft compared with WT (J, n = 7).
	Fig. 5. The Chordin and pSmad1/5/8 gradients self-regulate in dorsal half-embryos. The ventral side of albino embryos was marked at the 8-cell stage using the method described in Fig. S4. Embryos were bisected at stage 9, cultured until stage 12, fixed, and sectioned transversely. All embryos were siblings and images were processed identically. (A–C) pSmad1/5/8 immunostaining of whole embryo (A, n = 3), dorsal (B, n = 6) or ventral (C, n = 3 of 4 showing the phenotype) half-embryos. Note that the BMP gradient was reestablished in the dorsal half-embryo, while very strong uniform pSmad1/5/8 was present in the ventral half-embryo. (D–F) Chordin immunostaining of whole embryo (D, n = 3), dorsal (E, n = 10) or ventral (F, n = 7) half-embryo. The Chordin gradient was regenerated in the Brachet’s cleft of dorsal half-embryos (in 8 of 10), while no Chordin expression was detected in the ECM of the ventral half-embryo (in 6 of 7). Similar results were obtained in two independent experiments.
	Fig. 6. Chordin protein diffuses long-range in Brachet’s cleft of embryos transplanted with Spemann organizer tissue. (A) Diagram of experimental procedure. mGFP was injected in the donor embryo (four injections at the four-cell stage) to trace the lineage of grafted tissue. Secondary axes were complete (with heads, indicated with arrowheads in this photograph) in 75% of the cases (n = 6/8). (B–E) Two embryos transplanted at stage 10 and fixed at stage 12 stained with GFP and Chordin antibodies are shown in optical transverse sections. B and D show localization of the grafted organizer tagged with mGFP; borders are indicated by arrowheads. In C and E, a second gradient of Chordin was observed, with Chordin protein staining in Brachet’s cleft extending a considerable distance from the graft (arrowheads) (n = 19). This indicates that Chordin protein diffuses from the graft through Brachet’s cleft.
	Fig. S1. Chordin (Chd) depletion increases the BMP signaling (D-V) gradient. (A and B) pSmad1/5/8 immunostainings of injected embryos at stage 12 sectioned transversely between the animal–vegetal axis. pSmad1/5/8 signal is increased in the ventral side in Chd morpholino oligo (MO)-injected embryos (n = 6) compared with wild type (WT) (n = 6). (C) A secreted form of GFP does not diffuse in Brachet’s cleft (arrowhead) after overexpression (n = 5); this indicates that the observed long-distance diffusion of BMP–GFP fusions (zADMP-GFP and xBMP2-GFP) in Brachet’s cleft requires the BMP moiety. ADMP, anti-dorsalizing morphogenetic protein.
	Fig. S2. Schematic drawing of the anatomical structures observed in the embryos shown in Fig. 3. On the left, diagram of stage-12 embryo sectioned transversely between the animal–vegetal axis. On the right, a sagittally sectioned embryo; the dotted line indicates the plane of section used in the transverse sections shown throughout this study. Note that the archenteron cavity (definitive gut) provides an excellent reference point for the dorsal side. The red line indicates the Fibronectin-rich extracellular matrix (ECM) that separates the ectodermal and endomesodermal layers, called the “Brachet’s cleft” in Xenopus.
	Fig. S3. The Chordin antibody staining of ECM is specific. (A) Immunostaining with Chordin antibody of neurula (stage 14) wild-type (WT) embryo sectioned transversely. Signal is observed on the dorsal side in the notochord (where Chordin is transcribed, arrow) and in the spaces separating the presomitic mesoderm (pm) from ectoderm (upper arrowheads) and dorsal endoderm (lower arrowheads), in regions where Chordin is not actively transcribed (n = 7). (B) Transverse section of Chordin-depleted embryo at neurula (Chd MO) stained with Chordin antibody. Note that the lack of signal in ECM indicates that anti-Chordin staining is specific (n = 5). (C and D) Diagrams of the anatomical structures seen in neurula stage WT or Chd MO-injected embryos shown in A and B, re- spectively. Arch, archenteron; bc, blastocoel; endomes, endomesoderm; no, notochord. (E) Chordin staining in transverse section of WT embryo (n = 5) showing Chordin protein in Brachet’s cleft (arrowheads) and dorsal mesoderm (arrow). (F) Immunostaining of stage-12 embryo injected with Chd MO showing that Chordin signal was eliminated in both dorsal mesoderm and Brachet’s cleft, confirming the specificity of the antibody; note that some background staining remains in the ectoderm (n = 5).
	Fig. S4. The D-V polarity of the embryo can be predicted from the shape of the animal blastomeres at the eight-cell stage. Use of the vital dye Nile Blue sulfate allows marking of the ventral side of an albino embryo in preparation for bisection at blastula. (A) Animal (top) view of a pigmented eight-cell embryo showing the shape of the ventral cells spreading in a butterfly pattern and dorsal cells extending toward the equator. (B) A similar cell-shape pattern was observed in albino embryos, allowing a ventral blastomere to be marked with 20 mg/mL Nile Blue sulfate. (C) This pattern allows reliable marking of the ventral (or dorsal) side at the eight-cell stage as illustrated by this stage-10 embryo showing a ventral Nile Blue mark. (D–F) Bisection experiment in which pigmented or albino embryos were bisected at the blastula stage into dorsal and ventral halves (pigmented embryos are shown here). The dorsal half rescales to form a smaller but perfectly patterned embryo, whereas the ventral half lacks axial structures. In albino bisected embryos, all presumptive dorsal halves formed an axis (n = 20), whereas in presumptive ventral halves, only one (n = 12) formed an axis. The amount of Nile Blue sulfate should be kept to a minimum as it can quench fluorescence. Although Nile Blue sulfate was used in this study, other lineage tracers may be preferable in future studies.

References [+] :

Ambrosio, Crossveinless-2 Is a BMP feedback inhibitor that binds Chordin/BMP to regulate Xenopus embryonic patterning. 2008, Pubmed, Xenbase